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Mimicking B crystallin phosphorylation

at serine 45 and 59 in vivo

A thesis submitted to the

Graduate School

of the University of Cincinnati

in partial fulfillment of the

requirements for the degree of

Masters of Science

in the Department of Molecular and Developmental Biology

of the College of Medicine

Shawna K.B. Hottinger

B.S. Marshall University, May 2004

June 2009

Committee Chair: Jeffrey Robbins, Ph.D.

Abstract

The small heat shock protein B crystallin (CryAB) is involved in a myriad of cellular functions including chaperone activity, apoptosis, and protein degradation. Its prevalence in cardiac myopathies and protein aggregation disorders has generated immense interest in this protein. Recent in vitro studies advocate a role for phosphorylation in the regulation and solubility of CryAB. To determine the effects of phosphorylation on cardiac CryAB’s localization, aggregation, and function in vivo, this investigation used a cardiac-specific, inducible bi-transgenic system in which CryAB serine residues 45 and

59 were mutated singly and in combination to aspartic acid to mimic a constitutively phosphorylated state (CryABS45D, CryABS59D, CryABS45/59D). The data suggest that mimicking phosphorylation at serine 45 is benign, that a negative charge at serine 59 causes dilated hypertrophy of the heart, and that aspartic acid substitution at serine 45 and

59 causes formation of peri-nuclear intracellular aggregates and cardiomyopathy. Further in vivo studies of CryAB and its phosphorylation under normal and disease conditions are required in order to advance CryAB as a therapeutic target.

iii

Mimicking B crystallin phosphorylation at serine 45 and 59 in vivo

Table of Contents

Title Page ii

Abstract iii

Table of Contents v

Introduction 1-7

Rationale for Thesis Project 7-8

Significance 8

Experimental Approach 8-11

Results & Discussion 11-27

Founder Screening 11

tTA System 12

CryABS59D 12-17

CryABS45D 17-19

CryAB S45/59D 19-22

Experimental Approach Addendum 22-23

HSPB2 23-26

Conclusion 26-27

Materials & Methods 28-33

Funding 33

Figures 34-50

References 51-57

Introduction

Small heat shock proteins (sHSP) are a highly divergent family of proteins prevalent from bacteria to higher eukaryotes (Haslbeck et al., 2005). Although exhibiting great variance in sequence and mass, most family members exhibit the following commonalities: 1) a central, conserved domain of approximately 90 residues ( -crystallin domain) flanked by two variable domains (N- and C-terminal), 2) a molecular mass of

12-43kDa, 3) formation of large, dynamic oligomers, 4) stress-responsive induction, and

5) chaperone activity via binding and sequestration of denatured protein (Augusteyn,

2004; Haslbeck et al., 2005; Sun and MacRae, 2005a). Although often involved in disease prevention due to their molecular chaperone function, sHSP have received attention due to their association with a variety of pathological conditions (Sun and

MacRae, 2005b).

Initially studied due to its predominance in the vertebrate eye lens, B crystallin

(CryAB) is the most abundant sHSP in cardiac and skeletal muscle, constituting approximately three percent of total myofibril protein (Kato et al., 1991; Lutsch and al,

1997; Lutsch et al., 1997). Low level CryAB expression is constitutive in lens epithelium, heart, kidney, skeletal muscle, lung, and brain and can be upregulated in response to certain physiological stressors such as heat shock and hypertonic stress (Bhat et al., 1999;

Bhat and Nagineni, 1989; Dasgupta et al., 1992; Dubin et al., 1989; Horwitz, 1992; Iwaki et al., 1990; Iwaki et al., 1989; Klemenz et al., 1991). The CryAB gene shares an intergenic promoter region with another sHSP, myotonic dystrophy kinase binding protein (MKBP) otherwise known as HSPB2 (Iwaki et al., 1997). While intergenic promoters often indicate co-expression or expression-repression of the head-to-tail genes,

it has not been determined whether HSPB2 and CRYAB have overlapping regulatory elements (Iwaki et al., 1997). Accumulating data implicate CryABwildtype in a myriad of cellular functions including autokinase activity, cytoskeletal modulation, protein degradation, apoptosis, Golgi organization, DNA binding, and molecular chaperoning

(Boelens et al., 2001; den Engelsman et al., 2003; Djabali et al., 1999; Fujita et al., 2004;

Gangalum et al., 2004; Horwitz, 1992; Ito et al., 2002; Kamradt et al., 2001; Kantorow and Piatigorsky, 1994; Morrison et al., 2003; Pietrowski et al., 1994; Quinlan, 2002). The high conservation of the CryAB gene between species is considered an indication of stringent structural and functional restrictions (de Jong and Hendriks, 1986). This selective degree of sequence preservation can easily be seen through comparison of the human and murine amino acid sequence (Figure 1).

CryAB is associated with a number of pathologic conditions. A familial study in 1998 found that the presence of a missense mutation in CryAB, an arginine to glycine substitution at residue 120 (CryABR120G), cosegregated with the neuromuscular disorder desmin-related myopathy (DRM) (Vicart et al., 1998). A transgenic mouse model with adult, heart-specific expression of murine CryABR120G recapitulates the cardiomyopathy of the human disease, indicating causality (Wang et al., 2001a). One hundred percent of these animals die of heart failure between five and seven months of age (Wang et al.,

2001a). At a cellular level, these transgenics evidence decreased CryAB solubility, formation of aggresomes, presence of soluble amyloid oligomer, and cardiomyocyte toxicity (den Engelsman et al., 2005; Sanbe et al., 2004; Sanbe et al., 2005). In addition to the CryABR120G mutation, CryABwildtype has been found in inclusion bodies, intracellular accumulations of aggregated proteins, in many human disorders including

Alexander disease, Lewy body disease, Alcoholic liver disease, and Alzheimer’s (Head and Goldman, 2000; Iwaki et al., 1989; Kato et al., 2001; Lowe et al., 1992; Renkawek et al., 1994; Stege et al., 1999). CryAB’s presence in cardiac myopathies and protein aggregation disorders has generated immense interest in this protein. However, whether the presence of CryAB in these myopathies is a secondary effect, a protective or maladaptive response, or an underlying cause remains largely undetermined.

Mechanistic models of the in vivo activity of CryABwildtype and CryABR120G will be critical in defining CryAB’s role in human disease.

In spite of high gene conservation suggesting CryAB’s necessity and its promising expression profile during cardiac development leading to hypothesized functions in myogenic differentiation and cardiac morphogenesis, the CryAB/HSPB2 double knockout mouse (KO) was not embryonic lethal, showed normal Mendelian ratios in heterozygous matings, and did not display any overt cardiac malformations (Andley et al., 2001; Brady et al., 1997; Brady et al., 2001; de Jong and Hendriks, 1986; Dubin et al.,

1991; Gopal-Srivastava et al., 1995; Sugiyama et al., 2000). It did, however, develop cytoplasmic inclusion bodies in the lens and showed increased intracellular levels of desmin, a known binding partner of CryABwildtype in the heart (Andley et al., 2001;

Bennardini et al., 1992; Brady et al., 1997). These mice suffered late-onset postural defects due to progressive skeletal muscular dystrophy that manifested at seven weeks of age and resulted in death at approximately forty weeks (Morrison et al., 2004). Further characterization of the KO heart revealed that it exhibited no developmental, morphological, or functional defects until challenged by a stress condition, such as ischemia, under which it displayed increased necrosis and apoptosis (Morrison et al.,

2004). Investigators have speculated that the abundance of other cardiac sHSPs and their structural similarities to CryABwildtype and HSPB2 result in functional redundancy under unstressed conditions, but that the loss of CryABwildtype cannot be compensated for during stress (Morrison et al., 2004). Basal characterization and ischemia-reperfusion studies of the KO crossed with a transgenic CryABwildtype line (KOxCryABwildtype) suggest that

CryABwildtype and HSPB2 have the non-redundant roles of preserving cardiac mechanical structure and energetic balance respectively (Pinz et al., 2008).

Research concerning the importance of CryABwildtype phosphorylation is beginning to surface (Gaestel, 2002; Inaguma et al., 2001; Ito et al., 2001; Ito et al., 1997; Kamradt et al., 2002; Kato et al., 2001; Kato et al., 2002; Kato et al., 1998). CryABwildtype can be phosphorylated at serines 19, 45, and 59, depending on cell cycle state and signals from the extracellular environment (Ito et al., 1997; Kato et al., 1998). Serines 19, 45 and 59 can all be phosphorylated by 42/44 MAPK and p38 MAPK; however, serine 45 and 59 show preferential phosphorylation by 42/44 MAPK and p38 MAPK respectively (Ito et al., 1997; Kato et al., 1998). Although basal levels of phosphorylation are low,

CryABwildtype phosphorylation increases during a stress response (Eaton et al., 2001;

Golenhofen et al., 1998). Serines 19, 45, and 59 are located in the N-terminus, which contains hydrophobic regions putatively involved in binding of chaperone substrates (Das and Surewicz, 1995; Raman and Rao, 1994). These data suggest that alteration of the hydrophobic environment via phosphorylation could influence CryAB conformation and activity.

The preferential phosphorylation of serine 45 and 59 by distinct kinases is merely one indication that residue-specific phosphorylation may regulate distinct CryAB functions.

