Cardiac Myosin Binding protein-C Research Articles

A Gain-of-Function Mutation in the M-domain of Cardiac Myosin-binding Protein-C Increases Binding to Actin

The M-domain is the major regulatory subunit of cardiac myosin-binding protein-C (cMyBP-C) that modulates actin and myosin interactions to influence muscle contraction. However, the precise mechanism(s) and the specific residues involved in mediating the functional effects of the M-domain are not fully understood. Positively charged residues adjacent to phosphorylation sites in the M-domain are thought to be critical for effects of cMyBP-C on cross-bridge interactions by mediating electrostatic binding with myosin S2 and/or actin. However, recent structural studies revealed that highly conserved sequences downstream of the phosphorylation sites form a compact tri-helix bundle. Here we used site-directed mutagenesis to probe the functional significance of charged residues adjacent to the phosphorylation sites and conserved residues within the tri-helix bundle. Results confirm that charged residues adjacent to phosphorylation sites and residues within the tri-helix bundle are important for mediating effects of the M-domain on contraction. In addition, four missense variants within the tri-helix bundle that are associated with human hypertrophic cardiomyopathy caused either loss-of-function or gain-of-function effects on force. Importantly, the effects of the gain-of-function variant, L348P, increased the affinity of the M-domain for actin. Together, results demonstrate that functional effects of the M-domain are not due solely to interactions with charged residues near phosphorylatable serines and provide the first demonstration that the tri-helix bundle contributes to the functional effects of the M-domain, most likely by binding to actin.

Cross-Species Mechanical Fingerprinting of Cardiac Myosin Binding Protein-C

Cardiac myosin binding protein-C (cMyBP-C) is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 8 Ig- and 3 fibronectin III (FNIII)-like domains along with a unique regulatory sequence referred to as the MyBP-C motif or M-domain. We previously used atomic force microscopy to investigate the mechanical properties of murine cMyBP-C expressed using a baculovirus/insect cell expression system. Here, we investigate whether the mechanical properties of cMyBP-C are conserved across species by using atomic force microscopy to manipulate recombinant human cMyBP-C and native cMyBP-C purified from bovine heart. Force versus extension data obtained in velocity-clamp experiments showed that the mechanical response of the human recombinant protein was remarkably similar to that of the bovine native cMyBP-C. Ig/Fn-like domain unfolding events occurred in a hierarchical fashion across a threefold range of forces starting at relatively low forces of ∼50 pN and ending with the unfolding of the highest stability domains at ∼180 pN. Force-extension traces were also frequently marked by the appearance of anomalous force drops suggestive of additional mechanical complexity such as structural coupling among domains. Both recombinant and native cMyBP-C exhibited a prominent segment ∼100 nm-long that could be stretched by forces <50 pN before the unfolding of Ig- and FN-like domains. Combined with our previous observations of mouse cMyBP-C, these results establish that although the response of cMyBP-C to mechanical load displays a complex pattern, it is highly conserved across species.

Late gadolinium enhancement cardiac magnetic resonance imaging in prognostic assessment of hypertrophic cardiomyopathy

A 46-year-old man who was diagnosed with familial hypertrophic cardiomyopathy (HCM) related to a heterozygote MYBPC3 (cardiac myosin-binding protein-C) gene mutation at the age of 18 was admitted with cardiogenic shock. A progressive decline in functional capacity started 2 years earlier while successive cardiac magnetic resonance (CMR) imaging performed in 2006, 2010, and 2012 showed focal and progressive myocardial fibrosis ( Panel 1 A ). At admission, bedside echocardiography showed severe HCM and biventricular dysfunction. Femoro-femoral extracorporeal life support …

Myofilament Ca2 + desensitization mediates positive lusitropic effect of neuronal nitric oxide synthase in left ventricular myocytes from murine hypertensive heart

