DIFFERE NTIAL MICR OR NA REGULATI O N B Y P DE5 A N D P DE9 I N T HE HEART A N D T HEIR I NTRA -MYOCYTE CO MPART MENTATION

by

Kristen Kokkonen -Si m o n

A dissertati o n s u b mitte d t o J o h ns H o p ki ns U ni versity i n c o nf or mity wit h t he re q uire me nts

for the degree of Doctor of Philosophy.

Balti m ore, Maryla n d

Marc h, 201 9

© Kristen Kokkonen -Si m o n 2 0 1 9

All ri g hts reser ve d

ABSTRACT

Pr otei n ki nase G ( P K G) acti vati o n is pr otecti ve i n vari o us car di o vasc ular diseases, including ische mia, hyper tr o p hy, a n d heart fail ure. P K G is acti vate d by cyclic g ua n osi ne monophosphate (c G M P), which is produced by guanylyl cyclases and degraded by phosph o diesterases ( P D Es). I n t he heart, t here are t w o c G MP -selecti ve P D Es , P D E 5 a n d

P D E 9. I n hi bi ti o n of eit her P D E is protective in a mouse pressure -overl oa d -induced model of cardiac hypertrophy and failure, i mproving function and de creasi n g mala da pti ve re m o deli n g a n d pat h ol o gical m olec ular si g na li n g. H o we ver, d es pite t hese si milarities , t here is still m uc h t o be understood about the differential signaling afforded by these t wo enzy mes , i ncl u di n g t heir effec ts o n tra nscri pti o nal re g ulati o n a n d na n o -c o m part me nt pr otei n part neri n g.

T his t hesis re p orts t w o pri mary st u di es. T he firs t st u d y test e d t he hy p ot hesis t hat micro R N A and m R N A regulation by P D E5 and P D E9 re veals differe nt transcripto mic si g nat ures reflecti n g t heir na n o d o mai n c o ntr ol of c G M P. T o e xa mi ne t his, m R N A a n d mi R N A sequencing was perfor med on myocardi u m fr o m mice s u bjecte d t o press ure - overl oa d a n d treate d wit h eit her a P D E5 or P D E 9 i n hi bit or. In hi biti o n of eit her P D E si g nifica ntly alter e d m R N A tra nscri pti o n, wit h si milar path ways engaged by eac h tr eat me n t.

By c o nt rast, eac h i n hi bit or ha d a dra matically differe nt i m pact o n mi R N A expressi o n . P D E 5 i n hi biti o n broadly reduce d mi R N A e x pr essi o n , w hile P D E 9 i n hi biti o n ha d esse ntially n o i m pact o n mi R N As relat i ve t o press ure -o verl oa d. N ota bly, e ve n if a si milar sti m ulati o n pat h way was e ngaged, as bet ween P D E5 i n hi biti o n a n d s G C acti vati o n , t he mi R N A res ults were n ot f ully re plicati ve.

ii T he seco nd st u dy exa mine d where P D E5 a n d P D E 9 resi de i n t he car di o my ocyte, s pecifically i de ntifyi n g i nteracti n g pr otei ns i n t heir na n o d o mai n s through t wo approaches . I n t he first approach , I per for med c o -i m m u n o preci pitati o n of P D E9 f oll o we d by mass s pectr o metry . T his re veale d a cl uster of pr otei ns b o u n d t o P D E9 , i ncl u d i n g ma ny i n t he mit oc h o n drial me mbrane a n d a n u m ber of sarc o plas mic retic ul ar pr otei ns , which were lar gel y unchanged under pr o -hypertrophic gro wth c o n diti o ns . The second approach utilize d

AP E X2 -P D E fusion proteins . U p o n H 2 O 2 sti m ulati o n, A P E X 2 bi oti nylat es nei g h b ori n g pr otei ns , w hic h ca n be p ulle d d o w n a n d s u bjecte d t o mass s pectr o metry . AP E X2 -tagge d

P D E constructs were verifie d a n d o pti mize d , yet mass spectro metry a nalysis faile d t o i de ntify bi oti nylate d pr otei ns . O ng oi ng eff orts are re-e xa mi ni n g t hese s pectra t o i mprove i de ntificati o n of pr otei ns selecti vely bi oti nylate d by P D E5 and P D E9 .

A d vis or: Da vi d Kass, M D

Reader: Brian O’ Rourke, Ph D

iii ACKNO WLEDG MENTS

I w o ul d first a n d f ore m ost li ke t o t ha n k my a d vis or, Dr. Da vi d Kass. Wit h o ut his support and mentorship over the past 5 years, I w o ul d ne ver ha ve bec o me t he scie ntist t hat I a m n o w. Fr o m t he day I first a p pr oac he d hi m a b o ut a r otati o n, he has bee n h o nest a n d i nsi g htf ul, al ways tell i n g me his t h o u g hts a n d gi vi n g a d vice o n b ot h scie nce a n d life. I w o ul d als o li ke t o t hank my co m mittee me mbers: Brian O’ Rourke, Mark Anderson, and Brian

Foster, as well as Jin Zhang and Ryuya Fukunaga (for mer co m mittee me mbers ). I a m gratef ul f or t heir i n si g ht, as well as for David and Jin co ming back to rescue me fro m the

Dali Museu m befo re t he G R C b us left wit h o ut me.

I w o ul d ne xt li ke t o e x press my gratit u de t o my la b mates b ot h past a n d prese nt i n t he Kass la b i ncl u di n g: Grace Ki m, Bria n Li n, Britta ny Du nkerly -Eyri n g, D o n g Lee, S u mita

Mis hra, C hristia n Oei n g, Vir gi nia Ha h n, J o nat ha n Kir k, Tais hi Na ka m ura, a n d Ste ve Hs u. I n partic ular I w o ul d li ke t o t ha n k Mar k Ra ne k, w h o me nt ore d me w he n I was a r otati o n st u de nt a n d is still s o me o ne fr o m w h o m I fre q ue nt ly seek a dvice .

I w o ul d f urt her m ore li ke t o t ha n k t he Cell ular a n d M olec ular Me dici ne gra d uate pr o gra m, wit h o ut w h o m I w o ul d n ot be here at all. I partic ularly w o ul d li ke t o ac k n o wle d ge

C ollee n Gra ha m a n d Leslie Lic hter f or t heir s u p p ort o ver my years i n the C M M progra m.

Fi nally, I w o ul d li ke t o t ha n k my fa mily. I a m i m me nsely gratef ul t o my pare nts f or al ways encouraging me to pursue my passions, and supporting me every step of the way.

And of course, I don’t kno w what I would have done without my husband , Be n Si m o n, w h o t olerate d ma ny h o urs of scie ntific tal k t hat was mea ni n gless t o hi m , and encouraged me w he n e ver I felt fr ustrate d or defeate d.

i v T he w or k i n t his dissertati o n was s u p p orte d i n part by a R ut h L. Kirsc hstei n

National Research Service A ward In di vi d ual Pre d o ct oral Fell o ws hi p fr o m t he Nati o nal

L u n g, Heart, Bl o o d a n d L u n g I nstit ut e ( F 3 1 -H L134196).

v TABLE OF C O NTE NTS

PA G E

A bstract ...... ii

Ackno wledg ments ...... iv

List of T a bl es ...... vii

List of Fi g ur es ...... viii

C h a pt er 1: I ntr o d ucti o n ...... 1

C ha pter 2 : Mar ke d dis parity of micr o R N A m o d ulati o n by c G MP -selecti ve

P D E 5 vers us P D E 9 i n hi bit ors i n heart disease ...... 2 1

C ha pter 3 : I de ntificati o n of P D E 9 bi n di n g p artners in cardio myocytes via c o -i m munoprecipitation ...... 6 1

C ha pter 4 : Ge nerati o n a n d testi n g of n o vel m olec ular t o ols t o st u dy P D E 5 and P D E9 co mpart me ntati o n ...... 9 1

C h a pt er 5: General Discussion and Conclusions ...... 1 2 8

R e fere nces ...... 1 3 2

Curriculu m Vitae ...... 1 5 5

vi LIST OF TABL ES

PA G E

T a bl e 2 .1 : mi R N As differentially expressed in P O as co mpared to sha m ...... 5 0

T a bl e 2 .2 : mi R N As differentially expressed in P O +P D E5 -I as co mpared to P O ...... 5 3

T a bl e 2 .3 : mi R N As differentially expressed in P O +P D E5 -I as co mpared to P O ...... 5 7

T a bl e 2 .4 : Ro w legends for the heat map presented in Fi g ure 1. 2 D ...... 5 8

T a bl e 3 .1 : Pr otei ns si g nifica ntly e nric he d i n a d P D E 9 -FL A G as co mpared to adFL A G mass spectro metry sa mples ...... 7 9

T a bl e 3 . 2 : D A VI D pat h ways a nalysis of pr otei ns si g nifica ntly e nric he d i n adP D E9 -FL A G sa mples ...... 8 5

T a bl e 3 . 3 : Pr otei ns si g nifica ntly less ass ociate d wit h P D E 9 f oll o wi n g E T -1 treat me nt . . . . 8 7

T a bl e 3 . 4 : Pr otei ns si g nifica ntly m ore ass ociate d wit h P D E 9 f oll o wi n g E T -1 treat me nt . . . 8 9

T a bl e 4 .1 : Pri mers used for asse mbly of P D E5 and P D E9 GFP -AP E X2 fusion c o nstructs ...... 1 0 2

T a bl e 4 . 2 : Ge ne -specific pri mers used for sequencing of P D E5 and P D E9

G F P -A P E X2 fusion constructs ...... 1 0 3

T a bl e 4 . 3 : Si g nifica nt pr otei ns i n P D E 5 -G F P -A P E X2 analysis ...... 1 2 2

T a bl e 4 . 4 : Si g nifica nt pr otei ns i n P D E 9 -G F P -A P E X2 analysis ...... 1 2 3

T a bl e 4 . 5 : Path ways analysis for significant proteins fro m P D E5 -G F P -AP E X2 a nalysis ...... 1 2 6

T a bl e 4 . 6 : Pat h ways a nalysis f or si g nifi cant proteins fro m P D E9 -G F P -AP E X2 a nalysis ...... 1 2 7

vii LIST OF FI G U R ES

PA G E

Fi g ur e 1.1: Mechanis ms of P D E regulation , l ocalizati o n, a n d s u bstrate selecti vity ...... 3

Fi g ur e 1.2 : Co mparative structure and regula tion of cardiac P D Es ...... 5

Fi g ur e 1.3 : Cardiac myocyte regulation by P D E5 A and P D E9 ...... 1 2

Fi g ur e 1.4 : N O -independent P D E9 A modulation of cardia c stress res p o nses ...... 1 6

Fi g ur e 2 .1 : Phenotypic i mprove ment in P O mice after P D E5 -I and P D E9 -I ...... 2 8

Fi g ur e 2 . 2 : P D E5 -I and P D E9 -I a p plie d t o P O heart yiel d dis parate mi R N A pr ofiles . . . . 2 9

Fi g ur e 2 . 3 : Transcripto me for P D E5 -I and P D E9 -I s h o ws m any changes but fe w o verla p p ing ones ...... 3 2

Fi g ur e 2 . 4 : mi R N A dis parities fr o m differe nt P D E i n hi bit ors occ ur at late -sta ge p r ocessi ng ...... 3 5

Fi g ur e 2 . 5 : P he n oty pic i m pr o ve me nt i n P O mice f oll o wi n g G C -1 acti vati o n ...... 3 8

Fi g ur e 2 . 6 : Effect o n mi R N As fr o m alter nati ve sti m ulati o n of G C -1 or G C -2 A si g nali n g ...... 3 9

Fi g ur e 2 . 7 : P D E 5 i n hi biti o n i n t he n or mal heart a n d stresse d l u n g ...... 4 2

Fi g ur e 2 . 8 : P D E5 -I treat me nt d oes n ot alter mi R N A e x pressi o n i n n or mal m ouse lu ng ...... 4 4

Fi g ur e 3 .1 : P D E9 is enriched in adP D E9 -F L A G transduced adult ra b bit cardio myocytes ...... 7 0

Fi g ur e 3 . 2 : I P A path ways a nalysis of pr otei ns si g nifica ntly e nric he d i n P D E9 -FL A G sa mples sho ws many mitochondrial proteins ...... 7 3

Fi g ur e 3 . 3 : P D E 9 c ol ocalizes wit h S E R C A 2a i n a d ult ra b bit car di o my ocytes ...... 7 5

Fi g ur e 3 . 4 : P D E 9 bi n ds t o V D A C 2 i n a d ult ra b bit car di o my ocytes ...... 7 7

viii LIS T OF FI G U R E S

PA G E

Fi g ur e 4 .1 : P D E -G F P -A P E X2 fusion constructs express in neonatal cardiac my ocytes ...... 1 0 5

Fi g ur e 4 . 2 : G F P -AP E X2 -tagging of P D E5 and P D E9 near the catalytic do main d oes n ot i n hi bit c G M P hy dr olysis ...... 1 0 8

Fi g ur e 4 . 3 : G F P -A P E X 2 si g nal c ol ocalizes wit h res pecti ve P D E i n ne o na tal and adult cardio myocytes ...... 1 1 0

Fi g ur e 4 . 4 : N -acetylcystei ne i ncreases o verall bi oti nylati o n by A P E X 2, w hile cycl o he xi mi de has n o i m pact ...... 1 1 2

Fi g ur e 4 . 5 : Bi oti nylate d pr ot ei ns re mai n c o nstrai ne d t o t he l ocal P D E c o m part me nts in adult cardio myocytes ...... 1 1 4

Fi g ur e 4 . 6 : Bet ween -group analysis sho ws H 2 O 2 d oes n ot c o ntri b ute t o varia nce as much as expected ...... 1 1 7

Fi g ur e 4 . 7 : Co mparison of P D E9 AP E X2 data to P D E9 co -i m munoprecipitation data s h o ws litt le overla p ...... 1 1 9

i x

C H A P T E R 1: Introduction

P hosphodiesterases ( P D Es) are me mbers of an enzy me superfa mily that hydrolyze cyclic adenosine or guanosi ne 3 ¢, 5 ¢-monophosphate (c A M P or c G M P, respectively). In so d oi n g, t hey pr o vi de fi nely t u ne d a n d l ocalize d re g ulati o n of t hese sec ond messengers to c o ntr ol t heir r oles i n cell si g nali n g. Si nce t he disc o very of P D Es j ust o ver half a ce nt ury a g o, researc h has uncover e d a m ulti plicity of s u b gr o u ps a n d is of or ms, t he di verse r oles playe d by t hese e nzy mes, a n d t he t hera pe utic val ue t hat ca n be glea ne d by t heir selecti ve i n hi biti o n.

So me inhibitors are used widely to treat diseases such as pul monary hypertension an d erecti le dysf u ncti o n, b ut ot hers are bei n g i n vesti gate d acti vely wit h res ur ge nt i nterest i n t heir bi ol o gy a n d re g ulati o n. Bey o n d well -recognized differences a mong the pri mary me mbers of the P D E superfa mily, recent studies have revealed major differences i n t heir re g ulati o n ste m mi n g fr o m t he precise l ocalizati o n of t hese e nzy mes wit hi n cells. T his na n o d o mai n regulation depends on c ell ty pe, P D E s u bty pe, a n d is of or m a n d is yiel di n g ne w ways t o le vera ge t heir m o d ulati o n t o ac hie ve vari o us t hera pe utic g oals. T his re v ie w discusses ne w i nsi g hts i nt o c o m part me ntati o n of P D E si g nali n g wit h a f oc us o n t heir r ole i n t he heart i n b ot h healt h and disease. We refer the reader to several excellent revie ws on P D Es 1, 2 t hat pr o vi d e extensive and co mple mentary infor mation to what is presented here.

Cyclic Nucleotide P D Es: General Features: The P D E superfa mi ly co mprises 1 1 pri mary me mbers transcribed fro m 21 genes that in turn generate nearly 100 different isofor ms. Each me mber shar es a si milar str uct ure b ut is f u ncti o nally u ni q ue. T he e xte nsi ve n u m ber of is of or ms arises fr o m m ulti ple tra nscri pti o n start site s a n d alter nat i ve s plici n g, a n d t hese feat ures, al o n g wit h tiss ue - or cell -s pecific e x pressi o n a n d i ntracell ular l ocalizati o n, res ult i n

1

i n di vi d ualize d si g nali n g r oles 3 . Each P D E (and often isofor m) has different affin ities f or t he t w o cyclic n ucle oti des, fa v ori n g o ne or t he ot her t o s o me de gree. P D E 1, 2, 3, 1 0, a n d 1 1 hydrolyze both c A MP and c G M P, whereas P D E 4, 7, a n d 8 are c A M P s pecific a n d P D E 5 A,

6, a n d 9 A are c G M P s pecific. Of t hese, P D E 1 -5, P D E8, and P D E9 are expre sse d a n d f u ncti o nal i n t he heart.

Each P D E contains a conserved C -ter mi nal catalytic re gi o n as well as a varia ble

N ter mi n us. T he catalytic site c o nsists of a p pr o xi mately 3 5 0 c o nser ve d a mi n o aci ds.

Of t hese, a n i n varia nt gl uta mi ne resi d ue hel ps f or m b o n ds wit h b ot h cyclic n ucle oti des a n d P D E i n hi bit ors (false s u bstrates), a n d a hi g hly c o nser ve d p he nylala ni ne resi d ue ai ds partic ularl y i n b o n d f or mati o n t o i n hi bit ors 4, 5 . Together, these conserved hydrophobic residues act as cla m ps t o l oc k i n hi bit ors i n t he acti ve site. T he b o n ds f or me d deter mi ne t he orie ntati o n of t he i n hi bit or i n t he catalyti c site, ulti mately i m pacti n g h o w well it ca n i n hi bit t he P D E. H o we ver, varia ble resi d ues i n t he acti ve site als o pr o vi de s u bty p e i n hi bit or s pe cificity. F or e xa m ple, P D E 9 A has a Tyr 4 2 4 resi d ue, a site t hat is a P he i n all ot her is of or ms, all o wi n g f or t he de si g n of hi g hly s pecific P D E 9 A i n hi bit ors 6, 7 . T he varia ble N -ter mi nal re gi o n of eac h P D E provides f u ncti o nal a n d l ocalizati o n differe nces. I n a d diti o n, t he N -ter mi nal re gi o n ca n vary i n p osttra nslati o nal m o dificati ons (such as phosphorylation sites) t hat ulti mately i m pact

P D E acti vity a n d c o m ple xi n g wit h ot her pr otei ns i n si g nal os o mes. Fi g ur e 1.1 de picts t he maj or m olec ular feat ures t hat c o ntr ol P D Es, i ncl u di n g all osteric re g ulati o n by cyclic n ucle oti des (e.g., P D E 2 A; Fi g u r e 1 .1 a ), i ntracell ular tar geti n g by N -ter mi nal re gi o n -l ocalizi n g se q ue nces (e.g., P D E 4 A 1; Fi g u r e 1 .1 b ), a n d s u bstrat e selecti vity i n t he catalytic d o mai n (e.g., P D E 1 0 A 2; Fi g u r e 1 .1 c , d ). Fi g u r e 1 .2 sho ws sche matics f or t he ge neral structure a n d re g ulat ory m o d ulati o n f or all t he P D Es f o u n d i n heart.

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Fi g ur e 1.1: Mechanis ms of P D E regulati o n, l o c ali z atio n, a n d s u bstrate s el e cti vit y.

Fi g ur e 1.1: Mechanis ms of P D E regulation, localization, and substrate selectivity. (a)

All os teric re g ulati o n of P D E 2 A. T he di meric pr otei n s h o ws a n i nter m olec ular i nterface al o n g t he e ntire le n gt h of t he t w o m o n o mers, i ncl u di n g bi n di n g of t he t w o catalytic d o mai ns t hat i n hi bit cyclic n ucle oti de hy dr olysis. A n H -loop do main (residues 702 -7 2 3) f ol d s i n a p ositi o n t o bl oc k t he acti ve bi n di n g site. H o we ver, w he n c G M P bi n ds t o t he G A F -B re g ulat ory d o mai n ( These motifs are na m ed ‘ G A F do mains’ for their presence in c G M P - re g ulate d cyclic n ucle oti de P D Es, certai n a de nylyl cyclases a n d t he bacterial tra nscr i pti o n fact or F hl A) , t he H -l o o ps s wi n g o ut, se parati n g t he catalytic d o mai ns a n d e x p osi n g t he acti ve sites. T his res ults i n e n h anced c A M P hydrolysis. Panel adapted fro m Refere nce 8 wit h per mission 8 . ( b) N ter mi n us tar geti n g of P D E 4 A 1. P D E 4 is of or ms c o ntai n N T Rs i n t he first t wo exons t hat c o de f or effecti ve i ntracell ular zi p c o des, tar geti n g t he pr otei n t o s pecific

3

l ocati o ns i n t he cell. T he e xa m ple here is for the supershort for m of P D E4 A1, where the

N T R c o ntai ns t w o heli x re gi o ns se parate d by a li n ker. Calci u m i nteracti o n wit h a n as partat e resi d ue ( As p 2 1) tri g gers a c o nf or mati o nal c ha n ge i n heli x -2, all o wi n g t w o try pt o p ha ns

( Tr p 1 9 a n d 2 0) a n d o ne le uci ne ( L e u 1 6) t o i nsert i nt o t he G ol gi me m bra ne f or s pecific tar geti n g. Pa nel a da pte d fr o m Refere nce 9 wit h per missi o n 9 . ( c) c A M P place me nt i n t he catalytic site of t he d ual -s u bstrate esterase P D E 1 0 A 2. Bi n di n g is i n t he syn c o nf or mati o n.

Zi nc ( red ball ) coordinates bonds bet ween His529, His563, Asp564, and Asp674. The second, less clearly defi ne d metal ( Me -2) is s h o w n by a purple ball. Hydrogen bonds are denoted by d otte d li nes. T he i n varia nt Gl n 7 2 6 ma kes t w o hy dr o ge n b o n ds wit h N 1 a n d N 6 of c A M P, the adenine ring is e mbraced by the hydrophobic cla mp for med by Phe729 on one side and

P he 6 9 6 a n d Ile 6 9 2 o n t he ot her si de, a n d the cyclic phosphate group for ms one hydrogen b o n d wit h His 5 2 5 a n d t hree b o n ds wit h water m olec ules ( n ot s h o w n). ( d) c G M P place me nt i n t he catalytic site of P D E 1 0 A 2. c G M P bi n di n g i n v ol ves a n a p pr o xi mately 1 8 0° orie ntati o n fli p as c o m pare d t o c A M P, b ut t h e guanine is sand wiched in the sa me hydrophobic cla mp a n d t he p h os p hates occ u py a si milar l ocati o n. I n c o ntrast t o c A M P b i n di n g, t he i n varia nt

Gln726 makes only one hydrogen bond with N7 of c G MP. Panels c a n d d adapted fro m

Refere nce 2 wit h per missi o n. A b b re viati o ns: c A M P, cyclic a de n osi ne 3 ¢, 5 ¢-monophosphate; c G M P, cyclic g ua n osi ne 3 ¢, 5 ¢-monophosphate; G AF do main; N T R, N -te r mi nal re gi o n;

P D E, phosphodiesterase.

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Fi g ur e 1.2: Co mparative structure and regulation of cardiac P D Es.

Fi g ur e 1.2: C o mparative structure and regulation of cardiac P D Es.

Eac h P D E is s h o w n wit h c orres p o n di n g details pertai ni n g t o its re g ulat ory N -ter mi nal re gi o n. P h os p h orylati o n sites a n d t he ass ociate d ki nases are s h o w n, al o n g wit h cyclic n ucle oti de -- binding G AF do mains , upstrea m conserved regions ( U C Rs), trans me mbrane d o mai n ( T Ms), cal m o d uli n ( Ca M) bi n di n g sites, a n d me m bra ne a nc h ori n g d o mai ns. Ot her a b bre viati o ns: Ca M KII, calci u m -cal modulin dependent kinase -II; c G, cyclic g ua n osi ne 3 ¢, 5 ¢- monophosphate; E R K2, extracellu la r si g nal -re g ulate d ki nase 2; G A F d o mai n; P A S d o mai n;

P D E, p h os p h o diesterase; P K A, pr otei n ki nase A; P K B, pr otei n ki nase B.

5

Virt ually all P D E i n hi bit ors, w het her i de ntifie d by tra diti o nal s mall -m olec ule screening or designed fro m kno wn structural c he mistry, tar get t he catalytic site. Br oa d - base d i n hi bit ors were disc o vere d first, n ota bly t he xa nt hi ne deri vati ves s uc h as theop hylli ne, caffei ne, a n d 3 -is o b utyl -1 -met hyl xa nt hi ne (I B M X). T he o p hylli ne was t he earliest P D E -i n hi biti n g p har mace utical t o bec o m e a dr u g i n t he early t we ntiet h ce nt ury. It c o m bi ne d i n otr o pic, di uretic, a n d br o nc h os pas m olytic pr o perties.

H o we ver, it als o i n d uced arrhyth mias, including tachycardia, and could cause nausea, v o miti n g, or w orse, seiz ures. It was als o fairly wea k, a n d e ve n lo w doses could produce seri o us a d verse effects. T his lac k of p ote ncy a n d n o nselecti ve acti vity di mi nis he d its t hera peutic use. I B M X was use d wi dely i n cell ular a n d bi oc he mistry st u dies, b ut it i n hi bite d all P D Es e xce pt P D E 8 a n d P D E 9. Met h o ds t o better i de ntify a n d se parate

P D E isofor ms using anion exchange chro matography were reported in the 1970s, and wit h t he re p ort fr o m Ree ves et al. in 1987 1 0 , t he c urre nt syste m of P D E1 -P D E5 A was established. Subsequent work by Conti & Beavo 1 1 esta blis he d t he c urre nt s u perfa mily classificati o n. Selecti ve P D E se parati o n e na ble d i de ntificati o n of selec ti ve a nta g o nists, such as a mrinone for P D E 3 and rolipra m for P D E4. Ne wer efforts are taking advantage of molecular structural data to generate even more selective and po te nt

i n hi bit ors, wit h I C 5 0 values in the nano molar or pico molar range.

c A MP and c G MP ar e critical second messengers influencing m a ny si g nali n g pat h ways i n t he heart. c A M P is pr o d uce d by 1 of 1 0 a de nylyl cyclase ( A C) is of or ms ( 9 of which are trans me mbrane), wit h differe ntial e x pressi o n a m o n g cell ty pes. I n t he heart, Ca 2 + -i n hi bite d A C 5 a n d A C 6 do minate, whereas Ca 2 + -acti vate d A C 1 a n d A C 7 are e x presse d i n si n oatrial n o dal cells 1 2 . c A M P ca n i n t ur n acti vate pr otei n ki n ase A

( P K A) or t he g ua ni ne -nucleotide exchange factor Epac 1 3 , b ot h ca pa ble of m o difyi n g

6

cellular gro wth and function. P K A provides rapid response changes to stress by p h os p h orylati n g a br oa d variety of tar gets, i ncl u di n g t he v olta ge -gate d L -ty pe calci u m c ha n nel ( L T C C); t he calci u m cycli n g pr otei ns p h os p h ola m ba n ( P L B) a n d rya n o di ne rece pt or ( Ry R 2); t he my ofila me nt pr otei ns my osi n bi n di n g pr otei n C, titi n, tr o p o ni n I; a n d o t her pr otei ns. Phosphorylation of these proteins enhances Ca 2 + i nfl u x a n d cycli n g by t he sarc o plas mic retic ul u m ( S R) as well as sarc o mere c o ntractility, rela xati o n, a n d my ocyte diste nsi bility. E pac is ass ociate d wit h i ntracell ular Ca 2 + m o d ulati o n by calci u m -cal m o d uli n dependent kinase -II ( Ca M KII), t he p h os p hatase calci ne uri n, a n d

G T P -bi n di n g pr otei ns 1 4 . c A M P can also activate c A M P response -ele me nt bi n di n g pr otei n ( C R E B) via P K A -mediated phosphorylation to pro mote gene expression 1 5 .

Depending on the precise nature of c A M P sti mulation and its corresponding d o w nstrea m acti vat ors, c hr o nic sti m ulati o n ca n be detri me ntal t o b ot h car diac str uct ure a n d f u ncti o n. I n a d diti o n t o c o ntracti o n, c A M P als o re g ulates heart rate by affecti n g hy per p olarizati o n -acti vate d cyclic n ucle oti de gate d -c ha n nels ( H C Ns) 1 6 . I n mice, ge ne de leti o n of t he m ost pr o mi ne nt is of or m, H C N 4, res ults i n mar ke d sl o wi n g of heart rate a n d heart bl oc k 1 7 . H o we ver, c ha n nel gati n g of H C N 4 is re g ulate d n ot o nly b y c A M P 1 8 b ut a lso by P K A phosphorylation via a parallel path way 1 9 .

c G M P is produced by one of t wo g ua nylyl cyclases ( G Cs) 2 0 . T he first is a s ol u ble

G C (s G C), l ocate d b ot h i n t he c ytoplas m and at the plas ma me mbrane 2 1, 2 2 , t hat is acti vate d by nitric o xi de ( N O) pr o d u ce d by N O sy nt hase ( N O S). I n my ocytes, β 3 - adre nergic rece pt ors ( β 3 -A Rs) acti vate t his e nzy me 2 3 -2 5 . T he sec o n d G C is a n intracellular me mbrane -l ocalize d e nzy me t hat is c o u ple d t o t he natri uretic pe pti de

( N P) rece pt o r ( G C -A / B) a n d acti vate d u p o n r ece pt or a g o nis m. c G M P i n t ur n acti vates pr otei n ki nase G ( P K G), w hic h p h os p h orylates m ulti ple i ntracell ular tar gets.

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P K G is expressed by t wo genes, Pr kg 1 a n d Pr kg 2 , t he f or mer e xisti n g as t w o s plice varia nts --- Pr kg1a a n d Pr kg 1b . Of t hese, P K G 1 a pr otei n is t he m ost pr o mi ne ntly expressed in the cardiovascular syste m. In myocytes, targets of phosphorylation by

P K G include me mbers of the transient receptor potential channels 2 6, 2 7 , re g ulat or of G pr otei n si g nali n g pr ote i ns 2 8 -3 0 , tr o p o ni n I 2 5, 3 1 , my osi n bi n di n g pr otei n C 3 2 , P L B,

3 3 3 4 3 5 P DE5 A , the proteaso me , a n d t he mit oc h o n drial K A T P cha n nel . As t hese st u dies de monstra te, P K G acts as a bra ke t hat ca n c o u nter p hysi ol o gical a n d pat h ol o gical stress a n d pr o vi de car di o pr otecti o n i n hy pertr o p hie d, isc he mic, a n d faili n g hearts.

P D Es and the Co mpart mentation of Cyclic N u cl e oti d e Si g n ali n g: All c o m p o ne nts of the c A MP and c G MP s i g nal os o me resi de wit hi n disti n ct i ntracell ular c o m part me nts, w hic h i ncl u de l ocally distri b ute d P D Es. F or c A M P -P K A, t his c o m ple x c oalesces ar o u n d a fa mily of proteins kno wn as A -ki nase -anchoring pro tei ns 3 6 . T here is n o direct c orrelate f o r c G M P -

P K G, alt h o u g h G -ki nase -i nteracti n g pr otei ns ha ve been reported and are though t t o bi n d t o

P K G via its N ter mi n us c oile d c oil (le uci ne zi p per) d o mai n 3 7 -3 9 . These syste ms for c A M P and c G M P e na ble eac h of t he cyclic n ucle oti des t o re g ulate cell si g nali n g differe n tly de pe n di n g on ho w and where they were actuated .

Exploration of nanodo main cont r ol has de vel o pe d ra pi dly wit h a d va nces i n ge netically e nc o de d fl u oresce nt pr otei ns t hat ca n detect re gi o nally circ u mscri be d cyclic n ucle oti des or ki nase acti vity. M ost of t he se rely on Förster resonance en ergy tra nsfer

( F R E T), and several have been stably , ge netically i ntr o d uce d i nt o mice, all o wi n g is olati o n of mat ure cells c o ntai ni n g a c A M P - or c G MP -se nsitive se ns or 4 0 -4 3 . S o far, t he greatest success has been with cA MP -P K A wit h e xcelle nt re p orters f or b ot h. c G M P se ns ors ha ve bee n m ore diffic ult t o de vel o p, i n part because c G M P levels are often

8

very lo w and thus require greater reporter sensitivity. A successful P K G -acti vity se ns or has n ot yet bee n de vel o pe d. Still, e xisti n g pr o bes are pr o vi di n g i n vitr o a n d i n vi v o understanding of ho w co mpart mentation is regulated .

Studies with c A M P F R E T biosensors targeted to the plas ma me mbrane and the sarc o plas mic e n d o plas mic retic ul u m calci u m ( Ca 2 + ) A T Pase 2a ( S E R C A 2a) microdo ma i ns ha ve re veale d differe ntial re g ulati o n of β 1 - a n d β 2 -A Rs by P D Es i n healt hy car di o my ocytes, wit h P D E 2, 3, a n d 4 c o ntr olli n g c A M P e q ually fr o m t he β 1 -

A Rs a n d P D E 3 c o ntr olli n g c A M P pri marily fr o m t he β 2 -A Rs 4 3 . Ot her st u dies f o u n d that P D E3 and P D E4 are both responsible for controlling c A M P in the S E R C A2a - containing microdo main and regulate P L B phosphorylation 4 0 . I n a d diti o n, P D E 3 a n d

P D E4 act together to chaperone c A M P fr o m its sy nt hesis at t he me m bra ne t o

S E R C A2a. c G M P F R E T sensors are more li mited in signal strength and are so far best s uite d t o N P -pa t h way detecti o n. A ne w pr o be (calle d c G E S -D E 5 by t he a ut h ors) uses the P D E5 A c G MP binding do main coupled to green ( T -Sa p p hire) a n d re d ( Di mer 2) fluorescence peptides and sho ws a better signal -t o -n oise rati o t ha n pri or versi o ns w hile retai ni n g c G M P selecti v ity 4 4, 4 5 , whereas one that used a binding do main fro m

Plas modiu m falciparu m P K G has pr o vi de d a de q uate se nsiti vity e ve n if tar gete d t o s pecific cell ular c o m part me nts 4 6 . A nother non -F R E T c G MP sensor, Flinc G 4 7 , has bee n usef ul f or real -ti me c G M P dy na mic i ma gi n g a n d detecti o n of selecti ve P D E re g ulati o n i n ne o nata l myocytes 4 8 ; h o we ver, t he si g nal was n ot s ufficie nt f or detecti o n of c G M P i n a d ult cells. Ne w varia nts of Fli nc G may i m pr o ve u p o n t his li mitati o n 4 9 .

F R E T sensors for P K A isofor ms have revealed differences in signaling coupled t o P K A wit h re g ulat ory s u b u nit I or II (P K A -RI a n d RII). P K A -RI is acti vate d i n res p o nse t o pr osta gla n di n rece pt or ( E P 2 a n d E P 4) acti vati o n, d uri n g w hic h si g nali n g

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via P K A -RII is shut do wn through the E P3 receptor and G i-coupled mechanis ms. By c o ntrast, P K A -RII is acti vate d selecti vely i n res p o nse t o β -A R sti m ulati o n 5 0 .

C osti m ulati o n of c G M P wit h β -A R sti m ulati o n s hifte d acti vati o n t o P K A -RI, providing a novel mechanis m for regulating c A MP/c G MP crosstalk 5 1 . A n ot her t o ol is a ge netically tar gete d ki nase i n hi bit or, s uc h as o ne us e d t o bl oc k P K A at t he e n d o plas mic retic ul u m 5 2 . This intriguing approach re mains to be developed for ot her microdo mains or extended to P K G.

Several sensors have also been expressed stably in mice. c A M P -re p orti n g mice have been used to sho w reduced P D E3 control of β 2 -A R si g na li n g a n d rel ocalizati o n of P D E2 fro m β 1 -A R t o β 2 -A R after mild pressure overload 4 3 . Mice wit h c G M P sensors have been generated and appear most useful for studying the N P -si g nali n g path way 4 1, 4 2 . Plas ma me mbrane c G MP co mpart ments have also been detected using electrophysiological methods in co mbination with expression of c G M P -se nsiti ve i o n c ha n nels 5 3 .

U n dersta n di n g w here P D Es resi de i n t he cell i n or der t o act uate t heir c o ntr ol of cyclic n ucle oti des is als o critical. St u dies t o date ha ve pri marily f oc u se d o n c o - i m m u n o preci pitati o n of P D Es i n or der t o i de nti fy binding partners by mass spectro metry. A ne wer met h o d of a p pr oac hi n g na n o d o mai n i de ntificati o n is pr o xi mity pr ote o mics, w hic h has n ot yet bee n a p plie d t o P D Es. Wit h t his met h o d, a pr otei n of i ntere st is ta g ge d wit h eit her a bi oti n li gase or a per o xi dase a nd overex pressed i n cells.

U p o n i nc u bati o n wit h bi oti n a n d ot her a p pr o priate rea ge nts, t he f usi o n pr otei n will bi oti nylate its nei g h b ori n g pr otei ns wit hi n a s mall ra di us, facilitati n g p ull d o w n of t h ese bi oti nylate d pr otei ns a n d i de ntificati o n by mass s pe ctr o metry. Se veral bi oti n li gases have been engineered for this purpose ( BioI D 5 4 , Bi oI D 2 5 5 , TurboI D 5 6 , a n d

1 0

mi ni T ur b o 5 6 ), as well as se veral per o xi dases ( AP E X 5 7 and AP E X2 5 8 ). N ota bly, t he

peroxidases require H 2 O 2 sti m ulati o n i n a d diti o n t o bi oti n i n or der t o sti m ulate bi oti nylati o n, w hic h may be a dra w bac k f or s o me st u dies; h o we ver, t hey ca n als o be use d f or electr o n micr osc o py t o ac q uire hi g h res ol uti o n i ma ges of P D E l ocalizati o n wit h o ut t he nee d f or a n a nti b o dy, which cann ot be d o ne wit h t he bi oti n li gase ta gs 5 8, 5 9 .

