TNNT2 mutations in the binding region of TNT1 disrupt its role in contractile inhibition and stimulate cardiac dysfunction

Aditi Madana, Meera C. Viswanathana, Kathleen C. Woulfeb, William Schmidta, Agnes Sidora, Ting Liua, Tran H. Nguyena, Bosco Trinhc, Cortney Wilsonb, Sineej Madathild, Georg Voglerc, Brian O’Rourkea, Brandon J. Biesiadeckie,f, Larry S. Tobacmand, and Anthony Cammaratoa,g,1

aDepartment of Medicine, Division of Cardiology, Johns Hopkins University, Baltimore, MD 21205; bDepartment of Medicine, Division of Cardiology, University of Colorado Denver, Aurora, CO 80045; cDevelopment, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037; dDepartment of Medicine, University of Illinois College of Medicine, Chicago, IL 60612; eDepartment of Physiology and Cell Biology, The Ohio State University, Columbus, OH 43210; fThe Davis and Lung Research Institute, The Ohio State University, Columbus, OH 43210; and gDepartment of Physiology, Johns Hopkins University, Baltimore, MD 21205

Edited by Edwin W. Taylor, The University of Chicago, Chicago, IL, and approved June 15, 2020 (received for review January 28, 2020) is regulated by the movement of end-to-end- “closed” C-state position, which partially uncovers binding linked −tropomyosin complexes over the thin filament sur- sites. Subsequent binding of a small population of myosin cross- face, which uncovers or blocks myosin binding sites along F-. bridges further displaces Tpm to an “open” M-state position, The N-terminal half of (TnT), TNT1, independently pro- completely exposing neighboring binding sites, leading to coop- motes tropomyosin-based, steric inhibition of acto-myosin associa- erative, full filament activation. tions, in vitro. Recent structural models additionally suggest TNT1 This three-state model of contractile regulation is strongly sup- may restrain the uniform, regulatory translocation of tropomyosin. ported by biochemical and structural data (3, 4). However, it was Therefore, TnT potentially contributes to striated muscle relaxation; however, the in vivo functional relevance and molecular basis of developed in the absence of high-resolution structures of thin this noncanonical role remain unclear. Impaired relaxation is a hall- filament-bound Tn, and therefore the model has been incomplete in mark of hypertrophic and restrictive cardiomyopathies (HCM and important respects. Fortunately, a great deal of Tn’s structure on PHYSIOLOGY RCM). Investigating the effects of cardiomyopathy-causing muta- the thin filament has recently been revealed for the first time, via an tions could help clarify TNT1’s enigmatic inhibitory property. We incisive cryo-electron microscopy (cryo-EM) study by Yamada et al. tested the hypothesis that coupling of TNT1 with tropomyosin’s (9). Tn’s effects on the shifting position of Tpm along the regulated end-to-end overlap region helps anchor tropomyosin to an inhibi- thin filament are markedly more apparent. Three Tn regions—1) tory position on F-actin, where it deters myosin binding at rest, and an extended TnI C terminus, 2) the Tn tail (i.e., the TnT N-terminal that, correspondingly, cross-bridge cycling is defectively suppressed region), and 3) the Tn core domain (consisting of parts of all three under diastolic/low Ca2+ conditions in the presence of HCM/RCM subunits)—all interact directly with F-actin−Tpmsoastoinfluence lesions. The impact of TNT1 mutations on Drosophila cardiac perfor- mance, rat myofibrillar and cardiomyocyte properties, and human Tpm position. Furthermore, these regions seemingly have po- TNT1’s propensity to inhibit myosin-driven, F-actin−tropomyosin sitions on F-actin that, depending upon conditions, may impact motility were evaluated. Our data collectively demonstrate that re- moving conserved, charged residues in TNT1’s tropomyosin-binding Significance domain impairs TnT’s contribution to inhibitory tropomyosin posi- tioning and relaxation. Thus, TNT1 may modulate acto-myosin ac- Roughly two decades ago, troponin T (TnT) of the trimeric thin − tivity by optimizing F-actin tropomyosin interfacial contacts and by filament regulatory troponin complex, was shown to contribute ’ binding to actin, which restrict tropomyosin s movement to activating directly to the inhibition of skeletal and contrac- ’ configurations. HCM/RCM mutations, therefore, highlight TNT1 ses- tion, independent of I and C. The molecular basis of sential role in contractile regulation by diminishing its tropomyosin- this enigmatic role for TnT remains unknown. Hypertrophic and anchoring effects, potentially serving as the initial trigger of pathol- restrictive cardiomyopathies (HCM and RCM) are characterized ogy in our animal models and humans. by impaired relaxation and, thus, an inability of the heart muscle to properly “turn off.” Based on data gleaned from multiple troponin T | tropomyosin | Drosophila | cardiomyopathy | diastolic model systems used to investigate HCM and RCM TnT muta- dysfunction tions, and recently published thin filament structural models, we propose a mechanism that potentially underlies TnT’s heretofore triated muscle contraction is regulated by Ca2+- and myosin- poorly understood role in muscle relaxation, which, when com- Sdependent changes in the location of troponin (Tn) and promised, may cause disease. tropomyosin (Tpm) over the surface of the actin-based thin filament (1–4). Tn consists of a Ca2+ binding subunit, TnC, an inhibitory Author contributions: A.M., K.C.W., W.S., T.L., G.V., B.O., B.J.B., L.S.T., and A.C. designed subunit, TnI, and a subunit that connects the complex tightly to research; A.M., M.C.V., K.C.W., A.S., T.L., T.H.N., B.T., C.W., S.M., G.V., B.J.B., and A.C. performed research; A.M., M.C.V., K.C.W., A.S., S.M., G.V., and B.J.B. contributed new Tpm, TnT. Tpm is a semirigid, coiled-coil dimer that binds seven reagents/analytic tools; A.M., M.C.V., K.C.W., W.S., T.L., G.V., B.O., B.J.B., L.S.T., and A.C. successive actin protomers (5, 6). Tpm molecules link end to end to analyzed data; and A.M., K.C.W., W.S., T.L., S.M., B.J.B., L.S.T., and A.C. wrote the paper. form continuous strands that track along the winding, long-pitch The authors declare no competing interest. 2+ F-actin helix (5, 7, 8). In resting muscle, when intracellular Ca This article is a PNAS Direct Submission. is low, Tpm is constrained to an inhibitory, “blocking” B-state po- Published under the PNAS license. sition over myosin binding sites on F-actin (1–4). Hence, contrac- 1To whom correspondence may be addressed. Email: [email protected]. 2+ 2+ tion is suppressed. As Ca rises, Ca -bound TnC draws a TnI This article contains supporting information online at https://www.pnas.org/lookup/suppl/ regulatory region away from actin, releasing a steric constraint on doi:10.1073/pnas.2001692117/-/DCSupplemental. Tpm. This enables azimuthal movement of Tpm across F-actin to a