Neuronal nitric oxide synthase (NOS1 or nNOS) exerts negative inotropic and positive lusitropic effects through Ca2+ handling processes in cardiac myocytes from healthy hearts. However, underlying mechanisms of NOS1 in diseased hearts remain unclear. The present study aims to investigate this question in angiotensin II (Ang II)-induced hypertensive rat hearts (HP). Our results showed that the systolic function of left ventricle (LV) was reduced and diastolic function was unaltered (echocardiographic assessment) in HP compared to those in shams. In isolated LV myocytes, contraction was unchanged but peak [Ca2+]i transient was increased in HP. Concomitantly, relaxation and time constant of [Ca2+]i decay (tau) were faster and the phosphorylated fraction of phospholamban (PLN-Ser16/PLN) was greater. NOS1 protein expression and activity were increased in LV myocyte homogenates from HP. Surprisingly, inhibition of NOS1 did not affect contraction but reduced peak [Ca2+]i transient; prevented faster relaxation without affecting the tau of [Ca2+]i transient or PLN-Ser16/PLN in HP, suggesting myofilament Ca2+ desensitization by NOS1. Indeed, relaxation phase of the sarcomere length–[Ca2+]i relationship of LV myocytes shifted to the right and increased [Ca2+]i for 50% of sarcomere shortening (EC50) in HP. Phosphorylations of cardiac myosin binding protein-C (cMyBP-C282 and cMyBP-C273) were increased and cardiac troponin I (cTnI23/24) was reduced in HP. Importantly, NOS1 or PKG inhibition reduced cMyBP-C273 and cTnI23/24 and reversed myofilament Ca2+ sensitivity. These results reveal that NOS1 is up-regulated in LV myocytes from HP and exerts positive lusitropic effect by modulating myofilament Ca2+ sensitivity through phosphorylation of key regulators in sarcomere.

Characterization of the cardiac myosin binding protein-C phosphoproteome in healthy and failing human hearts

IntroductionCardiac myosin binding protein-C (cMyBP-C) becomes dephosphorylated in the failing heart and reduced phosphorylation-dependent regulation of cMyBP-C has been implicated in contractile dysfunction. To date, several phosphorylation sites have been identified for human cMyBP-C; however, a comprehensive characterization of the cMyBP-C phosphoproteome is lacking. This study aimed to characterize the cMyBP-C phosphoproteome using two different proteomic-based methods in explanted donor and end-stage failing hearts. MethodsThe first approach used to characterize the cMyBP-C phosphoproteome employed a strong-cation exchange chromatography (SCX) fractionation method (10 pooled samples, technical replicates=4) and the second employed a sodium dodecylsulfate polyacrylamide gel electrophoresis method (n=10; technical replicates=2). Each subsequently underwent titanium dioxide (TiO2) affinity chromatography to enrich for the tryptic phosphopeptides, which were analyzed using an LTQ-Orbitrap mass spectrometer. Moreover, recombinant C0–C2 fragment of mouse cMyBP-C incubated with PKA, PKC, CaMKII and CK2 was analyzed to identify the kinases involved with phosphorylation of cMyBP-C. ResultsSeventeen phosphorylation sites on cMyBP-C were identified in vivo, with the majority localized in the N-terminal domains C0–C2. The three most abundant phosphorylated sites, Ser284, Ser286 and Thr290, are located in the regulatory M-domain of cMyBP-C. Ser284 showed a significant reduction in phosphorylation in HF. ConclusionThis study demonstrates that cMyBP-C harbors more phosphorylation sites than previously known, with a total of 17 (9 novel) identified phosphorylation sites in vivo. Most sites were primarily located within the N-terminal side of the protein. The most highly phosphorylated site on cMyBP-C was Ser284 and this site showed decreased phosphorylation in the failing heart, which implicates importance for fine-tuning contractility. To date, the functional importance of Ser286 and Thr290 is unknown. In addition, 16 sites were identified after in vitro kinase incubation. The data have been deposited to the ProteomeXchange with identifier PXD000158.

Molecular modeling of disease causing mutations in domain C1 of cMyBP-C.