In a rat whole-heart ischemia-reperfusion model, it was found that CryABwildtype phosphorylation increased three fold at serines 45 and 59, but not at serine 19 in response to ischemia; however, neither oligomer size nor translocation of CryABwildtype to the Z lines of the myocardium was coupled with phosphorylation (Eaton et al., 2001;

Golenhofen et al., 1998). Although CryABwildtype was found to have cytoprotective effects in response to ischemia, the function of the endogenous differential phosphorylation in this model remained undetermined (Eaton et al., 2001). However, an in vitro experiment using cardiac myocytes infected with adenoviral constructs of nonphosphorylatable

(serine to alanine: CryABAAA) and phosphomimetic (serine to glutamic acid; CryABEEE and CryABAAE) CryAB determined that a negative charge at residue 59 was necessary and sufficient to confer endogenous levels of protection from stress-induced apoptosis in culture (Morrison et al., 2003). Two recent studies suggest that during cardiac ischemia

CryAB translocates to the mitochondria, becomes phosphorylated at serine 59, and remains at the mitochondria to mitigate damage during reperfusion (Jin et al., 2008;

Whittaker et al., 2009). In conjunction, these studies suggest that phosphorylation of serine 59 may mediate the role of CryABwildtype in cardiac protection from apoptosis in vivo.

In contrast, the specific role of phosphorylation of serine 45 is less clear. Serine 45 phosphorylation increases three fold in a whole-heart ischemia-reperfusion model as mentioned previously; the in vitro experiment using adenovirally infected cardiac myocytes did not investigate the effect of negative charge at residue 45 separately as it did for residue 59 (Eaton et al., 2001; Morrison et al., 2003). In addition, CryAB phosphorylation at serine 45 significantly increased in the brains of humans with

Alzheimer’s and Alexander’s disease, although phosphorylation at serine 59 was more prevalent (Kato et al., 2001). Intriguingly, CryAB phosphorylated at serine 45 was found only in the insoluble fractions in both Alzheimer’s and control brains (Kato et al., 2001).

Serine 45 phosphorylation involvement in the ubiquitin/proteasome pathway may explain this localization: in a cell culture model, dual aspartic acid substitution at serines 19 and

45 of CryAB enhanced interaction with FBX4, a component of the ubiquitin ligase SCF complex, and resulted in translocation to the insoluble fraction and stimulation of protein ubiquitination (den Engelsman et al., 2003). However, localization of serine 45 phosphorylated CryAB to the insoluble fraction could also be related to its co-localization with SC35 ‘nuclear’ speckles, a known reservoir for pre-mRNA splicing factors (den

Engelsman et al., 2005; Lamond and Spector, 2003; Spector et al., 1991). Overall, the function of serine 45 phosphorylation remains largely undetermined.

The CryABR120G missense mutation can be hyperphosphorylated in vivo, suggesting a possible role of phosphorylation in its pathogenesis (den Engelsman et al., 2005). In order to segregate the effects of the missense mutation from hyperphosphorylation, HeLa cells were infected with unmodified (CryABwildtype, CryABR120G), nonphosphorylatable (serine to alanine; CryABwildtype-AAA, CryABR120G-AAA) and hyperphosphomimetic (serine to aspartic acid: CryABwildtype-DDD, CryABR120G-DDD) adenoviral constructs. CryABwildtype,

CryABwildtype-AAA, and CryABR120G-AAA showed wildtype solubility patterns, whereas

CryABwildtype-DDD showed the decreased solubility characteristic of CryABR120G.

Interestingly, CryABR120G-DDD had decreased solubility even beyond that of unmodified

CryABR120G (den Engelsman et al., 2005). This discrepancy may merely be due to incomplete phosphorylation of the unmodified constructs in HeLa cells as opposed to

cardiomyocytes. However, the trend of increased negative charge in the hydrophopic N- terminal region of CryAB resulting in increased insolubility may very well contribute to the tendency of CryABR120G to aggregate and cardiomyopathy.

Rationale for Thesis Project

The above-mentioned studies suggest phosphorylation’s importance in the anti- apoptotic function, localization, and solubility of CryABwildtype. However, further research is needed to define the effects that differential phosphorylation has upon CryAB function in vivo under basal and disease conditions. I hypothesized that residue-specific and extent of phosphorylation regulates cardiac CryAB’s localization, aggregation, and function in vivo. I planned to define the mechanistic consequences of cardiac CryABwildtype phosphorylation in vivo using a cardiac-specific, inducible bi-transgenic system in which

CryAB serine residues 45 and 59 were mutated singly and in combination to aspartic acid to mimic a constitutively phosphorylated state (CryABS45D, CryABS59D, CryABS45/59D).

Although CryAB has low levels of phosphorylation under basal conditions, I predicted that the single-residue phosphomimetics would not have basal myopathy since

CryABwildtype phosphorylation levels increase under stress (Dasgupta et al., 1992;

Golenhofen et al., 1998; Klemenz et al., 1991). I hypothesized that the CryABS59D single residue phosphomimetic would be diffusely localized, mimicking the distribution of

CryABwildtype. I also predicted that no intracellular aggregate formations would be detected, and decreased cardiomyocyte apoptosis in response to stress would be observed. I expected that the CryABS45D single residue phosphomimetic would have nuclear speckle localization, no intracellular aggregate formation, and no change in

stress-induced cardiomyocyte apoptosis. I hypothesized that the CryABS45/59D double residue phosphomimetic would have peri-nuclear localization, would form intracellular aggregates, and would exhibit no change in stress-induced cardiomyocyte apoptosis.

Significance

As mentioned previously, CryAB is associated with a variety of pathologic conditions. The prevalence of increased phosphorylation of CryABwildtype and the

CryABR120G missense mutation in human disease suggests a possible role of phosphorylation in the etiology of CryAB-associated diseases. This investigation is necessary in order to differentiate whether CryAB phosphorylation is associative, protective, maladaptive, or causal in regards to cardiomyopathy in vivo under basal and disease conditions. As CryAB is a promising candidate for intervention dues to its involvement in many diseases, additional insight into the function of CryAB phosphorylation will facilitate development of therapeutic strategies for cardiomyopathies and protein aggregation disorders.

Experimental Approach

Differential phosphorylation of CryABwildtype can occur in cell culture lines under a variety of conditions (Gaestel, 2002; Inaguma et al., 2001; Ito et al., 2001; Ito et al.,

1997; Kamradt et al., 2002; Kato et al., 2001; Kato et al., 2002; Kato et al., 1998;

Morrison et al., 2004). An in vivo, cardiac-specific, inducible bi-transgenic model of altered CryAB protein was initiated. CryAB serine residues 45 and 59 were mutated singly and in combination to aspartic acid to mimic a constitutively phosphorylated state

(CryABS45D, CryABS59D, CryABS45/59D) in order to analyze this posttranslational modification in a more endogenous system (Figure 2). It is important to note that an untagged, murine CryAB sequence was used for generation of these transgenic models in order to minimize cross-species noise and artifacts. The phosphomimetic constructs were placed under regulation of the modified -myosin heavy chain ( MHC) promoter cassette MHCminTetO, and responder mice were generated as previously described

(Sanbe et al., 2003). These responder mice ( MHCminTetO:CryABmutant) were crossed with the previously characterized cardiac-specific transcriptional activator mice,

MHC:tTA line 55 to generate the double transgenic (Figure 2) (Sanbe et al., 2003). A

FVB/N wildtype background rather than the KO background was used in order to eliminate the loss of HSPB2 as a factor. A previously described constitutive CryABwildtype transgenic, CryABwildtype line 11, was used as a control for CryAB overexpression to confirm that any phenotype observed was not due simply to increased amounts of CryAB

(Wang et al., 2001b).

Inducible systems offer more precise, reversible, and flexible regulation of transgene expression than classic transgenic models, and tetracycline-based systems are currently the most commonly used (Sun et al., 2007). Precise control over transgene expression would permit imitation of the endogenous pattern of expression as CryAB endogenous protein expression and degree of phosphorylation increase under stress conditions (Bhat et al., 1999; Bhat and Nagineni, 1989; Dubin et al., 1989; Horwitz, 1992). An inducible strategy would also facilitate testing the reversibility of any deleterious effects caused by transgene expression. Despite the advantages of the tetracycline-off (tTA) and tetracycline-on (rtTA) systems, leaky expression can sometimes be limiting, especially in

models of toxic protein expression or those inherently hyper-sensitive to even minute levels of target protein expression (Sun et al., 2007). A tTA system was employed for this investigation because, in general, the rtTA system has not been successfully employed in the heart (Robbins et al., unpublished). Line 55, which carried the MHC:tTA construct, has no signs of myopathy during the first 4 months of life (Sanbe et al., 2003). However, tTA cardiomyopathy developed later in life, presumably due to the non-specific effects of the tTA on general transcriptional patterns (Sanbe et al., 2003). The characterization of the CryAB phosphomimetics therefore needed to be accomplished during this time span in order to avoid confounding effects of tTA cardiomyopathy on the phenotype. The three phosphomimetic responder line constructs (CryABS45D, CryABS59D, CryABS45/59D) were made by Howard Hughes Fellow Chet Villa (Robbins et al., unpublished).

Suitable phosphomimetic founder lines were selected on the following bases: 1) germ line transmission, 2) no RNA or protein expression in the absence of mating to the transactivator line (basal leak), 3) RNA and protein expression in the presence of the transactivator line in the absence of doxycycline treatment (expression), and 4) no RNA or protein expression in the presence of the transactivator line with doxycycline treatment

(tTA system) (Figure 2). RNA and protein expression were to be measured by RNA dot blot and Western blot analysis respectively. Alterations of function, structure, and protein localization were to first be assessed under non-stressed conditions. Functional changes were to be measured by echocardiography and structural alterations by histology and heart and body weight measurements. Protein localization was to be examined by immunohistochemistry and fractionation. After selection of appropriate lines and basal characterization of the CryAB phosphomimetic tTA system, re-assessment under

apoptotic stress would be done. Adriamycin injection was the main apoptotic model to be employed with the previously published transgenic Gαq model of peripartum apoptosis as an alternative (D'Angelo et al., 1997; Koh et al., 2002; Kumar et al., 1999; Lou et al.,

2006; Nakamura et al., 2000; Singal et al., 2000; Wu et al., 2002). In addition to the parameters previously mentioned, Western blot and immunohistochemistry would be used to assess alterations in intrinsic and extrinsic apoptotic pathway components.