P D E 5: P D E 5 A is a c G M P -specific P D E with three isofor ms, P D E5 A1 -3, all e x presse d i n hu mans 6 0 , alt h o u g h t here is as yet n o k n o w n differe nce i n t heir f u ncti o n or l ocalize d si g nali n g. P D E 5 A acti vity is m o d ulate d by c G M P bi n di n g t o G A F re g ulat ory d o mai ns a n d by P K G p h os p h orylati o n ( S 9 2 i n m o use, S 1 0 2 i n h u ma n), b ot h res ulti n g i n P D E acti vati o n 6 1,

6 2 (Fi g ur e 1.3 ). Of t he t w o pri mary pat h ways generating c G M P ( N O - a n d N P -dependent cascades), P D E5 A regulates the for mer selectively in myocytes 4 8, 5 3, 6 3, 6 4 . I n hi biti n g P D E 5 A counters maladaptive cardiac structural and functional re modeling in both hyp ertr o p hic 6 5 and ische mic heart disease 6 6 , bl oc ki n g m ulti ple pr o -h y pertr o p hic a n d fi br otic si g na li n g pat h ways by P K G -dependent mechanis ms 6 7 . D uri n g isc he mia -reperfusion, P D E5 A inhibitors reduce infarct size and cardio myocyte apoptosis; cardioprotection requires

6 8 -7 0 o pe ni n g of t he mit oc h o n dri al K A T P cha n nel . P D E 5 A n or mally l ocalizes at t he Z -disc of car di o my ocyte sarc o meres, b ut t his l ocalizati o n s hifts t o a m ore diff use patter n i n faili n g hearts a n d late -sta ge hy pertr o p hy a n d dilati o n, a n d if e n d ot helial N O S (e N O S or N O S 3) is p har mac ol o gically i n hi bite d or ge netically delete d 6 4, 7 1, 7 2 . N or mal P D E 5 A i ntracell ular l ocalizati o n ca n be rest ore d by directly sti m ulati n g s G C t o ge nerate c G M P e ve n i n t he prese nce of N O S i n hi biti o n. T his i n dicates t hat i ntra my ocyte P D E 5 A l ocalizati o n itself de pe n ds o n t he a vaila bility of its fa v ore d s u bstrate - s G C -deri ve d c G M P. T hese l ocalizati o n changes are unrelated to P K G -targeted S92 phosphorylation of P D E5 A 7 3 .

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Fi g ur e 1.3 : Cardiac myocyte regulation by P D E5 A and P D E9.

Fi g ur e 1.3: Cardiac myocyte regulation by P D E5 A and P D E9.

B ot h of t hese P D Es re g ulate c G M P very selecti vely. As de picte d, P D E 5 A c o ntr ols N O - sti m ulate d c G M P, a n d P D E 9 c o ntr ols N P -sti mulated c G MP. Both path ways converge on

P K G 1; h o we ver, t hese are als o li kely differe nt p o ols of t he ki nase ( he nce, t w o ic o ns are sho w n) wit h s o me o verla p pi n g f u ncti o nality b ut als o s o me very si g nali n g pat h way -s pecific

effects as well. T he fi g ure sho ws the i mpact of P K G1 in suppressing G q -c o u ple d pr otei n rece pt ors by R G S pr otei ns; bl oc ki n g T R P Cs; sti m ulati n g mit oc h o n drial pr otecti o n via K A T P channels; enhancing proteaso me function; and modifying multiple proteins in the sarco mere, i ncl u di n g T n, titi n, a n d My B P C. A b bre viati o ns: β -A R, β -adrenergic receptor; AngII, a n gi ote nsi n -II; c G M P, cyclic g ua n osi ne 3 ¢, 5 ¢-monophosphate; Cn, calcineurin; E T -1,

e n d ot heli n -1; G A F d o mai n; G q α , G -pr otei n( α q); G P C R, G pr otei n -- c o u ple d rece pt or;

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My B P C, my osi n bi n di n g pr otei n C; N F A T, n uclear fact or of acti vate d T cells; N O, nitric o xi de; N O S, nitric o xi de sy nt hase; N P, natri ureti c pe pti de; N P R, natri uretic pe pti de rece pt or;

P D E, phosphodiesterase; G C -A / B, g ua nylyl cyclase -A / B; P K G, pr otei n ki nase G; R G S, re g ulat or of G pr otei n si g nali n g; s G C, s ol u ble g ua nylyl cyclase; T n, tr o p o ni n I; T R P C, tra nsie nt rece pt or p ote ntial c ha n nel.

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P D E 5 A i n hi bit ors are use d cli nically t o treat erectile dysf u ncti o n a n d pul monary hypertension 3 . Alt h o u g h prec li nical 6 5, 6 6, 7 4 a n d s o me s mall -scale cli nical 7 5, 7 6 st u dies f o u n d P D E 5 A i n hi biti o n als o a meli orates vari o us f or ms of heart disease, ot her studies were neutral 7 7 , a n d c o n tr oversies a n d caveats regar di ng t his strategy persist.

O ne p ote ntial iss ue is t hat t he selecti vity of P D E 5 A f or N O -sti m ulate d c G M P c o ul d re d uce its efficacy i n sy n dr o mes s uc h as heart fail ure, i n w hic h N O si g nali n g is depressed by oxidant stress due to re duced N O synthesis 7 8, 7 9 , s G C acti vati o n 2 1, 8 0 , a n d c G M P ge nerati o n 8 1, 8 2 . A n ot her e xa m ple i n w hic h re d uce d N O si g nali n g occ urs is i n w o me n wit h l o w estr o ge n le vels. P D E 5 A i n hi biti o n i n fe male mice a meli orates heart

disease induced by excessive G q -pr otei n sti m ulati o n o r mec ha nical o verl oa d. T his treat me nt effect is l ost f oll o wi n g o variect o my b ut rest ore d if t hese a ni mals recei ve estrogen replace ment 8 3 - res ults t hat c o ul d ha ve i m p orta nt i m plicati o ns f or t he use of

P D E 5 A i n hi bit o rs i n p ost me n o pa usal w o me n. T o date, t he o nly m ultice nter trial of

P D E 5 A i n hi biti o n a n d heart fail ure was i n a gr o u p of patie nts wit h preser ve d ejecti o n fracti o n ( H F p E F), a n d t he res ults were ne utral 7 7 . H o we ver, t his c o h ort has m o dest N P acti vati o n 8 4 , a n d a maj ority i n t he st u dy re p orte dly di d n ot ha ve ve ntric ular hypertrophy. Left ve ntric ular ( L V) my ocar di u m fr o m H F p E F has l o w le vels of c G M P a n d P K G acti vity 8 5 , o p p osite t o heart fail ure wit h re d uce d E F, and because P D E5 A d oes n ot a p pear t o be u pre g ulate d as t he ca use, t his w o ul d li mit effects fr o m P D E 5 A inhibition. As discussed belo w, ne w data regarding another c G M P -tar g eti n g P D E,

P DE9 A 4 8 , may s he d f urt her li g ht i nt o t hese iss ues a n d i m pr o ve t hera pe utic tar geti n g.

P D E 9: P D E 9 A is a c G M P -s pecific P D E wit h t he hi g hest affi nity f or c G M P of a ny of t he

P D E super fa mily me mbers 8 6 . U ntil rece ntly, n ot hi n g was k n o w n a b o ut t he r ole of P D E 9 A

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i n t he heart, alt h o u g h it is e x presse d at t he m R N A le vel i n my ocar di u m 8 6, 8 7 . H o we ver, a rece nt st u dy deter mi ne d t hat P D E 9 A is e x presse d at pr otei n le vels i n h u ma n a n d ot her ma m malia n hearts, t hat it is f u ncti o nal, a n d t hat it s pecifically re g ulates c G M P res ulti n g fr o m

N P sti m ulati o n of G C -A / B 4 8 . P D E9 A protein expression is observed in a different s u bcell ular l ocati o n i n my ocytes, c o -stai ni n g wit h t he S R base d o n S E R C A 2a i m munohistoche mistry (Fi g ur e 1.4 A ). T his differs fr o m t he Z -dis k l ocalizati o n of P D E 5 A.

Expression is very lo w in nor mal mouse and hu man myocardiu m but is upregulated by heart fail ure (Fi g ur e 1.4 B ) wit h or wit h o ut re d uce d syst olic f u ncti o n, s u g gesti n g t hat tar geti n g t hese diseases wit h P D E 9 A i n hi bit ors may b e usef ul. P D E 9 A pr otei n e x pressi o n a n d acti vity are ele vate d i n h u ma n heart fail ure, n ota bly i n b ot h f or ms wit h re d uce d or preser ve d ejecti o n fracti o n , t he latter bei n g a disease w hic h has bee n particularly refract ory t o successful t hera py a n d i n w hic h P D E 5 A i n hi biti o n was pre vi o usly teste d a n d di d n ot i m pr o ve cli nical stat us 7 7, 8 8 .

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Fi g ur e 1.4: N O -independent P D E9A modula tio n of car diac stress res po nses.

Fi g ur e 1.4 : N O -independent P D E9 A modulation of cardiac stress responses.

Unlike P D E5 A, which hydrolyzes pri marily c G MP generated by N O -sti m ulate d s G C, rece nt work has sho wn P D E9 A targets NP -G C -A / B -- deri ve d p o ols of t his cyclic n ucle oti de. ( a)

B ot h P D Es are l ocalize d t o differe nt c o m part me nts i n t he car diac my ocyte, wit h P D E 5 A e x pressi o n c ol ocalizi n g t o t he Z -dis k (c ostai ni n g wit h α -acti ni n), w hereas P D E 9 A c ol ocalizes wit h T -t u b ular me m bra nes ( wit h S E R C A 2a). ( b) Expressio n i n t he n or mal heart is very l o w b ut is i ncrease d i n c o n diti o ns s uc h as h u ma n heart fail ure. ( c) Mice gl o bally lac ki n g P D E 9 A display protection to sustained pressure overload -induced cardiac stress, with enhanced cardiac FS and reduced hypertrophy ( L V mas s). Asteris ks i n dicate P < 0.01 as co mpar e d t o

W T T A C. ( d) U nli ke P D E 5 A i n hi biti o n, w hic h d oes n ot i m pr o ve car diac f u ncti o n de presse d

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by press ure o verl oa d if N O S is als o i n hi bite d ( L -N A M E), P D E 9 A i n hi biti o n is e q ually effecti ve wit h or wit h o ut c o nc o mita nt N O S i n hi biti o n. Fi g ure a da pte d wit h per mi ssi o n fr o m

Refere nce 4 8 . A b bre viati o ns: c G M P, cyclic g ua n osi ne 3 ¢, 5 ¢-m o n o p h os p hate; D C M, dilate d cardio myopathy; FS, fractional shortening; G A P D H, glyceraldehyde 3 -phosphate dehydrogenase; G C -A / B, g ua nylyl cyclase -A / B; L -N A M E, L -N G -Nitr oar gi ni ne met hyl est er;

L V, left ve ntric ular; N F, n o n -faili n g,; N O, nitric o xi de; N O S, nitric o xi de sy nt hase; N P, natri uretic pe pti de; P D E, p h os p h o diesterase; P F -9 6 1 3 ( P D E 9 i n hi bit or); SI L, sil de nafil,

P D E5 -i n hi bit or; T A C, tra ns -a ortic c o nstricti o n; W T, wil d ty pe.

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P D E9 A inhibition or gene deletion during murine cardiac hypertrophy induced by transaortic banding i mproved cardiac function and decreased fibrosis, car di o my ocyte size, a n d mala da pti ve si g nali n g (Fi g ur e 1.4 C ). T he f u ncti o nality of

P D E 9 A i n hi biti o n differs i m p orta ntly fr o m t hat d ue t o P D E 5 A s u p pressi o n.

P h os p h o pr ote o mic a nalysis re veale d t hat P K G acti vati o n by i n hi biti o n of P D E 5 A or

P D E 9 A le d t o se veral o verla ps b ut als o ma ny stri ki n g differe nces i n pr otei n phosp h orylati o n a n d tra nscri pti o nal re g ulati o n 4 8 . F or e xa m ple, w hereas i n hi biti n g eit her s u p presse d tra nscri pti o nal acti vity li n ke d t o N F A T t o tri g ger a nti hy pertr o p hic effects 2 7 , only P D E9 A suppression aug mented G A T A b i n di n g pr otei n 4, w hic h is linked to angiogenesis 8 9 . Mice s u bjecte d t o press ure o verl oa d a n d treate d wit h a selecti ve P D E 9 A i n hi bit or, b ut n ot a P D E 5 A i n hi bit or, we re protected even when

N OS was blocked by L -N G -Nit r oar gi ni ne met hyl ester ( L -N A M E) a d mi nistere d i n dri n ki n g water (Fi g ur e 1.4 D ). T his c o nfir me d at t he w h ole heart le vel w hat is olate d cell st u dies ha d re veale d a b o ut t he differe ntial tar geti n g of N P - versus N O -sti m ulate d c G M P p o ols by t he t w o P D Es.

Previ o u s cli nical st u dies of P D E 9 A i n hi bit ors p urs ue d t heir utility t o i m pr o ve ne ur oc o g niti ve f u ncti o n a n d were teste d i n sc hiz o p hre nia, H u nti n gt o n’s disease, a n d

Alz hei mer’s disease 9 0 -9 2 . T he i n hi bit ors were well t olera te d, a n d w hile ma ny precli nical st u dies had re p orted p ositi ve c o g niti ve effects i n m o use m odels of t hese dis orders 9 3, 9 4 , be nefits were n ot o bser ve d i n t he li mite d h u ma n data re p orte d t o date 9 5 . T he ne w fi n di n gs i n t he car di o vasc ular syste m re veal a n e ntirely differe nt p ote ntial use. Gi ve n t he i nteracti o n of P D E 9 A a n d t he N P si g nali n g syste m, a n d t he utility of valsarta n /sac u bitril, a c o m bi ne d a n gi ote nsi n -2 rece pt or bl ocker a nd ne prilysi n

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i n hi bit o r (t he latter bl oc ki n g N P pr ote olysis) 9 6 , P D E 9 A i n hi bit ors may be sy ne r gistic wit h t hese ne w dr u gs.

Cli ni c al Tr a nsl ation, Future Issues, and Conclusions: O ne of t he defi ni n g feat ures of

P D E p har mac ol o gy is t hat des pite str uct ural si milarities of t he catalytic site a m o n g t he differe nt s pecies of P D Es, t hey are s ufficie ntly u ni q ue t o e na ble ge nerati o n of hi g hly s pecific i n hi bit ors. Crystal str uct ures ha ve be en o btai ne d f or ma ny P D Es, w hic h is facilitati n g dr u g desi g n a n d disc o very. M ost of t hese ne w i n hi bit ors are effecti ve at na n o m olar c o nce ntrati o ns. F urt her, wit h t he e xce pti o n of P D E 2, i n hi bit ors are a vaila ble f or all k n o w n

P D Es affecti n g heart f u ncti o n, a n d they have been found to be safe for and are being tested in hu mans. Other than P D E3 and P D E5 A, these studies have not focused on cardiac i n dicati o ns, b ut t his is a c ha n gi n g la n dsca pe. T he maj or li mitati o n wit h t hese agents has been t he lac k of greater i sofor m -selecti ve i n hi biti o n, w hic h s o me belie ve may be p ossi ble, alt h o u g h t o date t his has bee n el usi ve. T he ele ga nce of i ntracell ular c o m part me ntalize d si g nali n g is t h us p ote ntially l ost w he n m ulti ple f or ms are m o d ula te d at t he sa me ti me. T he cli nical uti lity of P D E 3 i n hi bit ors li kely s uffere d beca use of t his, a n d i m pr o ve d selecti vity may be a ble t o alter t he o utc o me fr o m t hese a ge nts f or car diac dis or ders. A n ot her fact or is t hat or ga n selecti vity has bee n diffic ult, a lt h o u g h t his is hel pe d t o s o me e xte nt b y differe ntial e x pressi o n of vari o us P D Es i n t he tiss ues. T his has i m p orta nt cli nical i m plicati o ns, as m o difyi n g t he N O -si g nali n g pat h way by s G C acti vati o n, f or e xa m ple, will i m pact a br oa der variety of cell ty pes t ha n w o ul d occ ur by i n hi biti n g P D E 5 A --- w h o se tiss ue e x pressi o n is more selective. P D Es with broader expression may be less druggable unless isofor m selecti vity a n d t h us t he a bility t o better tar get effects are p ossi ble.

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A n ot her e v ol vi n g para di g m is t he use of m ulti ple P D E i n hi bit ors i n c o m bi nati o n wit h cyclic n ucle oti de sti m ulati o n pat h ways --- eac h at d oses t hat al o ne ha ve very m o dest i m pact b ut w he n c o m bi ne d yiel d greater cell -tar gete d si g nali n g a n d alterati o n i n f u ncti o n. T his is a tactic use d i n a ntica ncer t hera py, i n w hic h m ulti pr o n ge d attac ks can produce synergistic effects. If one could provide so me cyclase sti m ulati o n al o n g wit h t he selecti vity of P D Es, t he desire d res ult c o ul d be e n ha nce d si g nali n g i n t he cell a n d na n o d o mai n. T his a p pr oac h has yet t o be developed as a cli nical strate gy, b ut w it h a p pr o priate i nsi g ht i nt o w hic h c o m bi nati o ns are safe, t here is m uc h t o be sai d f or it. T o date, s mall m olec ules t hat acti vate P D Es selecti vely

(thereby degrading c A MP, c G MP, or both) have not been developed but mi g ht be m o d ulat ors of t he re g ulat ory N ter mi n us a n d may ha ve cli nical utility i n certai n setti n gs.

It has bee n 1 8 years since the last P D E superfa mily me mber ( P D E11 A) was i de ntifie d 9 7 . All P D Es ha ve bee n e x pl ore d, a n d ma ny ha ve bee n tar gete d wit h i n hi bi t ors f or t heir p ote ntial t hera pe utic val ue. E ve n s o, rat her t ha n a fiel d wit h m ost of its q uesti o ns a n d pr o ble ms s ol ve d, t his area re mai ns y o u n g a n d fres h, wit h acti ve researc h a n d o n g oi n g tra nslati o nal a p plicati o ns. T h e a p preciati o n of P D E na n ore g ulati o n, t he n ua nces t his pr o vi des f or m olec ular c o ntr ol, a n d h o w it mi g ht be leveraged to enhance the specificity and exte nt of drug res p o nses are all active areas of i n q uiry. We e x pect t his i nterest will s ur ge f urt her as ne we r a n d m ore effecti ve approaches are d e vel o pe d t o tar get a br oa d variety of diseases.

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C HAP T E R 2 : M ar k e d d is p arit y of m icro R N A m o d ul ati o n b y cG MP -s el e cti v e P D E 5

versus P D E9 in hi bit ors i n h e art d isease

M o difi e d fr o m: Kokkonen -Si m o n K M et al. Mar ke d Dis p arity of Micr o R N A M o d ulati o n by c G MP -selecti ve P D E 5 vers us P D E 9 I n hi bit ors i n Heart Disease. J CI I nsi g ht. 2 0 1 8. 3( 1 5): e121739.

INTRODUCTIO N

Micro R N As: Micr o R N As ( mi R N As) are s mall ri b o n ucleic aci ds t hat pr o vi de p ost - transcriptional control of messenge r R N A tra nslati o n a n d de gra dati o n 9 8 . First disc o vere d i n

1 9 9 3, t here are n o w o ver 2 4, 0 0 0 i de ntifie d mi R N As. T heir pri mary r ole is re pressi n g ge ne e x pressi o n by facilitati n g m R N A de gra dati o n, i n hi biti n g pr otei n tra nslati o n, or de gra di n g p oly pe pti des through co mple mentary binding to the 3’ U T R of tar get m R N As. Eac h mi R N A i mpacts around 1000 individual m R N As, generally clustering ar o u n d a t he matically c o here nt set of si g nali n g pat h ways. I m p orta ntly, t heir e x pressi o n c ha n ges wit h disease a n d t hera pe utic interventions, which has spa wned inte rest i n t heir use as b ot h dia g n ostic a n d t hera pe utic a ge nts 9 9, 1 0 0 .

Fr o m a syst e ms bi ol o gy pers pecti ve, t he p ositi o n of mi R N As is ge nerally place d a b o ve ge ne tra nslati o n. H o we v er, gro wing evidence de monstrating th eir m o dificati o n by i ntracell ular a n d e n vir o n me ntal si g nali n g ha ve le d t o t heir e x pl orati o n as mar kers of disease therapy , of partic ular i nterest t o t he eff orts t o better pers o nalize me dical treat me nt 1 0 1, 1 0 2 .

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H o we ver, s uc h data re mai ns fairly li mite d, a n d t he se nsiti vity of mi R N A si g nat ures t o treat me nts t hat s hare c o m m o n si g nali n g little e x pl ore d.

mi R N As as Bio markers and Therapeutics for Cardiovascular Disease: T he first re p orts of t he r ole of mi R N As i n hear t disease ca me i n 2 0 0 6 w he n t he Ols o n la b re p orte d enhance ment of mi R -2 0 8 i n pat h ol o gical hy pertr o p hy a n d t hera pe utic be nefits fr o m its re versal 1 0 3 -1 0 5 . Ot her si milar e xa m ples f oll o we d, i ncl u di n g s u p pressi n g mi R -1 22/132 or increasing mi R -1 or mi R -1 3 3 1 0 6 -1 1 0 , wit h n o w well o ver 2 0 0 0 st u d ies on various mi R N As and heart disease.

I n t he h u ma n heart, m ulti ple mi R N As ha ve bee n s h o w n t o be dysre g ulate d i n disease. M a ny circ ulati n g mi R N As have bee n proposed as heart failure bio markers 1 1 1 .

H o we ver, a c o nsiste nt heart fail ure mi R N A pr ofile has yet t o be i de ntifie d, des pite s o me overlap bet ween independent stu dies. mi R N As have also been sho wn to have so me pr o g n ostic val ue. I n cardiac resynchronization therapy ( C R T) , patie nts w h o res p o n d t o therapy have de monstrated specific mi R N A expression c ha n ges, i ncl u di n g mi R -2 6 b -5 p, 1 4 5 -

5 p, 9 2a -3 p, 3 0e -5 p, a n d 2 9a -3 p 1 1 2 . A d diti o nally, s pecific mi R N As ha ve bee n s h o w n t o c ha n ge i n D C M patie nts t hat res p o n d t o beta -blocker treat ment versus non -res p o nders, i ncl u di n g mi R -208a -3 p, 2 0 8 b -3 p, 2 1 -5 p, 1 9 9a -5 p, a n d 1a -3 p 1 1 3 . Several mi R N A -base d therapeutics have been proposed ; h o wever, none of these therapies have progressed beyond a ni mal m o dels, li kely d ue t o t he m ul ti plicity of mi R N A tar gets, c o ncer ns f or off -tar gets of t he t hera py i n ot her tiss ues, a n d diffic ulty of deli very t o t he heart.

Here, we teste d t he hy p ot hesis t hat mi R N A pr ofili n g may pr o vi de a usef ul t o ol t o disti n g uis h bet wee n mec ha nistically very si milar t hera pe utic i nter ve nti o ns. T o d o t his, we c o ntraste d t he effects of p har mac ol o gically i n hi biti n g eit her c G M P -selecti ve

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phosphodiesterase type 5 or type 9 ( P D E5 -I, P D E 9 -I). B ot h acti vate pr otei n ki nase G, t h o u g h t hey d o s o by m o d ulati n g differe nt c G M P p o ol s, t he f or mer c o u ple d t o nitric o xi de

( N O) sti m ulati o n of g ua nylyl cyclase -1 ( G C -1) a n d t he latter t o natri uretic pe pti de ( N P) sti m ulati o n of g ua nylyl cyclase -2 A 1 1 4 . I m p orta ntly, b o t h dr u gs p ote ntly a n d very si mil arly i mprove heart disease sti mulated by sustained pathological he modyna mic stress 1 1 4 . Des pi te t his, t he mi R N A pr ofiles were dra matically a n d s ur prisi n gly differe nt, wit h P D E 5 -I re d u ci n g a very broad array of mi R N As ass ociate d wit h t he disease state, w hereas P D E 9 -I ha d virt ually n o i m pact.

MET HODS

Car diac press ure -overload ( P O) model: Press u re overload was induced by trans -a ortic constriction, perfor med as previously described 1 1 4 . Size -, a ge - a n d sex -matc he d ( male)

C57 B L/6J mice (Jackson L a bs) were use d f or all dr u g i nter ve nti o n st u dies. S ha m c o ntr ols u n der we nt si milar s ur gery wit h o ut li gat ure place me nt. Mice were f oll o we d f or u p t o 6 wee ks after P O, a n d were c o -treate d wit h P D E 5 -I ( Sil de nafil, Sil, Pfizer, 2 0 0 m g / k g / day i n Bi oser v s oft die t), P D E 9 -I ( P F -0 4 4 4 9 6 1 3, Pfizer, 3 0 m g / k g t wice daily by oral ga va ge), B A Y 6 0 -2 7 7 0

( Bayer, 0.3 mg/kg/day once daily by oral gavage), or appropriate matche d ve hicle.

Treat me nt starte d 1 wee k after P O. F or t he dr u g i nter ve nti o n st u dies, a ni mals dyi n g pri or t o drug assign ment or who failed to develop disease after P O were excluded fro m analysis. The

G C -2 A overexpressor mice were generated as previously describ e d 1 1 5 , a n d s u bjecte d t o P O or sha m surgery. Ani mals were follo wed for 3 weeks after P O .

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Conscious mouse echocardiography: Intact heart morphology and function was deter mi ne d i n c o nsci o us mice by serial M -m o de tra nsthoracic echocardiography

(VisualSonics Vevo 2100, 18 -38 M Hz linear array transducer; SanoSite Incorporated).

I ma ges were o btai ne d a n d a nalyze d by a n i n di vi d ual bli n de d t o t he a ni mal c o n diti o n.

mi R N A s e q u e n ci n g: T otal R N A was is olate d fr o m left ve ntri cular myocardiu m using

Qiaz ol Rea ge nt a n d t he mi R Neasy kit ( Qia ge n) acc or di n g t o ma n ufact urer pr ot oc ol. R N A was analyzed on an Agilent Bioanalyzer for RI N values and concentrations. Only sa mples wit h RI N > 7 w ere used for sequencing. Libraries were prepared usi n g t he Ill u mi na Tr u Se q

S ma ll R N A Sa mple Prep Kit. Sequencing was perfor med on an Illu mina HiSeq 2500.

Ill u mi na’s C A S A V A 1. 8. 4 was use d t o c o n vert B C L files t o F A S T Q files. Se q ue nci n g rea d filteri n g a n d a d a pter tri m mi n g, rea d ali g n me nt, a n d ge nerati o n of c o u nt data a n d R P M data were done using the mi Rge progra m v1.0 1 1 6 , which incorporates Cutadapt v 1. 1 8 a n d B o wtie v1.1.1. Differential expression analysis was perfor med using Bioconductor’s D ESeq package

( v 1. 2 6. 0) 1 1 7 . mi R N As were filtere d acc or di n g t o t he f oll o wi n g: m ore t ha n 5 0 % of mice ha d rea ds f or a gi ve n mi R N A, a n d t he mi R N A was prese nt i n t he Mus musculus mi Rgene D B data base 1 1 8 . Heat maps were generated using the Morpheus progra m ( Broad Institute).

Clustering was perfor me d by the one minus Spear man rank correlation. The data discussed in this publication have been deposited in N C BI’s Gene Expression O m ni b us 1 1 9 , a n d are acce ssible through G E O Series accession nu mber GS E112056

(https:// w w w.ncbi.nl m.nih.gov/geo/query/acc.cgi?acc = GS E112056 ).

R N A sequencing: R N A sa mples were prepared and analyzed as described f or mi R N A -se q.

Libraries were generated using the TruSeq Stranded Total R N A sa mple preparation kit

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(Ill u mi na), a n d se q ue nce d o n a n Ill u mi na Hi Se q 2 5 0 0. Ill u mi na’s C A S A V A was use d t o c o n vert B C L files t o F A S T Q files. R N A -seq rea ds were tri m me d a n d ma p pe d t o t he

Ense mbl mouse reference geno me ( m m10) using HIS A T2 version 2.0.5 1 2 0 . Tra nscri pt c o u nts were q ua ntifie d usi n g R S E M v 1. 3. 0 wit h defa ult para meters 1 2 1 . Differe ntial e x pressi o n ( D E) a nalysis of g enes bet ween different treat ments was perfor med using R package D ESeq2 v1.18.1 1 2 2 . Path way enrich ment analysis was done using the Kyoto

Encyclopedia o f Genes and Geno mes ( K E G G) database and R package K E G G.db v3.2.3)

1 2 3 . The data discussed in this publication have been deposited in N C BI’s Gene Expression

O m ni b us 1 1 9 , a n d are accessi ble through G E O Series acc ession nu mber GS E112056

(https:// w w w.ncbi.nl m.nih.gov/geo/query/acc.cgi?acc = GS E112056 ).

Gene expression – q R T -P C R: T otal R N A was is olate d fr o m left ve ntric ular my ocar di u m, l u n g, or c ult ure d cells usi n g Qiaz ol Rea ge nt a n d t he mi R Neasy kit ( Qia ge n) acc or di n g t o manufacturer protoc ol. Re verse tra nscri pti o n t o c D N A was perf or me d usi n g t he mi Scri pt II

R T kit ( Qiagen). c D N A under went P C R a mplification for m R N A expression using Taq Man pr o bes f or atrial natri uretic pe pti de ( A N P) ( m o use # M m01255747_g1), brain or B -ty pe natri uretic pe pti de ( B NP) ( mouse # M m01255770_g1), Argonaute 2 ( Ago2) ( mouse

# M m00838341_ m1), D GCR8 ( mouse # M m01146851_ m1), Dicer1 ( mouse

# M m00521722_ m1), Drosha ( mouse # M m01310009_ m1), or glyceralde hyde -3 -phosphate dehydroge nase ( G AP D H) ( mouse #99999 9 1 5 _ g 1) ( A p plie d Bi osyste ms). F or mat ure mi R N A expression, Qiagen miScript probes were used for mi R -1a ( mouse MS00011004), mi R -101a ( mouse MS00011011), mi R -133a ( mouse MS00032305), mi R -1 4 5 ( m o use

MS00001631), mi R -195 ( mouse MS0000 1 7 9 2), mi R -199 ( mouse MS00 0 3 2 5 2 9), mi R -2 0 8 b

( mouse MS00011466), mi R -21 ( mouse MS00011487), mir -214 ( mouse MS00032571), mi R -

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26b ( mouse MS00001344), mir -27b ( mouse MS00001358), mi R -29 ( mouse MS00001372), mi R -29c ( mouse MS00001379), mi R -30b ( mouse MS00001386) , mi R -3 0 d ( m o use

MS0001174 6), mi R -34c ( mouse MS0000 1 4 2 2), mi R -451a ( mouse MS00002408), and R N U6 -

2 (hu man, MS00033740). For pre -mi R N A expression, Qiagen probes were used for pre - mi R -1a ( mouse MP00003990), pre -mi R -199 ( mouse MP00004970), pre -mi R -2 0 8 b ( m o use

M P00005082), pre -mi R -2 1a ( mouse MP00005103), and pr e-mi R -34c ( mouse MP00005628).

F or pri -mi R N A expression, Taq man probes were used for pri -mi R -1a ( m o use

# M m03306163_pri), pri -mi R -199 ( mouse #03306505_pri), pri -mi R -2 0 8 b ( m o use

# M m03308667_pri), pri -mi R -2 1a ( mouse # M m03306822_pri), a n d pri -mi R -3 4c ( m o use

# M m 03306660_pri). The threshold cycle value was deter mined using the crossing point method. Sa mples were nor malized to the G AP D H value for each Taq man run, and R N U6 -2 f or eac h mi Scri pt r u n.

St atisti c al A n al y sis: All val ues are prese nte d as mea n ± S E M. F or a nalysis of m ulti ple independent groups, a 1 -way A N O V A or Kr us kal Wallis test was use d wit h a p ost h oc

T u key ( or D u n ns) m ulti ple c o m paris o ns test t o assess gr o u p differe nces. P ost h oc testi n g was o nly perf o r me d if t he 1 W -A N O V A was s i g nifica nt ( p < 0. 0 0 1 i n al l cases). F or a nalysis of only t wo independent groups, an unpaired t wo -taile d T -test was perfor med. Analysis was perfor med using Graphpad Pris m soft ware ( Ver. 7a, 2016).

St u dy a p proval: All a ni mal st u di es perfor med in this paper were approved by either t he

J o h ns H o p ki ns Me dical I nstit uti o ns A ni mal Care a n d Use C o m mittee ( Balti m ore, M D, U S A) or t he University of Würzburg ani mal care co m mittee ( Würzburg, Ger many).

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RES ULTS

P D E5 -I and P D E9 -I a p pli e d t o P O h eart yiel d very dis parate mi R N A pr ofil es

Mice were subjected to sustained pressure -overload ( P O) for 5 weeks and further ra n d o mize d t o recei ve eit her t he P D E 5 i n hi bit or (sil de nafil) or P D E 9 i n hi bit or ( P F -

0 4 4 4 9 6 1 3), eac h i nitiate d o ne wee k after P O. Re p orte d he modyna mic and morpholo gical data fr o m these mice docu ments si milar reduction of hypertrophy and cardiac dilation, as well as reversal of abnor mal molecular signaling and fibrosis, and i mproved heart function 6 5,

1 1 4 . Salie nt data are pr o vi de d i n Fi g ur e 2 .1 .

Fi g ur e 2 . 2 dis plays mi R N A -se q res ults as v olca n o a n d heat -ma p pl ots f or t hree group co mparisons: P O +vehicle versus sha m -control, P O +P D E5 -I versus P O +vehicle, and

P O+P DE9 -I versus P O + Vehicle. Each analysis was derived fro m whole m y ocar dial tiss ue is olates o btai ne d at ter mi nal st u dy ( 5 wee ks after P O). Wit h P O, 6 3 mi R N As si g nifica ntly increased and 48 declined ( Fi g ur e 2 . 2 A, T a bl e 2 .1) . Ma ny mi R N As re p orte d t o be eit her enhanced ( mi R -2 0 8 b, 1 9 9, 1 9 9 b, 2 1a, 3 4 b, a n d 3 4c) or di mi nis he d ( mi R -1 3 3a, 1 3 3 b, a n d 1a) by P O were o bser ve d i n t his a nalysis. P D E 5 -I r educed mi R N A expression broadly ( Fi g ur e

2 . 2 B, T a bl e 2 .2 ) as see n i n t he v olca n o pl ot wit h t he vast maj ority of c ha n ges i n t he ne gati ve l o g -f ol d re gi o n. T hi s eit her reverse d or f urt her di minished expression of many of t he mi R N As altere d by P O, a n d l o were d o thers unchanged by P O. In stark contrast, P D E9 -

I mi ni mally altere d mi R N As ass ociate d wit h P O, i m pacti n g o nly 9, 5 of w hic h re verse d P O c ha n ges ( Fi g ur e 2 . 2 C, T a bl e 2. 3 ). T he ma g ni tude of the change of even these 9 was m o dest. A heat ma p usi n g hierarc hical c lustering aggregated P O and P O +P D E9 -I t o get her, each being quite different fro m either Sha m or P O + P D E5 -I gr o u ps ( Fi g ur e 2 . 2 D ). T he mi R N A na mes for eac h r o w are pr o vi de d i n T a b l e 2. 4 .

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Fi g ur e 2 .1: Phenotypic i mprove ment in P O mice after P D E5 -I a n d P D E9 -I. A Fractional Shortening B Fr a cti o n al S h ort e ni n g LV L V mass Mass 8 0 0 33Sha 0 0 0 0 m $ $ ** * Sha m $ T A C 6 0 0 $ T A C ** ** 22TA0 0 0 C+ SIL $ TA C+ SIL 4 0 0 TA C+PF-9613 TA C+PF-9613

11 0 0 0 2 0 0 L V Mass ( mg) LV Mass ( mg)

% Fractional Shortening 0

%Fractional00 Shortening 0 BB a a s s eli eli n n e e 11 W W e e e e k k 55 Weeks Weeks B a a s s eli eli n n e e 11 W W e e k 55 Weeks Weeks

L V- E D s C L V -E D s 4 * * Sha m Fractional Shortening 3 T A C 8 0 $ ShaShaT A m m C + Sil $ $ 2 PP O TA C+PF-9613 6 0 $ $ P O + Sil E Ds ( m m) P O + Sil - * * PP O+PF O+PF-9613-9613 L V 14 0 LV- E Ds ( m m)

2 0 0 BB a a s s eli eli n e 11 W W e e e e k k 5 Weeks Weeks

%Fractional0 Shortening B a s eli n e 1 W e e k 5 Weeks

Fi g ur e 2 .1: Phenotypic i mprove ment in P O mice after P D E5 -I and P D E9 -I.

C57 Bl6/J mice were studied as previously described 1 1 4 . Le ft ve ntric ular tiss ue was subsequently used for mi R N A -se q a nalysis. A) Fracti o nal s h orte ni n g. B) Left ve ntric ul ar mass. C) Left ve ntric ular e n d -syst olic di me nsi o n as a meas ure of dilati o n. All para meters were si g nifica ntly i m pr o ve d by treat me nt wit h eit her Sil ( P D E 5 -I) or P F -9613 (P D E9 -I). * p <0.05 versus P O group baseline measure ments. $ p <0.05 versus P O measure me nt as sa me ti me p oi nt. N = 5 -9 per gr o u p. Data is prese nte d as mea n ± S E M. Data were a nalyze d usi n g m ulti ple t -tests.

2 8 !

Figure 2.2: P D E5-I and P D E9-I applied to P O heart yield disparate mi R N A profiles.