www.pnas.org/cgi/doi/10.1073/pnas.2001692117 PNAS Latest Articles | 1of10 Downloaded by guest on September 25, 2021 myosin binding. To allow contraction, both Tpm and Tn must been investigated in vivo, however, nor have their effects been reposition properly. assessed across a full range of experimental models, and then Significantly for the current report, information regarding the considered in light of the new structural advances (9, 11). conformation of the end-to-end overlap of successive Tpms is Here, we posit that the K124N, R130C, and E136K hcTnT now available from the above work (9), and also from concur- substitutions diminish hcTNT1’s inhibitory properties by com- ring, independent, in silico studies (10, 11). The overlap domain promising its Tpm-anchoring role in contractile regulation, serving has a relatively fixed position on F-actin that approximates the as a trigger for cardiac remodeling. Specifically, we test the hy- Tpm B-state location, whether or not Ca2+ is present (9). Notably, pothesis, based on the aforementioned structural and biochemical the overlap region includes a TnT helix that closely interacts with results (9, 11–13, 18, 19, 23, 25, 34), that the TNT1−Tpm overlap both Tpm ends, and also, potentially, with actin. It thus may help region is essential for relaxation, and that, correspondingly, sup- anchor the overlap domain on actin and restrain its motion during pression of cross-bridge cycling under diastolic/low Ca2+ condi- regulatory repositioning. In light of these findings, the Tn tail, tions is defective when these missense mutations are present. First, from which this helix derives, takes on particular interest and is the we developed several Drosophila melanogaster models to scrutinize subject of the present study. the mutations’ impact on organ-, cellular-, and myofibrillar-level The primary switch regulating muscle contraction involves the function. We next ascertained the effects of the equivalent E136K TnI C terminus and the Tn core domain, which interact with each hcTnT variant on activation and, importantly, relaxation proper- other and with F-actin−Tpm in a Ca2+-sensitive manner. No ties of rat ventricular and cardiomyocytes. Finally, we matter its primacy, this switch is considered insufficient for con- employed a reductionist in vitro approach, using hcTNT1 pep- tractile activation; actions of myosin cross-bridges are also re- tides, to identify the immediate and completely Ca2+-independent quired. The switch is also insufficient for inhibition, which requires consequences of all three mutations on hcTNT1−Tpm-mediated other features of Tn, Tpm, and actin (9, 12–17). In particular, the inhibition of contraction. Our data collectively demonstrate that N-terminal half of TnT, otherwise known as TNT1, may play a removing conserved, charged residues in the TNT1−Tpm binding role in relaxation, to a degree that is an open subject of investi- domain impairs TnT’s involvement in inhibitory Tpm positioning, gation. This domain, corresponding to residues 1 to 156 of human muscle relaxation, and diastole across phyla and across multiple cardiac TnT (hcTnT), couples with Tpm through an evolutionarily levels of organization. The consistent findings support the func- conserved, highly charged binding element that spans residues 113 tional importance of the recently revealed Tpm overlap−Tn tail to 136 of hcTNT1 (SI Appendix,Fig.S1)(18–20). The α-helical region (9, 11) in thin filament-based contractile regulation, and TNT1 tail extends along, binds tightly to, and buttresses the earlier findings that TNT1 biases Tpm to a relatively inhibitory head-to-tail overlap of successive Tpm dimers (9, 11, 18, 21, 22). azimuthal location on F-actin (12, 13). We suggest a mechanism Additionally, it enhances the affinity of Tpm for F-actin as well as whereby TNT1 might strengthen F-actin−Tpm binding and in- the cooperativity of myosin cross-bridge binding to actin (18, crease energetic demands necessary for Tpm translocation and 23–25). Moreover, in the absence of TnI and TnC, both skeletal thin filament activation. Thus, it is plausible that pathological and cardiac forms of TNT1 bias Tpm toward an inhibitory position modifications to the N-terminal half of TnT more generally act by that impedes myosin S1 interaction with actin−Tpm−TNT1 fila- perturbing TNT1’s interaction with Tpm (11, 18, 19, 34) and/or ments (12, 13). These structural and biophysical findings in solution F-actin (9) and, in turn, Tpm’s intrinsic inhibitory association with suggest TNT1 may have an inhibitory role in thin filament regula- F-actin (8, 12, 13, 15, 35–41). tion. However, the functional importance of TNT1 in inhibition of contraction is unclear in intact muscle and in vivo. Is TnT1 ancillary Results to inhibition, or is it essential for normal relaxation? Overexpression of Mutant TnTs Induces Diastolic Dysfunction and Defective contractile regulation is frequently associated with Cardiomyopathy in D. melanogaster. Drosophila is an outstanding striated muscle pathology. Hypertrophic and restrictive cardio- animal model to study striated muscle physiology and patho- myopathies (HCM and RCM) are primary disorders of heart genesis. They age and reproduce quickly, are easily maintained, muscle (2, 26, 27). HCM is characterized by abnormal thickening lack genetic redundancy, and offer a wide repertoire of tools that and stiffening of the heart walls, cellular and subcellular disarray, enable extensive genetic manipulation. Additionally, over 75% and cardiac arrhythmias (26, 27). RCM is typified by restrictive of human disease have fly homologs including those un- physiology in the presence of normal or reduced diastolic ven- derlying cardiac disorders (42). The adult Drosophila heart, or tricular volumes, normal or reduced systolic volumes, and normal dorsal vessel, is a tubular structure, which bears resemblance to heart wall thickness (27). Impaired relaxation, diastolic dysfunc- the early vertebrate embryonic heart, and is made up of ∼80 tion, and hyperdynamic contractility are observed for both HCM cardiomyocytes (Fig. 1A). Thin filament conformational transitions and RCM and have been reported to be the earliest and pre- accompanying contractile regulation and cardiomyocyte Ca2+ han- dominant biomechanical abnormalities that are necessary to drive dling are also extremely well conserved between flies and mammals overt hypertrophic remodeling (26, 28). (43, 44), making this a unique system to rapidly explore the effects Mutations in TNNT2, which encodes hcTnT, account for 15% of Tn mutations on cardiac function. of all HCM-causing lesions, and ∼75% of these localize to hcTNT1 We employed the PhiC31 integrase system (45) to generate (18, 19, 29). A number of studies have characterized the molecular-, transgenic lines that permit overexpression of wild-type (WT) cellular-, and tissue-level effects of hcTNT1 mutations that flank the Drosophila TnT or the fly equivalent of the K124N, R130C, and Tpm-binding element (2, 18, 19, 29–33). Several missense mutations E136K cardiomyopathy-inducing TnT variants, specifically in the that affect highly conserved amino acids are also located within this dorsal vessel. PhiC31 technology ensured the transgenes, preceded element (SI Appendix,Fig.S1), yet they have received less attention. by a 5′ upstream activating sequence (UAS), integrated at an These include the K124N and R130C HCM and E136K RCM identical, predetermined genomic locus, and thus eliminated ge- substitutions, which remove or replace invariant charged residues netic variability introduced by random insertions. When these lines whose sidechains likely participate in, and therefore when mutated are crossed with a GAL4 transactivating -expressing strain, may affect, Tpm binding (11, 22). Supporting this, the R130C the progeny inherit both transgenes and ectopically express the substitution was shown to weaken hcTNT1’s computationally de- TnTs in the same pattern as GAL4 (46), and the results are directly rived Tpm interaction energy (11), and the K124N and R130C comparable. Initially, to confirm tissue-restricted expression and mutations decreased hcTnT’s affinity for Tpm (34). The K124N correct localization of transgenic TnT, Hand4.2-GAL4 Drosophila, substitution additionally sensitized myosin ATPase activity in thin which express GAL4 in the heart, were crossed with a line harboring filament-regulated acto-myosin assays. The mutations have never a that encodes His-tagged WT TnT, under the control of a

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.2001692117 Madan et al. Downloaded by guest on September 25, 2021 Fig. 1. In vivo effects of transgenic overexpression of mutant TnTs in the Drosophila heart. (A) Fluorescent micrograph (40×)ofatdtK-expressing heart tube PHYSIOLOGY imaged through the cuticle of a live animal. O2 and O3 denote ostia 2 and 3, which served as positional markers for consistent measurements of chamber areas from multiple flies. Dotted rectangular box depicts the region imaged in B. (Scale bar, 50 μm.) (B) Confocal micrographs of a dissected Hand > TnTWT.His fly heart, taken at 100× magnification, show colocalization of actin and transgenic His-TnT along cardiac thin filaments. (Scale bar, 5 μm.) (C) In vivo analysis of beating from 3-wk-old Hand > TnT flies revealed significantly reduced diastolic chamber areas in all mutants, and reduced systolic chamber areas in the R81C and E87K mutants, relative to control. Data are presented as mean ± SEM (n = 26; *P ≤ 0.05; ***P ≤ 0.001). (D) M-mode kymograms generated from high-speed videos of beating hearts overexpressing WT or mutant TnT and the fluorescent reporter, tdtK. Vertical red lines terminate at opposing edges of the heart wall, with the left line indicating diastolic and the right line indicating the systolic diameter.

UAS regulatory sequence. Anti-His−labeling and confocal micros- according to fly TnT; SI Appendix, Fig. S1), were individually copy of the progeny (i.e., Hand > TnTWT.His) revealed His-TnT was crossed with Hand virgin females that coexpressed tdtK (47); tdtK restricted to, and present throughout, the dorsal vessel. Further- is a red fluorescent protein that allows visualization of beating more, it precisely colocalized with phalloidin-labeled cardiac actin hearts directly through the cuticle of live, intact animals (48). (Fig. 1B). Uniform thin filament incorporation of transgenic TnT, Importantly, nontransgenic (Hand x w1118) and WT transgenic with no resolvable aggregation, was observed. (Hand > TnTWT) control hearts behaved indistinguishably from To test the in vivo effects of the cardiomyopathy-associated each other (SI Appendix,Fig.S2A), and, therefore, only the latter TnT variants, males from four transgenic lines, UAS-TnTWT, control was used for subsequent analyses. Female progeny were UAS-TnTK75N, UAS-TnTR81C,andUAS-TnTE87K (numbering aged to 3 wk, and their hearts were recorded using fluorescence

Fig. 2. In situ effects of transgenic overexpression of mutant TnTs on Drosophila cardiac function. (A) In situ analysis of 3-wk-old Hand > TnT hearts resolved cardiac restriction, decreased cardiac outputs, and reduced rates of relaxation in mutants relative to control. Data are presented as mean ± SEM (n = 24 to 36; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). (B) M-mode kymograms generated from high-speed videos of beating hearts that overexpressed WT or mutant TnT. Vertical red lines terminate at opposing edges of the heart wall, with the left line indicating diastolic and the right line indicating systolic diameter.