Results & Discussion

Founder Screening

The CryABS45D, CryABS59D, and CryABS45/59D phosphomimetic responder lines had 6, 16, and 13 transgenic founders out of 290, 430, and 419 embryo injections respectively (Table 1). To reduce breeding times, founders were bred directly to the transactivator line. The resultant nontransgenic (NTG), transactivator line 55 single transgenic (tTA), responder line single transgenic (CryABS45D, CryABS59D, CryABS45/59D), and transactivator/responder double transgenic (tTAxCryABS45D, tTAxCryABS59D, tTAxCryABS45/59D) offspring were screened on the above-mentioned criteria. For the CryABS45D, CryABS59D, CryABS45/59D responder lines, 4,

13, and 12 had germ line transmission respectively (Table 1). However, many responder lines were eliminated due to basal leak in the single transgenic or lack of expression in the double transgenic. Founder lines that were germline, had low single transgenic responder leak, and significant double transgenic expression were then tested for doxycycline responsiveness (data not shown).

tTA System

Unfortunately, the tTA system proved refractory for this set of inducible phosphomimetic responder lines. A few trends surfaced irrespective of the specific phosphomimetic construct.

When doxycycline was administered prenatally by dosing the mother’s water, only extremely high expressing lines (>20 fold compared to endogenous CryAB levels) would ever express the transgenic protein. The lower level lines (<20 fold) were permanently silenced and had no protein expression even 7 weeks after cessation of doxycycline treatment. These results appear to be unique for the CryAB transgenes as we and others have not seen that effect (Robbins et al., unpublished; Sanbe et al., 2003; Sanbe et al., 2005). In contrast, doxycycline given postnatally, either at weaning (4 wks of age) or later (8wks or 12wks), could silence expression of low level expressing lines (≤8 fold), but only partially repress high level expressing lines (≥ 8 fold)

(Figures 3, 8, 9, 11). One potential explanation for this observation is prenatal doxycycline treatment causes activation of a chromatin silencing mechanism such as methylation. This possibility could be explored by gene-specific methylation analysis via digestion of DNA with methylation sensitive restriction enzymes followed by Southern blot or PCR amplification or via bisulfate reaction-based methods such as methylation specific PCR.

CryABS59D

Two lines were maintained for the CryABS59D construct, line 148 and line 104. Line

148 had good correlation between transgenic mRNA expression and fold CryAB protein expression over endogenous. The single transgenic responder did exhibit basal leak in the absence of the transactivator and doxycycline treatment with levels of 1.3 for transgenic mRNA and 2.5 fold CryAB protein over endogenous (Figure 3). Relative to endogenous

levels, the double transgenic’s transgenic mRNA expression and fold CryAB protein expression were 15.1 and 15.5 respectively (Figure 3). In accordance with the trends mentioned previously, a prenatal doxycycline regimen yielded 1 fold protein expression that did not increase with cessation of treatment (Figure 3C,E). Postnatal doxycycline treatment for 4 weeks reduced protein expression levels to 5 fold (Figure 3C,E).

Increasing the duration of doxycycline treatment did not decrease protein expression further (data not shown). From a later set of embryo injections, line 104 characterization lagged behind that of line 148. Line 104 had 2.7 fold protein expression for the single transgenic responder and 4.2 fold protein expression for the double transgenic (Figure

3D,E). Neither line 148 or line 104 were ideal as both had leaky expression in the single transgenic lines, and line 148 double transgenics had CryAB expression levels above that of the CryABwildtype control levels previously shown to be benign (Wang et al., 2001b).

However, both lines were maintained since the other CryABS59D lines had proven even less desirable. It should be noted that CryABwildtype line 11 consistently had 7 fold protein expression as opposed to the previously published 8 fold, suggesting that inter- observational procedures resulted in slightly different quantification or that genetic drift may have selected for animals expressing lower quantities of CryABwildtype (Wang et al.,

2001b).

Circumstantial evidence, including the proportional relationship between transgenic mRNA expression and total CryAB protein increases as well as the shifted phosphoisoform distribution of CryAB by IEF, indicates that the increase in CryAB protein in

CryABS59D line 148 is solely due to the expression of transgenic CryABS59D. Although the transgenic protein was untagged, it was expected that it would be separable from the

endogenous protein through the use of phosphomimetic antibodies. However, the phosphomimetic CryABS59D is recognized by phospho-specific antibodies against both phosphorylated serine 59 and serine 45, suggesting that aspartic acid substitution effectively mimics native serine phosphorylation at residue 59 and that CryABS59D can be phosphorylated in vivo at serine 45 (Figure 4). Potentially, the increase in CryAB protein could be partially or entirely due to induced expression of the endogenous CryABwildtype in response to the expression of CryABS59D. This possibility was explored through 2D gel electrophoresis, IEF, and Western blot analyses. It was intended that a side by side comparison of phosphatase-treated and mock-treated samples would provide clarity as to the contribution of the endogenous CryAB and CryABS59D to total CryAB since the aspartic acid at site 59 of the phosphomimetic CryABS59D will remain charged. However, these experiments were inconclusive due to technical difficulties in phosphatase reactions

(data not shown). Expression of CryABwildtype, tTA, and CryABS59D line 148 caused no alteration in HSPB2 protein levels (data not shown).

IEF did reveal an increase in not only the amount of CryAB in double transgenic

CryABS59D mice, but also in the bi-, and tri-charged isoforms of CryABS59D and

CryABR120G as compared to nontransgenic and CryABwildtype line 11 (Figure 5). The

CryABS59D line 148 had a dramatic reduction in the nonphosphorylated isoform (p0) with concordant increases in the single- (p1), bi- (p2), and tri-charged (p3) isoforms, supporting the previous supposition that the increase in total CryAB protein is due to

CryABS59D transgene expression (Figure 5). Nontransgenic and CryABwildtype line 11 had similar phosphorylation profiles, especially when p0 and p1 isoforms were combined for analysis: p0/p1, p2, p3 of nontransgenic and CryABwildtype line 11 were 72%, 22%, 5%

and 75%, 20%, 5% respectively (Figure 5B). The same was true between CryABS59D and

CryABR120G with p0/p1, p2, p3 of 55%, 27%, 18% and 55%, 27%, 17% respectively

(Figure 5B). Comparison of these pairs shows a clear shift of increased charge for

CryABS59D and CryABR120G (Figure 5).

Unexpectedly, CryABS59D line 148 exhibited basal myopathy with 2.5 and 15.5 fold levels of CryAB expression in the single and double transgenics respectively. While hearts were indistinguishable by genotype at 8 weeks of age, by 4 months genotype was easily identifiable by examination of the heart (data not shown, Figure 6A). In the double transgenic of CryABS59D line 148, all four chambers of the heart were enlarged with the right atria evidencing calcification and disproportionate enlargement while single transgenic CryABS59D line 148 showed a very mild phenotype (Figure 6A, data not shown). While there was no difference in overall body weight at 5.5 months, the

CryABS59D line 148 double transgenics had significant increases in ventricular, right ventricular, left ventricular and septal, and left atrial weights compared to nontransgenic,

CryABwildtype line 11, and tTA age-matched controls (Figure 6B). Right atrial measurements failed normality tests likely due to the variance in calcification between

CryABS59D line 148 double transgenic animals; however, the right atrial weights were significantly increased compared to CryABwildtype line 11 by rank analysis (Figure 6B).

CryABS59D line 148 double transgenics evidenced panting with females affected to a greater extent than males (data not shown). M-mode echocardiography of CryABS59D double transgenic line 148 and control animals revealed significant functional deficits at

4 months that only worsened by 5 months (Figure 6C,D, Tables 2-5). At 4 months, there were significant increases in the diastolic intraventricular septum thickness and diastolic

left ventricular posterior wall thickness (Table 2). These were accompanied by trends of increased diastolic and systolic left ventricular internal dimensions, diastolic and systolic left ventricular volume, and left ventricular mass (Tables 2,3). Fractional shortening and posterior wall thickness exhibited significant and drastic decreases, while trends of decreased relative wall thickness and ejection fraction were noticeable (Table 3). By 5 months, the increases in diastolic and systolic left ventricular internal dimensions and left ventricular mass, as well as the decreases in relative wall thickness and ejection fraction had become significant (Tables 4,5). Systolic left ventricular internal dimensions and left ventricular mass increased significantly from 4 to 5 months (Tables 4,5). These structural and functional alterations are consistent with that of a progressive dilated hypertrophy.

CryABS59D line 148 double transgenic animals also had severe arrhythmias; however, no changes were noted by confocal in the amount or distribution of connexin 43, a gap junction protein involved in ventricular conduction, compared to nontransgenic animals

(data not shown) (Davis et al., 1994; Davis et al., 1995a; Davis et al., 1995b).

Although high levels (>10 fold) of CryABwildtype may cause cardiomyopathy, M-mode echocardiography of a CryABS59D line 148 single transgenic suggests the pathology is mutation-specific rather than a result of transgenic insertion, the level of CryAB overexpression, or tTA myopathy (Figure 6D, Tables 2-5). The CryABS59D line 148 single transgenic displayed intermediary trends in many parameters as measured by M- mode echocardiography, and the trends of decreased ejection fraction and fractional shortening were most notable (Tables 4,5). However, there were no significant differences in left ventricular internal dimensions, relative wall thickness, and left ventricular mass in the single transgenic animals (Tables 4,5). The 2.5 fold CryAB

expression of CryABS59D line 148 single transgenics is lower than the 7 fold overexpression of CryABwildtype shown to be tolerated without pathology (Table 2-5).