Cardiac myosin binding protein-C (cMyBP-C) is a multi-domain (C0–C10) protein that regulates heart muscle contraction through interaction with myosin, actin and other sarcomeric proteins. Several mutations of this protein cause familial hypertrophic cardiomyopathy (HCM). Domain C1 of cMyBP-C plays a central role in protein interactions with actin and myosin. Here, we studied structure-function relationship of three disease causing mutations, Arg177His, Ala216Thr and Glu258Lys of the domain C1 using computational biology techniques with its available X-ray crystal structure. The results suggest that each mutation could affect structural properties of the domain C1, and hence it’s structural integrity through modifying intra-molecular arrangements in a distinct mode. The mutations also change surface charge distributions, which could impact the binding of C1 with other sarcomeric proteins thereby affecting contractile function. These structural consequences of the C1 mutants could be valuable to understand the molecular mechanisms for the disease.

CARDIAC MYOSIN-BINDING PROTEIN C PHOSPHORYLATION ENHANCES CARDIAC RELAXATION

Cardiac myosin-binding protein-C (MyBPC3), a thick filament protein that inhibits myosin-actin interaction to slow cross-bridge cycling, has been implicated in diastolic dysfunction. MyBPC3 phosphorylation releases this inhibition; thus, we hypothesize that MyBPC3 phosphorylation promotes lusitropy

Helix Three (H3) of the M-Domain of Cardiac Myosin Binding Protein-C is not Essential for Functional Effects in Permeabilized Rat Trabeculae

Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein involved in the regulation of cardiac muscle contraction. Cardiac specific phosphorylation sites in the M-domain regulate binding of cMyBP-C to myosin and actin. Immediately downstream of the phosphorylation sites is a bundle of three α-helixes that are well conserved across all species and isoforms of MyBP-C, however the functional significance of these helices is unknown. Helix 3 (H3) bears high homology to the inhibitory peptide of troponin I, suggesting it as a potential binding site for interactions with actin. To understand the functional significance of H3, we produced a recombinant N-terminal protein containing domains C1, M and C2 (C1C2) with the H3 sequence deleted (H3KO). We then assessed effects of the mutant H3KO protein on contractile forces in permeabilized rat trabeculae and compared them to effects of the wild-type C1C2. Results showed that 5µM C1C2 significantly increased Ca2+-sensitivity of force (ΔpCa50=0.29±0.03), whereas 5µM H3KO had no effect (ΔpCa50=0.02±0.01) and 10µM H3KO only modestly increased Ca2+-sensitivity (ΔpCa50=0.11±0.02,). Similarly, whereas 10µM C1C2 activated force in the absence of Ca2+ (pCa 9.0), H3KO activated force in the absence of Ca2+ only at concentrations >20µM. Together, these results establish that H3 is not absolutely required for the functional effects of the N-terminal domains of cMyBP-C to either increase Ca2+-sensitivity of force or to activate tension in sarcomeres, but that H3 contributes to the overall efficacy of the N-terminus in mediating these effects. Potentially, H3 could dock the N-terminus of cMyBP-C with actin or extend/stabilize cMyBP-C interactions with actin or other regulatory proteins. This work was supported by NIH HL-080367 (SPH), NDSEG and AHA graduate fellowships (KLB), and AHA undergraduate fellowship (JKK).

Phosphorylation of Serine 282 in Cardiac Myosin Binding Protein-C Increases Myosin MgADP Release and MgATP Binding Rates in Mouse Myocardium

Cardiac myosin binding protein-C (cMyBP-C) modulates contractility in part through phosphorylation of serines (S) found in the cardiac-specific motif between domains C1 and C2. S282 phosphorylation appears to allow the hierarchical phosphorylation of S302 and S307 by protein kinase-A (PKA). We investigated whether S282 phosphorylation status influenced the effects of PKA on acto-myosin cross-bridge MgADP release rate (k-ADP) and MgATP binding rate (k+ATP) in myocardial strips. Transgenic mice expressing cMyBP-C with a single amino acid switch at S282 to glutamic acid (D) or alanine (A) were generated to mimic phosphorylated or unphosphorylated states, respectively. Mice were fed L-thyroxin for 10 days to ensure similar (>90%) α-myosin heavy chain (α-MyHC) background. Skinned papillary muscles were pretreated with alkaline phosphatase to reduce any differential in vivo phosphorylation, and examined by length perturbation analysis at maximum calcium activation (pCa 4.8) as MgATP concentration varied (0 to 5 mM). α-MyHC k-ADP and k+ADP increased 90% (212±22 vs. 111±9 s−1) and 45% (867±93 vs. 595±50 mM−1 s−1) for S282D vs. S282A, demonstrating that cMyBP-C S282 phosphorylation status significantly affects α-MyHC kinetics. Following PKA treatment, k-ADP and k+ADP values did not differ between S282D and S282A, due to PKA reducing k-ADP by 23% (163±19 s−1) and k+ATP by 45% (475±35 mM−1 s−1) for S282D, but increasing k-ADP by 23% (137±9 s−1) and decreasing k+ATP by 23% (457±52 mM−1 s−1) for S282A. These results suggest that S282 phosphorylation most significantly affects the initial functional status of the myocardium, prior to PKA-mediated effects from cMyBP-C or other phosphorylatable myofilament proteins.