88 0 0 A mi R-208b-3p D 66 0 0 3 0 33 0 Hypertrophy/Hypertrophy/ Fibrosis- Fibrosis Associated Associated U p U wit p h wit P hO, P D o O, w n D wit o h w n Dr u wit g h Dr u g 2 0 D oD w o n w wit n h wit P O, h U P p O, wit h U p Dr u wit g h Dr u g 22 0 0 mi R-34c-5p mi R-199a-5p Si gSi nifi g c nifi a nt c a nt mi R-21a-5p 1 0 -l o g 1 0( pv al) mi R-199a-3p/199b-3p S h a m S 5 h a m 1 S h a m 3 PP O+ O+ PF-9613 PF-9613 5 5 PP O+ O+ PF-9613 PF-9613P 1 1 O 3 S h a m 4 P O+ PF-9613 2 P O+ PF-9613 2 P O+ PF-9613 4 P O+ PF-9613 4 S h a m 2 P O 1 P O 2 P O 5 P O 4 P O + Sil P 1 O + Sil P 2 O + Sil 3 P O + Sil 4 P O+ PF-9613 3 P O+ PF-9613 3 P O + Sil 5 -l o g10( pval)1 0 -l o g10( pval) N oN n si o gn nifi si g c nifia nt c a nt 1 0 mi R-21a-3p

mi R-1a-3p -6 -4 -2 0 2 4 6 Log2Fold Change --6 6 - -4 4 - -2 2 0 0 22 44 6 6 Log2FoldLog2Fold Change Change N o. 1- 2 3

B 33 0 Hypertrophy/ Fibrosis Associated U p wit h T A C, D o w n wit h Dr u g 22 0 0

D o w n wit h T A C, U p wit h Dr u g N o. 2 4- 4 5 Si g nifi c a nt

-l o g 1 0( pv al) 11 0

-l o g10( pval) N o n si g nifi c a nt

--6 6 --4 4 - -2 2 00 2 2 4 4 6 6 N o. 4 6- 6 9 Log2FoldLog2Fold Change Change

C 33 0

22 0 N o. 7 0- 1 1 1

-l o g 1 0( pv al) 11 0 -l o g10( pval)

--6 6 - -4 4 - -2 2 0 0 2 2 4 4 6 6 Log2FoldLog2Fold Change Change R o w Mi n R o w M a x

Figure 2.2: P D E5-I and P D E9-I applied to P O heart yield disparate mi R N A profiles.

Left ventricle myocardiu m fro m mice subjected to sha m or P O surgery and subsequently

gi ve n eit her ve hicle, P D E 5-I ( Sil), or P D E 9-I ( P F- 9 6 1 3) were s u bjecte d t o mi R N A

sequencing and subsequent differential expression analysis (n =5 per group). A) V olca n o pl ot

of mi R N As altere d i n P O vers us s ha m, wit h mi R N As rele va nt t o car diac

! & - !

hypertrophy/fibrosi s la bele d. B) V olca n o pl ot of mi R N As altere d i n P O + Sil vers us P O. C)

V olca n o pl ot of mi R N As altere d i n P O + P F -9613 versus P O. For all v olca n o pl ots, dar k gray d ots i n dicate differe ntially e x presse d mi R N As; gree n tria n gles i n dicate mi R N As i ncrease d wit h P O, and decreased with drug treat me nt; re d tria n gles i n dicate mi R N As decrease d wit h

P O, a n d i ncrease d wit h dr u g treat me nt; a n d pi n k d ia m o n ds i n dicate mi R N As la bele d i n pa nel A t hat are ass ociate d wit h car diac hy pertr o p hy a n d fi br osis (le ge n d ca n be f o u n d i n pa nel 1 C). D) Heat ma p of a ll mi R N As c ha n ge d si g nifica ntly wit h P O f or all treat me nt gr o u ps, cl ustere d by b ot h r o ws ( mi R N As) a n d c ol u m ns (sa m ples). R o w la bels (i.e. mi R N A na mes) ca n be f o u n d i n T a bl e 2 .4 .

3 0

Transcripto me for each treat ment sho ws many changes but fe w ar e overla p pi ng

Gi ve n t he r ole of mi R N As, t hese res ults mi g ht pre dict mi ni mal tra nscri pt o me changes fro m P D E 9 -I whereas P D E5 -I treat me nt w o ul d be e x pecte d t o m ore br oa dly alter m R N A expression. This was tested by R N A -se q o n t he sa me s a m ples. T o o ur s ur prise, m ore than t wice as many genes were significantly altered by P D E9 -I ( 1 7 5 6) as c o m pare d t o

P D E5 -I ( 8 6 8) ( Fi g ur e 2 . 3 A ), 8 7 % a n d 7 3 % of t he m bei n g u ni q ue t o P D E 5 -I or P D E 9 -I treat me nt, res pecti vely. A m o n g t he s hare d ge nes, all b ut o ne c ha n ge d i n t he sa me dir ecti o n a n d ma g nit u de ( Fi g ur e 2 . 3 B ), t he o ne e xce pti o n bei n g Cdh20 encoding cadherin -2 0 prec urs or.

K E G G pat h way a nalysis f or t he P O c o n diti o n re veale d ty pical c ha n ges, i ncreasi n g e xtracell ular matri x, cyt os keletal, a n d hy pertr o p hy a n d heart fail ure -relate d ge nes, a n d decreasing metabolic path way related genes. While the specific genes altered by each treat me nt m ostly differe d, pat h way a nalysis yiel de d si milar cl usters, wit h t he n u m ber of ge nes altere d decli ni n g relati ve t o P O i n cas es t o le vels si milar t o S ha m c o ntr ol ( Fi g ur e 2 . 3 C a n d D ). T h us, des pite tar geti n g a si milar ki nase pat h way, P D E 5 a n d P D E 9 i n hi bi t ors i m pacte d ge nes very differe ntly, w hile still c o n ver gi n g o n si milar si g nali n g pat h ways altere d by pressure -overl oa d stress .

3 1 0

- 5

- 1 0

- 1 5

- 2 0

- 2 5

- 3 0

F i g ur e 2 . 3 : T ranscriptoNu mber - of 3 Genes 5 me for P D E5 -I and P D E9 -I sho ws many changes but fe w overlapping ones

A - 4 0 C 88 0 0 PP O O P O+PF -9613 - 4 0 P O + Sil P O + Sil - 6 0 660 0 P O + Sil - 8 0 PP O+PF O+PF-9613-9613 -100 44 0 0 -200 -300 22 0 0 Nu mber of Genes Nu mber of Genes 1531 2 3 4 6 3 4 00 (63.8 %) ( 9. 8 %) (26.4 %) Peroxiso me

Metabolic path ways Phagoso me Fatty aci d el o n gati o n Pyruvate Phagoso metabolis me m Fatty aci d de gra dati o n FocalFocal adhesionadhesion Citrate cycle ( T C A cycle) Glutathione metabolis m P PA R signaling path way Oxidative phosphorylation Cardiac muscle contraction -r e c e pt or i nt er a cti o n Glycolysis / b Gluconeogenesis et a si g n ali n g p at h w a y Dilated cardio myopathy E C M-receptor interaction - Dilated cardio myopathy E C M T GF-beta signaling path way -c yt o ki n e r e c e pt or i nt er a cti o n Che mokine signaling path way T G F CheCysteine mokineRegulation signaling and methionineof pathactin waycytoskeleton metabolis m R e g ul ati o n ofCell a ctiadhesion n c yt o s k molecules el etProtein o n ( C digestion A Ms) and absorption Cell adhesion moleculesPr ot ei n( diC A g e Ms) sti o n a n d a b s or pti o n Glyoxylate and dicarboxylate metabolis m Hypertrophic cardio myopathy ( H C M) V ali n e, l e u ci n e a n d i s ol e u ci n e d e gr a d ati o n Cytokine-cytokine receptor interaction C yt o ki n e Hypertrophic cardio myopathy ( H C M) 1.1. 0 0 B D 00 -5 5

1- 10 0

0.0. 5 5 1- 1 5 5 9613 Log2F C - 2- 20 0

P O+PF 2- 2 5 5

P O+PF-9613 Log2F C 3- 3 0 0

--1. 1. 0 0 -0. 0. 5 5 0.0. 5 5 1.1. 0 0 Nu mber -3 of 3 Genes 5 5 P O+P O+ Sil SilLog2F Log2F C Nu mber of Genes 4 0 - 4 0 T A C 4- 4 0 0 6- 6 0 0 T A C + Sil -8 8 0 0 TA C+PF-9613 --0. 5 5 Cdh20 1-100 0 0 2-2000 0 3-300 0 0 R 2 = 0. 8 9 3 9 wit h C d h 2 0 R 2 = 0. 8 9 3 9 wit h C d h 2 0 2 R R = 2 0.= 0.9 4 9 1 4 91 9 wit wit h h o o ut ut C d d h h 2 2 0 0 l o n g ati o n egradationegradation e Peroxiso me --1. 0 0 d d Peroxiso me

Metabolic path ways u s cl e c o ntr a cti o n m Fatty aci d el o n gati o n Pyruvate metabolis m M et a b oli c p at h w a y s Fatty aci d de gra dati o n Citrate cycle ( T C A cycle) GlutathioneFatt metabolis y a ci d m Pyruvate P metabolis PA R signaling m path way Oxidative phosphorylation F att y a ci d Citr at e c y clCardiac e ( T C A muscle c y cl e)contraction Gl ut at hi o n e m et a b oli s m P P A R signaling path way Oxidative phosphorylation Glycolysis / Gluconeogenesis C ar di a c Glycolysis/ Gluconeogenesis

Cysteine and methionine metabolis m

Glyoxylate and dicarboxylate metabolis m V ali n e, l e u ci n e a n d i s ol e u ci n e d e gr a d ati o n C y st ei n e a n d m et hi o ni n e m et a b oli s m Gl y o x yl at e a n d di c ar b o x yl at e m et a b oli s m V ali n e, l e u ci n e, a n d i s ol e u ci n e F i g ur e 2 . 3 : Transcripto me for P D E5 -I and P D E9 -I sho ws many changes but fe w overlapping ones

3 2

Sa mples fro m the sa me cohort of mice fro m Figure 1 . 1 were s u bjecte d t o R N A -sequen ci n g. A) R N A -seq analysis revealed 234 shared genes bet ween P D E5 -I ( Sil) a n d P D E 9 -I ( P F -9613), with more genes changed overall by P D E9 -I ( 1 7 5 6) t ha n P D E 5 -I ( 8 6 8). B)

Correlation analysis of fold changes of the genes shared bet ween P D E 5 -I and P D E9 -I. C) Ge n e n u m bers i n K E G G pat h ways i de ntifie d t o be upregulated in P O co mpared to sha m for P O, P O +P D E5 -I, and P O +P D E9 -I. D) Ge ne n u m bers i n K E G G pat h ways i de ntifie d t o be do wnregulated in P O co mpared to sha m for P O, P O +P D E5 -I, and P O +P D E9 -I. Stri pe d bars i n t he K E G G path ways graphs indicate pat h ways t hat are n ot si g nifica ntly differe nt fr o m s ha m.

3 3

P D E5 -I and P D E9 -I mediated mi R N A disparities occur at late -stage processing

Micro R N As are transcribed fro m the geno me and processed fro m a pri t o pre f or m i n t h e nucleus by Drosha and D G C R8. The pre -mi R N A is t he n e x p orte d t o t he cyt os ol, a n d converted to its mature for m by Dicer and its partner T R B P, and gro wing evidence supports ki nase si g nali n g c o ntr ol o ver t his pr ocess 1 2 4 . N o study has re p orted a s pecific i nflue nce of

P K G, so we tested whether different mi R N A profiles evolve fro m nuclear or cytosol processing. We focused on a subset of relevant mi R N As ( mi R -1, 1 9 9, 2 0 8 b, 2 1a, a n d 3 4c), each kno w n t o be i n v ol ve d wit h c ar diac hypertrophy and/or fibrosis, and all expressed in cardio myocytes 1 0 4, 1 2 5, 1 2 6 . Pre a n d pri -m i R N A le vels were si milar bet wee n treat me nts ( Fi g ur e

2 . 4 A, 2 . 4 B ), w hereas differe nces i n ex pressi o n a p peared i n t he mature mi R N A as found by mi R N A -se q ( Fi g ur e 1. 4 C ). T h us, t he dis parity i n mi R N A pr ofiles fr o m P D E 5 -I vers us

P D E9 -I i n t he P O -heart occ urre d at t he le vel of cyt os olic pr ocessi n g.

We f urt her teste d if eit her P D E 5 or P D E 9 i n hi biti o n m odifies ge ne ex pressi o n of the pri mary mi R N A processing proteins, Argonaute ( Ago2), microprocessor co mplex sub - u nit - D G C R8, Dicer1, and Drosha. There were no significant differences in expression for any of these genes bet ween the groups ( Fi g ur e 2 . 4 D ).

3 4 !

Figure 2.4: mi R N A disparities fro m different P D E inhibitors occur at late-stage processing

A pri-pri- mi mi R-1a-1 R- 1 a pri-pri- mi mi R-199a R-199a pri-pri- mi R-208b pri-pri- mi mi R- 2R-21a 1 a pri-pri- mi R- R-34c 3 4 c 2. 5 33 2.2. 5 5 33 2. 5 2.2. 5 5

2.2. 0 0 2.2. 0 0 2.2. 0 0 22 22 1.1. 5 5 1.1. 5 5 1.1. 5 5

1.1. 0 0 1.1. 0 0 1.1. 0 0 11 11 0.0. 5 5 0.0. 5 5 0.0. 5 5

00 0.0 0 00 pri- mi R-1a1/ G AP D H m R N A pri- mi R-21a/ G AP D H R N A pri- mi R-34c/ G AP D H R N A pri- mi R-199a/ G AP D H R N A pri- mi R-208b/ G AP D H R N A 0.0 0 0.0 0 pri- mi R-34c/ GAP D H m R NA pri- mi R-21a/ GAP D H m R NA pri- mi R-1a/ GAP D H m R NA pri- mi R-199a/ GAP D H m R NA pri- mi R-208b/ GAP D H m R NA B pre-pre- mi mi R-1a R-1a pre-pre- mi mi R-199a R-199a pre-pre- mi mi R-208b pre- mi mi R-21a R-21a pre-pre- mi mi R-34c R-34c 2.2. 5 5 1.1. 5 5 33 2.2. 5 5 2.2. 5 5

2.2. 0 0 2.2. 0 0 2.2. 0 0 1.1. 0 0 22 1.1. 5 5 ** 1.1. 5 5 1.1. 5 5 1.1. 0 0 1.1. 0 0 1.1. 0 0 0.0. 5 5 11 0.0. 5 5 0.0. 5 5 0.0. 5 5 pre- mi R-1a/ R N U6 R N A pre- mi R-21a/ R N U6 R N A pre- mi R-34c/ R N U6 R N A pre- mi R-199a/ R N U6 R N A 0.0 0 0.0 0 pre-00 mi R-208b/ R N U6 R N A 0.0 0 0.0 0 pre- mi R-21a/ R N U6 m R NA pre- mi R-34c/ R N U6 m R NA pre- mi R-208b/ R N U6 m R NA pre- mi R-1a/ R N U6 m R NA mi R- 1 a pre- mi R-199a/ R N U6 m Rmi NA R-199a mi R-208 b mi R-21a mi R-34c mi R- 1 a mi R- 1 9 9 a mi R- 2 0 8 b mi R- 2 1 a mi R- 3 4 c C 3 3 2.2. 0 0 2.2. 0 0 2.2. 0 0 2.2. 0 0

* 1.1. 5 5 1.1. 5 5 1.1. 5 5 1.1. 5 5 2 2 * * 1.1. 0 0 1.1. 0 0 * 1.1. 0 0 1.1. 0 0 11 * 0.0. 5 5 0.0. 5 5 *** *** 0.0. 5 5 0.0. 5 5 * **** mi R-1a/ R N U6 R N A

** mi R-21a/ R N U6 R N A ** *** mi R-34c/ R N U6 R N A mi R-208b/ R N U6 R N A ** mi R-199a/ R N U6 R N A *** 00 0.0 0 0.0 0 0.0 0 0.0 0 mi R-1a/ R N U6 m R NA mi R-21a/ R N U6 m R NA mi R-208b/ R N U6 m R NA mi R-34c/ R N U6 m R NA mi R-199a/ R N U6 m R NA D gcr8 Di c er 1 Dr os ha Ar gA o g n o2 a ut e 2 D c gr 8 Di c er 1 Fractional ShorteningDr os h a D 2.2. 0 0 2.2. 0 0 1.1. 5 5 1.1. 5 5 8 0 SSha h a m m 1.1. 5 5 1.1. 5 5 1.1. 0 0 1.1. 0 0 $ $ 6 0 P O O 1. 0 1.1. 0 0 1. 0 PP O O + + Sil 0.0. 5 5 0.0.* 5 5 * 0.0. 5 5 0.0. 5 5 4 0 PP O+ O+PF-9613 PF-9613 Ago2/ GAPDH mRNA Dicer1/ GAPDH mRNA Dgcr8/ GAPDH mRNA 0. 0 0.0 0 Drosha/0.0 0 GAPDH mRNA Ago2/ GAPDH0 mRNA 0.0 0 Dicer1/ GAP D H m R NA Drosha/ GAP D H m R NA Dgcr8/ GAP D H m R NA 2 0

%Fractional0 Shortening Figure 2.4: mi R N A disparities fro m different P D E inhibitorsBaseline occur 1 Weekat late-stage 5 Weeks processing

� � � �

A -C ) q R T -P C R a nalysis f or A) pri -mi R N As, B) pre -mi R N As, a n d C) mature mi R N As for a pa nel of mi R N As selecte d fr o m t he lar ger sequencing data set that are associated with cardiac hypertrophy and fibrosis (pink dia mond mi R N As fro m Figures 2 A, B, and C) . D) q R T -

P C R analysis for mi R N A processing genes. A nalysis was perf or med on the sa me sa mp les used for sequencing analysis (n =5 per group). * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 as co mpared to P O. Data is presented as mean ± S E M. Data was a nalyze d usi n g a o ne - way A N O V A wit h a D u n nett p ost h oc test, or a Kr us kal -W allis test wit h D u n n’s p ost h oc test.

3 6

Effect on mi R N As fro m alternative sti mulation of G C -1 or G C -2 A si g n ali n g

P D E5 -I pri marily regulates c G M P generated fro m G C -1, w hic h is acti vate d by N O.

T o f urt her test if t his pat h way is i m p orta nt f or br oa d suppression of mi R N As by P D E5 -I, we st u die d mice e x p ose d t o P O a n d treate d wit h a direct G C -1 acti vat or, B A Y 6 0 -2 7 7 0.

B A Y 6 0 -2770 treat ment also reduces cardiac hypertrophy, f ibrosis, and i mproves function si milar t o P D E 5 -I (Fi g ur e 2 . 5 )1 2 7 . Thirteen mi R N As were assayed, each kno wn to be rele va nt t o pr o - or a nti -hy pertr o p hic sig nali ng 1 0 5 -1 0 7, 1 2 8 -1 3 9 a n d di vi de d t he m i nt o t h ose si g nifica ntly i ncrease d or decreased by P O. B ot h P D E 5 -I a n d G C -1 acti vati o n dis playe d si milar re d ucti o ns of mi R N As t hat were i ncrease d by P O, ( p = N S, F i g ur e 2 . 6 A ). H o we ver, a m o n g t h ose t hat decli ne d wit h P O (a nti -hypertrophic mi R N As), P D E5 -I furt her reduced 6 o ut of 7, w hereas G C -1 acti vati o n i m pac te d n o ne ( p < 0. 0 5 by Fis her’s e xact test, F i g ur e

2 . 6 B ). T h us, tri g geri n g P K G by e n ha nci n g c G M P sy nt h esis via G C -1 acti vati o n or by i mpeding its hydrolysis by P D E5 -I ha d si milar effects o n mi R N As p ositi vely ass ociate d wit h hy pertr o p hy, b ut o nly P D E 5 -I i m pa cte d mi R N As t hat were a nti -hypertrophic. Thus, even differe nt ways of sti m ulati n g t he sa me pat h way d o n ot g uara ntee i de ntical mi R N A m o d ulati o n.

As myocardial P D E9 pri marily hydrolyzes c G MP coupled to G C -2 A 1 1 4 , a n alter nati ve t o P D E 9 -I is t o s ustai n sti m ulati o n of t his N P -receptor coupled cyclase. We used a ge netic m o del w ith cardio myocyte -targeted overexpression of G C -2 A, w hic h is als o protective against P O induced cardiac hypertrophy 1 1 5 . As wit h P D E 9 -I, t here were mi ni mal changes in pro - or a nti -hypertrophic mi R N As in P O -myocardiu m fro m G C -2 A overexpressors as co mpared to litter mate controls ( Fi g ur e 2 . 6 C ).

3 7 !

Figure 2.5: Phenotypic i mprove ment in P O mice follo wing G C-1 activation.

A B C Fi br o si s FractionalFr a cti o n al ShorteningS h ort e ni n g LVL V mass M a s s Fi br o si s 1Fi1 5 5 br o si s 88 0 0 22 5 5 0 0 Sha m ******** 1 5 $ 22 0 0 0 0 P O 66 0 0 SSha h a m m 11 0 0 ******** B A Y ******** 11 5 5 0 0 $ $ $ PP O O 44 0 0

% % ar e a **** 11 0 0 0 0 1 0 % Ar e a 55 PB O+BAYA Y 22 0 0 LV Mass5 (5 0 0 mg) LV Mass ( mg)

0 00 0 % ar e a %Fractional0 0 Shortening % Fr a cti o n al S h ort e ni n g 5

Figure 2.5: Phenotypic i mprove ment in P O mice0 follo wing G C-1 activation.

C57 Bl6/J mice were subjected to sha m or P O surgeries. P O mice were subsequently treated

wit h eit her ve hicle or B A Y 6 0- 2 7 7 0 ( G C- 1 acti vat or) a n d f oll o we d f or 3 wee ks 1 2 7 . A) Left

ve ntric ular mass. B) Fracti o nal s h orte ni n g. C) Fibrosis as quantified by Masson’s trichro me

stai n. All para meters were si g nifica ntly i m pr o ve d by treat me nt wit h B A Y 6 0- 2 7 7 0. ****

p <0.0001 versus sha m. $ p <0.05 versus P O, $$ p <0.01 versus P O. N =8-15 per group. Data

is prese nte d as mea n ± S E M. Data was a nalyze d usi n g a Kr us kal- Wallis test wit h D u n n’s

p ost h oc test.

� � � � !

U p wit h P O 2. 0 Figure 2.6: Effect on mi R N As fro m alternative sti mulation of G C-1 or G C-2 A signaling U p wit h P O D o w n wit h P O A U p wit h P O B D o w n wit h P O 2.2. 0 0 3.3. 5 5

1. 5 3.3. 0 0 1. 5 1. 5 2.2. 5 5

2.2. 0 0 SSha h a m m Sha m 1.1. 0 0 1. 0 P O PP O O ��* 1.1. 5 5 * P O+BAY PP O+BAY O+BAY ��* P O + Sil mi R/ R N U 6 m R N A

mi R/ R N1.1. U 6 0 0 m R N A

0. 5 miRNA/RNU6* RNA PP O O + + Sil Sil miRNA/RNU60. 5 RNA �� �� ��* �� ��* �� * * �� ��* * * ��* 0.0. 5 5 �� * ��* miRNA/RNU60. 5 RNA ��* * *�� * ��* ��* * * 0.0 0 0.0 0 * * * * 11 a a 1 0 1 a 1 3 3 a 1 4 5 2 6 b 3 0 b 4 5 1 a 1 9 9 a 2 0 8 b 22 1 a a 2 1 1 4 4 2 7 7 b b 3 4 4 c c 1 4 5 2 6 b 3 0 b 199a 208 b * 101a 133a 451a * mi R- 1 a C mimi R- R- 1 1 a mimi R-199a R- 1 90. 9 a 0 mimi R-208 R- 2 0 8 b b mimi R-21a R- 2 1 a mi R- R-34c 3 4 c 1.1. 5 5 2.2. 5 5 2.2. 0 0 2.2. 0 0 44 �� WT Sha m 1. 5 * �� WWT T S Sha h a m m 2.2. 0W 0 T P O * 2 1 a 2 1 4 2* 7 b 3 4 c 199a 1.1. 5 5 208 b 1.1. 5 5 33 1.1. 0 0 G C-2A OE Sha m 1.1. 5 5 WW T T P P O O G C-2A OE P O 1.1. 0 0 1.1. 0 0 22 1.1. 0 0 1. 0 GG C-2A C-2A OE OE Sha Sha m m 0.0. 5 5 0. 5 0. 5 1 0.0. 5 5 0. 5 0. 5 1 GG C- C-2A 2 A O OEE P OP O mi R-1a/ R N U6 R N A mi R-21a/ R N U6 R N A mi R-34c/ R N U6 R N A mi R-208b/ R N U6 R N A mi R-199a/ R N U6 R N A

mi R-1a/0.0 R 0 N U6 m R NA 0.0 0 0.0 0 0.0 0 00 mi R-21a/ R N U6 m R NA mi R-34c/ R N U6 m R NA mi R-199a/ R N U6 m R NA mi R-208b/ R N U6 m R NA 0. 5

Figure 2.6: Effect on mi R N As fro m alternative sti mulation of G C-1 or G C-2 A signalingmi R-1a/ R N U6 R N A 0. 0 A a n d B) Left ve ntricle my ocar di u m fr o m mice s u bjecte d t o s ha m or P O s ur gery a n d s u bse q ue ntly gi ve n eit her ve hicle, P D E 5-I ( Sil), or a n

s G C activator ( B A Y 60-2770) were subjected to q R T- P C R analysis for A) pro-hypertrophic mi R N As and B) a nti- hy pertr o p hic mi R N As.

Sil do wnregulates both categories of mi R N As, whereas B A Y do wnregulates only pro-hypertrophic mi R N As. N =4-11 per group. * p <0.05

co mpared to P O for Sil and B A Y analysis (s maller p-values are not differentiated by different sy mbols, though so me co mparisons were

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very si g nifica nt). C) Mice overexpressing G C -2 A a n d W T litter mate c o ntr ols were s u bjecte d t o s ha m or P O s ur gery ( n = 3 -5 per gr o u p).

M y ocar dial tiss ue was used for q R T -P C R analysis of mi R N As. * p <0.05, ** p <0.01, *** p <0.001 as co mpared to indicated group for G C -

2 A a nalysis. All data is prese nte d as mea n ± S E M. Data was a nalyze d usi n g a o ne -way A N O V A wit h a D u n nett p ost h oc test, or a Kr us kal -

Wallis test wit h D u n n’s p ost h oc test.

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P D E 5 i n hi biti o n i n t h e n or m al h e art a n d l u n g d o es not have t he sa me effect as i n disease d hearts

Gi ve n t he br oa d i m pact of P D E 5 -I o n mi R N A e x pressi o n, we w o n dere d if si milar effects would be obser ved under nor mal c o n diti o ns or if a stresse d or disease state was re q uire d. T o test t his, we e x p ose d C 5 7 Bl / 6J mice t o P D E 5 -I (sil de nafil) f or 5 wee ks a n d t he n assesse d a re prese ntati ve pa nel of 1 1 mi R N As t hat all decli ne d wit h t his t hera py i n t he

P O heart. Of t hese, 6 s h o w e d si g nifica nt c ha n ges ( mi R -1a, mi R -2 1a, mi R -2 6 b, a n d mi R -2 7 b decli ne d; mi R -1 3 3a a n d mi R -208 b i ncrease d), s o a maj ority ( 6 4 %) were disc or da nt wit h P O - induced changes (p =0.0039 by Fisher’s exact test, Fi g ur e 2 . 7 ).

P D E5 -I i m pacts t he e nzy me gl o bally, i ncl uding in the lung where P D E5 expression is pr o mi ne nt 1 4 0 . T o test if its effects o n mi R N A e x pressi o n were or ga n i n de pe n de nt, we exa mined lungs fro m nor mal and P O mice, assaying those kno wn to be highly ex presse d i n l u n g 1 4 1, 1 4 2 . All were confir med as being reduced in P O +P D E5 -I myocardiu m by qP C R and/or miR N A -se q, yet n o ne were altere d i n l u n gs by P D E 5 -I u n der eit her c o n diti o n

(Fi g ur e 2 . 7 B, Fi g ur e 2 . 8 ). T his i n dicates t hat P D E 5 -I d oe s n ot tri g ger a path way broadly s u p pressing mi R N A maturation machinery, as expected if Dicer or associated proteins were i n hi bite d, a n d its effects are m ore pr o mi ne nt i n t he heart a n d lar gely de pe n d o n t he e xiste nce of u n derlyi n g disease.

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Figure 2.7: P D E5 inhibition in the nor mal heart and stressed lung

A mimi R- R- 1 1 a a mimi R-101aR- 1 0 1 a mimi R-133a R- 1 3 3 a mimi R-26 R- 2 6 b b mimi R-30 R- 3 0 b b mimi R-451a R- 4 5 1 a 1.1. 5 5 1.1. 5 5 2.2. 0 0 1.1. 5 5 1.1. 5 5 1.1. 5 5 �** �� 1.1. 5 5 1.1. 0 0 **� �� 1.1. 0 0 1.1. 0 0 �** �� 1.1. 0 0 1.1. 0 0 1.1. 0 0 0.0. 5 5 0.0. 5 5 0.0. 5 5 0.0. 5 5 0.0. 5 5 0.0. 5 5 mi R-1a/ R N U6 R N A mi R-30b/ R N U6 R N A mi R-26b/ R N U6 R N A mi R-451a/ R N U6 R N A mi R-101a/ R N U6 R N A mi R-133a/ R N U6 R N A mi R-1a/ R N U6 R N A 0. 0 mi R-30b/0. 0 R N U6 R N A 0. 0 0.0 0 0. 0 0. 0 mi R-26b/0 R N U6 R N A 0 0 mi R-451a/ R N U6 R NA mi R-101a/0 R N U6 R NA 0 mi R-133a/ R N U6 R NA mi R-R-199a 1 9 9 a mimi R-208 R- 2 0 8 b mimi R-21a R- 2 1 a Do wnmimi R-27 R- with 2 7 b TA C mi R- R-34c 3 4 c 1.1. 5 5 1.1. 5 5 1.1. 5 5 1.1. 5 5 2.2. 0 0 **� �� 1. 5 1.1. 5 5 CC o o ntr ntr ol ol 1.1. 0 0 1.1. 0 0 1.1. 0 0 1.1. 0 0 * �** �� 1.1. 0 0 ***� � �� SilSil 0.0. 5 5 0.0. 5 5 0.0. 5 5 1. 0 0.0. 5 5 * *0.0. 5 5 mi R-34c/ R N U6 R N A mi R-27b/ R N U6 R N A mi R-21a/ R N U6 R N A mi R-208b/ R N U6 R N A mi R-199a/ R N U6 R N A 0. 0 0.0 0 0. 0 0.0 0 0.0 0 mi R-21a/0 R N U6 R N A mi R-27b/ R N U6 R N A mi R-34c/0 R N U6 R N A * mi R-199a/ R N U6 R NA mi R-208b/ R N U6 R NA * mi R-26 b mi R-29a 0. 5 mi R-29c mi R-30 b mi R-30 d mi R-451a B mi R- 2 6 b mi R- 2 9 a mi R- 2 9 c mi R- 3 0 b mi R- 3 0 d mi R- 4 5 1 a 1.1. 5 5 2.2. 0 0 1.1. 5 5 1.1. 5 5 1.1. 5 5 2.2. 0 0 miRNA/RNU6 RNA mi R-451a Sha m 0. 0 T A C 1.1. 5 5 1.1. 5 5 1. 5 1.1. 0 0 1.1. 0 0 1.1. 0 0 1.1. 0 0 S hT a A m C + Sil 2 1 a 2 1 4 2 7 b 3 4 c Sha m 195a 199a 208 b 1. 0 1.1. 0 0 1. 0 PP O O 0.0. 5 5 0.0. 5 5 0.0. 5 5 1. 0 0.0. 5 5 0.0. 5 5 0.0. 5 5 PP O O + + Sil Sil mi R-26b/ R N U6 R N A mi R-29a/ R N U6 R N A mi R-29c/ R N U6 R N A mi R-30b/ R N U6 R N A mi R-30d/ R N U6 R N A mi R-26b/ R N U6 R N A mi R-29a/ R N U6 R N A mi R-29c/ R N U6 R N A mi R-30b/ R N U6 R N A mi R-30d/ R N U6 R N A mi R-451a/ R N U6 R N A 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 mi R-451a/0.0 0 R N U6 R NA 0. 5 Figure 2.7: P D E5 inhibition in the nor mal heart and stressed lung

mi R-451a/0. 0 R N U6 R N A A) q R T- P C R was perfor med for various mi R N As in tissue fro m nor mal, non-stressed C57 Bl6/J mice treated with P D E5-I (Sil) for 5

wee ks ( n = 5 per gr o u p). Car diac mi R N As t hat n or mally decrease wit h P O (t o p r o w) a n d i ncrease wit h P O ( b ott o m r o w) assesse d fr o m left

ventricular tissue. Only mi R N As that were significantly decreased by Sil in Figure 4 were assayed. The dashed line on each graph represents

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t he le vel t o w hic h Sil re d uce d mi R e x pressi o n i n t he P O m o del. B) Lung tissue fro m C57 Bl6/J mice subjected to P O, P O +P D E 5 -I ( Sil), or sha m surgeries was subjected to q R T -P C R f or l u n g mi R N As ( n = 5 -6 per gr o u p). * p < 0. 0 5, ** p < 0. 0 1, *** p < 0. 0 0 1, **** p <0.0001 as c o m pare d t o c o ntr ol f or s ha m Sil heart a nalysis. Data is prese nte d as mea n ± S E M. F or A, data was a nalyze d usi n g t w o -taile d u n paire d t - tests. F or B, data was a nalyze d usi n g a o ne -way A N O V A wit h a D u n nett p ost h oc test, or a Kr us kal -Wallis test wit h D u n n’s p ost h oc te st.

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Figure 2.8: P D E5-I treat ment does not alter mi R N A expression in nor mal mouse

l u n g.

mi R-29a mi R-29c mi R-26 b mi R- 2 9 a mi R- 2 9 c mi R- 2 6 b 1.1. 5 5 2.2. 0 0 1.1. 5 5

1.1. 5 5 1.1. 0 0 1.1. 0 0

1.1. 0 0 0.0. 5 5 0.0. 5 5 0.0. 5 5 mi R-29c/ R N U6 R N A mi R-29a/ R N U6 R N A mi R-26b/ R N U6 R N A mi R-29c/0. R0 0 N U6 R N A mi R-29a/0.0 0 R N U6 R N A mi R-26b/0.0 R 0 N U6 R N A mi R-30 b mi R-30 d mi R-451a mi R- 3 0 b mi R- 3 0 d mi R- 4 5 1 a 1.1. 5 5 1.1. 5 5 2.2. 0 0 Do wn with TA C Sha m 1.1. 5 5 S h a m + Sil 5 w k 1.1. 0 0 1. 5 1.1. 0 0 CC o o ntr ntr ol ol * 1.1. 0 0 0.0. 5 5 0.0. 5 5 SilSil 1. 0 * 0.0. 5 5 * mi R-30d/ R N U6 R N A mi R-451a/ R N U6 R N A mi R-30b/ R N U6 R N A mi R-30b/ R N U6 R N A mi R-30d/ R N U6 R N A 0.0 0 0.0 0 mi R-451a/0.0 R 0 N U6 R NA * 0. 5 * miRNA/RNU6 RNA Figure 2.8: P D E5-I treat0. 0 ment does not alter mi R N A expression in nor mal mouse

2 1 a 2 1 4 2 7 b 3 4 c 195a 199a l u n g. 208 b

q R T- P C R was perfor med for various mi R N As in lung tissue fro m nor mal, non-stressed

C 5 7 Bl 6 /J mice treate d wit h P D E 5-I ( Sil) f or 5 wee ks ( n = 5 per gr o u p). N o si g nifica nt

changes were seen with P D E5-I treat ment. Data is presented as mean ± S E M. Data was

a nalyze d usi n g t w o-taile d u n paire d t-tests.

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DIS C US SI O N

To our kno wle d ge, t his st u dy is t he first t o directly c o m pare mi R N A si g nat ures fr o m very cl osely relate d a n d p he n oty pically si milar t hera pies ( P D E 5 a n d P D E 9 i n hi biti o n) t hat both effectively counter the sa me pathological hypertrophic heart disease by ulti mately acti vati n g t he s a me pr otei n ki nase ( P K G). T he differe nce bet wee n t he m lies pri marily i n t he s u b -cell ular l ocalizati o n of t he t w o P D Es, a n d t his ulti mately defi nes w here t he effect is act uate d. Des pite stri ki n gly si milar i m pr o ve me nt i n press ure -overload induced heart disea se and many co m mon features of both P D E5 -I and P D E9 -I, we f o u n d nearly o p p osite effects o n mi R N A e x pressi o n, t he f or mer s u p pressi n g m ost of t he m w hile t he latter ha d virt ually n o i m pact.

T his stu dy reveals several sur prisi ng a spects of mi R N As . First, it s h o ws t hat t heir e x pressi o n ca n be br oa dly a n d p ote ntly m o difie d by a cli nically wi dely use d p har mace utical, si multaneously reversing many mi R N A changes pathophysiologically relevant to pressure - overl oad i nduced heart disease. I m p orta ntly, t hi s effect re q uires p har mace utical bi ol o gy i n the appropriate intracellular co mpart ment, and cannot be predicted purely based on the pr otei n it affects. Sec o n dly, it dra matically de m o nstrates t hat bi ol o gical c o n diti o ns t hat appear phenotypic ally very si mila r ca n ha ve m arkedly disparate mi R N A expression profiles.

Lastly, it s h o ws t hat mi R N As altere d by disease d o n ot nee d t o be re verse d f or a treat me nt to effectively counter tissue and organ -level pathology and pathophysiology. This poses s o me qua ndaries f o r t heir use as bio markers for therapeutic responses.

B ot h P D E 5 a n d P D E 9 i n hi biti o n a u g me nt c G M P a n d acti vate P K G. T his ki nase acti vati o n c o nfers a nti -fi br otic a n d a nti -hypertrophic effects by blocking Gq -pr otei n rece pt or c o u ple d acti vati o n 2 8 , a n d i n hi biti n g tra nsie nt rece pt or p ote ntial ca n o nical i o n c ha n nel (ty pe 6) t o bl u nt n uclear fact or of acti vate d T -cells and seru m response factor

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regulated gene expression 6 5, 1 1 4, 1 4 3 . T he p o ols of P K G acti vate d by eit her P D E 5 or P D E 9 inhibition share these properties, but also display do wnstrea m phosphorylation profiles u ni q ue t o eac h i nter ve nti o n 1 1 4 . We f o u n d o nly t w o pri or st u dies re p ort i n g mi R N As altere d by P D E5 -I. Eac h i n v ol ve d very dif ferent models and did not assess mi R N A expression broadly. For exa mple, pig hearts subjected to cardiac arrest and resuscitation had reduced mi R -1 and increased mi R -1 3 3a wit h sil de nafil tr eat me nt 1 4 4 . Sil de nafil re d uce d mi R -1 i n t he c urre nt m o del as well. T he sec o n d st u dy e xa mi ne d a di p ose tiss ue mi R N A e x pressi o n i n ty pe

2 dia betic patie nts treate d wit h c hr o nic hi g h d ose P D E 5 -I, and found P D E5 -I decr ease d mi R -2 2 -3 p ex pressi o n 1 4 5 . A gai n, t he m o del a n d tiss ue e xa mi ne d are very differe nt t o t he prese nt w or k, a n d o nly selecti ve a nalysis was pr o vi de d. O ur o bser vati o n t hat P D E5 -I d oes n ot si m ply s u p press mi R N A le ve ls i n n or mal mice or ot her n o n -car diac tiss ue fr o m P O mice s h o ws t his is n ot a bla n ket effect, b ut m ore s pecific t o t he tiss ue a n d c o n diti o n. We fi n d n o reports of mi R N A changes with P D E9 -I. F urt her m ore, t he res u lts wit h P D E 9 -I are u n us ual i n t hat we als o f o u n d n o r e p orts of a t hera py c o nferri ng su bsta ntial be nefits agai nst disease yet wit h virt ually n o i m pact o n mi R N A si g nali n g c o u ple d wit h t he disease. T his d oes n ot mea n ge ne tra nscri pti o n is u naltere d, j ust t h at it is diss ociate d fr o m mi R N A re g ulati o n .