The gradation of the line 148 cardiomyopathy correlating with the amount of CryAB overexpression, 2.5 fold for the single transgenic and 15.5 fold for the double transgenic, suggests that the negative charge at site 59 of CryABS59D is the cause of CryABS59D line

148’s dilated hypertrophy. Additionally, tTA line 55 animals do not show pathology at this age by M-mode echocardiography (Table 2-5). Despite the previously mentioned differences in level of expression and charged isoform distribution between CryABS59D and CryABwildtype, both CryABS59D lines 148 and 104 had diffuse, non-nuclear protein localization and an absence of protein aggregates as predicted (Figures 3,5,7, data not shown). These data indicate that the putative CryABS59D cardiomyopathy is likely due to altered signaling or protein partners leading to a maladaptive response as opposed to a protein aggregation disorder such as in the case of the CryABR120G-related myopathy.

CryABS45D

Two lines were maintained for the CryABS45D construct, line 174 and line 165. Line

174 had a reasonable correlation between transgenic RNA expression and fold protein expression (Figure 8). The single transgenic responder did not exhibit basal leak in the absence of the transactivator and doxycycline treatment, having levels of 0.2 transgenic mRNA and 1 fold protein relative to endogenous (Figure 8). The double transgenic had

4.6 transgenic mRNA and 8 fold protein expression relative to endogenous mRNA and protein (Figure 8). Postnatal doxycycline treatment for 4 weeks efficiently reduced protein expression levels to 1 fold; however, transgenic protein expression did not

recover after cessation of treatment (Figure 8D, data not shown). Line 165 had 1.6 fold protein expression for the single transgenic responder and 11 fold protein expression for the double transgenic (Figure 9 A,B). Although the double transgenics of both line 174 and 165 had expression levels above that of the CryABwildtype line 11 control, the amounts were still within the range of CryABwildtype overexpression levels previously shown to be benign (Wang et al., 2001b). Similarly to the CryABS59D lines, circumstantial evidence suggests that the increase in total CryAB protein expression is mainly due to the expression of the phosphomimetic CryABS45D protein as opposed to increased expression of endogenous CryAB (data not shown). The phosphomimetic CryABS45D is also recognized by a phospho-specific antibody against phosphorylated serine 45, suggesting that aspartic acid substitution effectively mimics native serine phosphorylation at residue

45 (Figure 9C). The phosphomimetic CryABS45D lines were not tested with the phospho- specific antibody against phosphorylated serine 59. Neither line 174 or 165 displayed any overt cardiomyopathy despite high levels of CryABS45D protein expression in the double transgenics, supporting the previously mentioned possibility that the line 148 cardiomyopathy is CryABS59D-related.

CryAB protein localization in CryABS45D line 165 and line 174 double transgenics is similar to nontransgenic mice: diffuse and non-nuclear (Figure 10, data not shown).

These findings are counter to the prediction that mimicking a negative charge at residue

45 of CryAB would result in nuclear speckle localization. As this hypothesis was based on studies using adenoviral CryAB phosphomimetics in HeLa cells, these findings could be due to the artificial system or to discrepancies in phosphorylation between HeLa cells and cardiomyocytes. Adenoviral CryABwildtype is not phosphorylated at serine 19, 45, or

59 in HeLa cells (den Engelsman et al., 2005). In the heart, phosphorylation is higher at serine 19 and 59 than serine 45 (den Engelsman et al., 2005; Zhou et al., 2000). One model of CryAB nuclear trafficking proposes that phosphorylation at serine residue 59 is a pre-requisite for nuclear entry and that serine 45 is phosphorylated by a nuclear kinase after nuclear entry (den Engelsman et al., 2005). Absence of the pre-requisite negative charge at residue 59 could potentially lead to the diffuse localization seen for the

CryABS45D models if the protein is inhibited from nuclear entry. However, adenoviral

CryAB phosphomimetics in HeLa cells serve as the basis for this model so its applicability to cardiomyocytes is also suspect (den Engelsman et al., 2005).

Alternatively, serine 45 phosphorylation may have less of a role in the heart in general as it is less highly phosphorylated than the other two serines or it could have functions that do not alter its subcellular localization. Similar to the CryABwildtype and CryABS59D models, the CryABS45D transgenic lines had no intracellular aggregates.

CryABS45/59D

Two lines were maintained for the CryABS45/59D construct, line 45 and line 46. For line 45, the single transgenic responder did exhibit minimal basal leak with 1.2 fold protein expression, in the absence of the transactivator and doxycycline treatment (Figure

11A,B). The double transgenic had 8.5 fold protein expression over endogenous (Figure

11A,B). In accordance with the trends mentioned previously, a prenatal doxycycline regimen yielded 1 fold protein expression, which did not increase with cessation of treatment (Figure 11B, data not shown). Four weeks of postnatal doxycycline treatment reduced induced protein expression levels to 1.4 fold (Figure 11A,B). This treatment

followed by cessation of doxycycline for 4 weeks yielded 2.6 fold protein expression for the double transgenic, exhibiting its ability to recover (Figure 11B). Line 45 had expression levels within the range of the CryABwildtype control levels previously shown to be benign, although the double transgenic had higher levels of expression than the

CryABwildtype line 11 control (Wang et al., 2001b). In contrast, line 46 had severe basal leak in the single transgenic responder with 5.4 fold protein expression, and the double transgenic showed very high levels of expression that was 23 fold that of endogenous

CryAB (Figure 11B). A prenatal doxycycline regimen yielded 1 fold protein expression; however, protein expression increased noticeably within one week after cessation of treatment (~3 fold) and continued to do so in a gradient fashion thereafter (~14 fold at 4 weeks cessation) (data not shown). Similar to the CryABS45D and CryABS59D single residue phosphomimetics, the dual residue phosphomimetic CryABS45/59D is recognized by the phospho-specific antibodies against phosphorylated serine 45 and serine 59, suggesting that aspartic acid substitution effectively mimics native serine phosphorylation at these residues (Figure 11C). In parallel with the previous phosphomimetic constructs, circumstantial evidence suggests that the increase in total

CryAB protein expression is mainly due to the expression of the phosphomimetic

CryABS45/59D protein as opposed in increased expression of endogenous CryAB (data not shown).

In the CryABS45/59D transgenic lines, CryAB protein shows peri-nuclear localization and a punctuate, aggregate-like appearance (Figure 12, data not shown). The localization is thought to be specific to the CryABS45/59D construct rather than due to mere overexpression of CryAB since comparable levels of CryABwildtype overexpression to

CryABS45/59D line 45 do not have these characteristics (Sanbe et al., 2004; Sanbe et al.,

2005; Wang et al., 2001b). However, this pattern is reminiscent of the transgenic

CryABR120G model, which forms insoluble peri-nuclear aggresomes (Sanbe et al., 2004;

Sanbe et al., 2005; Wang et al., 2001b). Although not conclusive, these data suggest that hyperphosphorylation of CryABR120G could cause or be associated with CryABR120G dysfunction, since increasing the negative charge in the hydrophobic N-terminus of

CryAB results in a similar localization and aggregation. It would be informative to determine whether CryABS45/59D results in the formation of soluble amyloid oligomer as was observed in the CryABR120G model, since toxicity is correlated with soluble amyloid oligomer rather than insoluble aggregates in Alzheimer’s and cardiomyopathy

(Bucciantini et al., 2002; Kayed et al., 2003; Sanbe et al., 2004; Sanbe et al., 2005; Walsh et al., 2002a; Walsh et al., 2002b). However, it remains likely that the aggregates themselves play a role in toxicity progression or prevention in post-mitotic cells

(Bucciantini et al., 2002; Sanbe et al., 2004; Sanbe et al., 2005).

Line 46 double transgenic mice exhibited severe cardiomyopathy while the single transgenic had a mild cardiomyopathy (data not shown). Presence of cardiomyopathy for

CryABS45/59D line 45 was not determined. Although the expression levels of line 46 double transgenics exceeded the range of CryABwildtype control levels shown to be tolerated without phenotype, the line 46 single transgenic was well within this range with only 5.4 fold protein expression (Figure 11B) (Wang et al., 2001a; Wang et al., 2001b).

The severity of the CryABS45/59D line 46 myopathy correlating with levels of protein expression suggests specificity and is similar to the trends noted for CryABS59D line 148.

However, it remains unclear whether this pathology is due to the negative charge at

residue 59 of CryAB, to disruption of the N-terminal hydrophobic environment leading to decreased solubility and aggregation, or other causes. Interestingly, increases in phosphorylation of both serine 45 and 59, but not serine 19 were also observed after cardioplegia and cardiopulmonary bypass in humans: this increase in phosphorylation was negatively correlated with post-surgical cardiac function (Clements et al., 2007).

Experimental Approach Addendum

Due to the refractory tTA inducible system, inability to separate endogenous CryAB from the phosphomimetic protein, and availability of the KO as well as leaky CryABmutant responder lines, a new strategy was developed to explore the effect of CryAB phosphorylation at residue 59 and 45/59 in vivo. We planned to cross single transgenic phosphomimetic responder lines (CryABmutant) with basal leak into the KO background.