Detection of cardiac myosin binding protein-C (cMyBP-C) by a phospho-specific PKD antibody in contracting rat cardiomyocytes

Protein phosphorylation plays an important role in physiological processes, such as muscle contraction. Phospho-specific antibodies have become powerful tools to study these processes. Cardiac myosin binding protein-C (cMyBP-C) is one of the proteins that make up the contractile apparatus of cardiomyocytes. Phosphorylation of cMyBP-C is essential for normal cardiac function, since dephosphorylation of this protein leads to its degradation and has been associated with cardiomyopathy. One of the upstream kinases, which phosphorylate cMyBP-C, is protein kinase D (PKD). While studying the role of PKD in cMyBP-C phosphorylation, we tried to analyze phosphorylation of PKD with a phospho-specific PKD-Ser744/748 antibody. Contrary to the expected 115 kDa, a signal was found for a 150-kDa protein. By MALDI-TOF mass spectrometry, we identified this protein to be cMyBP-C. These data were confirmed by immunostaining using the p-PKD-Ser744/748 antibody, which displayed a striated pattern similar to the one observed for a regular cMyBP-C antibody. To our knowledge there are no antibodies commercially available for phosphorylated cMyBP-C. Thus, the p-PKD-Ser744/748 antibody can accelerate research into the role of cMyBP-C phosphorylation in cardiomyocytes.

Activation and Inhibition of F-Actin and Cardiac Thin Filaments by the N-Terminal Domains of Cardiac Myosin Binding Protein C

Myosin binding protein-C (MyBPC) has been known for over thirty years to inhibit actomyosin ATP hydrolysis and more recently has been shown to affect the calcium sensitivity of force in muscle fibers. The various domains of MyBPC have previously been shown to have complex interactions with other myofibrillar proteins. The C-terminal domains have been shown to bind to the thick filament and the N-terminal domains interact with the S2 region of myosin and with f-actin. The number of mutations in cardiac MyBPC (cMyBPC) are the second most prevalent of sarcomeric proteins in producing cardiomyopathies. We have used steady state kinetics to study the molecular mechanism by which the soluble N-terminal C0C1, C1C2 and C0C2 domains of mouse and human cMyBPC affect the activation of myosin ATP hydrolysis by f-actin and native porcine thin filaments.The N-terminal domains of cMyBPC inhibit the activation of the steady state ATPase myosin by f-actin. However, in the presence of native cardiac thin filaments.the N-terminal domains C1C2 and C0C2 of mouse and human cMyBPC produce biphasic effects on the steady state ATP hydrolysis of cardiac myosin-S1. That is, ATPase is activated at low ratios of cMyBPC-N-terminal domain to thin filament and is inhibited by higher ratios similar to the effects observed with f-actin. These data suggest that low ratios of cMyBPC N-terminal domains activate thin filaments in a mechanism similar to that of rigor myosin-S1 but higher ratios inhibit the ATPase rate by competing with myosin-S1-ADP-Pi binding to actin and thin filaments. Effects also appear species-dependent since the C0C1 domains of human cMyBPC produce similar effects as C1C2 and C0C2 on ATPase in the presence of thin filaments, but mouse C0C1 does not produce significant activation.