Madan et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 25, 2021 videography. We measured diastolic and systolic chamber areas organ-level phenotypes, including impaired relaxation and diastolic spanning regions flanking the second (O2) and third (O3) ostial dysfunction. inflow tracts (Fig. 1A) and observed significant decreases (Fig. 1C) for the mutants relative to control. The reduced diastolic areas are Restricted Cardiac Dimensions Result from Ca2+-Dependent and consistent with restrictive cardiac physiology and an inability of the Ca2+-Independent Effects of Mutant TnTs on Drosophila Cardiomyocytes. myocardium to relax properly. Corresponding M-mode kymo- The dorsal vessel of Drosophila is composed of a single layer of grams that depict mutation-induced, restricted heart wall motion cardiomyocytes, opposing pairs of which are joined by intercel- over time are shown in Fig. 1D. lular junctions to form the heart tube lumen. This arrangement To conduct a more comprehensive assessment of cardiac uniquely allows analysis of cardiomyocyte behavior, with single- dysfunction, we surgically exposed the dorsal vessels of 3-wk-old cell resolution, in the context of a functioning organ. Hence, it female control and mutant Hand > TnT flies under oxygenated renders the fly heart conducive for studying diastolic properties artificial hemolymph (AHL), and recorded high-speed movies of at the individual cellular level without the adverse effects associ- the spontaneously contracting heart tubes. Videos were analyzed ated with cardiomyocyte isolation (15). To investigate the mech- using Semiautomated Optical Heartbeat Analysis (SOHA) software anisms responsible for diastolic dysfunction and, specifically, those (49,50).Thisinsituanalysisverifiedthedefectsobservedinvivo underlying shortened cardiomyocytes and reduced resting volumes with respect to cardiac restriction for all three mutant lines, Hand > in the TnT mutants, beating hearts from 3-wk-old Hand > TnT TnTK75N, Hand > TnTR81C,andHand > TnTE87K vs. Hand > TnTWT females were exposed and treated sequentially with ethylene gly- (diastolic diameters: 65.6 ± 1.0 μm, 56.8 ± 1.2 μm, and 60.1 ± 0.8 col bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA)/ μmvs.70.7± 0.9 μm, respectively; systolic diameters: 43.4 ± 0.9 μm, EGTA,acetoxymethyl ester (AM)-, followed by blebbistatin-containing 38.3 ± 0.9 μm, and 40.8 ± 0.6 μmvs.48.0± 0.9 μm, respectively) AHL solutions. The cardiac tube diameters, which are directly (Fig. 2A). The resultant decreased cardiac volumes prompted proportional to cellular length, were measured during diastole and significantly lower cardiac outputs in the mutants relative to con- again after each treatment. As shown previously, WT dorsal vessel trol (94.1 ± 4.1 nL/min, 59.3 ± 3.0 nL/min, and 65.0 ± 5.2 nL/min diastole involves less than full 100% relaxation in this preparation vs. 131.4 ± 5.2 nL/min, respectively). The mutants additionally (15, 17, 51). This is evident from the effect of intracellular che- exhibited slower rates of cardiac relaxation (162.0 ± 5.0 μm/s, lation and complete dissociation of Ca2+ via EGTA/EGTA,AM, 131.2 ± 6.3 μm/s, and 121.6 ± 7.5 μm/s vs. 167.5 ± 10.7 μm/s, re- which increased the diameter by 2.6% compared to diastole, and spectively). Similar decreases in relaxation rates were observed from myosin inhibition via blebbistatin that caused another 3.7% when exposed hearts were forced to contract against a viscous load increase (Fig. 3). Therefore, prevention of cross-bridge binding in of AHL containing 15% Ficoll (wt/vol) (SI Appendix, Fig. S2B). diastole is incomplete, due to a small persistence of Tn−Ca2+,and However, since the impact of mutant TnT expression on systolic also because thin filament-based inhibition is, intrinsically, im- and diastolic diameters was similar for each mutant, there was no perfect. If the TNT1 mutations tend to allow unobstructed myosin significant effect on fractional shortening (SI Appendix, Fig. S2C). binding, then these phenomena might increase. This is what was There were also no differences observed in heart rhythm. Heart observed. The effects of both agents were significantly exaggerated period (HP, the time interval from the beginning of systole to the in the presence of the TNT1 mutations, particularly the effect of end of the following diastole) between genotypes showed signifi- blebbistatin (Fig. 3 B and C). After prior relaxation from EGTA/ cant differences, as did systolic intervals (SI). However, since they EGTA,AM, cellular lengthening due to blebbistatin was almost both increased to a similar extent per mutant, we did not observe twice as large for the mutants as for WT. These observations are any significant differences in SI/HP, an index that depicts the consistent with restrictive physiology and diastolic dysfunction that proportion of time spent generating active tension during the result largely from excessively disinhibited cross-bridge cycling, cardiac cycle, relative to control (SI Appendix,Fig.S2C). Overall, enhanced basal stress, and incomplete relaxation, at the car- both in vivo and in situ analyses demonstrated that our D. mela- diomyocyte level. Thus, the TnT variants cause Tpm and Tn to nogaster models, which express the analogous K124N, R130C, and block myosin binding inadequately, producing excessive myofi- E136K hcTnT mutations, recapitulate the earliest HCM/RCM brillar tension and myocyte shortening that restrict proper filling.