In order to eliminate the effects of HSBP2 loss, this combination would be bred to a constitutive HSPB2 transgenic (Figure 13). Re-engineering the single CryAB knockout was considered; however, the intergenic region between CRYAB and HSPB2 was only

958bp in human and even less than that in mouse (Iwaki et al., 1997). It was unclear whether a single knockout, free of complications, could be produced. As a constitutive

HSPB2 transgenic did not yet exist, the creation of one would provide the additional benefit of investigating this protein’s specialized functions. The HSPB2 cDNA was placed under regulation of the MHC promoter and mice were generated by the Robbins laboratory as previously described (Wang et al., 2001b). This double transgenic model in the KO background would eliminate the complications of endogenous CryAB and allow analysis of the phosphomimetic CryAB at expression levels comparable to the

phenotypically benign CryABwildtype overexpression lines. With this strategy it was predicted that CryABS59D lines 148 and 104 would yield 1.5 and 1.7 fold protein respectively and CryABS45/59D line 46 would yield 4.4 fold protein based on the basal leak characterized earlier and an assumption of endogenous protein as 1 fold (Figures 3,11).

CryABS45D lines 174 and 165 would not be suitable for this strategy as they had no basal leak; however, these CryABS45D lines did not have basal myopathy and were less refractory in regards to the tTA system (Figures 8, 9). As a control for unaltered CryAB protein, CryABwildtype line 11 would also be bred into the HSPB2 transgenic/KO background and was expected to have 6 fold protein (Figure 13). We also began to breed the KO into the FVB/N background in order to avoid strain effects as it was originally made in the BL6/C57 background (Brady et al., 2001).

HSPB2

MHC:HSPB2 line 193 was characterized in the wildtype FVBN background. It had low levels of protein expression at 2.5 fold (Figure 14, data not shown). However, it also had basal cardiomyopathy. Overtly, these mice exhibited edema, panting, bulging eyes, and spikier and less lustrous fur which are indicative of heart failure (data not shown,

Robbins et al., unpublished). Heart weight increased compared to nontransgenic mice, and these animals died around 7-9 months of age (Figure 14, data not shown). The increase in HSPB2 protein was due to transgenic protein production rather than an increase in endogenous protein as line 193 crossed into the KO background had 1.5 fold protein levels (data not shown). It was not determined whether line 193 in the KO background had cardiomyopathy. Interestingly, there were no increases in CryAB protein

expression in line 193 in either the wildtype or KO backgrounds (data not shown). This unexpected cardiomyopathy could be the result of an insertional artifact or an indication that overexpression of HSPB2 is not well tolerated. Analysis of a second line would have been beneficial in differentiating between these possibilities; however, another line was not available. Injection of 300 embryos yielded 3 transgenic founders, of which only the line 193 founder was germline (Table 1). The HSPB2 construct did have a noticeably lower percentage of transgenic founders and germline founders per pups born alive compared to the three CryAB constructs; however, the number of pups born alive was comparable to the CryAB transgenics, suggesting it is not the construct itself (Table 1).

The very low expression of transgenic HSPB2 in line 193 coupled with their development of cardiomyopathy serves as an indication that it is likely the overexpression of HSPB2 itself that is toxic.

Differences in resultant morbidity and mortality between transgenic expression of

CryAB and HSPB2 could relate to HSPB2’s unique expression, structure, and partners compared to CryAB and other sHSPs. HSPB2 is the most divergent member of the human sHSP family, having only approximately 30% amino acid sequence identity to other sHSPs with the -crystallin domain being the region of greatest sequence conservation (Hu et al., 2008). While CryAB and HSPB2 are both highly expressed in heart and muscle, HSPB2 lacks CryAB’s breadth of expression in other tissues (Bhat et al., 1999; Bhat and Nagineni, 1989; Dubin et al., 1989; Horwitz, 1992; Iwaki et al., 1997;

Iwaki et al., 1990; Iwaki et al., 1989). In addition, HSPB2 does not exhibit the enhanced gene transcription after heat stress that is characteristic of the sHSP family (Iwaki et al.,

1997). CryAB shows a more classic sHSP response with high levels of expression

induced in a variety of stress-response models (Dasgupta et al., 1992; Ito et al., 2002;

Kamradt et al., 2002; Klemenz et al., 1991; Renkawek et al., 1994). These differences in gene induction may be a potential reason that CryAB overexpression is well tolerated as opposed to HSPB2. HSPB2 has unique stress responsiveness, being upregulated in the skeletal muscle of myotonic dystrophy patients (Hu et al., 2008; Sugiyama et al., 2000).

Known to bind and activate myotonic dystrophy protein kinase (DMPK) under normal conditions, HSPB2’s upregulation in this disease is believed to be a compensatory mechanism for the reduced amount of DMPK (Hu et al., 2008; Sugiyama et al., 2000;

Suzuki et al., 1998). In contrast, CryAB does not interact with DMPK and is not upregulated in myotonic dystrophy (Hu et al., 2008; Sugiyama et al., 2000). Interestingly,

CryAB is known to complex with other sHSPs, but not with HSPB2 (Sugiyama et al.,

2000; Vicart et al., 1998). These data suggest that despite the small intergenic region, cryab and hspb2 are differentially regulated and functionally divergent (Sugiyama et al.,

2000; Vicart et al., 1998).

Unfortunately, little is known about HSPB2 function and much of the data available are from loss of function models. In skeletal muscle, HSBP2 can act as a chaperone for

DMPK, preventing its heat-induced inactivation (Suzuki et al., 1998). DMPK knockout mice have progressive skeletal myopathy and other abnormalities similar to the KO, suggesting that loss of HPSB2 is a contributing factor in the KO phenotype (Brady et al.,

2001; Reddy et al., 1996). HSPB2 is located at the Z lines and intercalated discs of the heart and has shown co-localization with mitochondria in cell lines (Shama et al., 1999;

Nakagawa et al., 2001). A more recent study concluded that HSPB2 maintains energetic balance in the heart (Pinz et al., 2008). It is possible that this balance is delicate and

susceptible not only to an insufficiency of HSPB2, but also a surplus.

Conclusion

I hypothesized that residue-specific and extent of phosphorylation regulates the protein localization, aggregation, and function of cardiac CryAB in vivo. I anticipated that the single-residue phosphomimetics would not have basal myopathy since

CryABwildtype phosphorylation levels increase under stress; however, CryABS59D line 148, and CryABS45/59D line 46 as well as the line 193 HSPB2 all had basal myopathy. Based on circumstantial evidence, I believe these cardiomyopathies to be specific to the expressed transgenes rather than artifacts caused by construct insertion, complications of the inducible system, level of transgene overexpression, or induction of endogenous protein.

No overt myopathy was evident in CryABS45D line 174 or 165, and characterization of

CryABS59D line 104 and CryABS45/59D line 45 was not complete enough to determine if myopathy existed. This investigation did not progress to the stage of apoptotic stress characterization. As predicted the CryABS59D single residue phosphomimetic had diffuse localization similar to CryABwildtype and no intracellular aggregate formation. It is therefore likely that CryABS59D the line 148 cardiomyopathy is due to altered signaling or protein partners. Contrary to my expectations, the CryABS45D single residue phosphomimetic did not have nuclear speckle localization, possibly due to different roles for CryAB in HeLa cells and cardiomyocytes. The CryABS45D single residue phosphomimetic did not display intracellular aggregates. The CryABS45/59D double residue phosphomimetic had the predicted peri-nuclear localization and aggregate-like appearance. The CryABS45/59D cardiomyopathy and the similarity of localization and

phosphoisoform profile between CryABS45/59D and CryABR120G suggest that CryABR120G’s enhanced phosphorylation may play a role in its DRM.

Were I to continue to explore the role of cardiac CryAB phosphorylation in vivo, I would engineer the following series of knock-in mice to investigate: gain-of-function

(triple phosphomimetic) CryABDDD, loss-of-function (triple nonphosphorylatable)

CryABAAA, and residue-specific CryABAAD, CryABADA, and CryABADD. Nontransgenic mice would serve as a control. This strategy would eliminate several of the shortcomings inherent in the first model such as complications of the tTA system, the lack of a loss-of- function comparison, ability of the two nonmutated serines to be phosphorylated endogenously, inability to separate endogenous from transgenic protein, and the general disadvantages of protein overexpression. It would also be desirable to address the importance of the extent of CryAB phosphorylation with respect to CryAB function. A parallel knock-in study using CryABR120G, CryABR120G-AAA, and CryABR120G-DDD would be a useful model for in vivo analysis of the role of CryAB phosphorylation in

CryABR120G’s DRM. Characterization of the heterozygous knock-in could be especially interesting for its physiological relevance, since affected humans with CryABR120G-DRM were heterozygous for the CryABR120G mutation and CryABR120G’s strong tendency to self-associate can be disrupted by overexpression of CryABwildtype (Sanbe et al., 2005;

Vicart et al., 1998).

Materials & Methods

Mice

General: FVB/N mice were used for all studies. Animals were housed in an AAALAC- approved, temperature controlled barrier facility. All experiments were approved by the

Children’s Hospital Research Foundation Animal Care Review Board. Genotyping was carried out by PCR using primers targeted to the CryAB and tTA transcripts and subsequent agarose gel electrophoresis. MLC primers were used as an internal control.

Transgenic CryAB phosphomimetic responder lines: These lines were engineered by

Howard Hughes Fellow Chet Villa in the Robbins’ Laboratory. In brief, mutations in

CryABwildtype cDNA sequence leading to single amino acid substitution were achieved with overlap extension PCR. The mutated CryABwildtype cDNA was inserted into the modified -myosin heavy chain ( MHC) promoter cassette MHCminTetO, and responder mice were generated as previously described (Sanbe et al., 2003).

Transgenic transactivator line: Kindly provided by the Robbins’ Laboratory, the cardiac- specific transcriptional activator mice αMHC:tTA line 55 has been previously characterized (Sanbe et al., 2003).