Calcium-Calmodulin Competes with Actin for Binding to the M-Domain of Cardiac Myosin Binding Protein-C

The regulatory M-domain of cardiac myosin binding protein-C (cMyBP-C) binds to myosin, actin, and to calmodulin when calcium is present (i.e., calcium-calmodulin), but it is unclear whether binding of all three ligands is independent or if binding interactions are competitive. Here we investigated whether calcium-calmodulin (Ca-Cam) binding to the M-domain affected the ability of the M-domain to bind to actin using cosedimentation binding and calmodulin-sepaharose pull-down assays. Results of actin cosedimentation binding assays showed that Ca-Cam significantly reduced specific binding of a recombinant protein containing three N-terminal domains of cMyBP-C (i.e., C1-M-C2, referred to as C1C2) when 10 μM calmodulin was present in the presence of calcium (pCa 3.0). In the absence of calcium (at pCa 10.0) calmodulin had no effect on C1C2 binding to actin. Increasing Ca-Cam concentrations to achieve higher molar ratios with respect to C1C2 further reduced the amount of C1C2 that bound to actin. Conversely, in calmodulin-sepharose pull-down experiments, binding of C1C2 to calmodulin was only modestly reduced in the presence of increasing concentrations of F-actin. Taken together, these results indicate that binding of Ca-Cam can compete with actin for binding to the M-domain. These results suggest a potential mechanism whereby the functional effects of cMyBP-C binding to actin can be regulated by calcium. This work supported by NIH HL080367.

Structural Dynamics of Actin-Myosin Bound and Unbound States of Cardiac Myosin Binding Protein-C Detected by Dipolar EPR

We have used site-directed spin labeling and pulsed dipolar electron-electron paramagnetic resonance (DEER) to resolve the structure and dynamics of flexible and disordered regions of myosin binding protein-C (MyBP-C)'s cardiac isoform, with implications for the pathophysiology of hypertrophic cardiomyopathy (HCM). N-terminal domains of cMyBP-C contain binding domains for several interaction partners in the myofilament, including myosin heavy chain subfragment 2 (S2) and actin. We engineered pairs of labeling sites in protein fragments of mouse cMyBP-C to measure with high resolution distance and disorder between (1) domains C0 and C1, flanking the flexible Pro/Ala-rich linker, and between (2) domains C1 and C2, flanking the partially disordered phosphorylation motif, using DEER. Changes in distance and disorder were assessed for double-Cys mutant cMyBP-C's free in solution and when bound to myosin S2 or actin, with or without cMyBP-C phosphorylation by protein kinase A (PKA). Understanding conformational transitions in the flexible and dynamic portions of cMyBP-C upon actin-myosin binding and phosphorylation provide new molecular insight into defining its modulatory role in muscle force development. (NIH-F32 to BAC; NIH-R01 to DDT)

Cardiac Myosin Binding Protein-C Influences the Rates of MgADP Release and MgATP Binding Differently for the Two Cardiac Myosin Isoforms Expressed in Mouse Myocardium

Transgenic mouse models lacking myosin binding protein-C (cMyBP-C) have been used extensively to examine the influence of cMyBP-C on myocardial performance. Mouse ventricles normally express predominately α-myosin heavy chain (α-MyHC), while cMyBP-C knockout models (-/- and t/t) express a mixture of α- and β-MyHC isoforms. Performance comparisons between control mice and these knockouts are complicated by differences in MyHC isoform profile. We tested whether cMyBP-C differentially affected MgATP-dependent kinetics in the two cardiac MyHC isoforms. We applied varying MgATP concentrations to chemically-skinned myocardial strips undergoing length perturbation analysis and were able to deduce acto-myosin crossbridge MgADP release rate (k-ADP) and MgATP binding rate (k+ATP). Knockout mice treated for 10 days with L-thyroxine (t/tT4) expressed 90/10% α/β-MyHC as did transgenic controls expressing wildtype cMyBP-C (WTt/t-T4). Values for k-ADP were not different between t/tT4 (95.7±3.5 s−1) and WTt/t-T4 (90.3±9.6 s−1), but k+ATP was significantly lower in the t/tT4(154±4 mM−1s−1) compared to WTt/t-T4 (302±18 mM−1s−1), P<0.01. Mice fed a diet of 6-n-propyl-2-thiouracil (PTU) expressed 100% β-MyHC. Values for k-ADP were significantly higher in t/tPTU (24.8±1.0 s−1) compared to non-transgenic controls (NTGPTU, 15.6±1.3 s−1), P<0.01, but k+ATP was not significantly different in t/tPTU (77.5±14.5 mM−1s−1) compared to NTGPTU (99.8±20.0 mM−1s−1). These findings in the structural context of an intact cardiac sarcomere suggest that the presence of cMyBP-C influences MgATP-dependent kinetics differently for the two cardiac myosin isoforms. cMyBP-C does not influence MgADP release rate of α-MyHC, but effectively doubles the MgATP binding rate. In contrast, the presence of cMyBP-C reduces the MgADP release rate in the β-MyHC isoform, but does not change MgATP binding kinetics.