P D E 5 a n d P D E 9 eac h prefere ntially re g ulate differe nt c G M P p o ols li n ke d t o eit her

N O or N P signaling, respectively. Ho wever, we found even targeting presu mably the sa me c G M P p o ol b ut by differe nt strate gies – e. g. G C -1 acti vati o n vers us P D E 5 -I – t hat mi R N A ex pressi o n pr ofiles c o nsiste ntly differe d w hile or ga n a n d tiss ue le vel i m pr o ve me nt of u n derlyi n g heart disease was very si milar 1 2 7 . T he s pecific differe nces were i ntri g ui n g, as u nli ke P D E 9 -I, or u pre g ulati o n of t he N P -ass ociate d G C -2 A cyclase, G C -1 acti vati o n a n d

P D E5 -I both consistently reduce expression of pro -hy pertr o p hic mi R N As t hat are ot her wise i ncrease d by P O. H o we ver, mi R N As t hat decli ne wit h P O are u naltere d by G C -1

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acti vati o n, b ut re d uce d f urt her by P D E 5 -I. Sta tistically, t his c o nsiste nt patter n was u nli kely d ue t o c ha nce, a n d s h o ws t hat re gi o nal ki nase si g nali n g wit hi n t he cyt os ol ca n yiel d directi o nally c o hesi ve c ha n ges i n mi R N A e x pressi o n. T his t o o may relate t o c o m part me nt differences bet ween P D E 5 a n d G C -1, wit h G C -1 resi di n g i n b ot h t he cyt os ol a n d at t he plas ma me mbrane 2 1 , whereas P D E5 nor mally localizes to the sarco mere 6 4, 1 1 4 . P D E 5 -I ca n o nly alter c G M P i n t he na n o -do ma i n i n w hic h it is e x presse d; w hile G C -1 sti m ulati o n generates c G M P that can also be regulated by other P D Es besides P D E5 1 4 6 , s o t heir i m pact is n ot i de ntical.

The mechanis m by which P D E5 -I br oa dly alters mi R N A e x pressi o n i n t he P O heart re mai ns u n k n o w n. Gi ve n t hat pri - a n d pre - mi R N As are si milar bet ween P D E5 -I and P D E9 -

I, t he dis parity see ms li kely t o resi de at t he le vel of cyt os olic Dicer / Ar g o na ute mi R N A processing and/or ribonucleases 1 2 4 . W hile ge ne e x pressi o n of t he rele va nt pr otei ns was u nc ha n ge d, t his d oes n ot precl u de t heir p ost -tra nslati o nal m o dificati o n . Our phospho - kino me analysis of both P D E5 -I and P D E9 -I treat me nt i n P O di d n ot fi n d c ha n ges i n mi R N A p r ocessi n g pr otei ns fr o m eit her 1 1 4 , t h o u g h lac k of detecti o n d oes n ot mea n t hey d o not exist. An exa mple where kinase modulation of mi R N A processing occurs is Argonaute

2 phosphorylation by casein kinase -1 al p ha 1 t hat br o a dly i m pacts mi R N A -gene suppression

1 4 7 . This study utilized a geno mic C RIS P R/ Cas9 knock -o ut strate gy t o i de ntify pr otei ns t hat bl u nt mi R N A pr ocessi n g. Of n ote, P K G was n ot i de ntifie d i n t his scree n. F urt her m ore, if

P K G had a general effect on Dicer/ Argonaute processing, one would expect P D E5 -I t o suppress mi R N A expression in other tissues and conditio ns (e.g. wit h or wit h o ut disease), but this was not observed. Whatever changes P D E5 can induce also appear t iss ue a n d c o n diti o n s pecific, as si milar br oa d -suppression was not observed in nor mal hearts or the l u n g. It is als o u nli kely t hat P D E 5 -I w o ul d ne cessarily a meli orate diseases ge nerated by

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artificial o vere x pressi o n of a pat h o ge nic mi R N A, or t hat its e x pre ssi o n / acti vity is altere d by s uc h m o dels. U nf ort u nately, t he o b vi o us re d ucti o nist m o del syste m - is olate d c ult ure d my ocytes - proved inadequate fo r re plicati n g mi R N A si g nat ures f o u n d i n vi v o. T his is li kely due to the absence of required environ mental cu es a n d stresses prese nt i n i ntact hearts.

Mec ha nistic dissecti o n of t he c urre nt si g nali n g w o ul d a p pear t o re q uire m ore c o m ple x i n vi v o ge netic m o d els w here via bility is ofte n a li mitati o n.

Wit h res pect t o w hy P D E 9 i n hi bit ors d o n ot affect mi R N A e x pressi o n i n t he P O m o del, t his t o o may relate t o w here its c G M P m o d ulati o n occ urs. P D E 9 f u ncti o nal i nteracti o n wit h natri uretic pe pti de rece pt or -c o u ple d c G MP and co -l ocalizati o n wit h sarcoplas mic reticular me mbranes suggest co mpart ments re moved fro m cytosolic mi R N A pr ocessi n g. It is n ot d ue t o a fail ure t o get t he i n hi bit or i nt o t he my ocyte, as we re p orte d p ote nt i n vi v o a n d i n vitr o effects o n c G M P si g na li n g fr o m P D E 9 i n hi biti o n, i ncl u di n g effects o n a nti -fibrotic gene expression and hypertrophy markers, that a re a bs e nt w he n t he ge ne f or t he pr otei n is sile nce d 1 1 4 . As sho wn here, P D E9 -I also has marked effects on gene ex pressi o n - it j ust d oes s o wit h o ut alteri n g mi R N As, a n d li ke P D E 5 -I i n hi biti o n, i m pacts p ost -tra nslat i o nal pr otei n p h os p h orylati o n.

In su m mary, we have sho wn ho w dra matically different mi R N A expression profiles ca n be ass ociate d wit h si milarly tar gete d ki nase acti vati o n strate gies t hat s uccessf ully treat t he i de ntical heart disease – one broadly suppress i n g e x pressi o n w hile t he ot her lea vi n g it virtually unchanged. The results indicate that i mproving myocardial and cha mber disease p he n oty pe ca n be ac hie ve d wit h o ut e n ga gi n g mi R N As, a n d t hat wit h or wit h o ut t his engage ment, gene expression i mpacting si milar si g n ali n g pa t h ways ca n still occ ur. Lastly, we reveal a barcode -li ke si g nat ure pr o vi de d by mi R N A pr ofiles t hat ca n disti n g uis h bet wee n

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treat me nts tar geti n g t he sa me o verall pat h way. T his may pr o vi de a usef ul t o ol f or dr u g develop ment and precision thera py.

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T a bl e 2 .1: mi R N As differentially expressed in P O as co mpared to sha m.

Left ventricle myocardiu m fro m mice subjected to sha m or P O surgery and subsequently gi ve n eit her ve hicle, P D E 5 -I ( Sil), or P D E 9 -I ( P F -9 6 1 3) was s u bjecte d t o mi R N A sequencing and subsequent differential expression analysis (n =5 per group). The log2 fold c ha n ge a n d p val ue are pr o vi de d f or eac h mi R N A, wit h ta ble s orte d by p val ue.

mi R N A I D log2 Fold Change (vs. sha m) p v al u e m m u -mi R -208 b -3 p 4.177277275 4. 1 5 E -6 9 m m u -m i R -4 5 5 -3 p 2.566344072 6. 0 1 E -2 3 m m u -mi R -1247 -5 p 2.245542141 1. 1 6 E -1 9 m m u -mi R -1 9 9 -5 p 1.987698664 3. 1 3 E -1 9 m m u -mi R -3 4c -5 p 2.081491061 4. 7 6 E -1 8 m m u -mi R -2 1a -5 p 1.778120295 1. 5 0 E -1 7 m m u -mi R -2 1 4 -3 p 1.846356429 1. 6 2 E -1 5 m m u -mi R -2 1 4 -5 p 1.803377612 2. 4 7 E -1 3 m m u -mi R -1 9 9 -3p/ m mu -mi R -199 b -3 p 1.531757504 8. 9 6 E -1 3 m m u -mi R -146 b -5 p 1.66286642 1. 4 1 E -1 1 m m u -mi R -2 1a -3 p 1.486612606 6. 2 2 E -1 0 m m u -mi R -1 2 7 -3 p 1.228474661 1. 7 8 E -0 8 m m u -mi R -2 2 4 -5 p 1.282069615 1. 1 7 E -0 7 m m u -mi R -199 b -5 p 1.25544462 2. 3 3 E -0 7 m m u -mi R -1 4 0 -3 p 1. 1 4 9507292 2. 3 9 E -0 7 m m u -mi R -3 4 b -5 p 1.713965516 1. 2 6 E -0 6 m m u -mi R -4 1 1 -5 p 1.093259429 1. 4 0 E -0 6 m m u -mi R -1247 -3 p 2.407271182 6. 2 6 E -0 6 m m u -mi R -9 -5 p -1.15246671 7. 2 8 E -0 6 m m u -mi R -4 5 5 -5 p -1.125269179 7. 2 9 E -0 6 m m u -mi R -2 9 8 -5 p 1.121706324 7. 9 4 E -0 6 m m u -mi R -5 0 1 -5 p 1 .857763818 2. 4 8 E -0 5 m m u -mi R -2 7 b -3 p 0.85679754 3. 0 9 E -0 5 m m u -mi R -3 0 0 -3 p 0.985183431 4. 5 5 E -0 5 m m u -mi R -4 9 9 -5 p -0.777585635 0.000147302 m m u -mi R -4 1 0 -3 p 0.89245254 0.000161098 m m u -mi R -497a -5 p 0.776615594 0.000174491 m m u -mi R -3 7 9 -5 p 0.982619158 0.0002713 6 3 m m u -mi R -5 4 1 -5 p 0.853822266 0.000285637 m m u -mi R -133a -5 p -0.796649968 0.000305674 m m u -mi R -9 2 b -3 p 0.883212541 0.000384535 m m u -mi R -2 2 2 -3 p 0.744534001 0.000496055

5 0

m m u -mi R -4 3 1 -5 p 0.882820558 0.000496317 m m u -mi R -1 4 0 -5 p 0.883350398 0.000507314 m m u -mi R -2 4 -3 p 0 .76897668 0.000551962 m m u -mi R -3 3 8 -3 p -0.837017936 0.000575003 m m u -mi R -5 7 4 -3 p 0.798361265 0.000606652 m m u -mi R -125 b -1 -3 p 0.812749712 0.000614638 m m u -mi R -5 8 2 -5 p -1.01850343 0.000615216 m m u -mi R -1 3 5 -5 p -0.950045305 0.000621182 m m u -mi R -2 3 b -3 p 0.719158 4 7 9 0 .000643536 m m u -mi R -1 9 5 -5 p 0.723228506 0.000698554 m m u -mi R -1 0a -5 p -0.769965209 0.000984668 m m u -mi R -2 9c -3 p -0.709761981 0.00102143 m m u -mi R -3 2 9 -3 p 1.094665852 0.001190208 m m u -mi R -3 0c -1 -3 p -0.765860358 0.001248326 m m u -mi R -3 0c -5 p -0.676552634 0.00133 4222 m m u -mi R -8 7 4 -3 p -0.793623431 0.001440808 m m u -mi R -1 3 6 -5 p 0.792994603 0.001450841 m m u -mi R -3 0e -5 p -0.599468785 0.001594309 m m u -mi R -3 0e -3 p -0.605251844 0.001700235 m m u -mi R -1 3 6 -3 p 0.863419406 0.002005438 m m u -mi R -3 4 b -3 p 0.945263719 0.002109153 m m u -mi R -9 9 -3 p 0.791626989 0.002221577 m m u -mi R -1 0 b -5 p 0.651387841 0.00242168 m m u -mi R -2 2 1 -3 p 0.668772841 0.002702869 m m u -mi R -1 4 4 -3 p -0.791623865 0.002710719 m m u -mi R -1 4 9 -5 p -0.647036271 0.002778003 m m u -mi R -208a -3 p -0.658540111 0.002872092 m m u -mi R -1a -3 p -0. 5 7 6 7 8815 0.002904741 m m u -mi R -2 7 b -5 p 0.677970616 0.00345167 m m u -mi R -3 0c -2 -3 p -0.643612613 0.003849693 m m u -mi R -1960 -0.875994383 0.003898051 m m u -mi R -2 1 0 -3 p 0.64683885 0.004630607 m m u -mi R -6 6 7 -3 p 1.204853494 0.004877288 m m u -mi R -3 0 b -5 p -0.612743013 0.00495248 4 m m u -mi R -133 b -3 p -0.595539655 0.006280098 m m u -mi R -4 8 3 -3 p -0.730210151 0.007147573 m m u -mi R -5 8 2 -3 p -0.601335273 0.008068592 m m u -mi R -1964 -3 p -0.641278554 0.008245143 m m u -mi R -301a -3 p 0.60618848 0.008330173 m m u -mi R -1 3 9 -3 p -0.697509549 0.008483423 m m u -mi R -1 9 5 -3 p 0.640570655 0.00867885

5 1

m m u -mi R -1 8 4 -3 p 0.718421494 0.00988326 m m u -mi R -200c -3 p -1.144613357 0.010961819 m m u -mi R -2 1 9 -5 p -0.951127563 0.011887967 m m u -mi R -1 5 4 -5 p 1.009780507 0.014955031 m m u -mi R -1 3 2 -3 p 0.502716542 0.014970729 m m u -mi R -9 9 b -5 p 0. 5 1 9 7 32556 0.01554761 m m u -mi R -1 4 5 -3 p -0.510560397 0.015906786 m m u -mi R -2 6 b -3 p -0.621098994 0.01596098 m m u -mi R -181a -1 -3 p -0.466649623 0.018218181 m m u -mi R -3098 -5 p -0.920489101 0.018330095 m m u -mi R -3098 -3 p -0.929468531 0.018787963 m m u -mi R -5 0 1 -3 p 0.62960178 0. 0 18971 3 5 6 m m u -mi R -3 2 8 -3 p -0.511540878 0.019431341 m m u -mi R -1198 -5 p -0.54981481 0.020920779 m m u -mi R -3 0a -3 p -0.441725334 0.02179809 m m u -mi R -2 1 2 -5 p 0.593630296 0.0218344 m m u -mi R -133a -3 p -0.449577112 0.021989004 m m u -mi R -8 7 1 -3 p -1.15694682 0.022021046 m m u -mi R -9 9 -5 p 0.53007521 0.022479183 m m u -mi R -3 4 1 -3 p 0.716012717 0.02354315 m m u -mi R -378a -3 p -0.425598087 0.024202638 m m u -mi R -486a -3 p -0.451965148 0.02424837 m m u -mi R -1 3 4 -5 p 0.683384586 0.025274159 m m u -mi R -3 3 1 -5 p -0.71529655 0.025670549 m m u -mi R -4 9 9 -3 p -0. 6 1 47284 1 5 0.025868154 m m u -mi R -5 7 4 -5 p 0.564633788 0.027924415 m m u -let -7 d -5 p -0.482804262 0.029207826 m m u -mi R -5 3 2 -5 p 0.529975705 0.034580381 m m u -mi R -1 0 0 -5 p 0.494306994 0.034768324 m m u -mi R -1 4 1 -3 p -0.577602833 0.034852473 m m u -mi R -2 4 -1 -5 p 0.556695732 0. 0 3 6 9 21798 m m u -mi R -1 5 2 -5 p 0.548148402 0.039963989 m m u -mi R -4 0 9 -3 p 0.539785248 0.04177907 m m u -mi R -1 8 7 -3 p -0.549552419 0.043913063 m m u -mi R -3 2 4 -5 p 0.522237481 0.044598747 m m u -mi R -1 4 4 -5 p -0.51562852 0.047280829 m m u -mi R -8 7 4 -5 p -0.663785168 0.04760991 m m u -mi R -1 8 5 -5 p -0.446271837 0.047679759

5 2

T a bl e 2 .2 : mi R N As differentially expressed in P O + P D E5 -I as co mpared to P O.

Left ventricle myocardiu m fro m mice subjected to sha m or P O surgery and subsequently gi ve n eit her ve hicle, P D E 5 -I ( Sil), or P D E9 -I ( P F -9 6 1 3) was s u bj ecte d t o mi R N A sequencing and subsequent differential expression analysis (n =5 per group). T he l o g 2 f ol d c ha n ge a n d p val ue are pr o vi de d f or eac h mi R N A, wit h ta ble s orte d by p val ue.

mi R N A I D log2 Fold Change (vs. P O) p v al u e m m u -mi R -101a -3 p -4.575787 6 9 6 2 .18 E -2 5 m m u -mi R -208 b -3 p -4.259710819 1. 9 3 E -2 4 m m u -mi R -142a -5 p -4.720138559 5. 5 9 E -2 4 m m u -mi R -2 1a -5 p -4.292238301 1. 2 9 E -2 2 m m u -mi R -1 4 4 -3 p -5.079271251 7. 1 7 E -2 0 m m u -mi R -1 3 6 -5 p -4.821509198 1. 3 9 E -1 5 m m u -mi R -1a -3 p -3.593613103 5. 6 0 E -1 5 m m u -let -7a -1 -3p/ m m u -let -7c -2 -3 p -4.305815692 1. 0 1 E -1 4 m m u -mi R -101 b -3 p -3.606449179 4. 2 6 E -1 4 m m u -mi R -208a -3 p -3.520515306 9. 0 9 E -1 4 m m u -mi R -301a -3 p -3.226350129 1. 0 1 E -1 3 m m u -mi R -1 5 3 -3 p -5.244158568 1. 3 7 E -1 3 m m u -mi R -3 2 -5 p -4.019856028 2. 9 0 E -1 3 m m u -mi R -126a -5 p -3. 4 6 6 65630 4 2. 9 5 E -1 3 m m u -mi R -148a -3 p -2.994665951 1. 6 2 E -1 2 m m u -mi R -190a -5 p -3.733256642 5. 8 3 E -1 2 m m u -mi R -3 4 0 -5 p -3.067304799 5. 9 6 E -1 2 m m u -mi R -142a -3 p -3.265370345 5. 9 6 E -1 2 m m u -mi R -2 1 5 -5 p -4.043439658 1. 2 5 E -1 1 m m u -mi R -9 8 -3 p -3.786218782 1. 3 0 E -1 1 m m u -mi R -4 5 5 -5 p -3.88221935 2. 6 0 E -1 1 m m u -mi R -1 9 -3 p -4.331984183 5. 1 5 E -1 1 m m u -mi R -1 3 6 -3 p -4.061067822 2. 9 3 E -1 0 m m u -mi R -450 b -5 p -3.780780242 3. 2 5 E -1 0 m m u -mi R -3 2 2 -5 p -2.91301546 5. 9 3 E -1 0 m m u -mi R -5 4 2 -3 p -3.517618215 1. 1 2 E -0 8 m m u -mi R -1 9 b -3 p -3.37716808 1. 7 1 E -0 8 m m u -mi R -1 4 4 -5 p -3.185355157 5. 6 8 E -0 8 m m u -mi R -1 4 5 -3 p -2.782872281 7. 2 1 E -0 8 m m u -mi R -4 9 9 -5 p -2.570804953 1. 9 3 E -0 7 m m u -mi R -1 4 3 -3 p -2.574191179 3. 8 0 E -0 7

5 3

m m u -mi R -2 9 b -3 p -2.783979064 3. 8 4 E -0 7 m m u -mi R -1 5 b -3 p -3.274581748 4. 0 8 E -0 7 m m u -mi R -1 9 9 -3p/ m mu -mi R -199 b -3 p -2. 2 58928351 4. 1 9 E -0 7 m m u -mi R -3 0 d -3 p -2.819186253 4. 3 6 E -0 7 m m u -mi R -3 3 5 -5 p -3.073154998 4. 9 1 E -0 7 m m u -mi R -1 9 2 -5 p -2.478450318 6. 6 4 E -0 7 m m u -mi R -1 4 7 -3 p -3.445852507 1. 6 1 E -0 6 m m u -mi R -3 2 9 -3 p -2.619721833 2. 8 6 E -0 6 m m u -let -7f -1 -3 p -3.073407632 3. 0 4 E -0 6 m m u -mi R -2 9c -3 p -2.297479573 3. 3 2 E -0 6 m m u -mi R -146 b -5 p -2.297222027 3. 6 5 E -0 6 m m u -mi R -2 0 1 -5 p -3.550553371 4. 1 5 E -0 6 m m u -mi R -2 2 3 -3 p -2.615866479 4. 3 9 E -0 6 m m u -mi R -199 b -5 p -2.504635063 5. 1 9 E -0 6 m m u -mi R -1 9 9 -5 p -1.763126499 1. 1 5 E -0 5 m m u -mi R -1a -1 -5 p -3.39147674 1 .29 E -0 5 m m u -mi R -5 8 2 -5 p -3.364000211 1. 4 9 E -0 5 m m u -mi R -2 1a -3 p -2.167642629 1. 6 3 E -0 5 m m u -mi R -101a -5 p -2.733260511 2. 0 3 E -0 5 m m u -mi R -1 9 5 -5 p -2.148722392 2. 5 8 E -0 5 m m u -mi R -1 3 5 -5 p -3.142603222 2. 6 6 E -0 5 m m u -let -7c -1 -3 p -2.510702219 3. 5 4 E -0 5 m m u -mi R -4 1 1 -5 p -1 .8030 5691 4. 0 3 E -0 5 m m u -mi R -1 5 -5 p -2.146684268 5. 1 8 E -0 5 m m u -mi R -2 6a -2 -3 p -4.061562476 5. 3 0 E -0 5 m m u -mi R -451a -2.15864161 5. 3 5 E -0 5 m m u -mi R -3 0e -5 p -2.153355798 6. 1 9 E -0 5 m m u -mi R -3068 -3 p -2.165959958 6. 5 2 E -0 5 m m u -mi R -4 8 8 -3 p -2.302146104 6. 7 0 E -0 5 m m u -mi R -3 6 2 -3 p -2.810144748 7. 4 7 E -0 5 m m u -mi R -376c -3 p -4.311137398 9. 5 9 E -0 5 m m u -mi R -3 0 b -5 p -1.975135773 9. 6 6 E -0 5 m m u -mi R -8 7 2 -5 p -2.407065203 0.000119065 m m u -mi R -3 3 5 -3 p -2.422673274 0.000129716 m m u -mi R -2 6 b -5 p -1.940699675 0.00013046 m m u -mi R -7a -1 -3 p -2.877452256 0. 0 0 0139076 m m u -mi R -2 1 8 -5 p -2.330485724 0.0001522 m m u -mi R -2 7a -3 p -1.69596849 0.000156031 m m u -mi R -2 4 -2 -5 p -1.830541263 0.000171641 m m u -mi R -4 8 3 -3 p 1.205677818 0.000201253 m m u -mi R -6 7 6 -5 p -2.382599291 0.000254347

5 4

m m u -mi R -1 4 0 -5 p -2.06288526 0.000259883 m m u -m i R -9 8 -5 p -2.032506534 0.000287761 m m u -mi R -1 6 -1 -3 p -3.109524923 0.0003418 m m u -mi R -2 7 b -3 p -1.742733471 0.0003486 m m u -mi R -3 4 b -5 p -2.304392315 0.000361079 m m u -mi R -1 0 b -5 p -1.767827241 0.000483953 m m u -mi R -3 4c -5 p -1.607260613 0.000679358 m m u -mi R -450a -5 p -2. 017319595 0.00089246 m m u -mi R -5 4 7 -3 p -2.324212207 0.001522837 m m u -let -7f -5 p -1.74726319 0.001620898 m m u -mi R -5 8 2 -3 p -1.803396153 0.001629021 m m u -mi R -1 5 2 -3 p -1.798922482 0.001724373 m m u -mi R -3 2 -3 p -3.01248029 0.002335911 m m u -mi R -376 b -5 p -1.934067313 0. 0 0 2376858 m m u -mi R -2 9 9 -3 p -2.983870311 0.002768105 m m u -mi R -106 b -5 p -1.856559443 0.003448093 m m u -mi R -2 0 3 -3 p -1.928163717 0.004164814 m m u -mi R -3068 -5 p -2.051851585 0.004832613 m m u -mi R -4 2 3 -5 p 1.427573041 0.005622449 m m u -mi R -5 9 8 -3 p -2.790289114 0.005771 3 1 2 m m u -mi R -1247 -3 p 1.078058216 0.005816182 m m u -mi R -3 2 8 -3 p 1.346733766 0.006049766 m m u -mi R -3 0a -5 p -1.543528495 0.006297692 m m u -mi R -148 b -5 p -1.389338531 0.006305273 m m u -mi R -2 1 9 -5 p -3.034733728 0.007325161 m m u -mi R -5 0 1 -3 p 1.546412839 0.007537389 m m u -mi R -7 4 4 -3 p -1.77447997 0.007649749 m m u -mi R -1 8 2 -5 p -1.465409276 0.007755117 m m u -mi R -8 7 4 -5 p 1.249901366 0.008429325 m m u -mi R -3 5 0 -3 p -1.585452909 0.008543687 m m u -mi R -1 5 2 -5 p -1.365001436 0.008651479 m m u -mi R -1 6 -2 -3 p -1.801419981 0.009177028 m m u -mi R -3 8 1 -3 p -1 .5216 41225 0.009260401 m m u -mi R -1 8a -5 p -2.235746329 0.00999135 m m u -mi R -208a -5 p 1.342510232 0.01018137 m m u -mi R -4 5 5 -3 p -0.839591689 0.010910983 m m u -mi R -4 1 1 -3 p -1.594941657 0.01213073 m m u -mi R -3 8 0 -3 p -2.819319513 0.013275558 m m u -mi R -4 2 3 -3 p 1.263230963 0. 0 13490 0 5 9 m m u -mi R -3 4 0 -3 p -1.421705584 0.01514416 m m u -mi R -1 5 0 -5 p 1.044478629 0.015674139

5 5

m m u -mi R -1 2 5 -5 p 1.175742371 0.015696524 m m u -let -7 d -3 p 1.090460392 0.018232638 m m u -mi R -2 6a -5 p -1.317301178 0.018334224 m m u -mi R -7236 -3 p -2.70124277 0.018645667 m m u -m i R -2 1 4 -5 p -0.967135919 0.018901065 m m u -mi R -3 3 8 -3 p -1.260644787 0.020013995 m m u -mi R -148a -5 p -1.394021505 0.020430572 m m u -mi R -9 -5 p -1.231035575 0.021472458 m m u -mi R -7 4 4 -5 p 1.255979317 0.02479594 m m u -mi R -9 3 -3 p 0.985683796 0.025067739 m m u -mi R -9 2a -3 p 1. 1 2 7 56043 8 0.025076345 m m u -mi R -3 7 9 -5 p -1.558712488 0.025156207 m m u -mi R -1 6 -5 p -1.40711933 0.026162994 m m u -mi R -4 9 0 -5 p 1.247672271 0.027533408 m m u -mi R -7a -5 p -2.331704002 0.02844339 m m u -mi R -200c -3 p 1.13668089 0.030329734 m m u -let -7 g -5 p -1.237526729 0.03234462 9 m m u -mi R -1 4 9 -5 p 0.942728598 0.033806779 m m u -mi R -9 9 -3 p -1.167097642 0.034474809 m m u -mi R -1 9 4 -5 p -1.577767761 0.036915266 m m u -mi R -1 4 1 -3 p -1.849075308 0.037001253 m m u -mi R -4 1 0 -3 p -1.135707973 0.038459548 m m u -mi R -1981 -5 p 1.093683482 0.039078004 m m u -mi R -2 4 -1 -5 p -1.062738184 0.039385136 m m u -mi R -3 1 -3 p -2.613782686 0.039462493 m m u -mi R -2 4 -3 p -0.981127044 0.040437228 m m u -mi R -125 b -2 -3 p -0.985936937 0.043431362 m m u -mi R -3 3 0 -3 p 1.173939541 0.047306434 m m u -mi R -1 5 -3 p -1.582116176 0.047921565

5 6

T a bl e 2 .3 : mi R N As di fferentially expressed in P O + P D E9 -I as co mpared to P O.

Left ventricle myocardiu m fro m mice subjected to sha m or P O surgery and subsequently gi ve n eit her ve hicle, P D E 5 -I ( Sil), or P D E 9 -I ( P F -9 6 1 3) was s u bjecte d t o mi R N A sequencing and subsequent differ e ntia l e x pressi o n a nalysis ( n = 5 per gr o u p). T he l o g 2 f ol d c ha nge a n d p val ue are pr o vi de d f or eac h mi R N A, wit h ta ble s orte d by p val ue.

mi R N A I D log2 Fold Change (vs. P O) p v al u e m m u -mi R -486a -3 p -0.852288229 0.000759706 m m u -mi R -1247 -5 p -0.829164673 0. 0 0 5 0 17167 m m u -mi R -133 b -3 p -0.602267066 0.007479807 m m u -mi R -208 b -3 p -0.726931451 0.012992257 m m u -mi R -4 8 3 -3 p 0.678439682 0.017241916 m m u -mi R -3098 -3 p 0.853667319 0.021434169 m m u -mi R -1 4 4 -3 p -0.476077278 0.022872759 m m u -mi R -146 b -5 p -0.734020656 0.024039032 m m u -mi R -5 0 1 -3 p 0.462887415 0.038742158

5 7

T a bl e 2 .4 : Ro w legends (i.e. mi R N A I Ds) for the heat map presented in Fi g ur e 1 . 2 D . mi R N As differentially expressed in P O as co mpared to sha m were clustered using Morpheus

( Br oa d I nstit ute) acc or di n g t o r o ws ( mi R N A) a n d c ol u m ns (sa m ples), or o nly r o ws ( mi R N A) by the one minus Spear man rank coefficient method.

Ro w Nu mber mi R N A I D 1 m m u -mi R -133 b -3 p 2 m m u -mi R -1 8 7 -3 p 3 m m u -mi R -1198 -5 p 4 m m u -mi R -133a -3 p 5 m m u -mi R -1 4 9 -5 p 6 m m u -mi R -3 2 8 -3 p 7 m m u -mi R -1 3 9 -3 p 8 m m u -mi R -8 7 4 -3 p 9 m m u -mi R -3 0c -1 -3 p 1 0 m m u -mi R -3 0c -2 -3 p 1 1 m m u -let -7 d -5 p 1 2 m m u -mi R -8 7 4 -5 p 1 3 m m u -mi R -1964 -3 p 1 4 m m u -mi R -200c -3 p 1 5 m m u -mi R -3 3 1 -5 p 1 6 m m u -mi R -1960 1 7 m m u -mi R -378a -3 p 1 8 m m u -mi R -181a -1 -3 p 1 9 m m u -mi R -486a -3 p 2 0 m m u -mi R -3098 -3 p 2 1 m m u -mi R -3098 -5 p 2 2 m m u -mi R -4 8 3 -3 p 2 3 m m u -mi R -8 7 1 -3 p 2 4 m m u -mi R -1 8 5 -5 p 2 5 m m u -mi R -1 4 1 -3 p 2 6 m m u -mi R -1 4 4 -5 p 2 7 m m u -mi R -1 4 4 -3 p 2 8 m m u -mi R -208a -3 p 2 9 m m u -mi R -2 1 9 -5 p 3 0 m m u -mi R -133a -5 p 3 1 m m u -mi R -3 0c -5 p 3 2 m m u -mi R -1 4 5 -3 p

5 8

3 3 m m u -mi R -3 0 b -5 p 3 4 m m u -mi R -1a -3 p 3 5 m m u -mi R -1 3 5 -5 p 3 6 m m u -mi R -2 9c -3 p 3 7 m m u -mi R -3 0e -5 p 3 8 m m u -mi R -4 9 9 -5 p 3 9 m m u -mi R -4 9 9 -3 p 4 0 m m u -mi R -2 6 b -3 p 4 1 m m u -mi R -3 0a -3 p 4 2 m m u -mi R -3 0e -3 p 4 3 m m u -mi R -4 5 5 -5 p 4 4 m m u -mi R -3 3 8 -3 p 4 5 m m u -mi R -9 -5 p 4 6 m m u -mi R -2 1 2 -5 p 4 7 m m u -mi R -1 3 4 -5 p 4 8 m m u -mi R -4 0 9 -3 p 4 9 m m u -mi R -1 8 4 -3 p 5 0 m m u -mi R -1 3 2 -3 p 5 1 m m u -mi R -2 3 b -3 p 5 2 m m u -mi R -1247 -3 p 5 3 m m u -mi R -1 4 0 -3 p 5 4 m m u -mi R -1247 -5 p 5 5 m m u -mi R -2 9 8 -5 p 5 6 m m u -mi R -2 2 1 -3 p 5 7 m m u -mi R -2 2 2 -3 p 5 8 m m u -mi R -1 2 7 -3 p 5 9 m m u -mi R -125 b -1 -3 p 6 0 m m u -mi R -2 1 4 -3 p 6 1 m m u -mi R -1 9 5 -3 p 6 2 m m u -mi R -5 7 4 -5 p 6 3 m m u -mi R -9 2 b -3 p 6 4 m m u -mi R -3 2 4 -5 p 6 5 m m u -mi R -5 0 1 -3 p 6 6 m m u -mi R -9 9 b -5 p 6 7 m m u -mi R -5 7 4 -3 p 6 8 m m u -mi R -5 0 1 -5 p 6 9 m m u -mi R -6 6 7 -3 p 7 0 m m u -mi R -1 0a -5 p 7 1 m m u -mi R -1 0 b -5 p 7 2 m m u -mi R -1 3 6 -3 p 7 3 m m u -mi R -1 3 6 -5 p

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7 4 m m u -mi R -1 9 5 -5 p 7 5 m m u -mi R -1 4 0 -5 p 7 6 m m u -mi R -199 b -5 p 7 7 m m u -mi R -2 1a -5 p 7 8 m m u -mi R -301a -3 p 7 9 m m u -mi R -2 1a -3 p 8 0 m m u -mi R -2 7 b -5 p 8 1 m m u -mi R -1 5 2 -5 p 8 2 m m u -mi R -2 7 b -3 p 8 3 m m u -mi R -146 b -5 p 8 4 m m u -mi R -2 4 -3 p 8 5 m m u -mi R -9 9 -5 p 8 6 m m u -mi R -208 b -3 p 8 7 m m u -mi R -4 1 1 -5 p 8 8 m m u -mi R -1 9 9 -3 p/ m mu -mi R -199 b -3 p 8 9 m m u -mi R -9 9 -3 p 9 0 m m u -mi R -3 4 b -5 p 9 1 m m u -mi R -3 7 9 -5 p 9 2 m m u -mi R -2 4 -1 -5 p 9 3 m m u -mi R -5 8 2 -3 p 9 4 m m u -mi R -5 8 2 -5 p 9 5 m m u -mi R -3 2 9 -3 p 9 6 m m u -mi R -1 5 4 -5 p 9 7 m m u -mi R -1 0 0 -5 p 9 8 m m u -mi R -2 2 4 -5 p 9 9 m m u -mi R -2 1 4 -5 p 1 0 0 m m u -mi R -1 9 9 -5 p 1 0 1 m m u -mi R -3 4c -5 p 1 0 2 m m u -mi R -3 0 0 -3 p 1 0 3 m m u -mi R -3 4 b -3 p 1 0 4 m m u -mi R -4 1 0 -3 p 1 0 5 m m u -mi R -497a -5 p 1 0 6 m m u -mi R -3 4 1 -3 p 1 0 7 m m u -mi R -2 1 0 -3 p 1 0 8 m m u -mi R -4 3 1 -5 p 1 0 9 m m u -mi R -5 4 1 -5 p 1 1 0 m m u -mi R -5 3 2 -5 p 1 1 1 m m u -mi R -4 5 5 -3 p

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C HAPTER 3 : Identification of P D E 9 bi n ding partners in cardio myocytes via

c o -i m munoprecipitation

INTRODUCTION

P h os p h o diesterases are hi g hly c o m part me ntalize d e nzy mes t hat ti g htly c o ntr ol cyclic n ucle oti de le vels i n l ocal na n o d o mai ns. I n car di o my ocytes, ma ny differe nt phosphodiesterase is of o r ms have been studied, with P D E3 and P D E4 pri mary a mong the m.

O ne of t he n o r mal f u ncti o ns of t hese t w o P D Es is t o mai ntai n a gra die nt of c A M P fr o m t he plas ma me m bra ne, w here it is pr o d uce d by a de nylyl cyclase, t o t he sarc o plas mic retic ul u m, w here it c a n i n teract wit h a n d acti vate P K A. It has bee n s h o w n, h o we ver, t hat t his s h uttli n g is l ost i n heart fail ure d ue t o rel ocalizati o n of t he P D Es a n d s u bse q ue nt diff usi o n of c A M P a way fr o m t he gra die nt, lea di n g t o a l oss of si g nali n g bet wee n t he b -a dre nergic r ece pt or a n d the sarcoplas mic reticulu m 4 0 . Si milarly, o ur la b has s h o w n t hat P D E 5, w hic h re g ulates c G M P, rel ocalizes fro m the sarco mere Z -disc t o t he cyt o plas m i n t he p rese nce of car diac press ure -o verl oa d, dilate d heart fail ure 7 2 , or f oll o wi n g p har mac ol o gical or ge netic bl oc ka de of nitric o xi de sy nt h ase, res ulti n g i n a s witc h of its n or mal c o ntr ol of nitric o xi de -deri ve d c G M P t o t hat of natri uretic pe pti de -derived c G MP 7 1, 7 3 . P D E 9 is als o c G M P selecti ve, w hic h as rece ntly re p orted 4 8, 1 4 8 , c o nfers f u ncti o nally differe nt control over c G M P and consequent

P K G acti vati o n i n t he heart. I n partic ular, P D E 9 hy dr olyzes natri uretic pe pti de deri ve d c G M P, s o t he ca pacity t o acti vate P K G u p o n its i n hi biti o n is i n de pe n de nt of nitric o xi de sy nt hesis.