Fig. 3. Ca2+-dependent and Ca2+-independent processes contribute to impaired myocardial relaxation in mutant flies. (A) Small-molecule compounds elicited significant, incremental increases in the diameters of 3-wk-old fly hearts. Incubation with EGTA/EGTA,AM-containing AHL chelated extracellular and intra- cellular Ca2+, halted contraction, and prompted increases in heart tube diameters from baseline (i.e., diastole). Subsequent incubation with blebbistatin inhibited acto-myosin attachments, leading to further increases in cardiac diameters. (B)Ca2+ chelation prompted a significantly greater increase in the diameter of hearts overexpressing the TnT variants vs. WT. (C) Blebbistatin treatment caused an exaggerated response across the wall of all mutant hearts, relative to control. Data are presented as mean ± SEM (n = 20; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.2001692117 Madan et al. Downloaded by guest on September 25, 2021 Single Myofibrils Isolated from upE87K Drosophila Show Increased length (2.1 ± 0.1 μmvs.2.2± 0.1 μm) (Fig. 5C). A rapid solution Resting Stiffness. To explore the effects of mutant TnT on the switching approach was then used to study the kinetics of myofi- myocyte’s basic contractile elements, and to examine TNT1’s brillar force development and relaxation following abrupt changes in potentially universal role in contractile inhibition across striated Ca2+ (58, 59). Substituting endogenous Tn with myc-cTnTE138K−Tn muscle types, we compared the structural and mechanical prop- significantly increased the resting tension of myofibrils relative to erties of skeletal, indirect flight muscle (IFM) myofibrils con- those containing myc-cTnTWT−Tn (5.86 ± 0.92 mN/mm2 vs. 2.78 ± taining TnTE87K to those of WT. IFMs provide ample material for 0.39 mN/mm2), without altering maximum tension (32.75 ± 3.98 physiological analyses, and their thin filaments demonstrate Ca2+- mN/mm2 vs. 33.59 ± 6.19 mN/mm2) or the rate constant of tension − −1 −1 dependent, Tn Tpm regulatory switching that is indistinguishable generation (kACT:2.59± 0.19 s vs. 2.60 ± 0.17 s )(Fig.5D and SI from vertebrates (43). Using CRISPR-Cas9 technology, we cre- Appendix,Fig.S3B and Table S2). Moreover, myofibrils with myc- ated a Drosophila knock-in line with the upE87K missense mutation. cTnTE138K−Tn had a significantly longer initial, linear phase of re- WT As all Drosophila TnT isoforms are encoded by a single gene, laxation, compared to those harboring myc-cTnT −Tn (tREL,LIN: upheld (up), and the mutation lies within a constitutive exon, these 64.59 ± 5.08 s vs. 48.48 ± 2.93 s) (Fig. 5D and SI Appendix,Fig.S3B), flies express 100% mutant TnT in every muscle. The E87K sub- which reflects a slower rate of cross-bridge detachment (59). stitution (corresponding to human E136K; SI Appendix,Fig.S1)is There were no significant differences in the rate constants of the but two residues proximal to the site of the well-characterized, initial or exponential phases of relaxation in myofibrils with myc- 101 101 E138K WT −1 Drosophila-specific E89K mutation known as up ; up is a cTnT −Tn vs. myc-cTnT −Tn (kREL,LIN: 2.47 ± 0.64 s vs. −1 −1 −1 relatively harsh mutation, which engenders severe defects in fly 4.38 ± 1.02 s ; kREL,EXP: 33.95 ± 2.56 s vs. 29.28 ± 1.50 s ), or −1 (52), and restrictive cardiac pathophysiology (53). in the rate constant of tension redevelopment (kTR: 3.36 ± 0.23 s − These phenotypes result from Tpm mispositioning and poor vs. 3.24 ± 0.35 s 1)(Fig.5D and SI Appendix, Table S2). Of note is B-state preservation during muscle relaxation. We postulated that that rat ventricular cardiomyocytes, expressing mutant cTnTE138K it might be possible to examine IFM myofibrils with the E87K via adenovirus-mediated gene transfer, exhibited reduced mutation, mechanically, unlike those with up101 which hypercontract lengths, higher contraction velocities, and prolonged relaxation times, and self-destruct, particularly with respect to passive stiffness and akin to what we observed in the fly models (SI Appendix,Fig.S4). adequacy of relaxation. Therefore, the fly skeletal and vertebrate cardiac data il- Similar to up101, upE87K homozygotes are viable and fertile. lustrate that the TnT variant leads to disinhibition of thin filaments at However, in contrast to the severely damaged fibers that typify rest and impairs relaxation. These results support our hypothesis that up101 IFM, upE87K displayed normal IFM morphology (Fig. 4A) HCM/RCM charge-altering mutations in hcTNT1’s Tpm-binding and, hence, were well suited for myofibril isolation. Relative to element may jeopardize its important role in contractile inhibition PHYSIOLOGY other insect muscles, individual, fully intact myofibrils are easily and relaxation. purified from the IFM for imaging and, despite their high passive stiffness and resistance to stretch, can be analyzed mechanically TNNT2 Cardiomyopathy Mutations Impair Human Cardiac TNT1’s to discern relative passive length−tension relationships (54, 55). Inhibitory Properties In Vitro. To examine the inhibitory proper- Whether measured in situ, along intact fibers, or ex vivo fol- ties of the K124N, R130C, and E136K hcTnT variants in a fully lowing isolation, upE87K myofibrils showed a minor, but signifi- defined reconstituted system, we employed a four-element in vitro cant reduction in sarcomere length relative to control (3.31 ± motility (IVM) assay. The translocation of fluorescently labeled 0.01 μm, 3.32 ± 0.01 μm vs. 3.37 ± 0.01 μm) (Fig. 4B). To rabbit skeletal F-actin, over beds of immobilized rabbit skeletal compare mechanical properties under low Ca2+ conditions, in- myosin, was recorded and filament sliding velocity was assessed dividual IFM myofibrils were incrementally stretched from their under different experimental conditions. We first examined the corresponding resting lengths using a glass microtool, a length- effects of Tpm and Tpm−hcTNT1WT on actin filament sliding controlled motor, and a calibrated cantilevered force probe (56), velocity over a range of myosin concentrations (12.5 μg/mL to and tension was determined (Fig. 4 C and D and SI Appendix, 100 μg/mL) (Fig. 6A). The addition of bovine cardiac Tpm de- Fig. S3A). We detected significantly higher resting tension in creased F-actin velocities, by 50% or more at the lower myosin upE87K myofibrils compared to control (2.17 ± 0.46 mN/mm2 vs. concentrations (i.e., 12.5 μg/mL and 25 μg/mL) assayed (Fig. 6A). 0.37 ± 0.09 mN/mm2) (Fig. 4D). To determine whether the el- With greater myosin concentration on the IVM surface, the heads evated stiffness resulted from uninhibited acto-myosin interac- cooperatively overcame part of this inhibition. However, even at tions at rest, we treated both mutant and control myofibrils with 75 μg/mL myosin, when maximum sliding velocity was reached, the myosin inhibitor, 2,3-butanedione monoxime (BDM) (57). F-actin was propelled at a velocity of 3.6 ± 0.2 μm/s, while BDM incubation prompted no change for control, but induced a F-actin−Tpm sliding velocity was 3.2 ± 0.2 μm/s (Fig. 6A). These drastic drop in upE87K myofibrillar stiffness (Fig. 4D). Post drug observations support the notion that, under subsaturating myosin treatment, the tension of the mutant myofibrils was indistinguishable conditions, Tpm can be inhibitory because its default azimuthal from control (Fig. 4D and SI Appendix,Fig.S3A). These findings are location along F-actin is dictated by numerous, highly favorable, consistent with an upE87K-dependent increase in myofibrillar resting interfacial electrostatic interactions that position Tpm such that it tension due to excessive, unobstructed cross-bridge binding that adversely affects acto-myosin cycling (8, 12, 13, 15, 35–41). Ad- can be relieved upon treatment with a myosin inhibitor. dition of hcTNT1WT to F-actin−Tpm drastically reduced filament sliding velocities across all myosin concentrations (Fig. 6A). At Ex Vivo Incorporation of Mutant Cardiac TnT Increases Resting Stiffness 75 μg/mL myosin, the average sliding speed decreased roughly and Impairs Relaxation of Mammalian Ventricular Myofibrils. To verify 50% relative to F-actin to 1.9 ± 0.3 μm/s (Fig. 6A). The inhibitory the pathological effects of the TNNT2 RCM mutation in a ver- effect was much larger than that resulting from Tpm alone and is tebrate model, we cloned, expressed, and purified WT and mutant consistent with earlier studies showing an hcTNT1WT-mediated rat myc-tagged cTnT. The recombinant were recon- stabilization of Tpm in the B state (12). Two-way ANOVA con- stituted into functional, trimeric Tn complexes. The endogenous firmed that, as expected, myosin concentration (P < 0.0001) and Tn of myofibrils isolated from the left ventricles of adult rats was the addition of Tpm or Tpm−hcTNT1WT (i.e., filament type, P < then replaced with exogenous Tn containing either WT or E138K 0.0001) significantly affected F-actin sliding velocities (Fig. 6A). rat cTnT (corresponding to human E136K; SI Appendix,Fig.S1). To test whether the TNNT2 mutations affect hcTNT1’s inhibitory The extent of exchange was similar for both mutant and WT action, we repeated the assay with hcTNT1K124N-, hcTNT1R130C-, cTnT-containing Tns (75.9 ± 1.8% vs. 73.5 ± 2.5%, respectively) and hcTNT1E136K-containing filaments. All three mutant fila- (Fig. 5 A and B), and the variant had no effect on sarcomere ments exhibited higher myosin-propelled sliding velocities relative

Madan et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 25, 2021 Fig. 4. Effects of the hcTnT RCM mutation on IFM myofibrillar properties. (A) Fluorescence (10×) and confocal micrographs (63×,100×) show no gross structural differences in IFMs (Top) or myofibrils (Middle and Bottom) between control and upE87K Drosophila. Top (10×) depicts hemithoraces that were flash- frozen and sagittally bisected to reveal IFM fiber morphology. Preparations were stained with Alexa-568 phalloidin to label actin. Z discs were distinguished by the expression of GFP-tagged Zasp52, a protein that binds to α-, thereby restricting GFP fluorescence to Z discs. Dotted rectangular boxes highlight the regions imaged below. were visualized along myofibrils of the hemithoraces, in situ, by confocal microscopy (Middle,63×). Myofibrils were also isolated for ex vivo imaging (Bottom, 100×). (Scale bars, 5 μm.) (B) upE87K sarcomeres, measured from hemithoraces (in situ) or along isolated myofibrils (ex vivo), were significantly shorter than control (n = 220 to 240). Hence, myofibril removal and isolation from IFMs did not affect mutant sarcomere length. (C) Representative image of an IFM myofibril held between a glass microtool and cantilevered force probe for mechanical assessment. (D) upE87K IFM myofibrils exhibited significantly higher resting tension relative to control, which was restored to WT values postincubation with BDM. Control myofibrils showed no difference in stiffness pre- and post-BDM incubation (n = 7 to 16). Data are presented as mean ± SEM (ns > 0.05; *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001).