Transgenic CryABwildtype line: Kindly provided by the Robbins’ Laboratory, the cardiac- specific αMHC: CryABwildtype line 11 has been previously characterized (Wang et al.,

2001b).

Transgenic CryABR120G line: Kindly provided by the Robbins’ Laboratory, the cardiac- specific αMHC: CryABR120G line 14 has been previously characterized (Wang et al.,

2001b).

Transgenic HSPB2 lines: The HSPB2 lines were engineered by the Robbins’ Laboratory as previously described (Wang et al., 2001b). In brief, the full-length HSPB2 cDNA was cloned using RT-PCR. The HSPB2 cDNA was placed under the -myosin heavy chain

( MHC) promoter and used to generate transgenic mice.

Double CryAB/HSPB2 knockout line: Kindly provided by the Robbins’ Laboratory, the

C57/Bl6 KO has been previously characterized and was being bred into the FVB/N background (Brady et al., 2001).

Doxycycline

A 0.1% doxycycline, 1% sucrose solution was administered via drinking water to mothers (prenatal) or animals post-weaning (4wks of age). Bottles were wrapped in aluminum foil to prevent degradation of doxycycline from light exposure and solutions were changed bi-weekly.

RNA

Whole heart RNA was isolated by guanidinium thiocyanate-phenol-chloroform extraction

(TRIzol, Invitrogen) from 1 to 5 hearts separately for each genotype examined. RNA levels were quantified using dot blots hybridized to transcript-specific oligonucleotides

against the human growth hormone (hGH) poly-A tail to assess transgenic mRNA expression and against mouse GAPDH for loading quantification and normalization.

Protein

Total CryAB protein levels were determined using SDS-PAGE or isoelectric focusing

(IEF) followed by transfer and Western blots with either a mouse monoclonal anti-total

CryAB primary antibody (Assay Designs Stressgen, SPA-222) or rabbit polyclonal anti- total CryAB primary antibody (Assay Designs Stressgen, SPA-223). Phosphorylation of endogenous CryAB protein and phosphomimetic CryAB protein was assessed similarly using primary mouse monoclonal antibodies against serine 45 phosphorylation (Affinity

BioReagents, PA1-011) and serine 59 phosphorylation (Affinity BioReagents, PA1-012).

Mouse monoclonal anti-GAPDH (Chemicon MAB374) and mouse monoclonlonal anti- actin ( -sarcomeric) (Sigma-Aldrich, A2172) primary antibodies with were used as internal loading controls. Goat anti-rabbit IgG HRP-conjugated secondary antibody

(Santa Cruz Biotechnology, SC-2004) and goat anti-mouse IgG HRP-conjugated secondary antibody (Santa Cruz Biotechnology, SC-2005) were used as appropriate.

Whole heart homogenate was prepared using RIPA buffer or 9M Urea buffer from hearts that had been snap-frozen in liquid nitrogen and stored at -80°C. The homogenates were centrifuged at 14,000 g at 4°C and the supernatant separated from the cellular debris. The supernatant was then suspended in Laemmli SDS-PAGE loading buffer, boiled and loaded into the gel matrix for either SDS-PAGE or IEF. Tris-HCl, polyacrylamide, and glycerol gels were used. Protein was transferred to PVDF membranes, probed with the

desired primary and secondary antibodies, and signal detected and quantified using the

ECF western blotting protocol (ECF, Amersham Life Sciences, Arlington Heights, IL).

Echocardiography (statistics)

Functional measurements were made by transthoracic M-mode echocardiography.

Animals were anesthetized with inhaled isofluorane (2%) delivered via mask, and images obtained using an HP Sonos 5500 system and a 10MHz transducer. Off-line measurements included end systolic and end diastolic dimensions of the left ventricle

(LVISD and LVIDD, respectively), interventricular septal thickness (IVSS and IVSD), and left ventricular posterior wall thickness (LVPW S and LVPW D). Values for fractional shortening (%FS), percent posterior wall thickness (%PWTH), left ventricular mass (LV mass), ejection fraction (%EF), and end systolic and diastolic values of relative wall thickness (RWTS and RWTD) and left ventricular volume (LV vol S and LV vol D) were calculated. Data are presented as the mean value ± SEM. For comparisons of multiple groups, one-way analysis of variance followed by the Bonferroni pairwise multiple comparison test was used. For all tests, p<0.05 was considered significant. Mice were age-matched and gender distribution was roughly comparable between genotypes.

Heart & body weights (statistics)

Total body weight was measured of the living animal immediately prior to sacrifice.

Hearts were excised, rinsed in ice-cold PBS, weighed, rapidly dissected. Heart weight was measured first as the two ventricles and septum without atria, and the atria, right ventricle, and left ventricle/septum were subsequently weighed. Hearts isolated for

weight measurements were not used in further experiments. Gender distribution was roughly comparable between genotypes. CryABS59D line 148 and age-matched

CryABwildtype line 11 and nontransgenic controls were 5.5 months of age at time of measurement and consisted of the cohort previously used for M-mode echocardiography.

HSPB2 line 193 transgenic mice and age-matched nontransgenic controls were 7 to 7.5 months of age at time of measurement. Data are presented as the mean value ± SEM. For comparisons of multiple groups, one-way analysis of variance followed by the Holm-

Sidak pairwise multiple comparison test was used. Since line 148 right atrial mean values failed normality tests, the median values were assessed by Kruskal-Wallis one way analysis of variance on ranks followed by the Dunn pairwise multiple comparison test was used. For comparisons of data from two groups, two-tailed, unequal variance t tests were used. For all tests, p<0.05 was considered significant.

Immunohistochemistry

Indirect immunofluorescent staining was carried out on myocardial paraffin sections.

Hearts were perfused (retrograde perfusion through apex) with cardioplegic buffer

(1XPBS, 5% Dextrose, 25mM KaCl), excised, fixed with 10% buffered formalin for 24-

48 hours, dehydrated in 70% alcohol, embedded in paraffin, and sectioned. Sections were air-dried, melted for 20 minute at 60°C, and deparafinized in a series of xylene and ethanol rinses. The immunostaining protocol included antigen retrieval, accomplished by controlled heating of sections in 0.1M glycine in PBS (pH3.5) for 30 minutes, then blocking (1XPBS, 0.5% BSA/10% goat serum, 0.01% Tween20), followed by overnight incubation with polyclonal rabbit anti-total CryAB primary antibody (Assay Designs

Stressgen, SPA-233F) at 4°C. Samples were subsequently incubated in the dark with polyclonal goat anti-rabbit IgG (highly cross-absorbed) conjugated with ALexa 488

(Invitrogen, A11008) for 1 hour at 4°C. Some samples were then incubated with

TOPRO3 nuclear stain for 20 minutes before mounting in Vectashield Hard Set (Vector

Laboratories) and examination by confocal microscopy. Others underwent a second round of incubations with monoclonal mouse anti-pan actin primary antibody (Biomedia

V10275) and polyclonal goat anti-mouse IgG (highly cross-absorbed) conjugated with

ALexa 568 (Invitrogen, A11004) before subsequent TOPRO3 staining, Vectashield Hard

Set mounting, and confocal examination.

Funding

This work was supported by the National Institutes of Health training grant

5T32HL07382.

MDIAIHHPWI RRPFFPFHSP SRLFDQFFGE MDIAIHHPWI RRPFFPFHSP SRLFDQFFGE 30

HLLESDLFST ATSLSPFYLR PPSFLRAPSW HLLESDLFPT STSLSPFYLR PPSFLRAPSW 60

IDTGLSEMRL EKDRFSVNLD VKHFSPEELK FDTGLSEMRL EKDRFSVNLD VKHFSPEELK 90

VKVLGDVIEV HGKHEERQDE HGFISREFHR VKVLGDVIEV HGKHEERQDE HGFISREFHR Strand 5 Strand 6.1------turn------120

KYRIPADVDP LTITSSLSSD GVLTVNGPRK KYRIPADVDP LTITSSLSSD GVLTVNGPRK --Strand 6.2 Strand 7 150

QVSGPERTIP ITREEKPAVA AAPKK QVSGPERTIP ITREEKPAVT AAPKK 175

Figure 1: Amino acid sequence alignment of murine and human CryAB. The image aligns the 175 amino acid sequence of murine (upper) and human (lower) CryAB. Proven phosphorylation sites serine 19, 45, and 59 are shown in orange. Arginine 120, site of the R120G mutation, is shown in red. Amino acid differences between the two species are shown in green. The conserved -crystallin domain is in italics. - strands involved in dimerization are underlined and denoted. Amino acid residue number is indicated at the lower right corner of every 30 amino acids.

A TetO mutant MHC tTA X MHCmin CryAB

B tTA

MHC tTA

TetO mutant MHCmin CryAB

mutant tTA CryAB

tTA MHC tTA MHCminTetO CryABmutant X tTA C tTA

MHC tTA

TetO CryABmutant MHCmin

tTA tTA

MHC tTA MHCminTetO CryABmutant X

D wildtype CryAB

wildtype MHC CryAB

Figure 2: Bi-transgenic tTA Phosphomimetic System. A) Hemizygous MHC:tTA were crossed with MHCminTetO:CryABmutant. B) Expected gene expression (green arrow) and protein production in resultant MHC:tTA, MHCminTetO:CryABmutant, and MHC:tTA x MHCminTetO:CryABmutant progeny in absence of doxycycline and C) presence of doxycycline (red triangle). Nontransgenic offspring not depicted. D) Constitutive MHC:CryABwildtype control. Promoters (light gray). Genes (dark gray). Constitutive transgenics and protein products (black type). Tet-responsive transgenic constructs and protein products (white type).