P D E5 and P D E9 are diffe re nt i n t h eir preferre d s u bstrate p o ol a n d l ocalizati o n, as well as i n t heir c urre nt cli nical a p plicati o ns. B ot h P D Es are c G M P -s pecific, alt h o u g h P D E 9

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has a hi g her s pecificity f or c G M P t ha n d oes P D E 5. Hi g hly -s pecific i n hi bit ors f or b ot h

P D Es e xist, wit h P D E 5 i n hi bit o rs pri marily bei n g use d t o treat erectile dysf u ncti o n a n d pul monary hypertension. P D E5 inhibitors have been tested in heart failure with preserved ejecti o n fracti o n i n t he R E L A X trial wit h ne utral res ults. T his o utc o me is hy p ot hesize d t o be d ue t o l o w c G M P a n d P K G acti vati o n wit h o ut c o nc o mita nt i ncreases i n P D E 5 e x pressi o n, t here by li miti n g t he i m pact of P D E 5 i n hi bit or treat me nt. P D E 9 i n hi bit ors ha ve bee n teste d in Alzhei mer’s, Parkinson’s, and schizophrenia, although pre -cli nical a n d early sta ge cli nical trials for cardiac indications are under way. P D E5 regulates N O -deri ve d c G M P, t h o u g h it s witc hes t o re g ulati o n of N P -deri ve d c G M P w he n it s hifts t o a cyt o plas mic l ocalizati o n.

P D E9 has recently been sho wn to specifically regulate N P -deri ve d c G M P. I n cardio m yocytes, P D E 5 n or mally l ocalizes t o t he Z -dis k, w hereas P D E 9 l ocalizes t o t he T - tubular me mbrane. Under so me conditions, P D E5 relocalizes to beco me cytoplas mic; ho wever, P D E9 is not currently kno wn to change localization under differing condition s.

Ho wever , bey o n d t he li mite d c o nf ocal i ma ges a vaila ble i de ntifyi n g differe nt l ocalizati o n of

P D E 5 vers us P D E 9 i n t he car diac my ocyte, n ot hi n g is k n o w n a b o ut t he cl uster of pr otei ns wit h w hic h P D E 9 i nteracts, h o w t his differs fr o m P D E 5, a n d w het her li ke P D E 5, P D E 9 u n der g oes i ntracell ular re distri b uti o n f oll o wi n g pat h ol o gical stress.

O ne way t o st u dy pr otei n c o m part me ntalizati o n is by c o -i m m u n o preci pitati o n f oll o we d by mass s pectr o metry, all o wi n g f or i de ntificati o n of P D E bi n di n g part ners a n d co mplexes. Th is d oes re q uire ha vi n g eit her a n e xcelle nt a nti b o dy or a ta g ge d P D E f or hi g h efficie ncy p ull d o w n, b ut ca n be perf or me d wit h relati ve ease u n der m ulti ple c o n diti o ns (i.e. basal versus disease). Our lab has previously perfor med this experi ment for P D E5 by pu lli n g do wn FL A G -tagged P D E5 fro m myocardiu m derived fro m a cardio myocyte -s pecific P D E 5 overexpressor mouse. Ho wever, as a mouse expressing a tagged P D E9 does not currently

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e xist, a n d t here are fe w relia ble a nti b o dies f or P D E 9, we o pte d t o ta ke t he a p pr oac h of i ntr o d u cing adenovirus expressing F L A G -ta g ge d P D E 9 t o is olate d a d ult ra b bit car di o my ocytes. By usi n g t his syste m varia nce ca n be re d uce d, as m ulti ple c o n diti o ns ca n be teste d wit hi n car di o my ocytes fr o m a si n gle a ni mal. A d diti o nally, ra b bit car di o my ocyt es ex press relati vely si milar P D Es t o t h ose f o u n d i n t he h u ma n, ma ki n g t he m a n i deal m o del syste m. I n t his e x peri me nt, we ai me d t o i de ntify t he c o m part me nt wit hi n w hic h P D E 9 resi de d i n b ot h n or mal a n d hy pertr o p hic c o n diti o ns.

MET HODS

Pl as mi ds a n d a de novir us: p AdLo x -P D E9 -F L A G was constructed by Mike Sasaki.

AdP D E9 -FL A G and adFL A G were generated by Welgen, Inc ( Worcester, M A).

A d ult ra b bit myocyte isolatio n a n d i nfectio n: A d ult ra b bit my ocytes were is olate d as previously described 1 4 9 . My ocytes were i nfecte d wit h a de n o vir us ( 1 0 M OI) at t he ti me of plati n g i n Me di u m 1 9 9 ( Si g ma). After 3 -4 hours, media was changed to Mediu m 1 9 9 c o ntai ni n g 2 % F B S, pe nicilli n -stre pt o myci n ( 1 0 0 U / m L), L -gl uta mi ne ( 0. 1 m g / m L), creati ne

( 5 m M), ta uri ne ( 5 m M), a n d L -car niti ne ( 2 m M). After o ver ni g ht i nc u bati o n, cells were sti m ulate d wit h e n d ot heli n -1 ( 1 0 0 n M, Si g ma) or ve hicle f or 2 4 h o urs i n Me di u m 1 9 9 c o ntai ni n g pe nicilli n -stre pt o myci n, L -gl uta mi ne ( 0. 1 m g / m L), creati ne ( 5 m M), ta uri ne ( 5 m M), a n d L -car niti ne ( 2 m M).

I m munofluorescence: Cells were transduced as descri bed. After 48 h ours of viral c o nstr uct e x pressi o n, cells were fi xe d i n 1 0 % f or mali n i n P B S, per mea bilize d wit h 0. 5 % sa p o ni n i n

P B S, a n d bl oc ke d wit h 3 % B S A i n P B S. Cells were t he n stai n e d o ver ni g ht i n 3 % B S A wit h

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0. 5 % sa p o ni n i n P B S at 4 °C f or FL A G (F 1 8 0 4, Si g ma , 1: 2 0 0 or 1 4 7 9 3, Cell Si g nali n g, 1: 2 0 0 ) or S E R C A2 A ( M A3919, Ther moFisher, 1:100) . Cells were t he n c o u nterstai ne d wit h a nti - ra b bit Ale xa 5 6 8 a n d a nti -mouse Alexa 488 -coupled secon da ry a nti b o dy ( Ther moFisher) at a

1: 5 0 0 dil uti o n . Har d -m o u nt Vectas hiel d wit h D A PI ( Vect or La bs) wa s use d t o preserve fluorescence after staining was co mplete. I maging was perfor med on a Leica T CS S P E confocal microscope using a 40 X oil -i m mersion objective .

I m munoprecipitation of F L A G -tagged Proteins: A d ult ra b bit car di o my ocytes were i nfecte d as descri be d. After sa m ples were c ollecte d, t hey were s o nicate d t o disr u pt cell me mbranes and spun do wn. Protein concentration was measured using the Pierce bici nc h o n i n ic aci d ( B C A) pr otei n assay ( T her m o Fis her). F or i m m u n o preci pitati o n, 1 0 0 µ L of F L A G M 2 ma g netic b ea ds ( Si g ma) were pre -was he d t wice wit h T B S a n d t he n i nc u bate d wit h 4 0 0 µ g of pr otei n. Bea ds were r otate d wit h lysate o ver ni g ht at 4 °C. F oll o wi n g i nc u bati o n, b ea ds were was he d t hree ti mes wit h T B S. El uti o n was perf or me d by r otati n g t he bea ds wit h a 3 X F L A G pe pti de s ol uti o n t wice f or 3 0 mi n utes eac h at 4 °C.

Wester n bl otti n g: After i m m u n o preci pitati o n was perf or me d, b ot h i n p ut a n d el uate sa mples were run under reducing and denaturing conditions on precast 4 -2 0 % Mi ni -

PR OT E A N Tris -Glyci ne T G X gels ( Bi o -Ra d). F oll o wi n g se mi -dry tra nsfer t o nitr ocell ul ose me m bra nes, bl otti n g was perfor med for P D E9 (custo m antibody, ProSci Inc, 1:1000),

F L A G ( F 1 8 0 4, Si g ma, 1: 1 0 0 0), or V D A C2 (ab37985, Abca m, 1:1000). LI -C O R secondary a nti b o dies were use d f or detecti o n at a 1: 1 0, 0 0 0 dil uti o n, a n d me m bra nes were i ma ge d o n a

LI -C O R Odyssey using I mage St u di o v 5. 0 s oft ware. T otal pr otei n stai ni n g f or n or malizati o n was perfor med using the LI -C O R R E V E R T stai ni n g kit.

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Mass Spectro metry Experi mental Design: For FL A G co -i m m u n o preci pitati o n st u dies, a d ult ra b bit car di o my ocytes were is olate d a n d cells were i nf ecte d wit h o ne of t w o vir uses: adFL A G or adP D E9 -F L A G. Wit hi n eac h vir us gr o u p, sa m ples we re eit her wit h E T -1 ( 1 0 0 n M, Si g ma) or ve hicle, f or a t otal of t w o sa m ples per vir us gr o u p. T he e x peri me nt was re peate d wit h n = 3 ra b bits f or a t otal of t hree i n de pe n de nt e x peri me nts. Ta n de m mass ta g labels were shuffled bet ween sa mples in different multi pl e xes t o mi ni mize pe pti de la beli n g bias (see bel o w).

Mass Spectro metry Peptide Preparation: F oll o wi n g sa m ple el uti o n fr o m F L A G ma g netic bea ds, met ha n ol -chlorofor m extr acti o n was perf or me d f or all sa m ples acc or di n g t o t he methods of Wessel and Flugge 1 5 0 . After e xtracti o n was c o m plete, sa mples were resuspended i n 9 M urea c o ntai ni n g 1 0 m M Tris -H Cl, a n d s u bse q ue ntly dil ute d 1: 6 i n 6 0 m M H E P E S p H

7. 5, a n d 0. 6 m M D T T, t o yiel d a fi nal di gesti o n b uffer c o nt ai ni n g 5 0 m M H E P E S, p H 7. 5

1. 5 M urea, a n d 0. 5 m M D T T. 1 µ g of try psi n/ Lys -C ( Pro mega V5 0 7 3) was a d de d t o eac h sa mple, and peptide digest proceeded overnight at 25 °C (t o re d uce lysi ne car ba mylati o n) wit h sa m ples s ha ki n g at 1 0 5 0 R P M. T he ne xt day, a n ot her 0. 5 µ g of try psi n was a d de d al o n g wit h 5 m M D T T, a n d di gest c o nti n ue d f or a n ot her 2 h o urs . 2 0 m M i o d oaceta mi de was t he n a d de d t o al kylate t he sa m ples f or 1 h o ur i n t he dar k. 0. 5 % v / v T F A was a d de d t o aci dify t he pe pti des, w hic h were t he n p urifie d o n C 1 8 s pi n c ol u m ns ( Pierce) acc or di n g t o manufacturer’s instructions and eluted using 0.1 % for mic aci d i n 6 0 % acet o nitrile. Fi nally,

sa mples were evaporated to dryness in an Eppendorf Vacufuge. Distilled deionized H 2 O was a d de d after t he first s pi n a n d sa m ples were subjected to a second round of evaporation to re move trace levels of for mic acid and ac et o nitrile.

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Peptide Labeling and 2 -Di mensional Liquid Chro matography Coupled to Tande m

Mass Spectro metry (2 D -L C -M S / M S): Sa mples were then resuspended in triet hyla m m o ni u m bicar b o nate p H 8. 5. Pe pti des were la bele d wit h T M T (ta n de m mass ta g) reagents ( Ther m o F is her), wit h la bels s h uffle d bet wee n sa m ples o n differe nt m ulti ple xes t o mi ni mize la beli n g bias. T M T la beli n g was d o ne wit h sa m ples br o u g ht u p i n 1 0 0 µ l T E A B a n d la bels br o u g ht u p i n 4 1 µ L a n hy dr o us acet o nitrile. Eac h la bel was a d de d t o its corresponding sa mple for 1 hour before being quenched with 8 µ L of 5 % hy dr o xyl a mi ne f or 1 5 mi n utes. Sa mples were then mixed and dried do wn and subsequently brought u p i n 2 m L of 1 0 m M T E A B a n d i njecte d o nt o a n A gile nt L C r u n ni n g a 2. 1 x 1 0 0 m m Waters B E H

C 1 8 c ol u m n w it h 1. 7 µ M particles at 2 5 0 µ L / mi n ute. T he first 1 2 fracti o ns were fl o w - t hr o u g h, wit h t he ne xt 8 4 sa m ples c ollecte d o n a n 8 5 -mi n ute gra die nt fr o m 0 -9 0 % acet o nitrile c o ntai ni n g 1 0 m M T E A B. These 84 fractions were concatenated into 12 fractions for L C -MS/ M S a nalysis.

L C -MS/ MS was perfor med on an Orbitrap Fusion Lu mos Tribrid Mass

Spectro meter ( Ther mo Fisher). Liquid chro matography was perfor med prior to MS / M S by bri n gi n g sa m ples u p i n b uffer A ( 2 % acet o nitrile, 0. 1 % f or mic aci d 9 8 % dei o nize d water), a n d t he n i njecti n g t he sa m ple o nt o a tra p pi n g c ol u m n i n b uffer A f or 5 mi n utes at 5 µ L / mi n bef ore s witc hi n g t o t he a nalytical c ol u m n ( Re pr osil C 1 8 3 µ M particle s wit h 1 0 0 a n gstr o m p ore size, pac ke d a p pr o xi mately 2 0 c m wit h 7 5 µ M i nter nal dia meter f use d silica Pic o F rits fr o m Ne w O bjecti ve ( W o b ur n, M A)) at 3 0 0 n L / mi n. T he s ol uti o n was t he n s witc he d t o

10 % buffer B (90 % acetonitrile/10 % deionized water and 0.1 % for m ic aci d). T he gra die nt t he n we nt fr o m 1 0 % t o 2 0 % b uffer B o ver 4 5 mi n utes a n d t he n t o 3 5 % at 7 7 mi n utes

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b ef ore j u m pi n g t o 1 0 0 % b uffer B at 8 0 mi n utes, h ol di n g f or 2 mi n utes, t he n ret ur ni n g t o 2 % buffer B in another 2 minutes before the liquid chro matograph y r u n e n de d at 9 0 mi n utes. T he fracti o ns were i njecte d c o nsec uti vely f oll o wi n g t his gra die nt sc he me.

M S was c o n d ucte d wit h t he f oll o wi n g i nstr u me nt setti n gs. M S 1 res ol uti o n was set t o

1 2 0, 0 0 0 wit h a tar get i o n c o u nt of 4 0 0, 0 0 0. T he prec urs or i o n is olati o n wi dt h was 0. 7

Dalt o ns. F or M S 2, res ol uti o n was set t o 5 0, 0 0 0 wit h a tar get i o n c o u nt of 1 0 0, 0 0 0. C oll is i o n e ner gy was set t o 3 8. Easy I C i nter nal cali brati o n was use d o n t he prec urs ors w hic h uses t he

E T D ion for mass correction on -t he -fly f or M S .

Protei n I de ntificatio n: Pea k list files (. R A W) were searc he d a gai nst a ra b bit data base of pre dicte d pr otei n se quences ( N C BI RefSeq, taxono my: Oryctolagus cuniculus, 2018, F AS T A for mat, 38559 sequences; RefSeq83_ Oryctolagus_cuniculus_180413_1.fasta;), u si n g Masc ot

Version: 2.2.0 ( Matrix Science) interfaced through Proteo me Discoverer 1.4 ( Ther mo). Peaks were filt er e d at a si g nal t o n oise rati o of 1. 5, deis ot o pe d, a n d searc he d wit h a pare nt i o n mass tolerance of 10 pp m and an MS2 mass tolerance of 0.02 Da . Try psi n was s pecifie d as t he enzy me and 1 missed cleavage was allo wed. Cysteine carba mido methylation and N -ter mi nal a mi ne la beli n g wit h T M T 1 0 -ple x rea ge nt were s pecifie d as fi xe d m o dificati o ns f or t he database search. Dyna mic modifications included N -py r o gl uta mi ne, o xi dize d met hi o ni ne, p h os p h orylati o n of seri ne, t hre o ni ne, a n d tyr osi ne, a n d T M T la beli n g of lysi ne . All searc hes were conducted with the reversed database search mode engaged. Percolator soft ware was use d f or pe pti de F D R ( q -val ue) calc ulati o n s. Masc ot o ut p ut files (. dat) ta b ulate d were i n

Pr ote o me Disc o verer. Of t he hi g h c o nfi de nce pe pti des ( q < 0. 0 1), 2 % of u nique s pectra had i nte nsities missi n g fr o m o ne or m ore c ha n nels, i n dicati ve of efficie nt T M T la beli n g a n d

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frag mentation. Spectra for whic h pe pti de c o -is olati o n i nterference wa s > 5 1 % were o mitte d fr o m a nalysis. Pr otei ns i de ntifie d by si n gle s pectra w er e li ke wise o mitte d.

Protein Quantification by T M T and Statistical Analysis: T M T re p orter i o n i nte nsities were integrated over 30 pp m using the most confident centroid method and corrected for p urity i n Pr ote o me Disc o verer 1. 4 ( T her m o). Missi n g la bele d c h a n nel i nte nsities ( < 2 % of s pectra) were i n versely pr o p orti o nal t o t he u ntra nsf or me d me dia n i o n i nte nsities acr oss t he c ha n nels. F or i n d i vi d ual s pectra f or w hic h missi n g data was < 5 0 %, t he missi n g val ue was re place d by t he me dia n val ue of t he l o west i nte n si ties i n eac h c ha n nel. F oll o wi n g missi n g val ue i m p utati o n , t hese si g nals were q ua ntifie d usi n g t he me dia n s wee p al g orit h m ori gi nally de scri be d by Her bric h et al 1 5 1 esse ntially as i m ple me nte d rece ntly by F oster et al 1 5 2 wit h a mi n or m o dificati o n. T M T re p orter i o n i nte nsities were 1) l o garit h mically -transfor med (base

2), 2) q ua ntile -n or malize d f or each channel , 3) me dia n -ce nter e d f or eac h i n di vi d ual s pectr u m acr oss c ha n nels, 4) protein abundance deter mined by ta ki n g t he me dia n val ue of t he l o garit h mically -transfor med median -ce ntere d i nte nsities f or all s pectra bel o n gi n g t o t hat pr otei n i n a gi ve n c ha n nel , a n d fi nally, 5) re -ce nter e d f or eac h c ha n nel by s u btracti n g t he me dia n pr ot ei n a b u n da nce. F oll o wi n g t he median s weep, differential protein abundance bet ween experi mental groups was assessed by means of an e mpirical Bayesian method, s pecifically, li near m o deli n g of micr oarrays ( LI M M A) 1 5 3, 1 5 4 wit h m ulti -group co mparison, as i m ple me nte d i n O mics E x pl orer ( Ql uc ore, L u n d, S we de n). Res ulti n g p -val ues were corrected for multiple co mparisons using the false discovery rate (q -val ue) met h o d of

Be nja mi ni -Hochberg 1 5 5 .

6 8

Pat h ways a nalysis: Pat h ways a nalysis f o r mass spectro metry data was perf or me d usi n g

D A VI D Bioinfor matics Resources 1 5 6 . For D A VI D analysis, K E G G path ways were used wit h defa ult a nalysis para meters. P ath ways analysis was also pe rfor m e d wit h I n ge n uity

Path ways Analysis soft ware ( Qiagen) using default para meters.

RES ULTS

P D E9 is enriched in sa mples overexpressing P D E9 -F L A G, seen both by Western blotting and mass spectro metry

After ra b bit my ocyte lysates fr o m cells tra ns d uce d wit h eit her adFL A G or adP D E9 -

F L A G under went i m munoprecipitation, both inputs and eluates were run on S DS -P A G E gels to evaluate the effectiveness of overexpression and pulldo wn. Results sho w that P D E9 is o v erexpressed in input sa mples and enriched in i m munopre ci pit ate d sa m ples (Fi g ur e 3 .1).

Further more, when mass spectro metry data was analyzed, P D E9 was found to be si g nifica ntly e nric he d i n t he a d P D E 9 -F L A G sa mples as co mpared to adF L A G controls, with a p pr oxi mately 4 -f ol d i ncrease (T a bl e 3 .1).

6 9 !

Figure 3.1: P D E9 is enriched in ad P D E9- F L A G transduced adult rabbit

cardio myocytes

� ���� � �� �� �� �� �� �� �� �� �� �� �� �� �� ��� ������� �� ���� �� �� �� �� �� �� �� �� �� �� � � �� �� ���� �� �� �� �� �� �� �� �� �� � ���� � � � ��

� ���� � �� ��� �� �� �� ��

� � � � ��� ���� � �� �� �� �� �� �� �� �� �� �� �� �� �� ��� ������� �� ���� �� �� �� �� �� �� �� �� �� �� � � �� �� ���� �� �� �� �� �� �� �� �� �� � ���� � � � ��

� ���� � �� ���� �� �� �� ��

�� � �� ��

Figure 3.1: P D E9 is enriched in ad P D E9- F L A G transduced adult rabbit

cardio myocytes

� � � �

Adult rabbit cardio myocytes were transduced with either adFL A G or adP D E9 -F L A G, t he n sti m ulate d wit h 1 0 0 n M E T -1 or vehicle for 24 hours. Sa mples under went i m m u n o preci pitati o n f or F L A G, a n d after el uti o n, b ot h i n p uts a n d el uates were s u bjecte d t o

S D S -P A G E and subsequent Western blotting for FL A G and t otal pr otei n. A) I n p ut sa m ples, a n d B) el uate sa m ples . A b bre viati o ns: E T -1, e n d ot heli n -1.

7 1

P D E9 associates pri marily with mitochondrial proteins, but also sarcoplas mic reticulu m proteins

Mass spectro metry data was analyzed by co mparing ad P D E9 -F L A G sa m ples t o adFL A G sa mples (T a bl e 3 .1). Fr o m t his a nalysis, 1 5 5 pr otei ns were f o u n d t o be si g nifica ntly e nric he d i n t he P D E 9 -F L A G sa m ples ( p < 0. 0 5). T hese pr otei ns were t he n s u bjecte d t o

D A VI D K E G G path ways analysis. The enriched path ways were predo minantly mit oc h o n drial i n nat ure, i ncl u di n g o x i dati v e p h os p h orylati o n, citric aci d cycle, a n d fatty aci d meta b olis m (T a bl e 3 .2 ). These findings were confir med by I P A path ways analysis as well

(Fi g ur e 3 .2 ). Sarcoplas mic reticulu m and sarco meric proteins were also significant a mong the proteins enriched wit h P D E 9, n ote d as calci u m si g nali n g a n d car diac m uscle c o ntracti o n pat h ways i n t he D A VI D a nalysis.

7 2 !

Figure 3.2: I P A path ways analysis of proteins significantly enriched in P D E9- F L A G

sa mples sho ws many mitochondrial proteins

������� � ���

� �� �� �����

��� ��� �� ��������

��� �� � �� � � ���� ��

Figure 3.2: Mitochondrial proteins are enriched in P D E9- F L A G pulldo wns

Proteins that were significantly enriched (p <0.05) in ad P D E9- F L A G mass spectro metry

sa mples as co mpared to ad F L A G were subjected to I P A path ways analysis. The resulting

pr otei n i nteracti o n net w or k is s h o w n here.

� � � �

P D E9 does not relocalize follo wing E T -1-sti mulated hypertrophy

After i nitial a nalysis of t he mass s pectr o metry data, we ne xt wa nte d t o deter mi ne whether or not P D E9 relocalizes follo wing cardiac hypertrophy. Rabbit myocytes were sti m ulat e d t o hypertrophy by applying 100 n M E T -1 for 24 hours in seru m -free me dia. After co mparison of adP D E9 -FL A G to adP D E9 -FLA G+ET -1 gr o u ps, a t otal of 9 7 pr otei ns were i de ntifie d as si g nifica nt. W he n t he 4 6 pr otei ns t hat were si g nifica ntly decrease d wit h E T -1 trea t me nt (T a bl e 3 .3 ) were s u bjecte d t o D A VI D pat h ways a nalysis, o xi dati ve phosphorylation and metabolic path ways were identified (p values of 2.44 E -1 8 a n d 2. 0 6 E -9, res pecti vely); h o we ver, ma ny ot her mit oc h o n drial pr otei ns were still i de ntifie d i n t he E T -1 tre at me nt gr o u p. T hi s i n dicates t here is li kely n o si g nifica nt rel ocalizati o n of P D E 9 f oll o wi n g induction of hypertrophy, although there may be so me dissociation fro m oxidative p h os p h orylati o n c o m ple xes. Of t he 5 1 pr otei ns t hat were e nric he d wit h E T -1 tr eat me nt

(T a bl e 3 .4 ), n o si g nifica nt pat h ways were i de ntifie d. All of t hese pr otei ns were n o vel t o t he

P D E9 -FLA G+ET -1 gr o u p (i.e. were n ot si g nifica nt w he n P D E 9 -FL A G was co mpared to

F L A G c o ntr ol sa m ples).

P D E9 colocalizes with S E R C A2a

Rabbit myocytes wer e tra n sduced with adP D E 9 -F L A G and subsequently subjected to i m munofluorescent staining with S E R C A2 A and F L A G antibodies. Confocal i maging sho wed that P D E9 colocalizes with S E R C A2 A under both nor mal and E T -1 -sti m ulate d c o n diti o ns (Fi g ur e 3 .3 ), vali dati n g t he mass spectro metry fin di n g. We si milarly s h o we d i n

2 0 1 5 t hat P D E 9 c ol ocalizes wit h S E R C A 2 A i n is olate d a d ult m o use car di o my ocytes 4 8 .

7 4

Fi g ur e 3 .3: P D E 9 c ol o c ali z es wit h S E R C A 2 a i n a d ult r a b bit c a rdio myocytes

FL A G SERCA2a D A PI Merge Zoo m adFLA G FL A G - adP DE9 FL A G - + + E T 1 adP DE9

F i g ur e 3 .3: P D E9 colocalizes with S E R C A2a under basal and hypertrophic co n ditio ns

Adult rabbit cardio myocytes were transduced with adF L A G or adP D E9 -F L A G, a n d t he n treate d wit h 1 0 0 n M E T -1 or ve hicle. Cells were fi xe d a n d stai ne d f o r F L A G a n d S E R C A 2a, a n d s u bjected to confocal microscopy. P D E9 colocalizes with S E R C A2a under both hypertrophic and basal conditions.

7 5

P D E9 co -i m munoprecipitates with V D A C2

V D A C2 is a mitochondrial outer me mbrane pore protein that was highly enriched i n t he P D E 9 -F L A G sa m ples ( 3. 7 8 f ol d). T o vali date t his fi n di n g, ra b bit my ocytes tra ns d uce d wit h eit her a d P D E 9 -F L A G or a d F L A G a n d treate d wit h eit her ve hicle or E T -1 were s u bjecte d t o i m m u n o preci pitati o n f or F L A G a n d s u bse q ue nt Wester n bl otti n g f or V D A C 2.

B ot h a d P D E 9 -FL A G groups sho wed enrich ment of V D A C2 (Fi g ur e 3 .4 ), w hic h ali g ne d with our mass spectro metry data sho wing that V D A C2 bound to P D E9 under both basal and hypertrophic c o n diti o ns (T a bl es 3 .1, 3 . 3 , a n d 3 .4 ).

7 6

Fi g ur e 3 .4: P D E 9 b inds to V D A C2 in adult rabbit cardio myocytes

adFLA G + + - - + + - - adP DE9 -FL A G - - + + - - + + E T 1 - + - + - + - + P DE9 7 0 k D a - 3 8 k D a - V DAC2

7 0 k D a -

T ot al 3 8 k D a - Pr ot ei n

2 5 k D a -

I n p ut El u at e

Fi g ur e 3 .4: P D E9 binds V D A C2 under both basal and hypertrophic conditions

Adult rabbit cardio myocytes were transduced with ad F L A G or ad P D E 9 -F L A G, a n d t he n treate d wit h eit her ve hicle or 1 0 0 n M E T -1. Sa m ples were c ollecte d and under went i m m u n o preci pitati o n f or F L A G, f oll o we d by Wester n bl otti n g f or F L A G, V D A C 2, a n d t otal pr otei n. I n p ut sa m ples are o n t he left, a n d el uates are o n t he ri g ht.

7 7

DISC USSI O N

T his st u dy re prese nts t he first i n -de pt h e x pl orati o n of P D E9 co mpar t me ntati o n i n car diac my ocytes. I nitial st u dies fr o m o ur la b i n dicate d P D E 9 was li kely l ocalize d t o mit oc h o n dria, w hic h t his mass s pectr o metry data set has c o nfir me d. We als o pre vi o usly sho wed colocalization with S E R C A2a in mouse adult myocy tes, w hic h was als o see n i n t his c o -I P data set. Pre vi o us st u dies e xa mi ni n g c G M P at mit oc h o n dria ha ve s h o w n t hat t here is indeed a mitochondrial pool of c G MP 1 5 7 , w hic h is res p o nsi ble f or acti vati n g car di o my ocyte a p o pt osis via release of cyt oc hr o me C i n a mit oc h o n drial per mea bility tra nsiti o n - independent fashion 1 5 8 . c G MP has also been sho wn to pro mote A T P synthesis a n d mitochondrial biogenesis by several different groups 1 5 9, 1 6 0 . H o we ver, t hese st u dies were perfor med using N O -pat h way tar geti n g rea ge nts, s o t he r ole of natri uretic pe pti de -deri ve d c G M P at mit oc h o n dria is u ncle ar. H o we ver, i n o ne of t hese st u dies, it was deter mi ne d t hat a p o ol of P K G was prese nt at t he mit oc h o n dria w hic h me diate d t he a p o pt otic effects of c G M P i n a Ca 2 + dependent manner 1 6 1 . It is p ossi ble t hat P D E 9 acts t o re g ulate t his p o ol of c G M P a n d subsequently P K G activity, thereby preventing cardio myocyte apoptosis and a shift to wards increased A T P synthesis and mitochondrial biogenesis under high c G M P c o n diti o ns.

Natri uretic pe pti de pr otecti ve si g nali n g has bee n li n ke d t o mit oc h o n dria i n t he disea se d heart, w here it bl oc ks t he mit oc h o n drial calci u m u ni p orter 1 6 2 , i n hi bits mit oc h o n drial

R OS production and subsequent Ca 2 + leak fro m the S R 1 6 3 , a n d pre ve nts t he mit oc h o n drial per mea bility tra nsiti o n p ore fr o m o pe ni n g 1 6 4 . H o w P D E 9 fits i n t o t hese pr otecti ve be nefits is u n k n o w n, a n d re mai ns t o be deter mi ne d.

7 8

T a bl e 3 .1: Proteins significantly enriched in ad P D E9 -F L A G as co mpared t o a d F L A G mass spectro metry sa mples.

Mass spectro metry sa mples were analyzed using LI M M A, co mparing adFL A G to adP D E9 -

F L A G. All pr otei ns wit h p < 0. 0 5 are s h o w n.

R ati o P D E 9 - Gene Na me Protein Na me P V al u e FLAG/FLAG U QCR10 Cytochro me b -c1 co mplex sub u nit 9 2. 0 1 E -0 5 4. 9 1 N A D H dehydrogenase [ubiquinone] N D UFS3 ir o n -s ulf ur pr otei n 3, mit oc h o n drial 2. 3 5 E -0 5 2. 7 6 N A D H dehydrogenase [ubiquinone] 1 N D UFA5 al p ha s u bc o m ple x s u b u nit 5 3. 5 1 E -0 5 2. 4 0 NA D H -ubiquinone oxidoreductase 75 N D UFS1 k Da s u b u nit, mit oc h o n drial 3. 8 1 E -0 5 2. 3 3 N D UFA4 Cytochro me c oxidase subunit N D UF A4 5. 0 1 E -0 5 4. 1 0 A T P sy nt hase s u b u nit ga m ma, ATP5F1 C mit oc h o n drial 5. 9 4 E -0 5 3. 2 8 U QCR Q Cytochro me b -c 1 c o m ple x s u b u nit 8 6. 7 1 E -0 5 2. 8 2 N A D H dehydrogenase [ubiquinone] 1 N D UFB4 beta s u bc o mplex subu nit 4 6. 9 6 E -0 5 2. 1 3 L O C100352363 Cytochro me c oxidase subunit 6 C 7. 0 3 E -0 5 2. 0 4 N A D H dehydrogenase [ubiquinone] 1 al p ha s u bc o m ple x s u b u nit 9, N D UFA9 mit oc h o n drial 7. 3 8 E -0 5 2. 8 2 N A D H dehydrogenase [ubiquinone] 1 N D UFA6 alpha subco mplex subun it 6 7. 8 1 E -0 5 2. 1 5 L O C100346096 A T P sy nt hase s u b u nit f, mit oc h o n drial 8. 5 0 E -0 5 2. 6 3 N A D H dehydrogenase [ubiquinone] N D UFS2 ir o n -s ulf ur pr otei n 2, mit oc h o n drial 0.00011 2. 2 4 Mit oc h o n drial 2 -o x o gl utarate / malate SL C25 A11 carrier pr otei n 0.00018 3. 3 0 M T C H2 Mit oc h o n drial carrier h o m ol o g 2 0.00023 3. 3 6 M GST3 Micr os o mal gl utat hi o ne S -tra nsferase 3 0.00024 3. 0 7 N A D H dehydrogenase [ubiquinone] 1 N D UFA7 al p ha s u bc o m ple x s u b u nit 7 0.00025 1. 9 1 I M MT MI C OS co mplex subunit MI C60 0.00028 5. 7 2 N A D H de hydrogenase [ u bi q ui n o ne] 1 N D UFA13 alpha subco mplex subunit 13 0.00031 2. 0 0 A T P sy nt hase s u b u nit al p ha, ATP5 A1 mit oc h o n drial 0.00034 2. 0 5 A T P sy nt hase F( 0) c o m ple x s u b u nit B 1, ATP5PB mit oc h o n drial -li ke 0.00056 2. 9 0

7 9

SL C25 A4 A DP/ A TP translocase 1 0.00058 5. 4 0 A CSL5 l o n g -c hai n -fatty -aci d -- C o A li gase 5 0.00060 2. 4 1 N A D H dehydrogenase [ubiquinone] 1 N D UFA12 alpha subco mplex subunit 12 0.00062 2. 5 4 P OP DC2 Popeye do main -c o ntai ni n g pr otei n 2 0.00064 2. 4 8 C HC H D3 MI C O S co mplex subunit MI C19 0.00064 2. 7 6 N A D H dehydr o ge nase [ u bi q ui n o ne] 1 N D UFA2 alpha subco mplex subunit 2 0.00073 2. 2 4 Cytochro me c oxidase subunit 4 isofor m C O X4 1, mit oc h o n drial prec urs or 0.00073 1. 6 9 Hi g h affi nity c G M P -s pecific 3', 5' -cyclic P DE9 A phosphodiesterase 9 A 0.00082 3. 9 2 V olta g e-dependent a ni o n -selecti ve V D AC2 c ha n nel pr otei n 2 0.00090 3. 7 8 C D GS H iron -sulfur do main -c o ntai ni n g CIS D1 pr otei n 1 0.00113 1. 9 3 N A D H dehydrogenase [ubiquinone] 1 N D UFA8 al p ha s u bc o m ple x s u b u nit 8 0.00113 1. 5 3 N A D H dehydrogenase [ubiquinone] N D UFV2 flav o pr otei n 2, mi t oc h o n drial 0.00123 1. 8 0 U B B P oly u bi q uiti n -B 0.00124 1. 6 0 Calci u m -bi n di n g mit oc h o n drial carrier SL C25 A12 pr otei n Aralar 1 0.00132 2. 7 7 MI C OS10 MI C OS co mplex subunit MI C10 0.00139 1. 7 8 A p o pt osis -i n d uci n g fact or 1, AIF M1 mit oc h o n drial 0. 0 0 1 4 1 1. 6 2 P H B Pr o hi biti n 0.00145 1. 6 5 N A D H dehydrogenase [ u bi q ui n o ne] 1 al p ha s u bc o m ple x s u b u nit 1 0, N D UFA10 mit oc h o n drial 0.00152 2. 1 4 Cytochro me c oxidase asse mbly protein C O X15 C O X15 ho molog 0.00157 2. 0 9 El o n gati o n fact or T u, mit oc h o n drial -li ke T UF M |el o n gati o n fact or T u, mit oc h o n drial 0.00162 1. 8 8 AP O OL MI C OS co mplex subunit MI C27 0.00164 2. 1 1 N A D( P) transhydrogenase, N N T mit oc h o n drial 0.00168 2. 7 8 ATP5 H A T P sy nt hase s u b u nit d, mit oc h o n drial 0.00170 1. 6 1 M Y H4 Myosin heavy chain 4 0.00176 5. 3 0 Cytochro me b -c 1 c o m ple x s u b u nit 6, L O C10034 3982 mit oc h o n drial 0.00205 1. 7 1 ST K38L Serine/threonine -pr otei n ki nase 3 8 -li ke 0.00214 2. 2 2 MP C2 Mit oc h o n drial pyr u vate carrier 2 0.00224 2. 6 9