to controls at nearly every myosin concentration assayed (Fig. 6 B– (1, 3, 60–62). Therefore, it was unexpected, in 2002, when two D). Two-way ANOVA again confirmed significant effects of groups separately demonstrated that the skeletal and cardiac myosin concentration, and each mutant Tpm−hcTNT1 relative to TNT1 fragments could reduce F-actin−Tpm-activated myosin S1 Tpm−hcTNT1WT, on F-actin sliding velocities, with nonsignificant ATPase activity and S1-actin binding, and prompt inhibitory interaction effects (Fig. 6 B–D). Thus, the rate of change of ve- conformational states of Tpm (12, 13). A likely candidate seg- locity for each mutant filament vs. WT, over the various beds of ment responsible for these effects is the Tpm-binding region of myosin tested, did not differ. Moreover, at a subsaturating myosin TNT1, which includes several charged residues and has high concentration, a relatively greater proportion of filaments with the sequence identity/similarity (SI Appendix, Fig. S1), suggesting it HCM hcTNT1K124N and RCM hcTNT1E136K peptides were mo- is critical to TnT function in all striated muscle. Here we ex- tile (SI Appendix,Fig.S5). The increase in sliding velocities and plored the importance and mechanism of TNT1-mediated sup- higher proportion of motile filaments that contain mutant pression of contractile activity by investigating how its disruption, hcTNT1 peptides confirm less complete hcTNT1-mediated thin due to naturally occurring K124N, R130C, and E136K hcTnT filament inhibition. These results are in accord with our findings mutations, influences relaxation across a host of distinct model overall, in Drosophila hearts and myofibrils, and rat myofibrils and systems. cardiomyocytes. We began by generating fly models to evaluate the effects of the TnT variants on whole heart function. Transgenic TnT in- Discussion corporated uniformly along Drosophila cardiac thin filaments Newly revealed structural details of the thin filament offer the (Fig. 1), and the mutant proteins engendered restricted heart prospect that investigations of its protein components can pro- tubes, reduced cardiac outputs, and slower relaxation rates (Figs. vide novel insight into how missense mutations provoke disease, 1 and 2), consistent with diastolic dysfunction and impaired re- and also into the more general mechanism of contractile regu- laxation that precede hypertrophy in humans (26, 28). Measure- lation. Since its discovery more than half a century ago, Tn’s ments of heart diameters following sequential treatment with ability to shut off muscle contraction has been attributed to TnI EGTA/EGTA,AM and blebbistatin revealed that the mutants’

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.2001692117 Madan et al. Downloaded by guest on September 25, 2021 PHYSIOLOGY

Fig. 5. Effects of the hcTnT RCM mutation on rat cardiac myofibrillar properties. (A) Myofibrils isolated from rat ventricles were incubated with myc-cTnTWT− or myc-cTnTE138K−containing Tn to replace endogenous Tn complexes. Western blotting was performed with an anti-TnT antibody to quantify the extent of Tn exchange. The first two lanes of the blot show mobility differences between purified recombinant rat myc-cTnT and endogenous rat cTnT, followed by rat myofibrillar cTnT composition after exchange with either myc-cTnTWT− or myc-cTnTE138K−containing Tn. (B) Tn exchange was quantified by dividing the signal intensity of the myc-tagged protein by the total intensity of cTnT (myc-cTnT + cTnT), which confirmed a similar extent of exchange for both myc- cTnTWT− and myc-cTnTE138K−containing Tn, within myofibrils (n = 4 biological replicates, 9 or 10 technical replicates each). (C) No difference was observed in sarcomere lengths along myofibrils exchanged with either myc-cTnTWT− or myc-cTnTE138K−containing Tn. (D) Analysis of mechanical properties of rat ven- tricular myofibrils exchanged with myc-cTnTWT− vs. myc-cTnTE138K−containing Tn revealed no difference in maximum tension generated. Significant dif-

ferences were observed in myofibrillar resting tension and in the duration of the linear phase of relaxation (tREL,LIN). However, the rate constant of this phase (kREL,LIN) was not affected (n = 4; six to eight myofibrils/replicate). Data are presented as mean ± SEM (*P ≤ 0.05).

cardiac restriction resulted from both Ca2+-dependent and up101 (E89K) flies that exhibit severe diastolic dysfunction and Ca2+-independent processes (Fig. 3). The former potentially IFM hypercontraction due to an impaired thin filament B state include altered Ca2+ handling and/or elevated Ca2+ sensitivity. (53), enabled mechanical evaluation of individual myofibrils The latter likely involve poor Tpm-based obstruction of myosin containing 100% mutant TnT. Mutant IFM myofibrils displayed binding sites, leading to a greater number of freely cycling a higher level of resting tension that was restored to WT levels by cross-bridges during both diastole and systole. Elevated Ca2+ the myosin inhibitor, BDM (Fig. 4). These results corroborated sensitivity of reconstituted thin filaments containing the K124N those obtained following cardiac-restricted overexpression of and R130C hcTnT HCM mutations was previously reported UAS-TnTE87K. We then established the generality of the results (34). However, at the cardiomyocyte level, a greater increase in in a vertebrate model system, by demonstrating elevated resting resting cell length was observed upon blebbistatin-mediated tension in isolated rat ventricular myofibrils with 75% of en- inhibition of myosin-dependent tension, in the effective ab- dogenous Tn replaced by Tn containing the rat equivalent of the sence of free intracellular Ca2+. Therefore, we propose that same, RCM-causing variant. Furthermore, relaxation kinetics TNT1 normally serves as an indispensable anchor that helps revealed a significant increase in the linear relaxation interval in restrain Tpm azimuthal motion, and the predominant cause myofibrils after Tn exchange, suggesting a slower rate of cross- underlying diastolic dysfunction is defective anchoring of the bridge detachment and force cessation (Fig. 5) (59). Likewise, Tpm overlap domain in the inhibitory position recently de- expression of the variant in rat ventricular cardiomyocytes in- scribed (9). Defective Tpm anchoring in a myosin-blocking creased relaxation times and reduced sarcomere lengths (SI position could also affect Ca2+ sensitivity: myosin binding en- Appendix, Fig. S4). These findings imply disinhibited thin fila- hances TnC’s affinity for Ca2+ (1), and, therefore, weakening of ments that permit excessive acto-myosin interactions, thereby the thin filament B state could drive both Ca2+-dependent and prolonging the time required for force termination, elevating Ca2+-independent effects of the TnT mutations, and represent myofibril stiffness, and abnormally shortening cardiomyocytes the ultimate cause of disease. at rest. To further investigate the E136K mutation, including its effect To study the effects of the TNT1 variant peptides on acto- on TNT1’s inhibitory role in skeletal muscle, we generated myosin interactions in a fully defined, reconstituted system, we upE87K knock-in Drosophila. These flies, which are reminiscent of conducted IVM assays using hcTNT1 peptides. In concordance

Madan et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 25, 2021 Fig. 6. In vitro effects of mutant hcTNT1 peptides on F-actin−Tpm sliding velocity. (A) IVM assays were performed over a range of myosin concentrations. The addition of Tpm to F-actin (dark gray line) reduced sliding velocities relative to F-actin alone (light gray line), at all myosin concentrations. This inhibitory effect was more pronounced upon addition of Tpm−hcTNT1WT (black line). Myosin concentration and the filament type (i.e., F-actin−Tpm ± hcTNT1WT) had significant effects on F-actin sliding velocity as determined by two-way ANOVA. (B–D) Filaments containing the three mutant hcTNT1 peptides (blue lines) showed higher sliding velocities relative to internal F-actin−Tpm−hcTNT1WT controls (black lines), at all myosin concentrations tested. Two-way ANOVA confirmed significant effects of filament type (WT vs. mutant hcTNT1-containing filaments) as well as myosin concentration, on F-actin−Tpm sliding velocity. Note, motility of the internal F-actin−Tpm−hcTNT1WT controls, from each experiment, did not significantly differ (SI Appendix, Fig. S8). Data are presented as mean ± SEM (n = 2 to 7 replicates; 20 to 30 filaments/replicate).