Tetrahydrobiopterin improves diastolic dysfunction by reversing changes in myofilament properties

Despite the increasing prevalence of heart failure with preserved left ventricular function, there are no specific treatments, partially because the mechanism of impaired relaxation is incompletely understood. Evidence indicates that cardiac relaxation may depend on nitric oxide (NO), generated by NO synthase (NOS) requiring the co-factor tetrahydrobiopterin (BH4). Recently, we reported that hypertension-induced diastolic dysfunction was accompanied by cardiac BH4 depletion, NOS uncoupling, a depression in myofilament cross-bridge kinetics, and S-glutathionylation of myosin binding protein C (MyBP-C). We hypothesized that the mechanism by which BH4 ameliorates diastolic dysfunction is by preventing glutathionylation of MyBP-C and thus reversing changes of myofilament properties that occur during diastolic dysfunction. We used the deoxycorticosterone acetate (DOCA)-salt mouse model, which demonstrates mild hypertension, myocardial oxidative stress, and diastolic dysfunction. Mice were divided into two groups that received control diet and two groups that received BH4 supplement for 7days after developing diastolic dysfunction at post-operative day 11. Mice were assessed by echocardiography. Left ventricular papillary detergent-extracted fiber bundles were isolated for simultaneous determination of force and ATPase activity. Sarcomeric protein glutathionylation was assessed by immunoblotting. DOCA-salt mice exhibited diastolic dysfunction that was reversed after BH4 treatment. Diastolic sarcomere length (DOCA-salt 1.70±0.01 vs. DOCA-salt+BH4 1.77±0.01μm, P<0.001) and relengthening (relaxation constant, τ, DOCA-salt 0.28±0.02 vs. DOCA-salt+BH4 0.08±0.01, P<0.001) were also restored to control by BH4 treatment. pCa50 for tension increased in DOCA-salt compared to sham but reverted to sham levels after BH4 treatment. Maximum ATPase rate and tension cost (ΔATPase/ΔTension) decreased in DOCA-salt compared to sham, but increased after BH4 treatment. Cardiac MyBP-C glutathionylation increased in DOCA-salt compared to sham, but decreased with BH4 treatment. MyBP-C glutathionylation correlated with the presence of diastolic dysfunction. Our results suggest that by depressing S-glutathionylation of MyBP-C, BH4 ameliorates diastolic dysfunction by reversing a decrease in cross-bridge turnover kinetics. These data provide evidence for modulation of cardiac relaxation by post-translational modification of myofilament proteins.

In vivo and in vitro cardiac responses to beta-adrenergic stimulation in volume-overload heart failure