8 0

Sarcoplas mic/endoplas mic reticulu m ATP2 A2 calci u m A T Pase 2 0. 0 0239 3. 0 0 Sorting and asse mbly machinery SA M M50 co mponent 50 ho molog 0.00245 1. 9 3 Pyruvate dehydrogenase kinase, isozy me P D K4 4 0.00249 2. 0 1 Me di u m -c hai n s pecific acyl -C o A ACA D M dehydrogenase, mitochondrial 0.00252 1. 5 7 C AS Q2 Calse q uestri n -2 prec urs or 0.00260 2. 9 9 Pr otei n -L -is oas partate ( D -as partate) O - PC MT1 met hyltra nsferase 0.00263 1. 4 7 Cytochro me c oxidase subunit II C O X2 ( mit oc h o n dri o n) 0.00271 1. 8 1 SL C25 A3 Phosphate carrier protein, mitochondrial 0.00277 3. 1 0 M Y H6 Myosin heavy chain 6 0. 0 0 2 8 8 2. 0 9 T U B A8 T u b uli n al p ha -8 c hai n 0.00288 1. 5 8 A T P sy nt hase s u b u nit beta, ATP5F1B mit oc h o n drial 0.00296 1. 4 8 HSP A9 Stress -7 0 pr otei n, mit oc h o n drial 0.00306 1. 5 3 A T P sy nt hase F 0 s u b u nit 8 M T -A T P8 ( mit oc h o n dri o n) 0.00316 2. 1 4 Is ocitrate de hy dr oge nase [ N A D P], I D H2 mit oc h o n drial 0.00364 1. 5 0 F A M210 A Protein F A M210 A 0.00367 1. 9 4 S R L Sarcalu menin precursor 0.00371 2. 2 9 C C T7 T -c o m ple x pr otei n 1 s u b u nit eta 0.00397 2. 3 0 cA MP -dependent protein kinase type II - PR KAR2A al p ha re g ulat ory s u b u nit 0. 0 0 4 1 0 1. 5 1 A T P sy nt hase F 0 s u b u nit 6, partial M T -A T P6 ( mit oc h o n dri o n) 0.00437 2. 0 1 P L N Cardiac phosphola mban 0.00455 2. 6 1 Car niti ne O -pal mit oyltra nsferase 1, CP T1 B m uscle is of or m, partial 0.00461 2. 1 8 P C Pyruvate carboxylase, mitochondrial 0.00468 1. 7 7 Py ruvate dehydrogenase kinase, isozy me P D K2 2 0.00473 1. 3 7 M T X2 Meta xi n -2 0.00479 1. 7 5 Cytochro me c oxidase subunit 7 A1, C O X7A1 mit oc h o n drial, partial 0.00497 2. 6 2 B A G fa mily molecular chaperone B A G2 re g ulat or 2 0.00603 1. 7 2 Cytochro me b -c 1 c o m ple x s u b u nit U QCRFS1 Ries ke, mit oc h o n drial 0.00621 1. 8 0

8 1

Hydroxysteroid dehydrogenase -li ke HS DL2 pr otei n 2 is of or m X 2 0.00739 2. 1 3 F A M162 A Protein F A M162 A 0.00757 1. 6 8 SL C8 A2 Sodiu m/calciu m exchanger 2 0.00767 1. 9 9 ATP5 ME A T P sy nt hase s u b u nit e, mit oc h o n drial 0.00772 1. 4 3 Sodiu m/potassiu m -transporting A T Pase ATP1 A1 s u b u nit al p ha -1 0.00785 2. 0 0 PY G M Glycogen phosphorylase, muscle for m 0.00808 1. 3 3 TL N1 Tali n -1 0.00834 1. 6 4 M OB2 M O B ki nase acti vat or 2 0.00838 1. 4 1 H G D H o m o ge ntisate 1, 2 -dioxygen ase 0.00876 3. 2 8 Pyruvate dehyrogenase phosphatase P DP1 catalytic s u b u nit 1 0.00882 1. 6 5 Cytochro me b -c 1 c o m ple x s u b u nit 2, U QCRC2 mit oc h o n drial 0.00905 1. 5 8 SL C25 A5 A DP/ A TP translocase 2 0.00910 2. 5 0 A CSL1 L o ng -c hai n -fatty -aci d -- C o A li gase 1 0. 0 0 9 6 0 1. 5 9 Mit oc h o n drial car niti ne /acylcar niti ne S L C 25 A20 carrier pr otei n 0.01001 2. 4 0 A T P -dependent 6 -phosphofructokinase, PF K M m uscle ty pe 0.01017 1. 5 5 Vesicle -associated me mbrane pr otei n - V AP A ass ociate d pr otei n A 0.01020 1. 6 4 MY O M2 Myo mesin -2 0.01033 1. 5 4 Isocitrate dehydrogenase [ N A D P] I D H1 cyt o plas mic 0.01052 2. 6 2 EHHADH Peroxiso mal bifunctional enzy me 0.01063 2. 4 3 Succinate dehydrogenase [ubiquinone] S D HB ir o n -s ulf ur s u b u nit, mit oc h o n drial 0.01101 1. 6 0 Cytochro me c oxidase asse mbly prot ei n C O X14 C O X 1 4 0.01115 1. 6 6 NIPS N AP2 Pr otei n Ni p S na p h o m ol o g 2 0.01144 1. 4 2 C O Q8A Aty pical ki nase A D C K 3, mit oc h o n drial 0.01175 1. 5 5 D -beta -hydroxybutyrate dehydrogenase, B D H1 mit oc h o n drial 0.01213 2. 1 6 N A D H dehydrogenase [ubiquinone] N D UFV3 fla v o pr otei n 3, mit oc h o n drial 0.01319 2. 1 2 EIF4 A1 E u kary otic i nitiati o n fact or 4 A -I 0.01331 2. 0 4 M TFP1 Mit oc h o n drial fissi o n pr ocess pr otei n 1 0.01343 1. 8 2 N DR G2 Pr otei n N D R G 2 0.01447 1. 6 4 L O C100353846 Al p ha -2 -macroglobulin 0.01470 2. 2 9 R A C -beta seri ne /t h reonine -pr o tei n A K T2 ki nase 0.01561 1. 7 3

8 2

GTP: A MP phosphotransferase A K3, A K 3 mit oc h o n drial 0.01570 1. 3 9 AC A A2 3 -ket oacyl -C o A t hi olase, mit oc h o n drial 0.01588 1. 3 1 Met hyl mal o nate -se mialdehyde AL D H5A1 dehydrogenase [acylating], mitochondrial 0.01694 1. 3 8 V olta ge -dependent a ni o n -selecti ve V D A C 1 c ha n nel pr otei n 1 0.01752 1. 7 6 Dyna min -li ke 1 2 0 k Da pr otei n, OP A1 mit oc h o n drial 0.01782 1. 5 5 T RI M72 Tri partite m otif -c o ntai ni n g pr otei n 7 2 0.01833 1. 2 8 Cyt oc hr o me c o xi dase s u b u nit 7 B, L O C100353320 mit oc h o n drial 0 .01847 1. 9 8 Inorganic pyrophosphatase 2, PP A2 mit oc h o n drial 0.01865 1. 5 4 N DUFAB1 Acyl carrier pr otei n, mit oc h o n drial 0.01893 1. 3 4 4 -hydroxy -2 -o x o gl utarate al d olase, H O GA1 mit oc h o n drial 0.02009 2. 1 4 CL T C Clat hri n hea vy c hai n 1 0.02079 2. 1 1 ATP5 MPL 6. 8 k Da mit oc h o n drial pr ote oli pi d 0.02101 1. 4 8 S ucci nyl -C o A li gase [ A D P -f or mi n g] S UCL A2 s u b u nit beta, mit oc h o n drial 0.02123 1. 5 1 P ABPC1 P olyade nylate -bi n di n g pr otei n 1 0.02196 2. 7 5 P H B2 Pr o hi biti n -2 0.02201 1. 5 3 L NPEP Le ucyl -cysti nyl a mi n o pe pti dase 0 .02213 2. 0 2 B V ES Bl o od vessel e picardial su bsta nce 0.02385 2. 1 2 Delta -1 -pyrr oli ne -5 -car b o xylate AL D H4A1 dehydrogenase, mitochondrial 0.02422 1. 4 3 Cyt oc hr o me c o xi dase s u b u nit 5 A, C O X5A mit oc h o n drial 0.02424 1. 4 7 R P L4 6 0 S ri b os o mal pr otei n L 4 0. 0 2 4 3 8 1. 4 7 Cytochro me c oxidase subunit 6 A2, C O X 6 A 2 mit oc h o n drial 0.02521 1. 6 0 PP M1B Protein phosphatase 1 B 0.02570 1. 4 1 Mit oc h o n drial i m p ort i n ner me m bra ne TI M M50 translocase subunit TI M50 0.02775 1. 5 1 F B P1 Fruct ose -1, 6 -bisphosphatase 1 0.02809 2. 0 7 N A D H dehydrogenase [ubiquinone] N D UFS6 ir o n -s ulf ur pr otei n 6, mit oc h o n drial 0.02909 1. 4 9 AC O X1 Peroxiso mal acyl -coenzy me A oxidase 1 0.02946 1. 8 5 Phosphoenolpyruvate carboxykinase, P C K1 cyt os olic [ G T P] 0.03063 2. 0 3 Is ocitrate dehydrogenase [ N A D] s u b u nit I D H3B be ta, mit oc h o n drial 0.03149 1. 4 2

8 3

A TPase fa mily A A A do main -c o ntai ni n g AT A D1 pr otei n 1 0.03189 1. 4 1 R H OT1 Mit oc h o n drial R h o G T Pase 1 0.03228 1. 4 1 M YBPC3 My osi n -bi n di n g pr otei n C, car diac -ty pe 0.03342 1. 7 8 V olta ge -dependent anion -selecti v e V D AC3 c ha n nel p r otei n 3 0.03477 1. 9 2 AF G3L2 AF G3 -li ke pr otei n 2 0.03485 1. 4 9 HIST1 H2 A A Hist o ne H 2 A ty pe 1 -A 0.03509 2. 0 4 SL C25 A10 Mit oc h o n drial dicar b o xylate carrier 0.03513 1. 4 3 ATP6 V1 E1 V -type proton A T Pase subunit E 1 0.03539 1. 5 3 Cytochro me c o xi das e s u b u nit 7 A - C O X7A2L relate d pr otei n, mit oc h o n drial 0.03565 1. 6 7 2 -oxoisovalerate dehydrogenase subunit BCKDHA al p ha, mit oc h o n drial 0.03607 1. 3 6 DT NA Dystr o brevi n al p ha 0.03893 1. 5 5 Pyruvate dehydrogenase (acetyl - transferring) kinase isozy me 1 , P D K1 mit oc h o n d rial 0.04277 1. 4 0 D olic hyl -diphosphooligosaccharide -- RP N1 pr otei n glyc osyltra nsferase su bu nit 1 0.04341 1. 3 6 R R AS2 Ras -relate d pr otei n R -Ras 2 0.04568 1. 3 8 RB M10 R N A -bi n di n g pr otei n 1 0 is of or m X 4 0.04647 1. 3 4 AL D OB Fruct ose -bisphosphate a l d olase B 0.04678 1. 4 3 Trif u ncti o nal e nzy me s u b u nit beta, HA D HB mit oc h o n drial 0.04678 1. 3 4 L P P Li p o ma -preferre d part ner 0.04694 1. 5 2 Calci u m -bi n di n g mit oc h o n drial carrier SL C25 A13 pr otei n Aralar 2 0.04740 1. 8 1 MY O M1 Myo mesin -1 0.04768 1. 4 4 RPS4 X 4 0 S ri b os o mal pr otei n S 4, X is of or m 0.04774 2. 1 9 Pr otei n -gl uta mi ne ga m ma - T G M2 gluta myltra nsferase 2 0.04831 1. 2 6

8 4

T a bl e 3 .2: D A VI D path ways analysis of proteins significantly enriched in ad P D E9 -

F L A G sa mples.

Pr otei ns si g nifica ntly e nric he d i n a d P D E 9 -FL A G sa mpl es as co mpared to adF L A G sa mples

(see Ta ble 1) u n der we nt pat h ways a nalysis usi n g t he D A VI D data base. All pat h ways wit h p <0.05 are sho wn.

K E G G Path way P V al u e Ge nes F ol d Enrich ment O xi dati ve 1. 1 8 E -2 6 N D UFB4, N D UF AB1, L OC10035236 3, 1 7. 0 7 phosphorylation L O C100353 3 2 0, U Q C R Q, N D U F S 6, L OC100343982, N D UFS3, ATP5 H, N D UFS2, N D UFS1, N D UF A4, N D UFA5, N D UFA2, N D UFA8, N D UFA9, N D UFA6, N D UFA7, N D UF A13, N D UF A10, L OC100346096, N D U F A 1 2, P P A 2, S D H B, C O X 2, ATP6 V1 E1, N D UF V2, ATP5 A1 Meta b olic 1. 2 9 E -1 8 AC O X 1, E H H A D H, N D U F A B 1, 3. 4 5 path ways L OC100352363, U QCR Q, L O C100353320, N D UFS6, RP N1, S UCL A2, N D UFS3, N D UFS2, ATP5 H, N D UFS1, ACAA2, ACA D M, AL D H5A1, FBP1, N D UF A13, PF K M, N D UF A10, N D UF A12, L OC100346096, N NT, N D U F B 4, A L D O B, H A D H B, L O C100343982, A CSL1, I D H2, AL D H4 A1, I D H1, B D H 1, A C S L 5, N D UF A4, BC K D H A, N D UF A5, N D UFA2, N D UFA8, N D UFA9, N D UFA6, H O GA1, N D UFA7, H G D, I D H 3 B, P C K 1, S D H B, P Y G M, C O X 2, ATP6 V1E1, N D UF V2, ATP5 A1, PC Bi osy nt hesis of 3. 2 8 E -0 7 BC K D HA, ACAA2, ACA D M, 5. 5 4 a nti bi otics E H H A D H, A L D O B, F B P 1, A K 3, I D H 3 B, P F K M, P C K 1, H A D H B, S D H B, I D H2, I D H1, S UCL A2 Car b o n 8. 0 7 E -0 7 AC O X1, AC A A2, CPT1B, AC A D M, 1 4. 8 5 meta b olis m ACSL1, E H HA D H, HA D HB, ACSL5 Fatty aci d 1. 2 1 E -0 6 S D H B, A C A D M, E H H A D H, A L D O B, 7. 7 0 degradati o n I D H 2, F B P 1, I D H 3 B, I D H 1, P F K M, S U CL A2, P C Fatty aci d 3. 1 8 E -0 6 A C O X 1, A C A A 2, C P T 1 B, A C A D M, 1 2. 2 0 meta b olis m ACSL1, E H HA D H, HA D HB, ACSL5

8 5

Citrate cycle 4. 8 4 E -0 6 S D H B, I D H 2, I D H 3 B, I D H 1, S U C L A 2, 1 5. 3 2 ( T C A cycle) P C, P C K 1 Car diac m uscle 1. 7 1 E -0 4 AC O X1, CPT1B, AC A D M, ACSL1, 8. 3 0 c o ntracti o n E H HA D H, PC K1, ACSL5 P P A R si g nali n g 3. 0 5 E -0 4 L OC100343982, C O X2, ATP1 A1, M Y H6, 7. 4 7 pat h w ay L O C100352363, L O C100353320, U QCR Q c G MP -P K G 6. 0 8 E -0 4 A TP2 A2, SL C25 A4, SL C25 A5, PL N, 4. 6 6 si g nali n g pat h way ATP1 A1, V D AC2, V D AC3, V D AC1, A K T2 Bi osy nt hesis of 0. 0 0 1 5 AL D OB, I D H2, I D H 3 B, I D H 1, P F K M, 7. 0 2 a mi n o aci ds P C Peroxiso me 0. 0 0 4 1 AC O X1, ACSL1, E H H A D H, I D H2, 5. 5 7 I D H1, ACSL5 Vali ne, le uci ne 0. 0 0 4 4 B C K D H A, A C A A 2, A C A D M, 7. 3 6 a n d is ole uci ne EHHADH, HADHB degradati o n A di p ocyt o ki ne 0. 0 0 7 4 CPT1B, A CSL1, A KT2, P C K1, A CSL5 6. 3 7 si g nali n g pat h way 2 -Oxocarbo xylic 0. 0 1 6 1 I D H2, I D H3B, I D H1 1 5. 0 7 aci d meta b olis m Calci u m si g nali n g 0. 0 1 7 7 A TP2 A2, SL C25 A4, SL C25 A5, PL N, 3. 3 2 path way V DAC2, V DAC3, V DAC1 Glyc olysis / 0. 0 4 5 4 AL D OB, FBP1, PF K M, PC K1 4. 9 5 Gluconeogenesis Propanoate 0. 0 4 6 7 AL D H5 A 1, E H H A D H, B D H1 8. 5 4 meta b olis m B uta n oate 0. 0 4 6 7 ACA D M, E H HA D H, SUCLA2 8. 5 4 meta b olis m Pe nt ose 0. 0 4 6 7 AL D OB, FBP1, PF K M 8. 5 4 phosphate path way

8 6

T a bl e 3 .3: Protei ns significa ntly less associate d with P D E9 f oll o wi n g E T -1 treat me nt. adP D E9 -FL A G sa mples were c o m pare d t o adP D E9 -FLA G+ET -1 sa m ples usi n g LI M M A.

All pr otei ns wit h p < 0. 0 5 a n d a rati o f or P D E 9 + E T -1 t o P D E 9 less t ha n 1 are s h o w n.

R ati o Gene Na me Pr o t ei n N a m e P V al u e P DE9+ET1/P DE9 N A D H dehydrogenase [ubiquinone] N D UFB4 1 beta s u bc o m ple x s u b u nit 4 0. 0 0 1 1 0. 6 0 N A D H dehydrogenase [ubiquinone] N D U F A 7 1 al p ha s u bc o m ple x s u b u nit 7 0. 0 0 1 8 0. 6 3 C O X6C Cytochro me c oxidase subunit 6 C 0. 0 0 2 4 0. 5 1 N A D H dehydrogenase [ubiquinone] N D UFS2 ir o n -s ulf ur pr otei n 2, mit oc h o n drial 0. 0 0 2 8 0. 6 2 N A D H dehydrogen ase [ u bi q ui n o ne] N D UFS3 ir o n -s ulf ur pr otei n 3, mit oc h o n drial 0. 0 0 4 3 0. 5 7 N A D H dehydrogenase [ubiquinone] N D UFA13 1 al p ha s u bc o m ple x s u b u nit 1 3 0. 0 0 5 6 0. 6 2 Cytochro me c oxidase asse mbly C O X14 pr otei n C O X 1 4 0. 0 0 6 2 0. 5 7 A T P sy nt hase s u b u nit f, ATP5 MF mit oc h o n d rial 0. 0 0 7 3 0. 5 8 Micr os o mal gl utat hi o ne S -tra nsferase M GST3 3 0. 0 0 8 9 0. 4 9 N A D H dehydrogenase [ubiquinone] N D UFS4 ir o n -s ulf ur pr otei n 4, mit oc h o n drial 0. 0 0 9 0 0. 6 1 N A D H dehydrogenase [ubiquinone] N D UFA8 1 al p ha s u bc o m ple x s u b u nit 8 0. 0 0 9 5 0. 5 5 N A D H dehydrog e nase [ u bi q ui n o ne] N D UFA6 1 al p ha s u bc o m ple x s u b u nit 6 0. 0 1 3 7 0. 6 4 MTC H2 Mit oc h o n drial carrier h o m ol o g 2 0. 0 1 6 0 0. 4 0 Mit oc h o n drial 2 -o x o gl utarate / malate SL C25 A11 carrier pr otei n 0. 0 1 6 0 0. 5 2 N A D H dehydrogenase [ubiquinone] 1 alpha subco mp le x s u b u nit 9 , N D UFA9 mit oc h o n drial 0. 0 1 6 9 0. 5 2 Isocitrate dehydrogenase [ N A D P], I D H2 mit oc h o n drial 0. 0 2 0 2 0. 7 4 C B R4 Carbonyl reductase fa mily me mber 4 0. 0 2 0 6 0. 6 5 N A D H dehydrogenase [ubiquinone] N D UFA12 1 al p ha s u bc o m ple x s u b u nit 1 2 0. 0 2 1 4 0. 5 7 U QCR10 Cyt ochro me b -c 1 c o m ple x s u b u nit 9 0. 0 2 2 2 0. 3 5 A CSL5 L o ng -c hai n -fatty -aci d -- C o A li gase 5 0. 0 2 3 9 0. 5 6 A T P sy nt hase F( 0) c o m ple x s u b u nit ATP5PB B 1, mit oc h o n drial -li ke 0. 0 2 4 0 0. 4 7

8 7

MP C2 Mit oc h o n drial pyr u vate carrier 2 0. 0 2 4 3 0. 4 8 N DR G2 Pr otei n N DR G2 0. 0 2 5 4 0. 6 8 Cytochro me c oxidase subunit 7 A2, C O X7A 2 mit oc h o n drial 0. 0 2 6 1 0. 6 3 A T P sy nt hase F 0 s u b u nit 8 M T -A T P8 ( mit oc h o n dri o n) 0. 0 2 8 7 0. 6 2 T UF M El o n gati o n fact or T u, mit oc h o n drial 0. 0 2 9 2 0. 7 0 N A D H dehydrogenase [ubiquinone] N D UFA5 1 alpha subco mplex sub u nit 5 0. 0 2 9 9 0. 6 2 Pyruvate dehydrogenase kinase, P D K4 isozy me 4 0. 0 3 0 0 0. 7 0 Ge neral tra nscri pti o n fact or 3 C G TF3 C4 p oly pe pti de 4 0. 0 3 0 0 0. 5 9 Cytochro me c oxidase subunit II C O X2 ( mit oc h o n dri o n) 0. 0 3 1 4 0. 7 1 U B B P oly u bi q uiti n -B 0. 0 3 2 4 0. 6 8 P H B Pr o hi biti n 0. 0 3 3 2 0. 7 4 Cyt oc hr o me c o xi dase s u b u nit 5 A, C O X5A mit oc h o n drial 0. 0 3 3 9 0. 6 6 Cytochro me b -c 1 c o m ple x s u b u nit U QCRFS1 Ries ke, mit oc h o n drial 0. 0 3 5 3 0. 7 3 Sorting and asse mbly machinery SA M M50 co mponent 50 ho molog 0. 0 3 6 5 0. 6 9 MI C OS10 MI C OS co mp le x s u b u nit M I C 1 0 0. 0 3 7 6 0. 6 4 A T P synthase subunit ga m ma, ATP5F1 C mit oc h o n drial 0. 0 3 7 6 0. 5 0 Cytochro me c oxidase subunit 7 A1, C O X7A1 mit oc h o n drial 0. 0 3 9 0 0. 4 7 Cyt oc hr o me c o xi dase s u b u nit 7 B, C O X7B mit oc h o n drial 0. 0 3 9 4 0. 3 9 R A N G T P -bi n di n g n uclear pr otei n Ra n 0. 0410 0. 7 1 F A M162 A Protein F A M162 A 0. 0 4 3 3 0. 7 1 Cytochro me c oxidase subunit 6 A2, C O X6A2 mit oc h o n drial 0. 0 4 3 5 0. 3 4 AP O OL MI C OS co mplex subunit MI C27 0. 0 4 6 1 0. 6 4 U QCR Q Cytochro me b -c 1 c o m ple x s u b u nit 8 0. 0 4 6 8 0. 5 4 Cytochro me c oxidas e s u b u nit 4 C O X4 isofor m 1, mitochondrial precursor 0. 0 4 8 2 0. 7 2 A TPase fa mily A A A do main - AT A D1 c o ntai ni n g pr otei n 1 0. 0 4 8 7 0. 7 3

8 8

T a bl e 3 .4: Pr ot ei ns si g nifi c a ntl y m or e associated with P D E9 f oll o wi n g E T -1 treat me nt. adP D E9 -F L A G sa mples were co mpared to adP D E 9 -FLA G+ET -1 sa m ples usi n g LI M M A.

All pr otei ns wit h p < 0. 0 5 a n d a rati o f or P D E 9 + E T -1 t o P D E 9 greater t ha n 1 are s h o w n.

R ati o Gene Na me Protein Na me P V al u e P DE9+ET1/P DE9 Kerati n, ty pe II cyt os keletal 5 K R T5 isofor m X2 0. 0 0 0 5 2. 9 3 NCCRP1 F -b o x o nly pr ot ei n 5 0 0. 0 0 1 3 3. 7 5 K R T16 Kerati n, ty pe I cyt os keletal 1 6 0. 0 0 2 0 2. 3 1 Aflatoxin B1 aldehyde reductase A KR7 A3 me m ber 3 0. 0 0 4 2 1. 9 7 KL HL41 Kelc h -li ke pr otei n 4 1 0. 0 0 4 5 1. 6 5 C L U Clusteri n precurs or 0. 0 0 5 7 1. 9 8 NI D1 Nidogen -1 0. 0 0 8 3 1. 3 7 S100 A14 Pr o tei n S 1 0 0 -A 1 4 0. 0 0 8 7 2. 0 1 F -acti n -ca p pi n g pr otei n s u b u nit C APZ A1 al p ha -1 0. 0 1 0 2 2. 3 5 A N XA1 A n ne xi n A 1 0. 0 1 0 3 1. 6 7 HSP B7 Heat s h oc k pr otei n beta -7 0. 0 1 0 9 1. 3 4 AC OT13 Acyl -coenzy me A thioesterase 13 0. 0 1 1 6 1. 7 8 K R T71 Kerati n, ty pe II cyt os keletal 7 1 0. 0 1 1 8 2. 1 7 Al p ha -a minoadipic se mialdehyde A ASS sy nt hase, mit oc h o n drial 0. 0 1 2 3 1. 3 3 I D E I ns uli n -degrading enzy me 0. 0 1 2 7 1. 7 8 C HR DL2 Chordin -li ke pr otei n 2 0. 0 1 2 8 8. 8 3 Xi n acti n -bi n di n g re peat -c o ntai ni n g XI R P1 pr otei n 1 0. 0 1 3 4 1. 6 2 Kerati n, ty pe II cyt os kelet al 5 K R T5 isofor m X1 0. 0 1 3 9 1. 6 1 K R T14 Kerati n, ty pe I cyt os keletal 1 4 0. 0 1 4 3 1. 8 1 Ankyrin repeat do main -c o ntai ni n g A N KR D33 pr otei n 3 3 0. 0 1 7 4 1. 7 8 K R T80 Kerati n, ty pe II cyt os keletal 8 0 0. 0 1 8 3 1. 5 3 M AP4 Micr ot u b ule -ass ociate d pr otei n 4 0. 0 1 8 4 1. 5 4 K R T17 Kerati n, ty pe I cyt os keletal 1 7 0. 0 1 9 3 2. 2 8 AA M DC Mth938 do main -c o ntai ni n g pr otei n 0. 0 1 9 6 1. 4 0 C har ge d m ulti vesic ular b o dy pr otei n C H MP5 5 0. 0 1 9 7 1. 7 4

8 9

OBSC N O bsc uri n 0. 0 2 2 7 1. 5 2 Serine/threonine -pr otei n p h os p hatase 2 A catalyti c s u b u nit PPP2C A al p h a is of or m 0. 0 2 3 1 1. 7 7 K R T1 Kerati n, ty pe II cyt os keletal 1 0. 0 2 3 9 1. 5 2 K R T13 Kerati n, ty pe I cyt os keletal 1 3 0. 0 2 3 9 1. 8 1 K A RS Lysi ne -- t R N A li gase 0. 0 2 4 1 1. 3 2 Pr otei n ki nase C a n d casei n ki nase P ACSI N3 s u bstrate i n ne ur o ns pr otei n 3 0. 0 2 7 6 1. 4 9 G S N Gels oli n 0. 0 2 7 9 2. 3 0 N R AP Ne b uli n -relate d -anchoring protein 0. 0 2 9 4 1. 6 5 P RSS2 Try psi n -2 0. 0 3 3 5 1. 8 9 E Z R Ezri n 0. 0 3 4 4 1. 2 4 E u kary otic tra nslati o n i nitiati o n EIF3 H fact or 3 s u b u nit H 0. 0 3 5 5 1. 4 5 P GER1 Pr ostagla ndi n -E( 2) 9 -reductase -li ke 0. 0 3 6 0 1. 3 1 Y WHAE 1 4 -3 -3 pr otei n e psil o n 0. 0 3 7 3 1. 4 5 F ABP5 Fatty aci d -bi n di n g pr otei n, e pi der mal 0. 0 3 7 6 1. 4 4 E N O2 Ga m ma -e n olase 0. 0 3 8 6 2. 1 3 G TPase I M AP fa mily me mber GI M D1 GI M D1 0. 0 3 9 7 1. 7 4 SERPI NB12 Ser pi n B 1 2 0. 0 4 1 3 1. 8 2 A MY2A Pa ncreatic al p ha -a mylase 0. 0 4 3 7 1. 7 0 M AP1B Micr ot u b ule -ass ociate d pr otei n 1 B 0. 0 4 4 1 1. 2 3 A R G1 Argi nase -1 0. 0 4 4 4 1. 4 6 E u kary otic tra nslati o n i nitiati o n EIF4 H fact or 4 H 0. 0 4 6 0 1. 2 2 Late nt -transfor ming gro wth factor L T BP1 beta -bi n di n g pr otei n 1 0. 0 4 6 2 1. 6 0 SPRR1 A C o r nifi n al p ha -li ke 0. 0 4 6 8 1. 7 9 SERPI N A12 Serpin A12 0. 0 4 8 6 1. 6 2 Heterogeneous nuclear SY NCRIP ribonucleoprotein Q 0. 0 4 8 6 1. 4 7 U 1 s mall n uclear ri b o n ucle o pr otei n S NRPA A 0. 0 4 9 0 1. 3 8

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C HAPTER 4 : Ge neratio n a n d testi ng of novel molec ular tools to st udy P D E5 and

P D E9 co mpart mentation

INTRODUCTION

To date, fe w studies have been perfor med exa mining the precise cellular c o m part me nts wit hi n w hic h P D E 5 a n d P D E 9 resi de i n car diac my ocytes. T his is li kely d ue i n part t o a lac k of pri or m olec ular t o ols wit h w hic h t o i de n tify t hese s mall, c o m ple x d o mai ns wi t hi n t he cell. A pre vi o us st u dy wit hi n t he Kass la b (Ma nli n g Z ha n g, u n p u blis he d) exa mined the proteo me associated with pulldo wn of P D E5 fro m adult mouse cardiac myocytes, but no co mparable study has been pe rfor med for P D E 9 d ue t o t he lac k of a mouse expres si n g ta g ge d P D E 9 pr otei n (alt h o u g h i n C ha pter 2 of t his t hesis we n o w prese nt pulldo wn data for F L A G -tagged P D E9 overexpressed in adult rabbit cardio myocytes).

W hile p ull d o w n i de ntifies direct bi n di n g part n ers ( or bi n di n g c o m ple xes) of t he pr otei n of i nter est, t he br oa der e n vir o n me nt wit hi n w hic h t he pr otei n of i nterest resi des is n ot f ully i de ntifie d by t his a p pr oac h. F urt her m ore, t here ca n e ve n be diffic ulties i de ntifyi n g bi n di n g partners depending on the c o n diti o ns use d f or cell lysis. If a str o n g deter g e nt is use d, pr otei n i nteracti o ns are i nterr u pte d, a n d bi n di n g part ners ca n n ot be i de ntifie d. G o o d i de ntificati o n of bi n di n g part ners a n d c o m ple xes is als o de pe n de nt o n efficie nt p ull d o w n of t he pr otei n of i nterest. If n ot d o ne well, t his may lea d t o i de nt ificati o n of fe wer bi n di n g part ners d ue t o l o w protein abundance that cannot be detected by a mass spectro meter. Therefore, to fully identify P D E co mpart ments, a novel molecular approach was needed to conci sely e xa mi ne its cell ular na n o d o mai n.

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I n 2 0 1 2, t h e la b of Alice Ti n g first pre miere d t he A P E X ta g by s h o wi n g its utility as a ge netically -e nc o da ble ta g t hat all o ws f or electr o n micr osc o pic i ma gi n g of a pr otei n of i nterest wit h o ut t he nee d f or a n a nti b o dy 1 6 5 . T hey t he n e x pa n de d u p o n t his t he f oll o wi n g year, where they described an elegant use of A P E X for spatially restricted proteo mic ma p p i n g 5 7 . Si n ce t his ti me, t he la b has f urt her o p ti mized these methods and introduced

A P E X 2, a n i m pr o ve d versi o n of A P E X wit h hi g her catalytic acti vity, all o wi n g f or st u dies i n cell ular micr o d o mai ns t hat are partic ularly se nsiti ve t o pr otei n o vere x pressi o n, s uc h as t he endoplas mic reticulu m and mitochond rial outer me mbrane 1 6 6 .

A P E X2 is an ascorbate peroxidase derived fro m the soybean that carries several m utati o ns all o wi n g t he pr otei n t o be m o n o meric as well as gi vi n g it hi g her catalytic acti vity.

It c onverts various phenol derivatives into phenoxyl radicals, which can then tag nearby electr o n -ric h a mi n o aci ds, na mely cystei ne, histi di ne, try pt o p ha n, a n d tyr osi ne. T his partic ular f u ncti o n of A P E X 2 all o ws for proteo mic mapping, as when cells overexpressi n g

a n A P E X 2 f usi o n pr otei n are pr o vi de d wit h bi oti n -p he n ol a n d H 2 O 2 , pr otei ns wit hi n a 2 0 n m ra di us of t he f usi o n pr otei n are ta g ge d by bi oti n -p he n o xyl ra dicals, facilitati n g stre pta vi di n p ull d o w n of ta g ge d pr otei ns a n d s u bse q ue nt i de ntificati o n by mass s p ectr o metry. N ota bly, t his ta g gi n g reacti o n occ urs very q uic kly, wit h la beli n g ta ki n g o nly o ne mi n ute, all o wi n g f or m o nit ori n g of hi g hly dy na mic cell ular pr ocesses. A P E X 2 als o

catalyzes H 2 O 2 -dependent poly me rizati o n of di a mi n o be nzi di ne ( D A B), w hic h ca n s u bse q ue ntly react wit h t he stai ni n g rea ge nt os mi u m t o ge nerate c o ntrast t hat ca n be visualized by electron microscopy to provide high -res ol uti o n i ma ges of pr otei n s u b -cell ular l ocalizati o n.

Se veral ot her si mi lar ta gs e xist , i ncl u di n g Bi oI D 5 4 , Bi oI D 2 5 5 , T ur b oI D, a n d mi ni T ur b o 5 6 . T hese f our ta gs are all varia nts of bi oti n li gase, w hic h directly tra nsfers t he

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bi oti n m oiety t o pr otei ns ( u nli ke t he bi oti n -phenoxyl radical produced by A P E X2). BioI D and BioI D2 were de vel o pe d by t he R o u x la b at t he U ni versity of S o ut h Da k ota, wit h t he creati o n of B ioI D2 evolving fro m a desire for a s maller and more efficient ligase. These e nzy mes re q uire o nly bi oti n t o perf or m la beli n g e x peri me nts, b ut nee d a p pr o xi mately 2 4 h o urs t o c o m plete la beli n g. T his ti me fra me li mits t heir a p plicati o n t o static cell ular syste ms, as dy na mic cell res p o nses t o sti m uli will be l ost. T ur b oI D a n d mi ni T ur b o were de vel o pe d by the Ting lab as i mprove ments upon the BioI D enzy mes, na mely i mproving the labe li n g ti me by d ecreasi n g it t o o nly 1 0 mi n utes as c o m pare d t o t he 2 4 h o urs nee de d f o r Bi oI D. T hese e nzy mes are als o bi oti n li gases, li ke Bi oI D a n d Bi oI D 2, b ut u n der we nt directe d e v ol uti o n t o be hi g hly efficie nt. T hese ne w ta gs fr o m t he Ti n g la b a p pear pr o mi si n g f or f ut ur e st u dies of dy na mic cell ular syste ms, as t hey ha ve relati vely fast l a beli n g ti me a n d o nly re q uire bi oti n.

MET HODS

Generation of G F P -AP E X2 -tagged P D E Constructs and Adenoviruses: P D E 5, P D E 9, and GFP -AP E X2 were subcloned into pc D N A3.1 + ( Ther m o Fis her Scie ntific) utilizi n g

Ne w E n gla n d Bi o La bs Hi Fi cl o ni n g. Briefly, pc D N A 3. 1 + was di geste d wit h N heI a n d X h oI

( Ne w England BioLabs) and gel purified. The P D E5, P D E9, and GFP -A P E X2 inserts were a m plifie d wit h pri mers desi g ne d usi n g t he N E B uil der desi g n t o ol ( Ne w E n gla n d Bi o La bs) and synthesized by Integrated D N A Technologies. GFP -A P E X 2 was a m plifie d fr o m pc D N A3 Connexin43 -G F P -A P E X 2, w hic h was a gift fr o m Alice Ti n g ( A d d ge ne plas mi d

#49385; http://n2t.net/addgene:49385; R RI D: Addgene_49385). A short lin ker was i ncl u d ed bet ween the P D E and GFP -A P E X 2 fra g me nts c o nsisti n g of t he pe pti de sequence

G GS G G T. Q5 high fidelity poly merase ( Ne w England Bio Labs) was used for P C R

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a mplification. P C R frag ments were gel purified, and asse mbly and bacterial transfor mation was t he n perf or med according to N E B Hi Fi protocol. Sequencing pri mers were designe d using the GenScript sequencing pri mer design tool (https:// w w w.genscript.co m/tools/dna - sequencing -pri mer -desi g n). All cl o ni n g a n d se q ue nci n g pri mer s equences can be found i n

T a bl e 4 .1 a n d 4 .2 , respectively. Adenoviruses for G F P -AP E X2, P D E5 -G F P -A P E X 2, a n d

P D E9 -G F P -A P E X2 were generated by Welgen, Inc ( Worcester, M A).

Neonatal rat myocyte isolation and transfection: Ne o natal rat my ocytes were is olate d as previ ously descri be d 6 5 . N R C Ms were c ult ure d f or 2 4 h o urs i n hi g h gl uc ose ( 4. 5 g / L) D M E M wit h 1 0 % F B S, L -gl uta mi n e ( 4 m M), a n d pe nicill i n /stre pt o myci n ( 1 0 0 U / m L) pri or t o st u dy.

For plas mid transfection, N R C Ms were transfected overnight using Lipofecta mine 3000

( Ther mo Fisher Scientific) according to manufacturer protocol. For adenovirus infection,

N R C Ms were infecte d wit h a de n o vir u s ( 1 0 M OI) i n plai n D M E M f or 3 -4 h o urs. Me dia was t he n c ha n ge d bac k t o hi g h gl uc ose D M E M c o ntai ni n g 1 0 % F B S, pe nicilli n -stre pt o myci n, a n d gl uta mi ne after b ot h tra nsfecti o n a n d i nfecti o n. Cells were gi ve n at least 4 8 h o urs t o ex press t he fus ion constructs p ri or t o sa m ple c ollecti o n or fi xati o n.