Table 1: Transgenic Founder Screening

Construct MHCminTetO: MHCminTetO: MHCminTetO: MHC: CryABS45D CryABS59D CryABS45/59D HSPB2

Embryos Microinjected 290 430 419 300

Females Implanted (died) 11 (1) 16 (1) 16 11

Females Pregnant 6 10 9 6

Total pups born (dead) 37 (1) 89 (3) 38 (5) 32 (2) pups per injection concentration 4ng/ul - - 22 -

3ng/ul (1) 13 5 0

2.5ng/ul - - - 14 (1)

2ng/ul 3 4 2 (5) 16 (1)

1ng/ul 33 71 (3) 9 -

Transgenic Founders 6 16 13 3

% per pups born alive 16.7% 18.6% 39.4% 10%

Germline Founders 4 13 12 1

% per pups born alive 11.1% 15.1% 36.4% 3.3%

A R120G tTAxS59D line 148 S59D line 148 tTA NTG

GAPDH

R120G tTAxS59D line 148 S59D line 148 tTA NTG

hGH

Line 148 kD 50 B C 37 Actin Transgene RNA Expression 20 25 15.1 CryAB 15 20 NTG S59D S59D S59D KO kD 10 50 5 Actin 5 37 1.3 hGH/GAPDH 0.2 0 0 25

R120G tTA NTG S59D tTAxS59D 20 CryAB line 148 line 148 NTG tTAxS59D tTAxS59D tTAxS59D KO kD 50 Actin Line 104 37 D CryAB 25 CryAB NTG tTA tTAxS59D 20

NTG tTAxS59D tTAxS59D tTAxS59D KO DFC DFC-Rec

CryAB Protein Expression E 18 15.5 16 14 12 10 7 8 5 4.2 6 2.5 2.7 4 1 1 1 1 2

TotalCryAB/NTG 0

Figure 3: Expression analysis of CryABS59D line 148 and 104. A) RNA Dot blot line 148. The upper panel is GAPDH in vertical duplicate. The lower panel is hGH in vertical duplicate. All animals are 8 weeks of age except for the tTA, which is 8 months. B) RNA Dot blot analysis (n of 1-5). C) Representative Western blots of line 148 for total CryAB protein. Equal ug amounts of total heart homogenate were loaded for each sample. Samples are in horizontal duplicate, excepting the knockout. D) Representative Western blot of line 104 for total CryAB protein. Lanes 1, 4, and 6 are loaded with 30ug total heart homogenate, lanes 3 and 5 with 15ug, and lane 2 with 60ug. E) Protein expression analysis (n of 1-3). Doxycycline (Dox) from conception (DFC). Dox postnatal regimen (DPN). Recovery experiments on DFC animals involving cessation of doxycycline for 4 weeks (DFC-Rec). Nontransgenic (NTG). Double CryAB/HSPB2 KO (KO). CryABwildtype overexpression line 11 (WTOE). CryABS59D responder lines (S59D). Kilodaltons (kD).

A Line 148

anti-CryAB anti-p59 CryAB kD kD

50 50 Actin

37 37

25 25 20 CryAB 20

NTG NTG tTAxS59D NTG NTG tTAxS59D

B Line 148 anti-CryAB anti-p45 CryAB kD kD

50 50 Actin 37 37

25 25

CryAB 20 20 NTG tTAxS59D NTG tTAxS59D

Figure 4: CryABS59D is recognized by phospho-specific antibodies and can be phosphorylated in vivo at serine 45. A) Westerns of duplicate SDS-PAGE gels probed with anti-total CryAB and anti-p59 CryAB antibody. Lane 1 is a molecular weight marker. Lanes 2 and 4 of each have 7ug total heart homogenate loaded. Lane 3 has 10ug. B) Westerns of duplicate SDS-PAGE probed with anti-total CryAB and anti-p45 CryAB antibody. Lane 1 is a molecular weight marker. Lanes 2 and 3 of each have 10ug and 5ug total heart homogenate loaded respectively. Nontransgenic (NTG). CryABS59D responder lines (S59D). A Kilodaltons (kD).

A Line 148

KO R120G tTAxS59D WTOE NTG

B Phosphoisoform Distribution 100% 12 90% 22 32 80% 35 p0 70% 43 60% 34 p1 50% 43 37 40% p2 30% 27 27 20% p3 20 22 10% 17 18 0% 5 5 phosphoisofrm species Percent R120G tTAxS59D WTOE NTG

Figure 5: Increase in bi-, and tri-charged isoforms of CryABS59D line 148 and CryABR120G. A) Western blot for total CryAB protein of an IEF gel loaded with equivalent amounts of total heart homogenate of each genotype. B) Diagram of percent phosphoisoform distribution. Nontransgenic (NTG). Double CryAB/HSPB2 KO (KO). CryABR120G overexpression line 14 (R120G). CryABwildtype overexpression line 11 (WTOE). CryABS59D responder line (S59D). Non- (p0), single- (p1), bi- (p2), and tri-charged (p3) isoforms of CryAB.

NTG tTAxS59D A

Heart and Body Weight

45 * B 40 NTG 35 30 WTOE 25 20 * tTA 15 * * 10 tTAxS59D 5

Weight Weight(g,cg,mg) 0

C D

NTG

WTOE

tTA

tTAxS59D

S59D

Figure 6: CryABS59D line 148 have basal myopathy. A) Whole heart images of age-matched nontransgenic (NTG) and CryABS59D line 148 double transgenic (tTAxS59D) hearts. Images were taken at same magnification fields. B) Histogram of mean body and heart weight measurements (n of 3 for NTG, tTAxS59D; n of 4 for WTOE, tTA). *significant compared to NTG, CryABwildtype overexpression line 11 (WTOE), and tTA (p<0.005). For right atria (RA), median values are given with increases in CryABS59D double transgenics being significant compared to WTOE. Right ventricle (RV), Left ventricle (LV), and Left atria (LA). C) Representative M-mode echocardiography images at 4 months and D) 5 months.

Table 2: CryABS59D line 148 M-mode echocardiography measurements at 4 months. Genotype NTG WTOE tTA tTAxS59D

n 6 6 6 5

IVS D (mm) 0.678±0.020 0.700±0.015 0.688±0.032 0.800±0.021*

LVID D (mm) 4.22±0.060 4.16±0.092 4.23±0.172 4.95±0.211

LVPW D (mm) 0.563±0.018 0.627±0.021 0.607±0.016 0.734±0.032*

IVS S (mm) 1.01±0.091 1.07±0.045 1.06±0.043 1.09±0.045

LVID S (mm) 2.82±0.048 2.67±0.058 2.64±0.077 3.95±0.224

LV PW S (mm) 0.857±0.032 0.935±0.025 0.913±0.042 0.872±0.066

*significant difference compared to nontransgenic (p< 0.5). Nontransgenic (NTG) CryABwildtype line 11 (WTOE). CryABS59D responder line (S59D). Diastole (D). Systole (S). Interventricular septal dimension (IVS). Left ventricular internal dimension (LVID). Left ventricular posterior wall thickness (LVPW).

Table 3: CryABS59D line 148 M-mode echocardiography calculated values at 4 months. Genotype NTG WTOE tTA tTAxS59D

n 6 6 6 5

% FS 33.15±1.06 35.83±0.77 37.36±1.44 20.18±3.30*

RWT D 0.132±0.0054 0.152±0.0087 0.145±0.0076 0.148±0.0058 LV vol D (ul) 79.41±2.66 76.95±3.88 81.00±7.60 116.83±11.48

RWT S 0.307±0.014 0.353±0.014 0.345±0.018 0.226±0.029 LV vol S (ul) 30.07±1.27 26.30±1.37 25.86±1.87 69.27±8.73

%PWTH 52.96±4.39 49.46±4.57 50.57±4.39 19.81±8.49*

LV mass (mg) 92.1±3.1 97.±2.9 99.3±10.0 160.9±15.9 %EF 62.01±1.49 65.76±1.01 67.56±1.66 40.53±5.47

*significant difference compared to nontransgenic (p< 0.5). Nontransgenic (NTG) CryABwildtype line 11 (WTOE). CryABS59D (S59D). Diastole (D). Systole (S). Fractional shortening (%FS). Relative wall thickness (RWT). Left ventricular volume (LV vol). Posterior wall thickness (%PWTH). Ejection fraction (%EF).

Table 4: CryABS59D line 148 M-mode echocardiography measurements at 5 months. Genotype NTG WTOE tTA S59D tTAxS59D n 3 3 3 1 3

IVS D (mm) 0.637±0.048 0.603±0.034 0.677±0.038 0.740 0.857±0.095

LVID D (mm) 4.32±0.127 4.26±0.067 4.38±0.228 4.55 5.55±0.149*£

LVPW D (mm) 0.633±0.054 0.583±0.033 0.620±0.027 0.670 0.797±0.083

IVS S (mm) 1.03±0.025 1.00±0.103 1.04±0.085 1.00 1.02±0.117

LVID S (mm) 2.70±0.102 2.76±0.045 2.75±0.148 3.24 4.83±0.219*€

LV PW S (mm) 1.00±0.081 0.903±0.082 1.00±0.095 0.900 0.937±0.102

*significant difference compared to nontransgenic (NTG), £ NO significant difference compared to CryABS59D responder line (S59D), € significant difference compared to 4 month measurement (p< 0.5). CryABwildtype line 11 (WTOE). Diastole (D). Systole (S). Interventricular septal dimension (IVS). Left ventricular internal dimension (LVID). Left ventricular posterior wall thickness (LVPW).