with previous work, Tpm is inhibitory in this context, and WT tail complexed with overlapping Tpms, along thin filaments and in hcTNT1 exaggerates this inhibition (12, 13). Both sliding speeds isolation (9, 11). Taking into account these structures, previous and the number of motile filaments were affected, particularly findings, and our current results, we offer a plausible mechanism under subsaturating myosin conditions (Fig. 6A and SI Appendix, for TNT1’s role in anchoring the Tpm overlap region and, hence, Fig. S5). Therefore, in addition to structural evidence that implies its contribution to contractile inhibition, as well as the molecular TNT1 can restrict Ca2+-dependent motion of the Tpm end-to-end origins of cardiomyopathy. Numerous electrostatic contacts be- overlap domain along Tn-regulated thin filaments (9), we verified tween successive F-actin protomers and Tpm, along its entire earlier IVM data that suggest hcTNT1WT alone can likewise temper length including the end-to-end overlap, seemingly establish an myosin-based shifts in Tpm and, ergo, enhance Tpm’s inhibitory energetically favorable configuration where Tpm impedes acto- effects (12, 13). Filaments reconstituted with each of the three myosin associations and inherently promotes relaxation (8, 12, mutant hcTNT1 peptides were, nevertheless, propelled at consid- 13, 15, 35–41). TNT1 binds to the Tpm overlap domain via a series erably higher velocities compared to F-actin−Tpm−hcTNT1WT of conserved amino acids (11, 18–20). This high-affinity interac- (Fig. 6 B–D). These observations indicate inefficient blocking of tion is predicted to occur through integrated hydrophobic net- acto-myosin activity due to less effective inhibitory Tpm positioning works and multiple, closely packed salt bridges that involve several and, thus, impaired hcTNT1-mediated thin filament regulation by invariant residues (11). When bound, TNT1 enhances the affinity the HCM/RCM variants. and the interaction energetics of Tpm for F-actin (11, 18, 21–25). Cryo-EM and computationally driven docking protocols have We submit that TNT1 binding to Tpm could, in turn, optimize produced contemporary models of the N-terminal, α-helical TNT1 the accessibility of charged amino acid sidechains of the Tpm

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.2001692117 Madan et al. Downloaded by guest on September 25, 2021 end-to-end overlap, and its surrounding regions, to their oppo- Measurement of EGTA/EGTA,AM-Induced and Blebbistatin-Induced Changes in sitely charged binding partners along adjacent actin protomers Cardiac Dimensions. Semiintact preparations of beating hearts from twenty > (16). Precise TNT1−Tpm coupling may thereby, at least in part, 3-wk-old, female Hand TnT flies were filmed and assessed prior to and – following incubations with EGTA/EGTA,AM-, and, subsequently, blebbistatin- account for the enhanced affinity of Tpm for F-actin (18, 23 25). containing AHL, as delineated previously (15, 17, 51). EGTA is a high-affinity Additionally, putative interactions directly between TNT1 and Ca2+ chelator, EGTA,AM is a cell-permeant form of EGTA, and blebbistatin is a actin could also fortify F-actin−Tpm binding. The augmented small-molecule myosin inhibitor. F-actin−Tpm attractive forces may, consequently, stabilize Tpm’s intrinsic inhibitory positioning and heighten the energetic de- Drosophila IFM Myofibril Isolation and Mechanics. IFM myofibrils were isolated mands required to uniformly displace Tpm for activation. Earlier using a modified version of a published protocol (65). Myofibrils were pel- studies have shown HCM-causing mutations that remove or re- leted at 850 × g at 4 °C for 5 min and resuspended in 300 μL of bath solution place charged residues located throughout and adjacent to TNT1’s (66). A drop of suspended myofibrils was plated on a 15 °C chamber of an inverted microscope, and 1 mL of bath solution was added. Myofibrils were lifted conserved Tpm-binding element can reduce the affinity of TNT1 horizontally between a glass microtool, controlled by a length-controlled for Tpm (18, 34) and of Tpm for F-actin (18). These findings are motor (Mad City Labs), and a calibrated cantilevered force probe (compli- consistent with an interface between TNT1 and Tpm that can be ance of 12.26 μm/μN) (56). Then, 7 to 16 myofibrils were stretched incremen- disrupted by amino acid substitutions (11). Perturbations which tally to determine resting tension at different sarcomere lengths at pCa 9. weaken TNT1−Tpm interaction may dampen TNT1’s effect on Tension was also assessed in the presence of 50 mmol/L BDM. Data were F-actin−Tpm contacts, or disrupt TNT1−actin associations and, collected and analyzed using customized LabView software. thus, ease restrictions on Tpm motion that compromise normal inhibition of cross-bridge binding at rest. Additionally, by buttress- Ex Vivo Exchange of Myc-cTnT within Rat Myofibrils. Rat myc-tagged WT Tnnt2 − and the RCM mutant (E138K) were cloned, expressed, purified, and recon- ing the Tpm Tpm overlap, TNT1 increases cooperative activation stituted as Tn complexes (67). Myofibrils isolated from Sprague–Dawley rat (12, 24), and likely inactivation, of the thin filament. Mutations that ventricles (68) were resuspended in a solution containing 15 μM recombi- reduce TNT1−Tpm binding may influence the latter by diminishing nant Tn complex and incubated overnight at 4 °C to replace endogenous Tn the cooperative propagation of inhibitory positioning of Tpm to the (69, 70). The extent of exchange was quantified via Western blot analysis. next Tpm along the actin filament, resulting in a greater number of exposed myosin binding sites at rest, and impaired relaxation as- Vertebrate Myofibril Mechanics. A small bundle of Tn-exchanged ventricular sociated with early cardiac remodeling and disease. In sum, we myofibrils was mounted between two microtools, one of which was at- μ μ propose a molecular basis for the noncanonical, regulatory role of tached to a calibrated cantilevered force probe (8.67 m/ N). Myofibrils were activated and relaxed using standard procedures of rapid solution

TnT, acting to reinforce inhibitory positioning of the end-to-end PHYSIOLOGY switching to investigate contraction and relaxation kinetics (58, 59, 71). Data overlap domain of Tpm. This additional layer of control comple- were collected and analyzed using customized LabView software. ments TnI-mediated inhibition and is necessary for normal muscle relaxation, and its disruption potentially accounts for the most Immunohistochemistry and Fluorescence Imaging of Drosophila Muscle. Confocal proximal, disease-inducing effects of specific TNNT2 mutations. and fluorescence microscopy of heart tubes and IFMs were performed as de- scribed previously (14, 15). GFP-Zasp52−expressing flies were generated via Materials and Methods standard crosses. In situ and isolated IFM myofibrils were stained with Alexa- Detailed information regarding materials and methods is available in SI 568 phalloidin (1:100 in phosphate-buffered saline), mounted on a glass slide, × × Appendix. and imaged at 63 and 100 , respectively, with a Leica TCS SPE RGBV confocal microscope (Leica Microsystems). The average distance between adjacent GFP- tagged Z discs, from stained hemithoraces and isolated myofibrils imaged Construction of UAS-TnT Transgenes and Transgenic Drosophila. A full-length exclusively at 100×, was measured using Fiji (n = 220 to 240 sarcomeres). fly TnT (up) construct, RE18550, was obtained from the Drosophila Genomics Resource Center and cloned into the pUASTattB vector. HCM/RCM mutations, hcTNT1 Expression and Purification. The TNNT2 sequence encoding residues 1 K75N, R81C, E87K, were introduced via the QuikChange II Site-Directed Mu- to 156 of hcTNT1 was cloned into a pET15b expression vector. K124N, R130C, tagenesis Kit (Agilent Technologies, #200523). Transgenic lines were gener- and E136K substitutions were introduced using site-directed mutagenesis ated using the PhiC31 integrase system (14, 45). To create homozygous upE87K (described above). Peptides were expressed and purified as outlined in Hinkle flies, a point mutation was introduced into the TnT-encoding up gene via et al. (25). CRISPR-Cas9 technology, following published protocols (63).