A d ult ra b bit myocyte isolatio n a n d i nfectio n: A d ult ra b bit my ocytes were i s olate d as previously described 1 4 9 . My ocytes were i nfecte d wit h a de n o vir us ( 1 0 M OI) a t t he ti me of p lati n g i n Me di u m 1 9 9 ( Si g ma). After 3 -4 hours, media was changed to Mediu m 199 c o ntai ni n g 2 % F B S, pe nicilli n -stre pt o myci n ( 1 0 0 U / m L), L -gl uta mi ne ( 0. 1 m g / m L), creati ne

( 5 m M), ta uri ne ( 5 m M), a n d L -car niti ne ( 2 m M). After o ver ni g ht i nc u bati o n, cells were sti m ulate d wit h e n d ot heli n -1 ( 1 0 0 n M, Si g ma) or ve hicle f or 2 4 h o urs i n Me di u m 1 9 9

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c o ntai ni n g pe nicilli n -stre pt o myci n, L -gl uta mi ne ( 0. 1 m g / m L), creati ne ( 5 m M), ta uri ne ( 5 m M), a n d L -car niti ne ( 2 m M).

Bi oti n yl ati o n pr ot o c ol: Bi oti nylati o n wa s perfor med acc or di n g t o t he p u blis he d pr ot oc ol fr o m H u n g et al 1 6 7 , wit h se veral de viati o ns. M o dificati o ns t o t he p u blis he d pr ot oc ol were a d diti o n of N -acetyl -cystei ne (t o i ncrease bi oti nylati o n) a n d cycl o he xi mi de (t o re d uce n o n - c o m part me nt s pecific bi o ti nylati o n). N -acetyl -cystei ne ( 5 0 0 µ M, Si g ma) a n d cycl o he xi mi d e

( 1 0 0 µ M, Si g ma) were a d de d 2 h o urs pri or t o a d diti o n of H 2 O 2 . All ot her parts of t he protocol were perfor med as reco m mended.

Assess ment of c G M P levels in neonatal myocytes: For c G M P assay sa m ples, ne o natal rat my ocytes were first tra nsfecte d wit h plas mi d or i nfecte d wit h vir us. Cells were t he n treate d t he f oll o wi n g day wit h a n s G C acti vat or, B A Y 6 0 -2 7 7 0 ( 1 0 n M, Bayer Healt hcare

P har mace uticals), atrial natri uretic pe pti de ( 1 µ M, Si g ma A 1 6 6 3), and/or endothe li n -1 ( 1 0 0 n M, Si g ma E 7 7 6 4) f or 4 8 h o urs pri or t o t he assay. Cyclic G M P le vels were meas ure d usi n g t he c G M P Direct Bi o Tra k EI A kit ( G E Healt hcare) acc or di n g t o ma n ufact urer pr ot oc ol.

Plates were rea d at b ot h 4 5 0 a n d 6 3 0 n m usi n g a M olec ular Devices S pectra Ma x M 5 micr o plate reader. F or back -calc ulati o n o f c G M P le vels f or t he 4 5 0 n m rea d, a se mi -l o g fit was used. For back -calc ulati o n of c G M P le vels f or t he 6 3 0 n m rea d, a 4 -para meter fit was used. Nor malization was perfor med as reco m mended by th e manufacturer. Statistical analysis was perfor med using Graph P a d Pris m versi o n 7, wit h c o m paris o ns d o ne usi n g a o ne -way A N O V A or Kruskal -Wallis test wit h p ost -h oc D u n nett’s tests. A n o n -para metric

Kr us kal -Wallis test was use d if t he Br o w n -F orsyt he test re t ur ne d a val ue less t ha n 0. 0 5.

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I m munofluorescence: Cells were tra nsfecte d or transduced as descri bed. After 48 h ours of f usi o n c o nstr uct e x pressi o n, cells were fi xe d i n 1 0 % f or mali n i n P B S, per mea bilize d wit h

0. 5 % sa p o ni n i n P B S, a n d bl oc ke d wit h 3 % B S A i n P B S. Cells we re t he n stai ne d o ver ni g ht i n 3 % B S A wit h 0. 5 % sa p o ni n i n P B S at 4 °C f or bi oti nylate d pr otei ns wit h Ale xa 5 5 5 - c o u ple d stre pta vi di n ( # S 3 2 3 5 5, T her m o Fis her Scie ntific, 1: 1 0 0 0), P D E 5 (a nti b o dy # 2 3 9 5,

Cell Si g nali n g Tec h n ol o gy, 1: 2 0 0), or P D E 9 ( c ust o m a nti b o dy , Pr o Sci I nc, 1: 2 0 0). Cells were t he n c o u nterstai ne d wit h a nti -ra b bit Ale xa 5 6 8 -coupled secondary antibody ( Ther mo Fisher) at a 1: 5 0 0 dil uti o n for P D E5 and P D E9 if necessary. Hard -m o u nt Vectas hiel d wit h D A PI

( Vector Labs) was used to preserve fluorescence a fter stai ni n g was c o m plete. I ma gi n g was perfor med on a Leica T CS S P E confocal microscope using a 40 X oil -i m mersi o n o bjecti ve.

G F P was i maged as a marker for the fusion protein.

I m munoprecipitation of b i oti n yl at e d p r ot ei ns: A d ult ra b bit car d io myocytes were i nfecte d a n d bi oti nylate d as descri be d. After sa m ples were c ollecte d, t hey were s o nicate d t o disrupt cell me mbranes and spun do wn. Protein concentration was measured using the

Pierce 660 -n m protein assay ( Ther mo Fisher). Fo r i m m u n o preci pitat i o n, 1 0 0 µ L of My O ne

Streptavidin C1 Dyna Beads ( Ther mo Fisher) were pre -was he d t wice wit h RI P A b uffer

( Si g ma), a n d t he n i nc u bate d wit h 2 7 5 µ g of pr otei n. Bea ds were r otate d wit h lysate f or t w o h o urs at r o o m te m perat ure. F oll o wi n g i nc u bati o n, beads were wash e d t wice wit h R I P A b uffer, o nce wit h 1 M K Cl, o nce wit h 0. 1 M Na 2 C O 3 , o nce wit h 2 M urea i n 1 0 m M Tris -H Cl, a n d t wice a gai n wit h RI P A b uffer. El uti o n was perf or me d by b oili n g t he bea ds f o ur ti mes i n

2 X Novex Tris -Glycine S DS sa mple buffer co ntai ni n g 2 m M bi ot i n ( Si g ma) a n d 2 0 m M

D T T ( Si g ma).

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Wester n bl otti n g: Sa mples were run under reducing and denaturing conditions on precast

4 -2 0 % Mi ni -PR OT E A N Tris -Glyci ne T G X gels ( Bi o -Ra d). F or i m m u n o preci pitati o n sa m ples, b ot h i n p ut a n d el uate sa m ples were r u n. F oll o wi n g se mi -dry tra n sfer t o nitr ocell ul ose me m bra nes, bl otti n g was perf or me d f or bi oti nylate d pr otei ns usi n g a LI -C O R stre pta vi di n c o u ple d a nti b o dy ( LI -C O R 926 -3 2 2 3 0). T otal pr otei n stai ni n g f or n or malizati o n was perfor med using the LI -C OR REV E R T stai ni n g kit. Me mbranes were i maged on a LI -

C O R Odyssey using I mage Studio v5.0 soft ware.

M ass spectro metry e x p eri m e nt al d esi g n: A d ult ra b bit car di o my ocytes were is olate d a n d cells were i nfecte d wit h o ne of t hree vir uses: a d G F P -AP E X2, adP D E5 -G F P -AP E X2 , or adP D E9 -G F P -A P E X 2. All sa m ple s were treate d wit h bi oti n -p he n ol ( Si g ma). Wit hi n eac h

vir us gr o u p, sa m ples were eit her wit h E T -1 ( 1 0 0 n M, Si g ma) or ve hicle a n d wit h H 2 O 2 ( 1 m M, Si g ma) or vehicle for a total of four sa mples per virus group. The experi ment was re peate d wit h n = 3 ra b bits f o r a t otal of t hree i n de pe n de nt e x peri me nts. Ta n de m mass ta g la bels were s h uffle d bet wee n sa m ples i n differe nt m ulti ple xes t o mi ni mize pe pti de la beli n g bias (see bel o w).

M ass spectro metry p e pti d e p re paratio n: F oll o wi n g sa m ple el uti o n fr o m s tre pta vi di n ma g netic bea ds, met ha n ol -chlorofor m extraction was perfor med for all sa mples according to the methods of Wessel and Flugge 1 5 0 . After e xtracti o n was c o m plete, sa m ples were resuspended in 9 M urea containing 10 m M Tris -H Cl, a n d s u bse q ue ntly dil ute d 1: 6 i n 6 0 m M

H E P E S p H 7. 5, a n d 0. 6 m M D T T, t o yiel d a fi nal di gesti o n b uffer c o ntai ni n g 5 0 m M

H E P E S, p H 7. 5 1. 5 M urea, a n d 0. 5 m M D T T. 1 µ g of try psi n/ Lys -C ( Pro mega V5073) was added to each sa mple, and peptide digest proceeded overnight at 25 °C (t o re d uce lysi ne

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car ba mylati o n) wit h s a m ples s ha ki n g at 1 0 5 0 R P M. T he ne xt day, a n ot her 0. 5 µ g of try psi n was a d de d al o n g wit h 5 m M D T T, a n d di gest c o nti n ue d f or a n ot her 2 h o urs. 2 0 m M i o d oaceta mi de was t he n a d de d t o al kylate t he sa m ples f or 1 h o ur i n t he dar k. 0. 5 % v / v T F A was a d de d t o aci dif y t he pe pti des, w hic h were t he n p urifie d o n C 1 8 s pi n c ol u m ns ( Pierce) acc or di n g t o ma n ufact urer’s i nstr ucti o ns a n d el ute d usi n g 0. 1 % f or mic aci d i n 6 0 % acetonitrile. Finally, sa mples were evaporated to dryness in an Eppendorf Vacufuge. Distille d dei o nize d H 2 O was added a fter t he first s pi n a n d sa m ples were s u bjecte d t o a sec o n d r o u n d of e va p orati o n t o re m o ve trace le vels of f or mic aci d a n d acet o nitrile.

P e pti d e la b eli n g a n d 2 -d i m e nsi o n al li q ui d c hro matography c o u pl e d t o ta n d e m m ass sp ectro metry (2 D -L C -M S / M S): Sa mples we re then resuspended in triethyla m moniu m bicar b o nate p H 8. 5. Pe pti des were la bele d wit h T M T (ta n de m mass ta g) rea ge nts

( T her m o Fis her), wit h la bels s h uffle d bet wee n sa m ples o n differe nt m ulti ple xes t o mi ni mize la beli n g bia s. T M T la beli n g was d o ne wit h sa m ple s br o u g ht u p i n 1 0 0 µ l T E A B a n d la bels br o u g ht u p i n 4 1 µ L a n hy dr o us acet o nitrile. Eac h la bel was a d de d t o its c orres p o n di n g sa mple for 1 hour before being quenched with 8 µ L of 5 % hy dr o xyl a mi ne f or 1 5 mi n utes. Sa mples were t he n mi xe d a n d drie d d o w n a n d s u bse q ue ntly br o u g ht u p i n 2 m L of

1 0 m M T E A B a n d i njecte d o nt o a n A gile nt L C r u n ni n g a 2. 1 x 1 0 0 m m Waters B E H C18 c ol u m n wit h 1. 7 µ M particles at 2 5 0 µ L / mi n ute. T he first 1 2 fracti o ns were fl o w -t hr o u g h, wit h t he ne xt 8 4 sa m ples c ollecte d o n a n 8 5 -mi n ute gra die nt fr o m 0 -9 0 % acet o nitrile c o ntai ni n g 1 0 m M T E A B. These 84 fractions were concatenated into 12 fractions for L C -

M S / M S a nalysis.

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L C -MS/ MS was perfor med on an Orbitrap Fusion Lu mos Tribrid Mass

Spectro meter ( Ther mo Fisher). Liquid chro matography was perfor med prior to MS/ MS by bri n gi n g sa m ples u p i n b uffer A ( 2 % acet o nitrile, 0. 1 % f or mic aci d 9 8 % dei o nize d water), a n d t he n i njecti n g t he sa m ple o nt o a tra p pi n g c ol u m n i n b uffer A f or 5 mi n utes at 5 µ L / mi n bef ore s witc hi n g t o t he a nalytica l c ol u m n ( Re pr o sil C 1 8 3 µ M particles wit h 1 0 0 a n gstr o m p ore size, pac ke d a p pr o xi mately 2 0 c m wit h 7 5 µ M i nter nal dia meter f use d silica Pic o Frits fr o m Ne w O bjecti ve ( W o b ur n, M A)) at 3 0 0 n L / mi n. T he s ol uti o n was t he n s witc he d t o

1 0 % b uffer B ( 9 0 % acet o nitri le/10 % deionize d water a n d 0. 1 % f or mic aci d). T he gra die nt t he n we nt fr o m 1 0 % t o 2 0 % b uffer B o ver 4 5 mi n utes a n d t he n t o 3 5 % at 7 7 mi n utes bef ore j u m pi n g t o 1 0 0 % b uffer B at 8 0 mi n utes, h ol di n g f or 2 mi n utes, t he n ret ur ni n g t o 2 % b uffer B i n a n ot her 2 mi n utes bef ore t he liquid chro matography run ended at 90 mi n utes. T he fracti o ns were i njecte d c o nsec uti vely f oll o wi n g t his gra die nt sc he me.

M S was c o n d ucte d wit h t he f oll o wi n g i nstr u me nt setti n gs. M S 1 res ol uti o n was set t o

1 2 0, 0 0 0 wit h a tar get i o n c o u nt of 4 0 0, 0 0 0. T he pr ec urs or i o n is olati o n wi dt h was 0. 7

Dalt o ns. F or M S 2, res ol uti o n was set t o 5 0, 0 0 0 wit h a tar get i o n c o u nt of 1 0 0, 0 0 0. C ollisi o n e ner gy was set t o 3 8. Easy I C i nter nal cali brati o n was use d o n t h e precursors which uses the

E T D i o n f or mass c orrecti o n o n -t h e-fly f or M S.

Pr ot ei n id e ntifi c ati o n: Pea k list files (. R A W) were searc he d a gai nst a ra b bit data base of predicted protein sequences ( N C BI RefSeq, taxono my: Oryctolagus cuniculus, 2018, F AS T A f o r mat, 38559 sequences; RefSeq83_ Oryctolagus_cu niculus_180413_ 1.fasta;), usi n g Masc ot

Version: 2.2.0 ( Matrix Science) interfaced through Proteo me Discoverer 1.4 ( Ther mo). Peaks were filtere d at a si g nal t o n oise rati o of 1. 5, deis ot o pe d, a n d searc he d wit h a pare nt i o n mass tolerance of 10 pp m and an MS 2 mass t olera nc e of 0. 0 2 Da. Try psi n was s pecifie d as t he

9 9

enzy me and 1 missed cleavage was allo wed. Cysteine carba mido methylation and N -ter mi nal a mi ne la beli n g wit h T M T 1 0 -plex reagent were specified as fixed m o dificati o ns f or t he database search. Dyna mic m o dificati o ns i ncl u de d N -pyr o gl uta mi ne, o xi dize d met hi o ni ne, p h os p h orylati o n of seri ne, t hre o ni ne, a n d tyr osi ne, a n d T M T la beli n g of lysi ne. All searc hes were conducted with the reversed database search mode en gaged. Percolator soft ware was use d f or pe pti d e F D R ( q -val ue) calc ulati o ns. Masc ot o ut p ut files (. dat) ta b ulate d were i n

Proteo me Discoverer. Of the high confidence peptides (q < 0.01), 2 % of unique spectra had intensities missing fro m one or more channels, i n dicati ve of efficie nt T M T la beli n g a n d fra g me ntati o n. S pec tra f or w hic h pe pti de c o -isolation interference was > 5 1 % were o mitte d fr o m a nalysis. Pr otei ns i de ntifie d by si n gle s pectra were li ke wise o mitte d.

Pr ot ei n q uantification by T M T and st atisti c al a n al ysis: T M T re p orter i o n i nte nsities were i nt egrated over 30 pp m using the most confident centroid method and corrected for p urity i n Pr ote o me Disc o verer 1. 4 ( T her m o). Missi n g la bele d c ha n nel i nte nsities ( < 2 % of s pect ra) were i n versely pr o p orti o nal t o t he u ntra nsf or me d me dia n i o n i nte nsities acr oss t he c ha n nels. F o r i n di vi d ual s pectra f or w hic h missi n g data was < 5 0 %, t he missi n g val ue was re place d by t he me dia n val ue of t he l o west i nte nsities i n eac h c ha n nel. F oll o wi n g missi n g val ue i m p utati o n , these signals were quantified using the median s weep alg orit h m ori gi nal ly descri be d by Her bric h et al 1 5 1 esse ntially as i m ple me nte d rece ntly by F oster et al 1 5 2 wit h a mi n or m o dificati o n. T M T re p orter i o n i nte nsities were 1) l o gari t h mically -tra nsfor med (base

2), 2) q ua ntile -n or malize d f or each channel , 3) me dia n -ce nter e d f or eac h i n di vi d ual s pectr u m across channels, 4) protein abundance deter mi ne d by ta ki n g t he me dia n val ue of t he l o garit h mically -transfor med median -ce ntere d i nte nsit ies f or all s pectra bel o n gi n g t o t hat pr otei n i n a gi ve n c ha n nel , a n d fi nally, 5) re -ce nter e d for eac h c ha n nel by s u btracti n g t he

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median protein abundance. Follo wing the median s weep, differential protein abundance bet ween experi mental groups was assessed by means of a n e m pirical Bayesia n met h o d, s pecifically, li near m o deli n g of micr oarrays ( LI M M A) 1 5 3, 1 5 4 wit h m ulti -group co mparison, as i m ple me nte d i n O mics E x pl orer ( Ql uc ore, L u n d, S we de n). Res ulti n g p -val ues were correcte d f or m ulti ple c o m paris o ns usi n g t he false disc o very rate ( q -val ue) met h o d of

Be nja mi ni -Hochberg 1 5 5 .

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T a bl e 4 .1 : Pri mers used for asse mbly of P D E5 and P D E9 GFP -A P E X2 fusion c o nstr ucts.

All se q ue nces are liste d i n t he 5’ à 3’ orie ntati o n.

Pri mer Sequence A m pli c o n Pl as mi d

F or war d: tata g ggagacccaagctggctagcAT G GAAC GA GC G G GCCCC P D E5 P D E5 -G F P -

Reverse: cgccgctgccgcc GTCCC GCTT GCCCT G GCT AP E X2

For war d: caagcgggacggcggcagcggcggcaccAT G GT GA GCAA G G GC GA G G F P - P D E5 -G F P -

Reverse: tttaaacgggccctctagactcgagTT A G GC ATC A GC A A ACCC A A G AP E X2 AP E X2

For w ar d: tataggga gacccaagctggctagcAT G G GATCC G GCTCCTCC P D E9 P D E9 -G F P -

Reverse: cgccgctgccgcc G GCACA GTCTCCTTCACT G AP E X2

For war d: agactgtgccggcggcagcggcggcaccAT G GT GA GCAA G G GC GA G G F P - P D E9 -G F P -

Reverse: tttaaacgggccctctagactcgagTT A G GC ATC A GC A A ACCC A A G AP E X2 AP E X2

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T a bl e 4 . 2 : Ge ne -specific pri mers used for sequencing of P D E5 and P D E9 G F P -

A P E X2 fusion constructs.

All se q ue nces are liste d i n t he 5’ à 3’ orie ntati o n.

Pri mer Sequence Ge ne

TCTTTTACTAGCTCCAAGAGCC Mouse P D E5

TCCCACCATTTCCTGATTTC Mouse P D E5

TGGGAT ATTAAGTGGCTCCA T C Mouse P D E5

CGGTAATTTTAAGGGTTTGGG Mouse P D E5

CAAGCACTGGTCAAAATGATG Mouse P D E5

AAAAGAACTTAACATGGAGCCG Mouse P D E5

TGGCATCTATGAACCCAACTT Mouse P D E5

TCCTGGTAGCAGCCCTGG Hu man P D E9

CTCAGCATCTCATTGGGCTC Hu man P D E9

ATCAGGTGGGATGTTGGAGA Hu man P D E9

T GCA GCA TGATCTCCTCAAC Hu man P D E9

TATATCATGGCCGACAAGCA G F P

CTGGTAGTGGTCGGCGAG G F P

CGCTAACAACGGTCTTGACA AP E X2

AGAAATCGGCGTAGCTCAAA AP E X2

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RES ULTS

Generation of the P D E -G F P -A P E X2 fusion constructs

D ue t o t he d ual f u ncti o ns of A P E X 2, we deci de d t o e m pl oy t hi s n o vel ta g i n or der to explore the cardiac nanodo mains of P D E5 and P D E9. Fusion constructs were generated in the pc D N A3.1 + backbone in which GFP and AP E X2 were fused to the C -ter mi n us of eit her P D E 5 or P D E 9. A s h ort li n ker was i ncl u de d bet we e n t he P D E a n d t he ta gs t o all o w f or se parati o n a n d fle xi bility bet wee n t he differe nt m oieties of t he f usi o n pr otei n (Fi g ur e

4 .1 A ). Follo wing co mpletion of the pc D N A3.1 + constructs, adenoviruses were made for further use in cardio myocyte studies, as expre ssi o n le vels ar e m uc h hi g her wit h a de n o viral transduction than plas mid transfection for both neonatal and adult cardio myocytes. The vir uses were s h o w n t o e x press well i n ne o natal car di o my ocytes, wit h G F P si g nal clearly visi ble u p o n li ve -cell e pifl u oresce nt i ma gi n g (Fi g ur e 4 .1 B ). Cell lysates were als o pr o be d by

Wester n bl otti n g, w hic h s h o we d all vir uses t o be e x pressi n g pr otei ns of t he e x pecte d m olec ular wei g hts (Fi g ur e 4 .1 C ).

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Fi g ur e 4 .1: P D E -G F P -A P E X 2 fusion constructs express in neonatal card i a c my ocytes.

A P DE5 -G F P -APEX2 C M V P DE5 G F P APEX2 ( 1 5 1 k D a)

P DE9 -G F P -APEX2 C M V P DE9 G F P APEX2 ( 1 2 1 k D a)

B adP DE5 -G F P -APEX2 adP DE9 -G F P -APEX2

C -APEX2 - adP DE5 -G F P -APEX2 - adP DE9 -G F P 160 k Da - G F P -125 k Da 125 k Da -

T ot al Pr otei n

F i g ur e 4 .1: P D E -G F P -A P E X2 fusion constructs express well in neonatal cardiac my ocytes.

A) Desi g n of t he f usi o n c o nstr ucts, wit h e x pecte d m olec ular wei g hts. B) Li ve cell G F P i maging of neonatal myocytes transduced with adP D E5 or adP D E -G F P -A P E X 2. C) S D S -

P A G E blotting for G F P in neonatal myocytes expressing either ad P D E5 or ad P D E9 -G F P -

A P E X 2. Cells were transduced with increasing a mounts of virus, and after 48 hours sa mples were c ollecte d a n d s u bjecte d t o S D S -P A G E and subsequent blotting for G F P t o i de ntify

1 0 5

f usi o n pr otei ns. B ot h a d P D E 5 -G F P -A P E X 2 (left) a n d a d P D E 9 -G F P -A P E X 2 (ri g ht) a p pear at t he c orr ect m olec ular wei g ht. N ota bly , as the a mount of virus increases s o d oes cell deat h, causing the decrease in fusion protein expression seen at higher a m o u nts of vir us.

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Fusion of the G F P -A P E X 2 m oi et y d o es n ot i n hi bit P D E -catalyzed c G M P hydrolysis

The pri mary function of phosphodiesterases is to catalyze hydrolysis of cyclic n ucle oti des i nt o t heir n o n -cyclic for ms. P D E5 and P D E9 pre fere ntially hy dr oly ze cyclic

G M P, generated either by soluble or receptor -coupled guanyl yl cyclases t hat are sti m ulate d by nitric o xi de a n d natri uretic pe pti de, res pecti vely. As t he G F P a n d A P E X 2 m oieties were f use d t o t he C -ter mi nal e n d ne xt t o t he cataly tic d o mai n of eac h P D E, it was critical t o e ns ure t hat cyclic n ucle oti de hy dr olysis was u ni m pe de d by t he a d diti o n of t hese relati vely b ul ky ta gs. I n or der t o deter mi ne w het her t he P D E f usi o n c o nstr ucts were f u ncti o nal, neonatal rat myocytes were transfecte d or tra ns d uce d wit h eit her a c o ntr ol G F P -A P E X 2 construct, or P D E5 or P D E9 -G F P -A P E X 2. Ce lls were subsequently sti mulated for 48 hours wit h a s ol u ble g ua nylyl cyclase acti vat or, B A Y 6 0 -2 7 7 0, a n d atrial natri uretic pe pti de ( A N P) t o ma xi mize c G M P le vels i n t h e cell. After t his, c G M P assays were perfor med to assess whether P D E overexpression redu ce d c G M P le vels. c G M P le vels were si g nifica ntly re d uce d by overexpression of P D E9, and border -line significantly reduced by P D E5 overexpression, but were unaffected by t he c o ntr ol G F P -A P E X 2, i n dicati n g t hat t he catalytic d o mai n of both P D E5 and P D E9 is una ffecte d by t he a d diti o n of t he G F P a n d A P E X 2 ta gs (Fi g ur e

4 .2 ).

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Fi g ur e 4 . 2: G F P -AP E X2 -tagging of P D E5 and P D E9 near t he catalytic do mai n d o es n ot i n hi bit c G M P h y dr ol ysis

200000 C o ntr ol E T 1 150000 E T 1 A/ B a d G A E T 1 A/ B

100000 adP5 GA ET1 A/B p = 0. 0 7 ** adP9 GA ET1 A/B 50000

f mol c G M P/total protein **** *** 0

Fi g ur e 4 .2: P D E5 and P D E9 tagged with GFP -A P E X2 retai n catalytic activity

Neonatal rat myocytes were transfected or transduced with a G F P -AP E X2, P D E5 -G F P -

AP E X2, or P D E9 -G F P -A P E X 2. Cells were t he n sti m ulate d wit h E T -1 ( 1 0 0 n M), atrial natri ur etic pe pti de ( A N P, 1 µ M), a n d a n s G C acti vat or ( B A Y 6 0 -2 7 7 0, 1 0 n M). A N P a n d

B A Y treat me n t was use d t o sti m ulate c G M P pr o d ucti o n by b ot h t he N P a n d N O pat h ways, and is denoted in the figure legend by “ A/ B.” c G M P levels were assayed by E LIS A, and nor malized t o t otal pr otei n. Dat a sho wn represent co mbined transfection and transduction sa m ples ( 2 i n de pe n de nt e x peri me nts), wit h a n n of 2 -4 per e x peri me nt, per gr o u p. Data is presented as mean ± S E M. Data were analyzed by Kruskal -Wallis test wit h p ost h oc D u n n’s m ulti ple c o m paris o n testi n g. ** p < 0. 0 1, *** p < 0. 0 0 1, ** ** p <0.0001 versus E T1 A/ B gr o u p. A b bre viati o ns: E T -1, e n d ot heli n -1; A / B, A N P / B A Y 6 0 -2770 ; G A, G F P -AP E X2 ;

P 5, P D E 5; P 9, P D E 9.

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P D E5 and P D E9 GFP -A P E X2 fusion co nstr ucts co -localize with their corresponding endogenous P D E in isolated neonatal and adult cardiac myocytes

C orrect cell ular s u bl ocalizati o n of t he f usi o n c o nstr u cts was critical f or t he proposed la beli n g a n d p ull d o w n e x peri me nts wit h A P E X 2, as wit h o u t t he c orrect l ocalizati o n, t he resulting proteo me would provide inaccurate infor mation. In order to assess whether the f usi o n c o nstr ucts l ocalize c orrectly wit hi n t he cell, i m m u n ofl u oresce nt stai ni n g a n d c o nf ocal i ma gi n g was e m pl oye d. Ne o natal rat or a d ul t ra b bit car di o my ocytes were is olate d a n d transduced with virus, and subsequently stained for either P D E5 or P D E9. Upon confocal i ma gi n g, c ol ocalizati o n was see n bet we en G F P ( marker for the f usi o n pr otei n) a n d t he appropriate P D E for both P D E5 and P D E9 in ne o natal a n d a d ult my ocytes (Fi g ur e 4 .3, A a n d B res pecti vely) .

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Figure 4.3: G F P- A P E X2 signal colocalizes with respective P D E in neonatal and adult

cardio myocytes

� �� � � � �� �� � � �� �� � ����� ����� � �� � �� ���� � � � �� ������� � � � � �� � � � ��� � � � �� � ��� � � � ��

� � � � � � � � � �� ����� ��� � � ��� � ���

Figure 4.3: P D E and G F P signal colocalize in myocytes expressing the G F P- A P E X2

f usio n protei ns

Cells were transduced with adenovirus expressing P D E5 or P D E9- GFP- AP E X2. After 48

hours, cells were fixed and stained for either P D E5 or P D E9. Confocal i maging was

perf or me d, wit h i ma ges pr ocesse d i n Fiji. Scale bars re prese nt 2 5 µ M. A) Ne o natal my ocytes

and B) adult myocytes expressing P D E5- GFP- AP E X2 (top of each panel) and P D E9- GFP-

A P E X 2 ( b ott o m of eac h pa nel).

� � � � �

Biotinylation by the P D E fusion cons tr ucts is i m prove d by t h e a d diti o n of N -a c et yl cysteine, and unaffected by cyclohexi mide

Initial test studies of the A P E X2 fusion constructs sho wed so me background

bi oti nylati o n i n t he a bse nce of H 2 O 2 , i n dicati n g basal acti vity of A P E X 2. We hy p ot hesize d that a d diti o n of a n a nti o x i da nt may l o wer bac k gr o u n d bi oti nylati o n by re d uci n g o xi dati o n a n d ge nerati o n of free ra dicals ( partic ularly t he bi oti n -p he n o xyl ra dical) i n t he cell. We therefore e mployed a co m mon antioxidant, N -acetyl cystei ne ( N A C), a n d c o m pare d o verall bi oti nylati o n le vels via Wester n bl ot f or t he rec o m me n de d bi oti nylati o n pr ot oc ol vers us t he a da pte d pr ot oc ol wit h t he a d diti o n of N A C. Wester n bl otti n g s h o we d a dra matically i ncrease d le vel of bi oti nylati o n wit h a d diti o n of N A C as c o m pare d t o t he p u blis he d pr ot oc ol. W h il e there was also increased background biotinylation in the absence of H 2 O 2 , the overall increase in a mount of biotinylated proteins out weighed this background increase

(Fi g ur e 4 .4 A a n d B , left si de).

We f urt her m ore wa nte d t o o pti mize t he bi oti nylati o n p r o tocol to reduce non - co mpart ment -s pecific bi oti nylati o n. D ue t o t he nat ure of pr otei n sy nt hesis a n d tra ns p ort wit hi n t he cell, tra ns p ort pr otei ns a n d ri b os o mal mac hi nery will i ne vita bly be la bele d by

A P E X2 as the fusion proteins ar e synthesized and transp or te d t o t heir fi nal cell ular l ocati o n.

We hy p ot hesize d t hat a d diti o n of cycl o he xi mi de ( C H X), a pr otei n sy nt hesis i n hi bit or, w o ul d re d uce t his n o n -specific labeling, hence giving cleaner and therefore more accurate mass spectro metry res ults. Wester n bl otti n g f or bi oti nylate d pr otei ns s h o we d t hat C H X ha d n o i m pact o n o verall a m o u nts of bi oti nylati o n, u nli ke t he a d diti o n of N A C, a n d t hat t his was t he sa me w het her C H X was e m pl oye d i n t he p u blis he d pr ot oc ol f or bi oti nylati o n, or t he m o di fie d pr ot oc ol wit h t he a d d iti o n of N A C ( Fi g ur e 4 .4 A a n d B , ri g ht si de).

1 1 1

Fi g ur e 4 . 4: N -acetylcysteine ( N A C) increases overall biotinylation by A P E X2, while cyclohexi mide has no i mpact

A B C H X - - - - + + + + C H X - - - - + + + + N A C - - + + - - + + N A C - - + + - - + + B P + + + + + + + + B P + + + + + + + +

H 2 O 2 - + - + - + - + H 2 O 2 - + - + - + - +

Stre ptavi di n Stre ptavi di n

T ot al T ot al Pr ot ei n Pr ot ei n

Fi g ur e 4 .4: N A C increases biotinylation by G F P -AP E X2 -tagged P D E5 a n d P D E 9.

Ne o natal rat my ocyte s were transduced with adP D E5 or adP D E9 -G F P -A P E X 2. After 4 8 h o urs, a m o difie d bi oti nylati o n pr ot oc ol was perf or me d i n w hic h cells were treate d wit h N - acetylcystei ne ( N A C, 5 0 0 µ M ) a n d / or cycl o he xi mi de ( C H X, 1 0 0 µ M ) t w o h o urs pri or t o

a d d iti o n of H 2 O 2 . Sa m pl es were then collected and run on an S DS -P A G E gel, a n d s u bse q ue ntly bl otte d f or stre pta vi di n (t o detect bi oti nylate d pr otei ns) a n d t otal pr otei n. A) adP D E5 -G F P -AP E X2 transduced cells and B) adP D E9 -G F P -A P E X2 transduced cells. Le ft of t he re d li ne o n eac h pa nel are sa m ples t hat were n ot treate d wit h C H X, w hile ri g ht of t he ri g ht li ne are sa m ples t hat were treate d wit h C H X. A b bre viati o ns: C H X, cycl o he xi mi de;

N A C, N -acetylcystei ne; B P, bi oti n p he n ol.

1 1 2

Bioti nylate d protei ns re mai n locally co nstrai ne d withi n a p pro priate cell ular nanodo mains

T he bi oti n -phenoxy l ra dical s generated by AP E X2 d uri n g t he bi oti nylati o n pr ot oc ol ha ve a s h ort half -life, he nce li miti n g t he la beli n g ra di us t o a p pr o xi mately 2 0 n m fr o m t he f usi o n pr otei n. I n or der t o deter mi ne w het her t he bi oti nylati o n was i n dee d re mai ni n g l ocally c o nstrai ne d wit hi n the P D E co mpart men t, transduced adult rabbit cardio myocytes were stai ne d f or bi oti nylate d pr otei ns wit h a n Ale xa fl u or -stre pta vi di n f usi o n a nti b o dy, a n d subsequently i maged via confocal microscopy. Colocalization was seen bet ween G F P

( mar ki n g t h e f usi o n pr otei n) a n d bi oti ny late d pr otei ns f or b ot h P D E 5 a n d P D E 9 ( Fi g ur e

4 .5 ). T his i n dicates t hat bi oti nylati o n is re mai ni n g wit hi n t he c o m part me nt l ocal t o t he f usi o n pr otei n, w hic h is critical f or acc urately i de ntifyi n g resi de nt pr otei ns of t hese c o m part me nts.

1 1 3

Fi g ur e 4 .5: Biotinylated proteins re main constrained to the local P D E co mpart ments in adult cardio myocytes

A Bi oti n G F P D A PI M erge Zoo m B P APEX2 - G F P - 2 O 2 adP DE5 BP+ H

B Bi oti n G F P D A PI M erge Zoo m B P APEX2 - G F P - 2 2 O O 2 2 adP DE9 BP+ H BP+ H

Fi g ur e 4 .5: Bi oti n yl at e d pr ot ei ns c ol o c ali z e wit h t h e G F P -AP E X2 -tagged P D Es

Adult rabbit cardio myocytes were transduced w it h eit her A) P D E 5 -G F P -A P E X 2 or B)

P D E9 -G F P -A P E X 2. 4 8 h o urs later, bi oti nylati o n was perf or me d a n d cells were fi xe d a n d stai ne d f or bi oti nylate d pr otei ns usi n g a n Ale xa Fl u or -ta g ge d stre pta vi di n. Cells were i ma ge d by c o nf ocal, a n d i ma ges pr ocesse d i n Fiji. A b bre viati o ns: B P, bi oti n -p he n ol.

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M a ss s pectro metry of bioti nyla ted proteins for both P D E5 and P D E9 -A P E X2 fusion co nstr ucts i de ntifies fe w protei ns

Rabbit myocytes were transduced with either ad G F P -AP E X2 or adP D E5 or P D E9 -

G F P -A P E X 2, a n d s u bjecte d t o t he m o difie d bi oti nylati o n pr ot oc ol i ncl u di n g N A C a n d

C H X. I m munopre ci pitati o n was perf or me d t o is olate bi oti nylate d pr otei ns, w hic h t he n u n der we nt p urificati o n, di gesti o n, la beli n g, a n d fracti o nati o n f or L C -M S / M S. Statistical a nalysis of t he s u bse q ue nt i de ntifie d pe pti des t hat were of hi g h -confidence was perfor med in

w hic h a sa m ple wit h bi oti n p he n ol a n d H 2 O 2 was c o m pare d t o a sa m ple wit h o nly bi oti n p he n ol (i.e. a d P D E 5 -G F P -AP E X2 +biotin phenol + H 2 O 2 versus adP D E5 -G F P -AP E X2 + bi oti n p he n ol). T his a nalysis i de ntifie d fe w pe pti des as si g nifica nt a n d ha d a hi g h false disc overy r ate, i n dicati n g l o w c o nfi de nce i n t he fi n di n gs. T his was tr ue f or b ot h P D E 5 a n d

P D E 9 res ults, wit h 2 4 pr otei ns i de ntifie d f or P D E 5 ( T a bl e 4 . 3 ) a n d 5 7 pr otei ns i de ntifie d f or P D E 9 ( T a bl e 4 . 4 ), b ot h at p < 0. 1. Pat h ways a nalys is f or pr otei ns i de ntifie d wit h P D E 5 -

G F P -A P E X 2 f o u n d car b o n meta b olis m, a nti bi otic sy nt hesis, a n d glyci ne, seri ne, a n d threonine metabolis m path ways were enriched ( T a bl e 4 . 5 ). F or P D E 9 -G F P -A P E X 2, mitochondrial path ways were expected to be enriched, b ut we re n ot ( T a bl e 4 . 6 ).