Hearts in volume overload (VO) undergo progressive ventricular hypertrophy resulting in chronic heart failure that is unresponsive to β-adrenergic agonists. This study compared left ventricular (LV) and isolated cardiomyocyte contractility and β-adrenergic responsiveness in rats with end-stage VO heart failure (HF). Adult male Sprague-Dawley rats were studied 21weeks after aortocaval fistula (ACF) or sham surgery. Echocardiography revealed decreased fractional shortening accompanied by increased LV chamber diameter and decreased eccentric dilatation index at end-stage ACF compared to sham. Hemodynamic measurements showed a decrease in the slope of end-systolic pressure–volume relationship, indicating systolic dysfunction. Isolated LV myocytes from ACF exhibited decreased peak sarcomere shortening and kinetics. Both Ca2+ transient amplitude and kinetics were increased in ACF myocytes, with no change under the integrated Ca2+ curves relating to contraction and relaxation phases. Increases in ryanodine receptor and phospholamban phosphorylation, along with a decrease in SERCA2 levels, were observed in ACF. These changes were associated with decreased expression of β-myosin heavy chain, cardiac troponin I and cardiac myosin binding protein-C. In vivo inotropic responses to β-adrenergic stimulation were attenuated in ACF. Interestingly, ACF myocytes exhibited a similar peak shortening to those of sham in response to a β-adrenergic agonist. The protein expression of the gap junction protein connexin-43 was decreased, although its phosphorylation at Ser-368 increased. These changes were associated with alterations in Src and ZO-1. In summary, these data suggest that the disconnect in β-adrenergic responsiveness between in vivo and in vitro conditions may be associated with altered myofilament Ca2+ sensitivity and connexin-43 degradation.

Abstract 19028: Ablation of the Calpain-Targeted Site in Cardiac Myosin Binding Protein-C is Cardioprotective

Rationale: Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function. We recently demonstrated a direct correlation between cMyBP-C dephosphorylation and its degradation during ischemia-reperfusion (I-R) injury. Strikingly, cMyBP-C phosphorylation protects the heart from I-R injury. However, the mechanism of cMyBP-C-mediated cardioprotection is unknown. Objective: To determine if preventing cMyBP-C proteolysis and cleavage confers cardioprotection during I-R injury. Methods and Results: cMyBP-C is a substrate for calpain and dephosphorylation makes cMyBP-C more susceptible to proteolysis. In patients with congestive heart failure, increased calpain activity was associated with cMyBP-C degradation. During ischemia proteolysis leads to the cleavage and release of the predominant N-terminal fragment (40 kDa) in association with increased calpain activity. MS/MS analysis identified a motif in the cMyBP-C M-domain responsible for the release of the 40 kDa fragment, 272-TSLAGAGRR-280, which we term the calpain-targeted site (CTS). Next, we used cardiac-specific transgenic mice expressing an ablated CTS (cMyBP-C[[Unable to Display Character: &amp;#8710;]]CTS) which were bred into cMyBP-C null (t/t) mice. We hypothesized that ablation of the CTS would confer resistance to calpain-mediated proteolysis and protect the heart from I-R injury. An animal that transgenically expressed the normal cardiac isoform, cMyBP-CWT, served as a control. Histological, electron microscopic and immunofluorescence analyses confirm that cMyBP-C[[Unable to Display Character: &amp;#8710;]]CTS incorporates normally into the cardiac sarcomere and results in rescue of the cMyBP-Ct/t phenotype compared to control. Echocardiography and in vivo hemodynamic studies revealed that cMyBP-C[[Unable to Display Character: &amp;#8710;]]CTS mice have normal cardiac function similar to control mice. Altogether, these data suggest that cMyBP-C[[Unable to Display Character: &amp;#8710;]]CTS is benign. Compared to cMyBP-CWT, cMyBP-C[[Unable to Display Character: &amp;#8710;]]CTS protein is protected from calpain-mediated degradation. Finally, we determined that ablation of the CTS preserved cMyBP-C stability and cardiac function during I-R injury. Conclusions: Our data suggest that preserving cMyBP-C stability by thwarting calpain-mediated proteolysis protects the heart from I-R injury.