In Vitro Motility. Sliding speeds of Alexa-568-phalloidin-labeled rabbit skel- Fly Husbandry. Flies were raised at 25 °C on standard medium. Heart- etal F-actin (10 nmol/L), and filaments decorated with 300 nmol/L of both restricted transgene expression was achieved by the GAL4-UAS bipartite bovine cardiac Tpm and WT or mutant hcTNT1, over purified, full-length expression system (46), using the Hand4.2-GAL4 cardiac driver line. rabbit skeletal myosin (72) were measured via IVM assays (73); 20 to 30 fil- aments per replicate were manually tracked and measured over a range of Drosophila In Vivo Cardiac Analysis. Adult hearts coexpressing transgenic UAS- myosin concentrations (12.5 μg/mL to 100 μg/mL) at 30 °C, pH 7.2, and an TnT constructs and the tandem-tomato (tdtK) fluorescent protein (47, 48) ionic strength of 37 mmol/L, in two to seven replicate experiments using the were visualized through the cuticle of live flies. Twenty-six 3-wk-old animals Fiji plugin, MTrackJ. were anesthetized, glued to a coverslip, and their cardiac tubes recorded at 250 frames per second using a Hamamatsu Orca Flash 4.0 CMOS camera Statistical Analysis. Statistical analyses were performed using GraphPad Prism (Hamamatsu Photonics K.K.) on a Zeiss Imager.M1 microscope (Carl Zeiss Mi- software (v8.1.3). Details of analyses, and raw data (as scatter plots), are × croscopy) with a 25 air lens. Chamber areas were determined by outlining the provided in SI Appendix, Figs. S6 and S7. region of the dorsal vessel spanning Ostia 2 and 3 in Fiji (https://imagej.net/Fiji). Data Availability. All data, associated protocols, methods, and sources of materials Drosophila In Situ Cardiac Analysis. The heart tubes of 24 to 36 3-wk-old, are available in the main text or SI Appendix. female Hand > TnT flies were surgically exposed under oxygenated AHL at 25 °C (64). Imaging of beating, semiintact preparations, and analysis of ACKNOWLEDGMENTS. This work was supported by NIH Grants R01HL124091 cardiac performance using SOHA software, were performed as described (A.C.), R01HL063774 (L.S.T.), 2K12 HD057022-11 (K.C.W.), R01HL114940 (B.J.B.), previously (49, 50). and R01HL108917 and R01HL137259 (B.O.).

1. L. S. Tobacman, Thin filament-mediated regulation of cardiac contraction. Annu. Rev. 3. M. A. Geeves, “Thin filament regulation” in Comprehensive Biophysics,E.H.Egelman,Y.E. Physiol. 58, 447–481 (1996). Goldman, E. M. Ostap, Eds. (Academic, Oxford, United Kingdom, 2012), Vol. 4, pp. 251–267. 2. J. C. Tardiff, Thin filament mutations: Developing an integrative approach to a 4. W. Lehman, Thin filament structure and the steric blocking model. Compr. Physiol. 6, complex disorder. Circ. Res. 108, 765–782 (2011). 1043–1069 (2016).