Table 5: CryABS59D line 148 M-mode echocardiography calculated values at 5 months. Genotype NTG WTOE tTA S59D tTAxS59D n 3 3 3 1 3

% FS 37.45±1.57 37.12±1.89 37.23±0.80 28.65 13.06±1.65*

RWT D 0.147±0.0088 0.137±0.0088 0.140±0.0058 0.150 0.143±0.0176 LV vol D (ul) 84.27±5.78 81.19±3.11 87.27±10.34 94.83 135.99±16.08†

RWT S 0.370±0.035 0.340±0.035 0.363±0.023 0.280 0.193±0.029*£

LV vol S (ul) 27.21±2.47 26.40±1.13 28.47±3.72 42.38 109.43±11.46 %PWTH 59.33±6.94 54.36±6.57 63.24±15.46 36.06 17.27±0.93 LV mass (mg) 100.0±15.2 88.9±7.9 105.0±14.5 122.9 212.3±20.5*£€

%EF 67.65±2.03 67.30±2.42 67.42±1.04 55.3 27.62±3.27*

*significant difference compared to nontransgenic (NTG), † significant difference compared to CryABwildtype line 11 (WTOE), £ NO significant difference compared to CryABS59D (S59D), € significant difference compared to 4 month measurement (p< 0.5). Diastole (D). Systole (S). Fractional shortening (%FS). Relative wall thickness (RWT). Left ventricular volume (LV vol). Posterior wall thickness (%PWTH). Ejection fraction (%EF).

Longitudinal Transverse NTG tTAxS59D NTG tTAxS59D

CryAB

2° only

Figure 7: CryABS59D line 104 protein localization is diffuse and non-nuclear. Four month old, age- matched nontransgenic (NTG) and double transgenic line 104 (tTAxS59D) males. Primary antibody was rabbit anti-total CryAB pAb with secondary antibody goat anti-rabbit pAB ALexa 488 (green) (upper panels). Secondary only antibody control (lower panels). 60x magnification.

A tTAxS45D line 174 S45D tTA NTG B Transgene RNA Expression 5 4.6 4.5 4 GAPDH 3.5 2.9 3 2.5

2 1.5

hGH/GAPDH 1

0.5 0.2 0.2 tTAxS45D line 174 S45D tTA NTG 0 0 R120G tTA NTG S45D tTAxS45D line 174 line 174 hGH

C Line 174 kD

50 GAPDH

20 CryAB tTAxS45D S45D NTG WTOE

D CryAB Protein Expression

9 8 8 7 7 6 5 4 3 TotalCryAB/NTG 2 1 1 1 1 1 0

Figure 8: Expression analysis of CryABS45D line 174. A) RNA Dot blot. The upper panel is GAPDH in vertical duplicate. The lower panel is hGH in vertical duplicate. All animals are 8 weeks of age except for the tTA which is 8 months of age. B) RNA Dot blot analysis is based on an n of 1 to 3. C) Representative Western blot of line 174 for total CryAB protein. 2.5 ug of total heart homogenate loaded per well except for WTOE line 14 for which only 1ug was loaded. D) Protein expression analysis based on an n of 1 to 3. Doxycycline postnatal regimen (DPN). Nontransgenic (NTG). CryABS45D responder lines (S45D). CryABwildtype overexpression line 14 for panel C and line 11 for histogram D (WTOE). Kilodaltons (kD).

Line 165 A kD 50

Actin 37

CryAB 20 NTG S45D S45D tTAxS45D KO

B CryAB Protein Expression 12 11 10 8 8 7

4 1.6 2 1 1 1 1 1 CryAB/NTG Total 0

Line 165 kD

C 50 Actin

CryAB 20

S45D tTAxS45D KO

Figure 9: Expression analysis and phospho-specific antibody recognition of CryABS45D line 165. A) Representative Western blot for total CryAB. 5ug and 10 ug amounts of total heart homogenate were loaded for each sample, excepting the knockout for which only 10ug was loaded. B) Protein expression analysis of line 174 and line 165 based on an n of 1 to 3. C) Western of SDS-PAGE probed with anti-p45 CryAB antibody. Lanes 1, 3 and 5 each have 10ug and lanes 2 and 4 have 5ug total heart homogenate loaded respectively. All animals for the westerns are 8-8.5wks of age except for the KO which is 8.5 months of age. Nontransgenic (NTG). CryABwildtype overexpression line (WTOE). Double CryAB/HSPB2 KO (KO). Postnatal doxycycline regimen (DPN). Kilodaltons (kD).

Longitudinal Transverse NTG tTAxS45D NTG tTAxS45D

CryAB Actin

2° only Actin

Figure 10: CryABS45D line 165 protein localization is diffuse and non-nuclear. Four month old nontransgenic (NTG) male and five month retired male breeder from double transgenic line 165 (tTAxS45D) were compared. Primary antibody rabbit was rabbit anti-total CryAB pAb with secondary antibody goat anti-rabbit pAB ALexa 488 (green) (upper panels). Secondary only antibody control (lower panels). Primary mouse anti-pan actin mAb with secondary goat anti-rabbit pAb Alexa 568 (red) (upper and lower panels). Topro3 stain (blue). Sections shown were cut from the septum and subjected to immunohistochemistry. 20x magnification.

A Line 45 kD 50 Actin

25 CryAB 20 WTOE tTA NTG S45/59D tTAxS45/59D tTAxS45/59D DPN

B 25 CryAB Protein Expression 23

10 8.5 7 5.4

CryAB/NTGTotal 5 2.6 1 1 1.2 1 1.4 0

C Line 45 anti-p45 CryAB anti-p59 CryAB kD kD

50 50 Actin Actin

37 37

25 25

CryAB CryAB 20 20

R120G NTG tTAx KO R120G NTG tTAx KO S45/59D S45/59D

Figure 11: Expression analysis of CryABS45/59D line 45 and line 46 and phospho-specific antibody recognition of CryABS45/59D line 45. A) Representative Western blot of line 45 for total CryAB protein. Equal ug amounts of total heart homogenate were loaded for each sample. B) Protein expression analysis of line 45 and line 46 (n of 1 to 3). C) Westerns of duplicate SDS-PAGE of line 45 probed with anti-p45 CryAB and anti-p59 CryAB antibodies. Lane 1 is a molecular weight marker. Lanes 3 and 5 each have 14ug total heart homogenate loaded. Lane 2 has 0.7ug and lane 4 has 7ug. Doxycycline (Dox) from conception (DFC). Doxycycline postnatal regimen (DPN). Recovery experiments on DPN animals involving cessation of doxycycline for 4 weeks (DPN-Rec). Nontransgenic (NTG). CryABwildtype overexpression line (WTOE). CryABR120G overexpression line (R120G). Double CryAB/HSPB2 KO (KO). Kilodaltons (kD).

Longitudinal Transverse A NTG tTAxS45/59D NTG tTAxS45/59D

CryAB

Actin

2° only Actin

Longitudinal Transverse B NTG tTAxS45/59D NTG tTAxS45/59D

CryAB

D 2° only

Figure 12: CryABS45/59D line 45 has peri-nuclear and aggregate-like CryAB localization. A) Four month old male nontransgenic (NTG) and three month old double transgenic line 45 male (tTAxS45/59D). Primary antibody rabbit anti-total CryAB pAb with secondary antibody goat anti-rabbit pAB ALexa 488 (green) (upper panels). Secondary only antibody control (lower panels). Primary mouse anti-pan actin mAb with secondary goat anti-rabbit pAb Alexa 568 (red) (upper and lower panels). Topro3 stain (blue). Sections shown were cut from the septum and subjected to immunohistochemistry. 20x magnification. B) Four month old male nontransgenic (NTG) and three month old double transgenic line 45 male (tTAxS45/59D). Primary antibody rabbit anti-total CryAB pAb with secondary antibody goat anti-rabbit pAB ALexa 488 (green) (upper panels). Secondary only antibody control (lower panels). 60x magnification. 48

A HSPB2

MHC HSPB2 Wildtype Background

B HSPB2 mutant CryAB

MHC HSPB2 MHCminTetO CryABmutant CryAB/HSPB2 Knockout X

C HSPB2

HSPB2 CryAB/HSPB2 Knockout MHC

CryABmutant

TetO mutant MHCmin CryAB CryAB/HSPB2 Knockout

wildtype CryAB

CryABwildtype MHC CryAB/HSPB2 Knockout

Figure 13: Breeding Strategy & Transgenic HSPB2 construct. A) Hemizygous MHC:HSPB2 would be generated and first analyzed in the wildtype background. B) The MHC:HSPB2 would then be bred to leaky CryABmutant responder lines in the CryAB/HSPB2 knockout background. C) These double transgenics would be characterized alongside MHC:HSPB2, CryABmutant, and CryABwildtype single transgenic controls all in the CryAB/HSPB2 knockout background. Gene expression (green arrow). Promoters (light gray). Genes (dark gray). Genetic background (white boxes). Constitutive transgenics and protein products (black type). Leaky Tet-responsive transgenic constructs and protein products (light gray type).

A HSPB2 Protein Expression

3 2.5 2.5 2

1.5 1 1 0.5

0 Total HSPB2/NTG Total NTG Line 193

B 40.0 Heart and Body Weight

35.0 30.0 25.0 20.0 * NTG 15.0 * Line 193 10.0

Weight Mean(g,mg) cg, 5.0 0.0 Body (g) Heart (cg) RV (mg) LV&Septum (cg)

Figure 14: MHC:HSPB2 line 193 has basal myopathy. A) Protein expression analysis (n of 2). B) Histogram of mean body and heart weight measurements (n of 2 for NTG; n of 3 for line 193). Right ventricle (RV). Left ventricle (LV). *significant compared to nontransgenic (NTG) (p<0.05).

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