Furt her a nalys is was perf or me d t o deter mi ne p ote ntial reas o ns f or t he l o w

c o nfi de nce fi n di n gs. I n o ne a nalysis, t he relati ve effect size of H 2 O 2 was e xa mi ne d t o deter mi ne if t he e x pecte d effects (i.e. i ncrease d bi oti nylati o n) wer e act ually occ urri n g i n t he

sa m ple set (F i g ur e 4 .6 A -C ). These analyses sho wed that H 2 O 2 was ha vi n g a s maller effect size t ha n a ntici pate d, alt h o u g h it was see n t o ha ve a greater effect i n t he P D E 5 a n d P D E 9 data sets t ha n t he c o ntr ol G F P -A P E X 2 data set ( mea n F rati os of 1. 2 2, 1. 3 7, a n d 0. 9 6 f or

P D E 5, P D E 9, a n d G F P -A P E X 2 c o ntr ol res pecti vely). H o we ver, e xa mi nati o n of t he sa mples sent for mass spectro metry by Western blotting sho wed adequate increases in

1 1 5

bi oti nylati o n bet wee n bi oti n p he n ol a n d bi oti n p he n ol wi t h H 2 O 2 gr o u ps, s o it is u ncertai n w hy t h is effect appeared mini mal after analysis of the mass spec data. E T -1 treat me nt was s h o w n t o ha ve t he lar gest effect size f or all data sets (Fi g ur e 4 .6 A -C ). A d diti o nal a nalyses of t he data set e xa mi ne d sa m ple cl usteri n g, w hic h i n dicate d t hat sa m ples were f or mi n g u ni q ue clusters. So me sa mple groups clustered together, such as ad P D E9 -G F P -A P E X 2 + bi oti n

phenol + H 2 O 2 and adP D E9 -G F P -APEX2+ET -1 + bi oti n p he n ol (Fi g ur e 4 .6 D a n d E ).

1 1 6 !

Figure 4.6: Bet ween-group analysis sho ws H 2 O 2 does not co ntri b ute to varia nce as

much as expected

ad G A adP5 GA adP9 GA � 1. 5 � 2. 5 � 2. 0

2. 0 1. 5 1. 0 1. 5 1. 0 1. 0 0. 5 M e a n F R ati o M e a n F R ati o M e a n 0. F R 5 ati o 0. 5

1. 4 1 0. 9 6 1. 0 0 2. 1 8 1. 2 2 1. 0 0 1. 7 3 1. 3 7 1. 0 0 0. 0 0. 0 0. 0 E T- 1 H 2 O 2 Err or E T- 1 H 2 O 2 Err or E T- 1 H 2 O 2 Err or � � � �

� �� �� � � � � � � ��� �� � �

� � ��� �� � �� �� � � � �

Fi g ur e 4. 6: H 2 O 2 is not the pri mary contributor to variance bet ween groups

Mass spec sa mple groups were analyzed using LI M M A for factors contributing to variance,

which sho wed that E T-1 was the pri mary contributor to variance bet ween sa mple groups,

w hile H 2 O 2 ha d a relati vely m o dest effect. T his was tr ue f or A) G F P- A P E X 2, B) P D E 5-

GFP- AP E X2, C) P D E9- GFP- AP E X2, although GFP- AP E X2 sho wed no i mpact of H 2 O 2 .

Sa mples were also analyzed by principle co mponents analysis, which sho wed sa mples within

groups were generally clustered together for both D) P D E5- GFP- AP E X2 and E) P D E9-

GFP- AP E X2. N =3 per sa mple group for panels D and E. Abbreviations: E T-1, endothelin-

1; B P, bi oti n p he n ol.

� � � � �

Co mparison of P D E9 -F L A G co -i m munoprecipitation data to P D E9 -G F P -A P E X 2 mass spectro metry data sho ws little overlap

We next wanted to deter mine whether or not the A P E X2 mass spectro metry data w o ul d o verla p wit h o ur pre vi o usly c ollecte d c o -i m munoprecipitation data for P D E 9. T he c o -i m munoprecipitation data sho wed P D E9 loca lizes at t he mit oc h o n dria -sarcoplas mic retic ul u m i nterface, p ulli n g d o w n ma ny mit oc h o n drial e ner getics pr otei ns as well as s o me S R pr otei ns ( C ha pter 2 o f t his t hesis). W he n t his data set was c o m pare d t o t h e A P E X 2 data set, o nly 6 pr otei ns were f o u n d t hat o verlapped bet ween the t wo, one of which was P D E9

(Fi g ur e 4 .7 ). The re maining five proteins were C O X4, EIF4 A1, MI C10, T L N1, and

V D A C1. Three of these pr otei ns ( C O X 4, MI C 1 0, a n d V D A C 1) are mit oc h o n drial, w hi le

EI F 4 A 1 is i n v ol ve d i n i nitiati o n of pr otei n tra n slati o n, a n d T L N 1 is part of t he c osta mere i n cardio myocytes.

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Fi g ur e 4 .7: Co mparison of P D E9 AP E X2 data to P D E9 co -i m munoprecipitation dat a s h o ws littl e o v erl a p

C o -I P APEX2

1 4 9 6 5 1

p < 0. 0 5 p < 0. 1 F D R = 3 % F D R = 8 9 %

Fi g ur e 4 .7: Mi ni m al overlap bet ween A P E X2 and co -I P data sets for P D E9

After dat a a nalysis of t he P D E 9 -G F P -A P E X 2 data set, si g nifica nt pr otei ns ( p < 0. 1) were co mpared to significant proteins fro m the P D E9 -FL A G co -i m m u n o preci pitati o n e x peri me nt ( p < 0. 0 5). O nly 6 pr otei ns o verla p pe d bet wee n t he data sets, i n dicati n g little congruence bet w een experi mental approaches. A b bre viati o ns: F D R, false disc o very rate.

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DISC USSI O N

T his st u dy is t he first ti me t hat a pr o xi mity pr ote o mics a p pr oac h has bee n teste d t o exa mine P D E co mp art me nts. W hile t his i dea still h ol ds merit a n d c o ul d be hi g hly infor mative, modifications are clearly need e d. A P E X 2 -ta g ge d P D E 5 a n d P D E 9 i nitially l o o ke d pr o misi n g, as t hey a p pear t o acti vely de gra de c G M P, l ocalize c orrectly, a n d bi oti nylate well. H o we ver , t he mass s pectr o metry data di d n ot pr o vi de m u c h i nsi g ht, wit h relati vely fe w pr otei ns i de ntifie d as si g ni fica nt a n d a hi g h false disc o very rate a m o n gst t hese fe w. F urt her m ore, a m o n g t he pr otei ns i de ntifie d i n t he P D E 9 -G F P -A P E X 2 data set, o nly si x o verla p pe d wit h t he P D E 9 -FL A G co -i m m u n o preci pitati o n d ata set. T he reas o ns f or these results are unkno wn, as sa mpl es s h o we d a de q uate i ncreases i n bi oti nylati o n by Wester n bl otti n g. It is p ossi ble t hat t he pr ot oc ol m o dificati o ns t hat we i ntr o d uce d (t he a d diti o n of

N A C and C H X) may have caused unforeseen diffic ulties wit h mass s pec, b ut t his is u n k n o w n wit h o ut f urt her t esti n g. Sa m ple pr ocessi n g a n d data a nalysis pr ocee de d i de ntically to the workflo w used for the P D E9 -FL A G co -i m m u n o preci pitati o n e x peri me nt, w hic h yie l de d hi g h q uality data wit h a l o w false disc o ve ry rate, s o t hese are n ot t he li kely c ul prits. If

AP E X2 were t o be teste d a gai n f or st u dies of P D E c o m part me ntati o n, o ne m o dificati o n t hat c o ul d be ma de is t o utilize a m ore li pi d -s ol u ble f or m of H 2 O 2 s uc h as tert -b utyl per o xi de t o i ncrease t he a m o u nt of p er o xi de wit hi n t he cell t hat ca n acti vate bi oti nylati o n, as

1 6 8 me mbrane per meability of H 2 O 2 is li mite d . We would also reco m mend searching the data f or t he s pecific bi oti nylati o n m o dificati o ns i n or der t o get m ore i nf or mati ve res ults. T his process is currently under way for the current data set, and may al ter t he res ults prese nte d here.

F or a ny future st u dies usi n g pr o xi mity la beli n g tec h ni q ues t o e xa mi ne P D E c o m part me ntati o n, we w o ul d rec o m me n d t hat a differe nt ta g be use d – f or e xa m ple,

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TurboI D or mini Turbo, as these do not require the high dose of H 2 O 2 f or acti vati o n a n d s u bse q ue nt la beli n g, yet sti ll ha ve relati vely s h ort la beli n g ti mes. A d diti o nally, t he m o difie d bi oti nylati o n pr ot oc ol use d here i n w hic h N A C was i ncl u de d w o ul d n o l o n ger be nee de d,

si nce H 2 O 2 is n ot use d f or eit her of t hese bi oti n li gas es. Alt h o u g h t he a bility t o perf or m electr o n mi cr osc o py st u dies is l ost wit h t hese ta gs, t he pr o xi mity pr ot e o mics w or kfl o w is si m pler. We w o ul d als o rec o m me n d a d di n g cycl o he xi mi de t o t he sa m ples (si milar t o w hat was d o ne here). By a d di n g cycl o he xi mi de, la b eling of proteins near ne wly synthesized fusion pr otei ns (eit her at t he ri b os o me as it’s bei n g sy nt hesize d, or i n vesicles as it’s bei n g tra ns p orte d) is a v oi de d, pr o vi di n g clea ner mass s pec data sets. H o we ver, it is u n k n o w n whether cyclohexi mide would sign ifica ntly alter t he catalytic efficie ncy of ta g s ot her t ha n

A P E X 2, s o t his w o ul d nee d t o be teste d.

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T a bl e 4 . 3: Si g nifi c a nt pr ot ei ns i n P D E 5 -G F P -A P E X 2 a n al ysis.

Mass spectro metry data was analyzed by LI M M A, co mparing biotin -p he n ol t o bi oti n -p he n ol wit h H 2 O 2 sa m ples. All pr otei ns wit h a p < 0. 1 are s h o w n, alt h o u g h t he hi g hest p val ue was

0. 0 4 6 4.

F ol d C h a n g e G e n e Na me Protein Na me wit h H 2 O 2 P V al u e A N XA1 A n ne xi n A 1 2. 4 1 0. 0 0 1 7 DP YSL2 Dihydropyri midinase -relate d pr otei n 2 1. 8 3 0. 0 0 6 0 Di hy dr oli p oyllysi ne -resi d ue succinyltransferase co mponent of 2 - oxoglutarate dehydrogenase co mplex, D LS T mit oc h o n drial 1. 6 3 0. 0 0 8 9 P GA M2 Phosphoglycerate mutase 2 1. 3 9 0. 0 0 9 5 F U S R N A -bi n di n g pr otei n F U S 1. 7 0 0. 0 0 9 9 R ABEP1 Ra b G T Pase -bi n di n g effect or pr otei n 1 3. 0 3 0. 0 1 1 3 DI R AS2 G T P -bi n di n g pr otei n Di -Ras 2 3. 0 4 0. 0 1 6 9 P PI A Pe pti dyl -pr olyl cis -trans iso merase A 1. 8 8 0. 0 2 3 0 CT N NA1 Cate ni n al p ha -1 1. 7 7 0. 0 2 6 5 RS U1 Ras suppressor protein 1 1. 5 0 0. 0 2 6 7 A K 2 A de nylate ki nase 2, mit oc h o n drial 1. 5 7 0. 0 2 7 4 RT CB tR NA -s plici n g li gase Rtc B h o m ol o g 1. 6 1 0. 0 2 7 5 C F L1 C ofili n -1 1. 9 5 0. 0 2 8 2 A N XA2 A n ne xi n A 2 1. 7 2 0. 0 3 1 6 S EP T10 Se pti n -1 0 1. 5 0 0. 0 3 4 4 26S proteaso me non -A T Pase regulatory PS M D4 s u b u nit 4 1. 5 1 0. 0 3 5 0 M S N M oesi n 1. 4 6 0. 0 3 5 5 TL N1 Tali n -1 1. 5 4 0. 0 3 5 5 C D C 3 7 Hsp90 co -chaperone Cdc37 1. 8 3 0. 0 3 9 0 P GA M1 Phosphoglycerate mutase 1 1. 2 9 0. 0 3 9 6 E S D S -f or myl gl utat h ione hydrolase 1. 4 1 0. 0 4 4 4 C K AP4 Cyt os kelet o n -ass ociate d pr otei n 4, partial 1. 2 6 0. 0 4 4 7 T F G Pr otei n T F G 2. 4 5 0. 0 4 5 2 L NPEP Le ucyl -cysti nyl a mi n o p e pti dase 1. 6 1 0. 0 4 6 4

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T a bl e 4 . 4 : Si g nifi c a nt pr ot ei ns i n P D E 9 -G F P -A P E X 2 a n al ysis.

Mass spectro metry data was analyzed by LI M M A, c o m pari n g bi oti n -p he n ol t o bi oti n -p he n ol wit h H 2 O 2 sa m ples. R o ws hi g hli g hte d i n gray i n dicate pr otei ns t hat o verla p pe d wit h t he

P D E9 -FL A G co -i m m u n o preci pitati o n data set. All pr otei ns wit h p < 0. 1 are s h o w n.

Gene Na me Protein Na me F ol d Change with H2 O2 P V al u e SL C25 A1 Tricar b oxylate tra ns p ort 1. 5 0 0. 0 0 6 4 pr otei n, mit oc h o n drial UBE2 M NE D D8 -conjugating enzy me 1. 4 0 0. 0 1 3 1 U bc 1 2 EI F4 A1 E u kary otic i nitiati o n factor 1. 4 0 0. 01 8 2 4 A -I KP N A3 I m p orti n s u b u nit al p ha -4 1. 5 1 0. 0 2 3 3 C YFIP1 C yt o plas mic F MR1 -i nteracti n g 1. 6 6 0. 0 2 3 5 pr otei n 1 ATP AF1 A T P sy nt hase mit oc h o n drial F 1 1. 5 8 0. 0 3 1 7 co mplex asse mbly factor 1 S DPR Ser u m de pri vati o n -response 1. 9 2 0. 0 3 5 8 pr otei n A KR7 A3 Aflatoxin B1 aldehyde reductase 1. 3 6 0. 0 3 7 4 me m ber 3 A KR1C5 Pr osta gla n di n -E( 2) 9 -reductase - 1. 6 3 0. 0 3 8 0 li ke H NR NPAB Heterogeneous nuclear 1. 5 2 0. 0 4 3 6 ribonucleoprotein A/ B T ME M126A Trans me mbrane protein 126 A 1 . 3 4 0. 0 4 4 1 M S N M oesi n 1. 5 8 0. 0 4 5 1 N A RS As paragi ne -- t R N A li gase, 1. 5 6 0. 0 4 5 5 cyt o plas mic L2H G DH L -2 -hy dr o xy gl utarate 1. 2 9 0. 0 4 8 6 dehydrogenase, mitochondrial FL NB Fila mi n -B 1. 3 8 0. 0 4 9 4 GST M3 Gl utat hi o ne S -transferase Mu 3 1. 6 5 0. 0 5 1 3 AL D H1A1 Reti na l dehydrogenase 1 1. 7 8 0. 0 5 3 2 M RPS10 2 8 S ri b os o mal pr otei n S 1 0, 1. 3 1 0. 0 5 7 4 mit oc h o n drial GSP T1 Eukary otic pe pti de c hai n release 1. 3 6 0. 0 5 7 5 factor G TP -bi n di n g s u b u nit E RF3 A T HY N1 Thy mocyte nuclear protein 1 1. 5 1 0. 0 5 9 6 MI C OS10 MI C OS co mplex subunit 1. 2 8 0. 0 6 0 0 M I C1 0

1 2 3

D2H G DH D -2 -hydr oxyglutarate 1. 5 4 0. 0 6 0 7 dehydrogenase, mitochondrial CA M K2B Calci u m /cal m o d uli n -dependent 1. 2 6 0. 0 6 2 7 pr otei n ki nase ty pe II s u b u nit beta C O X4 Cytochro me c oxidase 3. 91 0. 0 6 3 4 s u b u nit 4 is of or m 1, mitoc ho n drial prec ursor F A M120 A C o nstit uti ve c oacti vat or of 1. 6 3 0. 0 6 4 8 PP A R -ga m ma -li ke pr otei n 1 I D E I ns uli n -degrading enzy me 1. 3 9 0. 0 6 5 4 EIF5 A2 E u kary otic tra nslati o n i nitiati o n 1. 4 4 0. 0 6 5 5 fact or 5 A -2 G N AI2 G ua ni ne n ucle oti de -bi n di n g 1. 6 7 0. 0 6 8 2 pr otei n G(i) s u b u nit al p ha -2 C APRI N1 Ca pri n -1 1. 6 0 0. 0 7 2 0 T L N1 T ali n -1 1. 4 0 0. 0 7 6 7 PR MT1 Pr otei n ar gi ni ne N - 1. 3 1 0. 0 7 6 9 met hyltra nsferas e 1 PT GES2 Pr osta gla n di n E sy nt hase 2 1. 3 0 0. 0 7 7 1 R PS12 4 0 S ri b os o mal pr otei n S 1 2 1. 2 2 0. 0 7 7 9 PR KAA2 5' -A M P -acti vate d pr otei n ki n ase 1. 3 6 0. 0 7 8 2 catalytic s u b u nit al p ha -2 AR H GAP1 Rho G TPase -acti vati n g pr otei n 1. 2 4 0. 0 7 9 8 1 W A RS Try pt o p ha n -- t R N A li gase, 1. 2 6 0. 0 8 0 3 cyt o plas mic HS D17B10 3 -hydroxyacyl -C o A 1. 3 8 0. 0 8 1 6 dehydrogenase type -2 RPL10 A 6 0 S ri b os o mal pr otei n L 1 0a 1. 3 3 0. 0842 NF KB2 Nuclear factor N F -ka p pa -B 1. 4 9 0. 0 8 4 8 p 1 0 0 s u b u nit D H X15 Pre -mR NA -s plici n g fact or 1. 3 3 0. 0 8 7 8 A T P -dependent R N A helicase D H X 1 5 UBE2 O ( E 3 -i n de pe n de nt) E 2 u bi q uiti n - 1. 4 4 0. 0 8 9 3 conjugating enzy me G N AI1 G ua ni ne n ucle oti de -bi n di n g 1. 5 4 0. 0 9 0 1 pr otei n G(i ) s u b u nit al p ha -1 FA M98A Pr otei n F A M 9 8 A 1. 5 8 0. 0 9 0 2 M RPL16 3 9 S ri b os o mal pr otei n L 1 6, 1. 4 3 0. 0 9 1 2 mit oc h o n drial C F L1 C ofili n -1 1. 3 5 0. 0 9 1 3 ST AT3 Si g nal tra ns d ucer a n d acti vat or 1. 2 8 0. 0 9 1 5 of tra nscri pti o n 3

1 2 4

GF M1 El o n gati o n fact or G, 1. 2 5 0. 0 9 2 1 mit oc h o n drial C B R1 Carbonyl reductase [ N A DP H] 1 1. 3 4 0. 0 9 3 9 MI C26 MI C OS co mplex subunit 1. 3 0 0. 0 9 5 6 MI C26 P D E9A Hi g h affi nit y c G M P -s p e cifi c 1. 8 8 0. 0 9 6 6 3', 5' -c y cli c phosphodiesterase 9 A DP YSL2 Dihydropyri midinase -relate d 1. 3 0 0. 0 9 6 6 pr otei n 2 M P P1 55 k Da erythrocyte me mbrane 1. 9 3 0. 0 9 6 7 pr otei n V DA C1 V olta ge -dependent anion - 1. 2 6 0. 0 9 7 2 selective channe l pr ot ei n 1 EI F3 B E u kary otic tra nslati o n i nitiati o n 1. 2 5 0. 0 9 7 3 fact or 3 s u b u nit B RS U1 Ras suppressor protein 1 1. 5 6 0. 0 9 7 5 C D C4 2 Cell di visi o n c o ntr ol pr otei n 4 2 1. 6 2 0. 0 9 8 8 ho molog DCT N3 Dy nacti n s u b u nit 3 1. 4 1 0. 0 9 9 1

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T a bl e 4 . 5 : Path ways analysis for significant proteins fro m P D E5 -G F P -A P E X 2 a n al ysis .

Proteins fro m Table 1 were subjected to K E G G path ways analysis utilizi n g t he D A VI D data base.

F ol d Ter m P V al u e Ge nes Enrich ment Carbon metabolis m 0. 0 0 2 1 D L S T, E S D, P G A M 1, P G A M2 1 4. 1 6 Bi osy nt hesis of a nti bi otics 0. 0 1 2 7 DLST, P G A M1, P G A M2, A K2 7. 4 8 Glyci ne, seri ne a n d threonine metabolis m 0. 0 9 7 7 P GA M1, P GA M2 1 8. 3 8

1 2 6

T a bl e 4 . 6 : Pat h ways a nalysis fo r significant proteins fro m P D E9 -G F P -A P E X 2 a n al ysis .

Pr otei ns f r o m Ta ble 2 were s u bjecte d t o K E G G pat h ways a nalysis utilizi n g t he D A VI D data base.

F ol d Ter m P V al u e Ge nes Enrich ment C D C42 , G N AI 2, G N AI 1, C F L 1, Axon guidance 0. 0 0 2 5 DP YSL2 8. 3 9 C D C 4 2, C A M K 2 B, M S N, F L N B, Proteoglycans in cancer 0. 0 1 3 3 ST AT3 5. 2 2 Leukocy te tra nse n d ot helial mi grati o n 0. 0 1 5 4 C DC42, G N AI2, G N AI1, MS N 7. 3 6 O xyt oci n si g nali n g G N AI2, G N AI1, C A M K2B, path way 0. 0 3 1 5 PR KAA2 5. 5 9 Che mokin e si g nali n g path way 0. 0 4 1 3 C DC42, G N AI2, G N AI1, ST AT3 5. 0 2 Pert ussis 0. 0 4 4 6 G N AI2, G N AI1, CFL1 8. 6 2 Gastric a ci d secreti o n 0. 0 4 5 7 G N AI2, G N AI1, C A M K2B 8. 5 0 Circa dia n e ntrai n me nt 0. 0 6 1 0 G N AI2, G N AI1, C A M K2B 7. 2 3 Re g ulati o n of acti n cyt os kelet o n 0. 0 7 0 6 C D C42, CFL1, C YFIP1, MS N 4. 0 3 Melanogenesis 0. 0 7 1 1 G N AI2, G N AI1, C A M K2B 6. 6 2 Gl uca g o n si g nali n g path way 0. 0 7 3 8 PR MT1, CA M K2B, PR KAA2 6. 4 9 Cholinergic synapse 0. 0 7 3 8 G N AI2, G N AI1, C A M K2B 6. 4 9 M RPL16, M RPS10, RPS12, Riboso me 0. 0 7 3 8 RPL10 A 3. 9 6 Ra p1 sig na li n g pat h way 0. 0 8 3 1 C DC42, TL N1, G N AI2, G N AI1 3. 7 6

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C H AP T E R 5: General Discussion and Conclusions

I n t hi s dissertati o n, I st u die d t he c o m part me ntati o n of P D E 5 a n d P D E 9 i n t he heart . First, I e xa mi n e d tra nscri pt o mic re g ulati o n i n t he heart u nder pre ss ure -overl oa d stress by eac h of t hese t wo different c G MP -selecti ve P D Es , testi n g t heir differe ntial i m pact o n both overall gene expression and on micro R N A expression. I th e n e xa mi ne d t he l ocalize d nanodo mains i n isolated cardiac myocytes usi n g c o -i m munoprecipitation a n d bi oti nylati o n strate gies c o u ple d wit h mass spectro metry .

I n c ha pter t w o , I teste d t he hy p ot hesis t hat i n hi biti o n of eit her P D E 5 or P D E 9 i n a mouse pressure -overload model of heart failure would lead to mi R N A and m R N A changes t hat were s pecific t o na n o d o mai n c o ntr ol by P D E 5 a n d P D E 9. I n t his st u dy, I de monstrated t hat des pit e si milar p he n oty pic i m pr o ve me nt f oll o wi n g P D E 5 a n d P D E 9 i n hi biti o n i n t he mouse pressure overload model, there is a virt ual ly bi nary differe nce i n t heir i m pact o n mi R N A e x pressi o n. P D E 5 i n hi biti o n br oa dly do wnregulate d mi R N As, re ver si n g m ost t hat are enhanced by pressure -overload stress and lo wering many others that were not the mselves increased by the disease stress. By c o ntrast, P D E 9 i n hi biti o n alter e d al m ost n o mi R N As when co mpared to the pressure -o verl oa d heart treate d wit h ve hicle. Des pite t his stri ki n g differe nce, R N A -se q a nalysis of m R N A ex pressi o n revealed b ot h treat me nts tar gete d many genes that con verged o n si milar si g nali n g p ath ways . H o we ver, t he ge nes t he mselves were largely differe nt, further revealing underlying dispar ities i n t he si g nali n g i mpact fro m t he t w o differe nt met h o ds t o sti m ulate t he c G M P / P K G si g nal os o me. W it h res pect t o mi R N A expression , I f o u n d t he phenotype arises at t he le vel of mat ure mi R N As, as earlier f or ms of mi R N As ge nerate d wit hi n t he n ucle us were si milar bet wee n P D E - i n h ibit or treat ment groups. T h e difference in processing was not ascribable to changes in

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expression of mi R N A processing genes , t h o u g h other mechanis ms including post - tra nsl ati o nal c ha n ges i n t heir acti vity re mai n t o be deter mi ne d . Further more, by de mons trati n g t hat t he stri ki n g car diac mi R N A pr ofile change foll o wi n g P D E 5 i n hi biti o n was both organ and disease s pecific, it is u nli kely t hat its i m pact re q uires a ge neral alterat i o n of funda mental mi R N A processing . I f urt her s h o we d t hat tar geti n g c G MP/P K G ac ti vati o n e ve n wit hi n t he sa me pat h way - N O -s G C -P D E 5 – b ut by differe nt mea ns d oes n ot tra nsl ate t o i de ntical c ha n ges i n mi R N A expression . S pec ifically, acti vati n g s G C res ults i n do wnregulation of mi R N As that were increased by p ress ure -o verl oa d stress ( pr o - hypertrophic ), si milar t o c ha n ges wit h P D E 5 i n hi biti o n . By c o ntrast, mi R N As t ha t decli ne d wit h press ure -o verl oa d (a nti -hy pe rtr o p h ic) were f urt her re d uce d by P D E 5 i n hi biti o n, b ut broadly unchanged by s G C sti mulation. T his s peaks furt h er t o t he critical r ole of co mpart me ntalizati o n e ve n of differe nt co mponents wit hi n the sa me signaling path way , a n d h o w t his ca n alter do wnstrea m tra nscri pti o nal c ha nges. T he res ults s u g gest t hat usi n g mi R N As as a bio marker for phenotypic i mprove ment follo wing t he ra py , as teste d here f or pat h ol o gical car diac hypertr o p h y, ca n be mislea di n g, as I de m o nstrate t hat se veral differe nt treat me nts t hat c o nfer near ly i de ntical p he n oty pic i mprove ment of t he disease d heart d o so wit h dra matically different mi R N A expression profiles.

I n c ha pter t hree , I defi ne d the nanodo main of P D E9 in cardiac myocytes by co - i m m u n o preci pitati o n a n d mass s pectr o metry t o i de ntify bi n di n g p artners of P D E9 under b ot h n or mal a n d hy pertr o p hic c o n diti o ns. I d eter mi ne d t hat P D E 9 resi des pri marily at t he mit oc h o n dria, a n d t hat it als o i nteracts wit h pr otei ns i n t h e sarc o plas mic retic ul u m. T his li kely pl aces P D E 9 at t he d ya d ic cleft, where the S R and mitochondrial me mbranes ar e p hysically cl ose. T his re gi o n plays a key r ole i n calci u m -sensing and A TP ho meosta sis .

Pat h ways a nalysis of s i g nifica nt pr otei ns i de ntifie d meta b olic pat h ways ass ociate d wit h

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mit oc h o n dria s uc h as o xi dati ve p h os p h orylati o n, citric aci d cycle, a n d fatty aci d o xi dati o n, as well as calci u m si g nali n g pat h ways ass ociate d wit h t he sarc o plas mic retic ul u m. N o si g nifica nt alterati o ns were see n wit h e n d ot heli n treat me nt t o sti m ulate hy pe rtr o p hy, i n dicati n g t hat

P D E9 does not relocalize under hypertrophic condition s.

I n c ha pter f o ur , I developed novel reagents to study P D E5 and P D E 9 nanodo mains i n a d ult car di o my ocytes utilizi n g t he p roxi mity proteo mics approach of A P E X2 tagging.

C onstructs were generated expressing G F P -AP E X2 -tagged P D E5 and P D E9, which were deter mi ne d t o acti vely hy dr olyze c G M P, l ocalize t o t heir c orrect s u bcell ular d o mai n, a n d bi oti nylate near by pr otei ns. T he b i oti nylati o n pr ot oc ol was o pti mize d by t he a d diti o n of t he a nti o xi da nt N -acety lcystei ne a n d t he pr otei n sy nt hesis i n hi bit or cycl o he xi mi de, t o i ncrease o verall bi oti nylati o n a n d decrease off -tar get bi oti nylati o n, res pecti vely. Des pite t he clear s uccess of the underlying experi ment to produce a substant ial i ncrease i n bi oti nylate d pr otei ns upon A P E X 2 acti vati o n , t he mass s pect ro metry and subsequent bioinfor matics w ere n ot o pti mize d t o detect resi d ues t hat were m o difie d by bi oti n. T his nee ds t o be correcte d t o detect t h ose pr ot ei ns t hat were tr uly altere d , a n d o n g oi n g eff orts ai m t o d o t h is.

T he data I di d o btai n is m ore c o nsiste nt wit h t he t otal pr otei n gels f or t hese e x peri me nts after t he bi oti n p ul l-d o w n, which sho wed appro xi mately e q ual pr otei n acr oss t he vari o us sa m ples, regardless o f bi oti nylati o n le v els . I hypothesize t hat n o n -bi oti nylate d pr otei ns t hat were bound t o t he s pecifically bi oti nylate d pr otei ns were als o p ulle d d o w n, a n d t he ref ore I w as u na ble t o deter mi ne w hic h were tr uly bi oti nylate d . T h is li kely le d t o a very hi g h false disc overy rate , fe w hits, a n d litt le o verla p wit h t he F L A G -p ull d o w n da ta. T his will re q uire further bioinfor matics modifications, which are under way. A n ot her feat ure of A P E X 2 is its a bility t o pr o vi de electr o n mic r osc o pic detecti o n wit h o ut t he nee d for a n a nti b o dy. T his work re mains to be perfor med, but can also provide nanodo mai n l ocalizati o n data.

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I n s u m mary, I ha ve s h o w n t hat i n hi biti o n of P D E 5 a n d P D E 9 ha ve hi g hly differe ntial i m pacts o n mi R N A re g ulati o n i n a m o use heart fail ure m o del des pite si milar p he n oty pic i m pr o ve me nt, i n dicati n g t hat c o m part me ntati o n of e nzy mes is critic al f or t heir do wnstrea m signaling effects. This study further de monstra te d t hat mi R N As are n ot a relia ble bi o mar ker rea d o ut f or treat me nt efficacy i n heart fail ure. I ha ve als o s h o w n t hat

P D E 9 is l ocalize d t o mit oc h o n dria i n car di o my ocytes, w here it i ntera cts wit h ma ny meta b olic pr otei ns. It als o i nterfaces wit h t he sarc o plas mic retic ul u m t o p ote ntially re g ulate calci u m si g nali n g. T his data i n dicates t hat t hera pe utically tar geti n g P D E 9 may be of be nefit i n meta b olically -li nke d diseases.

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C URRIC UL U M VI T A E

Krist e n Kokkonen -Si m o n 7 2 0 R utla n d A ve, R oss 8 5 8 • Balti m ore, M D 2 1 2 0 5 • k k o k k o n 1 @j h mi.e d u • ( 7 1 9) 6 4 8 -4432

Birt h D at e: A pril 2 2, 1 9 9 1 Birt h Pl a c e: Sa n J ose, C A

EDUCATIO N Johns Hopkins University Balti m ore , M D P h D, Cell ular a n d M olec ular Me dici ne May 201 9

U niversity of Colora do B o ul der, C O B A, M olec ular, Cell ular a n d De vel o p me ntal Bi ol o gy; Bi oc he mistry May 2013 G P A: 3. 9 1; H o n ors: s u m ma c u m la u de

R E S EARC H EXPERIE NCE Johns Hopkins Uni v ersit y Balti m ore, M D Graduate student with Dr. David Kass A u g ust 2 0 1 3 – Prese nt

U niversity of Colora do B o ul der, C O Undergraduate research assistant with Dr. Leslie Lein wand January 2011 – J uly 2 0 1 3

TEAC HI NG EXPERIE NCE Johns Hopkins University Balti m ore, M D Pollard Scholar for Graduate Student Genetics Course October 2014 – Dece mber 2014

U niversity of Colora do B o ul der, C O Teachin g Assistant, Molecular, Cellular and Develop mental Biology A u g ust 2 0 1 2 – Dece mber 2012

U niversity of Colora do B o ul der, C O Learning Assistant, General Che mistr y A u g ust 2 0 1 0 – Dece mbe r 2 0 1 0

GRA NTS A ND A WARDS R ut h L. Kirsc hstei n Pre -D oct or al National Research Service A ward A u g ust 2 0 1 6 - Prese nt Nati o nal Heart, L u n g, a n d Bl o o d I nstit ute, Nati o nal I nstit utes of Healt h Grant F31 H L134196; t otal a m o u nt of $ 1 3 0, 7 2 8

Keysto ne Sy mposia Futur e of Science Fund Scholarship January 2018 Travel A ward fro m Keystone Sy mposia on Molecular and Cellular Biology

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P UBLICATI O NS 1. Ra ne k MJ, Kokkonen -Si m o n K M , H ole wi ns ki RJ, Lee DI, C he n A, Pi nilla Vera M, D u n kerly - Eyri n g B, Patel C, Na ka m ur a T, Z h u G, Be dja D, Sasa ki M, Va n Ey k J E, P o well J D, Kass D A (2019) P K G -Modified TS C2 Regulates m T O R C1 Activity to Counter Adverse Cardiac Stress. Nat ure. 5 6 6( 7 7 4 3): 2 6 4 -2 6 9. 2. Z ha n g QJ, Tra n T A, Wa n g M, Ra ne k MJ, Kokkonen -Si m o n K M , Ga o J, L u o X, Ta n W, Kyry c he n k o V, Lia o L, X u J ( 2 0 1 8) Hist o ne lysi ne di met hyl -de methylase K D M3 A c o ntr ols pathological cardiac hypertrophy and fibrosis. Nature Co m munications. 9(1):5230. 3. Kokkonen -Si m o n K M , Sa beri A, Na ka m ura T, Ra ne k MJ, Z h u G, Be dja D, K u h n M, Halushka M K, Lee DI , Kass D A ( 2 0 1 8) Marked Disparity of micro R N A Regulation by c G MP - selecti ve P D E 5 vers us P D E 9 I n hi bit ors i n Heart Disease. J CI I nsi g ht. 3( 1 5). 4. Hs u S, Kokkonen -Si m o n K M , Kir k J A, K ol b T M, Da mic o R L, Mat hai S C, M u k herjee M, S ha h A A, Wi gley F M, Mar g ulies K B, Ha ssoun P M (2018 ) Ri g ht Ve ntric ular My ofila me nt F u ncti o nal Differe nces i n H u ma ns Wit h Syste mic Scler osis – Associated Versus Idiopathic P ul m o nary Arterial Hy perte nsi o n. Circ ulati o n. 1 3 7( 2 2): 2 3 6 0 -7 0. 5. Na ka m ura T, Z h u G, Ra ne k MJ, Kokkonen -Si m o n K , Z ha n g M, Ki m G E, Ts ujita K, Kass D A ( 2 0 1 8) Pre ve nti o n of P K G -1 α Oxidation Suppresses A nti hy pertr o p hic / A ntifi br otic Effects Fr o m P D E 5 I n hi biti o n b ut n ot s G C Sti m ulati o n. Circ ulati o n: Heart Fail ure. 1 1( 3):e 0 0 4 7 4 0. 6. Kokkonen K , Kass D A (2017) Nanodo main Regu lati o n of Car diac Cyclic N ucle oti de Si g nali n g by Phosphodiesterases. Annual Revie w of Ph ar macology and Toxicology. 57:455 -7 9.

ABSTRACTS 1. K M Kokkonen -Si m o n , G E Ki m, D B F oster, a n d D A Kass ( 2 0 1 8) P D E 5 a n d P D E 9 C o m part me ntalizati o n i n Car diac My ocytes. A bst racts of the Gordon Conference o n Cyclic Nucleotide Phosphodiesterases. 2. K M Kokkonen -Si m o n , DI Lee, T Na ka m ura, G Z h u, D Be dja, a n d D A Kass. P D E 5 a n d P D E9 inhibition differentially regulate micro R N A expression during pressure -overl oa d -induced heart fail ure ( 2 0 1 8) A bstracts of t he Keyst o ne Sy m p osia o n Heart Fail ure: Cr ossi n g t he Tra nslati o nal Di vi de. 3. MJ Ra ne k, K M Kokkonen , DI Lee, RJ H ole wi ns ki , V A gra wal, C Vir us, D A Ste ve ns, M Sasa ki, H Z ha n g, M M Ma n ni o n, P P Rai ner, R C Pa ge, J C Sc hisler, J E Va n Ey k, M S Willis, a n d D A Kass ( 2 0 1 7) N o vel R ole of P K G i n Pr otei n Q uality C o ntr ol by Re g ulati n g C hi p. A bstracts of 8 t h International Conference on c G MP G enerators, Effectors and Therapeutic I mplications. 4. MJ Ra ne k, K M Kokkonen , RJ H ole wi ns ki, DI Lee, T Na ka m ura, G Z h u, M Sasa ki, J E Va n Eyk, and D A Kass ( 2 0 1 6) Pr otei n Ki nase G is a Re d o x Se ns or t hat C o ntr ols A ut o p ha gic Fl u x by Phosphorylating Tuberin. Abstracts of the A merican Heart Association Scientific Sessions.

PATE NTS 1. D A Kass, MJ Ra ne k, K M K o k k o ne n, J D P o well, C Pate l. Engineere d T S C 2. U S A p pl. N o.: 62/532,909. File d 2 0 1 7. 2. D A Kass , B D u n kerly -Eyring, K M Kokkonen, MJ R a ne k. Targeting TS C2 S1366 ( mouse , rat ) S136 5 (hu man) by gene engineering or p h os p h orylati o n t o re g ulate m T O R si g nali n g casca des f or thera pe uti c in dicati o ns. File d 2 0 1 8.

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