Madan et al. PNAS Latest Articles | 9of10 Downloaded by guest on September 25, 2021 5. S. E. Hitchcock-DeGregori, B. Barua, “Tropomyosin structure, function, and interac- 41. W. Lehman, M. Orzechowski, X. E. Li, S. Fischer, S. Raunser, Gestalt-binding of tions: A dynamic regulator” in Fibrous Proteins: Structures and Mechanisms,D.A.D. tropomyosin on actin during thin filament activation. J. Muscle Res. Cell Motil. 34, Parry, J. Squire, Eds. (Springer, 2017), pp. 253–284. 155–163 (2013). 6. X. E. Li, W. Lehman, S. Fischer, The relationship between curvature, flexibility and 42. L. T. Reiter, L. Potocki, S. Chien, M. Gribskov, E. Bier, A systematic analysis of human persistence length in the tropomyosin coiled-coil. J. Struct. Biol. 170, 313–318 (2010). disease-associated gene sequences in Drosophila melanogaster. Genome Res. 11, 7. S. E. Hitchcock-DeGregori, Tropomyosin: Function follows structure. Adv. Exp. Med. 1114–1125 (2001). Biol. 644,60–72 (2008). 43. A. Cammarato et al., Drosophila muscle regulation characterized by electron mi- 8. K. C. Holmes, W. Lehman, Gestalt-binding of tropomyosin to actin filaments. J. Muscle croscopy and three-dimensional reconstruction of thin filament mutants. Biophys. J. Res. Cell Motil. 29, 213–219 (2008). 86, 1618–1624 (2004). 9. Y. Yamada, K. Namba, T. Fujii, Cardiac muscle thin filament structures reveal calcium 44. W. B. Limpitikul, M. C. Viswanathan, B. O’Rourke, D. T. Yue, A. Cammarato, L-type regulatory mechanism. Nat. Commun. 11, 153 (2020). calcium channels are a major source of plasmalemmel calcium influx for Drosophila 10. M. Orzechowski, X. E. Li, S. Fischer, W. Lehman, An atomic model of the tropomyosin cardiomyocytes. Biophys. J. 116, 152a–153a (2019). cable on F-actin. Biophys. J. 107, 694–699 (2014). 45. A. C. Groth, M. Fish, R. Nusse, M. P. Calos, Construction of transgenic Drosophila by 11. E. Pavadai, M. J. Rynkiewicz, A. Ghosh, W. Lehman, Docking troponin T onto the using the site-specific integrase from phage phiC31. Genetics 166, 1775–1782 (2004). tropomyosin overlapping domain of thin filaments. Biophys. J. 118, 325–336 (2020). 46. A. H. Brand, N. Perrimon, Targeted as a means of altering cell fates 12. L. S. Tobacman et al., The troponin tail domain promotes a conformational state of the and generating dominant phenotypes. Development 118, 401–415 (1993). thin filament that suppresses myosin activity. J. Biol. Chem. 277, 27636–27642 (2002). 47. N. C. Shaner et al., Improved monomeric red, orange and yellow fluorescent proteins de- 13. R. Maytum, M. A. Geeves, S. S. Lehrer, A modulatory role for the troponin T tail rived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004). – domain in thin filament regulation. J. Biol. Chem. 277, 29774 29780 (2002). 48. M. P. Klassen et al., Age-dependent diastolic heart failure in an in vivo Drosophila 14. M. C. Viswanathan, A. C. Blice-Baum, W. Schmidt, D. B. Foster, A. Cammarato, Pseudo- model. elife 6, e20851 (2017). acetylation of K326 and K328 of actin disrupts Drosophila melanogaster indirect 49. M. Fink et al., A new method for detection and quantification of heartbeat param- flight muscle structure and performance. Front. Physiol. 6, 116 (2015). eters in Drosophila, zebrafish, and embryonic mouse hearts. Biotechniques 46, 15. M. C. Viswanathan et al., Distortion of the actin A-Triad results in contractile disin- 101–113 (2009). – hibition and cardiomyopathy. Cell Rep. 20, 2612 2625 (2017). 50. A. Cammarato, S. Ocorr, K. Ocorr, Enhanced assessment of contractile dynamics in “ ‐ ” ’ 16. W. Schmidt, A. Cammarato, The actin A Triad s role in contractile regulation in Drosophila hearts. Biotechniques 58,77–80 (2015). health and disease. J. Physiol., 10.1113/JP276741 (2019). 51. W. A. Kronert et al., Prolonged cross-bridge binding triggers muscle dysfunction in a 17. M. C. Viswanathan et al., A role for actin flexibility in thin filament-mediated con- Drosophila model of myosin-based hypertrophic cardiomyopathy. eLife 7, e38064 (2018). tractile regulation and myopathy. Nat. Commun. 11 , 2417 (2020). 52. E. Fyrberg, C. C. Fyrberg, C. Beall, D. L. Saville, Drosophila melanogaster troponin-T 18. T. Palm, S. Graboski, S. E. Hitchcock-DeGregori, N. J. Greenfield, Disease-causing mutations engender three distinct syndromes of myofibrillar abnormalities. J. Mol. mutations in cardiac troponin T: Identification of a critical tropomyosin-binding re- Biol. 216, 657–675 (1990). – gion. Biophys. J. 81, 2827 2837 (2001). 53. M. C. Viswanathan, G. Kaushik, A. J. Engler, W. Lehman, A. Cammarato, A Drosophila 19. A. Hinkle, L. S. Tobacman, Folding and function of the troponin tail domain. Effects of melanogaster model of diastolic dysfunction and cardiomyopathy based on impaired cardiomyopathic troponin T mutations. J. Biol. Chem. 278, 506–513 (2003). troponin-T function. Circ. Res. 114,e6–e17 (2014). 20. J. P. Jin, S. M. Chong, Localization of the two tropomyosin-binding sites of troponin T. 54. M. Kulke et al., Kettin, a major source of myofibrillar stiffness in Drosophila indirect Arch. Biochem. Biophys. 500, 144–150 (2010). flight muscle. J. Cell Biol. 154, 1045–1057 (2001). 21. P. Jackson, G. W. Amphlett, S. V. Perry, The primary structure of troponin T and the 55. Y. Hao, S. I. Bernstein, G. H. Pollack, Passive stiffness of Drosophila IFM myofibrils: A interaction with tropomyosin. Biochem. J. 151,85–97 (1975). novel, high accuracy measurement method. J. Muscle Res. Cell Motil. 25, 359–366 (2004). 22. J. R. Pearlstone, L. B. Smillie, The binding site of skeletal alpha-tropomyosin on 56. F. Colomo, N. Piroddi, C. Poggesi, G. te Kronnie, C. Tesi, Active and passive forces of troponin-T. Can. J. Biochem. 55, 1032–1038 (1977). isolated myofibrils from cardiac and fast skeletal muscle of the frog. J. Physiol. 500, 23. L. E. Hill, J. P. Mehegan, C. A. Butters, L. S. Tobacman, Analysis of troponin- 535–548 (1997). tropomyosin binding to actin. Troponin does not promote interactions between 57. E. M. Ostap, 2,3-Butanedione monoxime (BDM) as a myosin inhibitor. J. Muscle Res. tropomyosin molecules. J. Biol. Chem. 267, 16106–16113 (1992). Cell Motil. 23, 305–308 (2002). 24. S. Schaertl, S. S. Lehrer, M. A. Geeves, Separation and characterization of the two 58. C. Tesi, F. Colomo, S. Nencini, N. Piroddi, C. Poggesi, The effect of inorganic phosphate functional regions of troponin involved in muscle thin filament regulation. Bio- on force generation in single myofibrils from rabbit skeletal muscle. Biophys. J. 78, chemistry 34, 15890–15894 (1995). 3081–3092 (2000). 25. A. Hinkle, A. Goranson, C. A. Butters, L. S. Tobacman, Roles for the troponin tail 59. C. Tesi, N. Piroddi, F. Colomo, C. Poggesi, Relaxation kinetics following sudden Ca2+ domain in thin filament assembly and regulation. A deletional study of cardiac tro- reduction in single myofibrils from skeletal muscle. Biophys. J. 83, 2142–2151 (2002). ponin T. J. Biol. Chem. 274, 7157–7164 (1999). 60. D. J. Hartshorne, S. V. Perry, V. Davies, A factor inhibiting the adenosine triphosphatase 26. A. C. Garfinkel, J. G. Seidman, C. E. Seidman, Genetic pathogenesis of hypertrophic activity and the superprecipitation of actomyosin. Nature 209,1352–1353 (1966). and . Heart Fail. Clin. 14, 139–146 (2018). 61. D. J. Hartshorne, S. V. Perry, M. C. Schaub, A protein factor inhibiting the magnesium- 27. D. Masarone et al., Epidemiology and clinical aspects of genetic cardiomyopathies. activated adenosine triphosphatase of desensitized actomyosin. Biochem. J. 104, Heart Fail. Clin. 14, 119–128 (2018). 907–913 (1967). 28. E. M. Green et al., A small-molecule inhibitor of sarcomere contractility suppresses 62. S. Perry, H. Cole, J. Head, F. Wilson, “Localization and mode of action of the inhibitory protein hypertrophic cardiomyopathy in mice. Science 351, 617–621 (2016). component of the troponin complex” in Cold Spring Harbor Symposia on Quantitative Biol- 29. L. Thierfelder et al., Alpha-tropomyosin and cardiac troponin T mutations cause familial – hypertrophic cardiomyopathy: A disease of the sarcomere. Cell 77,701–712 (1994). ogy, J. Gordon, Ed. (Cold Spring Harbor Laboratory Press, 1973), Vol. 37, pp. 251 262. ’ ‐ ‐ 30. D. Szczesna et al., Altered regulation of cardiac muscle contraction by troponin T 63. S. J. Gratz, C. D. Rubinstein, M. M. Harrison, J. Wildonger, K. M. O Connor Giles, CRISPR mutations that cause familial hypertrophic cardiomyopathy. J. Biol. Chem. 275, Cas9 genome editing in Drosophila. Curr. Protoc. Mol. Biol. 111, 31.2.1-31.2.20 (2015). 624–630 (2000). 64. G. Vogler, K. Ocorr, Visualizing the beating heart in Drosophila. J. Vis. Exp., 1425 31. T. Miller et al., Abnormal contractile function in transgenic mice expressing a familial (2009). hypertrophic cardiomyopathy-linked troponin T (I79N) mutation. J. Biol. Chem. 276, 65. D. M. Swank, Mechanical analysis of Drosophila indirect flight and jump muscles. – 3743–3755 (2001). Methods 56,69 77 (2012). 32. K. Harada, J. D. Potter, Familial hypertrophic cardiomyopathy mutations from dif- 66. J. S. Walker, L. A. Walker, K. Margulies, P. Buttrick, P. de Tombe, Protein kinase A ferent functional regions of troponin T result in different effects on the pH and Ca2+ changes calcium sensitivity but not crossbridge kinetics in human cardiac myofibrils. – sensitivity of cardiac muscle contraction. J. Biol. Chem. 279, 14488–14495 (2004). Am. J. Physiol. Heart Circ. Physiol. 301, H138 H146 (2011). 33. E. P. Manning, P. J. Guinto, J. C. Tardiff, Correlation of molecular and functional ef- 67. P. L. Engel et al., Identification of a region of important in signaling fects of mutations in cardiac troponin T linked to familial hypertrophic cardiomyop- cross-bridge-dependent activation of cardiac . J. Biol. Chem. 282, – athy: An integrative in silico/in vitro approach. J. Biol. Chem. 287, 14515–14523 (2012). 183 193 (2007). 34. B. Gangadharan et al., Molecular mechanisms and structural features of cardiomyopathy- 68. M. Y. Jeong et al., Histone deacetylase activity governs diastolic dysfunction through causing troponin T mutants in the tropomyosin overlap region. Proc. Natl. Acad. Sci. U.S.A. a nongenomic mechanism. Sci. Transl. Med. 10, eaao0144 (2018). 114, 11115–11120 (2017). 69. B. Brenner, T. Kraft, L. C. Yu, J. M. Chalovich, Thin filament activation probed by fluores- 35. J. H. Brown et al., Structure of the mid-region of tropomyosin: Bending and binding cence of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-labeled sites for actin. Proc. Natl. Acad. Sci. U.S.A. 102, 18878–18883 (2005). troponin I incorporated into skinned fibers of rabbit psoas muscle. Biophys. J. 77, 36. X. E. Li et al., Tropomyosin position on F-actin revealed by EM reconstruction and 2677–2691 (1999). computational chemistry. Biophys. J. 100, 1005–1013 (2011). 70. M. She, D. Trimble, L. C. Yu, J. M. Chalovich, Factors contributing to troponin ex- 37. B. Barua, M. C. Pamula, S. E. Hitchcock-DeGregori, Evolutionarily conserved surface change in myofibrils and in solution. J. Muscle Res. Cell Motil. 21, 737–745 (2000). residues constitute actin binding sites of tropomyosin. Proc. Natl. Acad. Sci. U.S.A. 108, 71. F. Colomo, S. Nencini, N. Piroddi, C. Poggesi, C. Tesi, “Calcium dependence of the 10150–10155 (2011). apparent rate of force generation in single striated muscle myofibrils activated by 38. J. von der Ecken et al., Structure of the F-actin-tropomyosin complex. Nature 519, rapid solution changes” in Mechanisms of Work Production and Work Absorption in 114–117 (2015). Muscle, H. Sugi, G. H. Pollack, Eds. (Springer, 1998), pp. 373–382. 39. B. Barua, P. M. Fagnant, D. A. Winkelmann, K. M. Trybus, S. E. Hitchcock-DeGregori, A 72. S. S. Margossian, S. Lowey, “Preparation of myosin and its subfragments from rabbit periodic pattern of evolutionarily conserved basic and acidic residues constitutes the skeletal muscle” in Methods in Enzymology, N. O. Kaplan, S. P. Colowick, Eds. (Aca- binding interface of actin-tropomyosin. J. Biol. Chem. 288, 9602–9609 (2013). demic, 1982), Vol. 85, pp. 55–71. 40. M. J. Rynkiewicz et al., Tropomyosin must interact weakly with actin to effectively 73. S. J. Kron, J. A. Spudich, Fluorescent actin filaments move on myosin fixed to a glass regulate thin filament function. Biophys. J. 113, 2444–2451 (2017). surface. Proc. Natl. Acad. Sci. U.S.A. 83, 6272–6276 (1986).

10 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.2001692117 Madan et al. Downloaded by guest on September 25, 2021