Cardiac myosin binding protein-C restricts intrafilament torsional dynamics of actin in a phosphorylation-dependent manner

We have determined the effects of myosin binding protein-C (MyBP-C) and its domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multidomain modulator of striated muscle contraction, interacting with myosin, titin, and possibly actin. Cardiac and slow skeletal MyBP-C are known substrates for protein kinase-A (PKA), and phosphorylation of the cardiac isoform alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin structural dynamics, we labeled actin at C374 with a phosphorescent dye and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy of the TPA decay, indicating restriction of the amplitude of actin torsional flexibility by 15-20° at saturation of the TPA effect. PKA phosphorylation of slow skeletal and cardiac MyBP-C relieved the restriction of torsional amplitude but also decreased the rate of torsional motion. In the case of fast skeletal MyBP-C, its effect on actin dynamics was unchanged by phosphorylation. The isolated C-terminal half of cardiac MyBP-C (C5-C10) had effects similar to those of the full-length protein, and it bound actin more tightly than the N-terminal half (C0-C4), which had smaller effects on actin dynamics that were independent of PKA phosphorylation. We propose that these MyBP-C-induced changes in actin dynamics play a role in the functional effects of MyBP-C on the actin-myosin interaction.

Comparison of Velocity Vector Imaging Echocardiography With Magnetic Resonance Imaging in Mouse Models of Cardiomyopathy

Myocardial strain imaging using echocardiography can be a cost-effective method to quantify ventricular wall motion objectively, but few studies have compared strain measured with echocardiography against magnetic resonance imaging (MRI) in small animals. We compared circumferential strain (CS) and radial strain (RS) measured with echocardiography (velocity vector imaging [VVI]) to displacement encoding with stimulated-echo MRI in 2 mouse models of cardiomyopathy. In 3-month-old mice with gene targeted deficiency of cardiac myosin-binding protein-C (cMyBP-C(-/-), n=6) or muscle LIM protein (MLP(-/-), n=6), and wild-type mice (n=8), myocardial strains were measured at 3 cross-sectional levels and averaged to obtain global strains. There was modest correlation between VVI and MRI measured strains, with global CS yielding stronger correlation compared with global RS (CS R(2)=0.4452 versus RS R(2)=0.2794, both P<0.05). Overall, strain measured by VVI was more variable than MRI (P<0.05) and the limits of agreement were slightly, but not significantly (P=0.14), closer for global CS than RS. Both VVI and MRI strain measurements showed significantly lower global CS strain in the knockout groups compared with the wild type. The VVI (but not MRI) CS strain measurements were different between the 2 knockout groups (-14.5±3.8% versus -6.6±4.0%, cMyBP-C(-/-) versus MLP(-/-) respectively, P<0.05). Measurements of left ventricular CS and RS are feasible in small animals using 2-dimensional echocardiography. VVI and MRI strain measurements correlated modestly and the agreement between the modalities tended to be greater for CS than RS. Although VVI and MRI strains were able to differentiate between wild-type and knockout mice, only global CS VVI differentiated between the 2 models of cardiomyopathy.

Abstract 97: Ablation of Calpain-Targeted Site in Cardiac Myosin Binding Protein C Is Cardioprotective

Rationale: Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function. We recently demonstrated a direct correlation between cMyBP-C dephosphorylation and its degradation during ischemia-reperfusion (I-R) injury. Strikingly, cMyBP-C phosphorylation protects the heart from I-R injury. However, the mechanism of cMyBP-C-mediated cardioprotection is unknown. Objective: To determine if preventing cMyBP-C proteolysis and cleavage confers cardioprotection during I-R injury. Methods and Results: cMyBP-C is a substrate for calpains and dephosphorylation makes cMyBP-C more susceptible to proteolysis. In patients with heart failure, such proteolysis leads to the cleavage and release of the predominant N'-fragment (40 kDa) in association with increased calpain activity. MS/MS analysis identified a cardiac-specific phosphorylation motif responsible for the release of the 40 kDa fragment, 272-TSLAGAGRR-280, which we term the calpain-targeted site (CTS). Next, we utilized cardiac-specific transgenic mice expressing cMyBP-C[[Unable to Display Character: △]]CTS in which CTS motif was ablated and bred into cMyBP-C null (t/t) mice. We hypothesized that ablation of the CTS motif would confer resistance to calpain-mediated proteolysis and thus protect the heart from I-R injury. An animal that transgenically expressed the normal cardiac isoform, cMyBP-CWT, served as a control. Histological, electron microscopic and immunofluorescence analyses confirm that cMyBP-C[[Unable to Display Character: &amp;#8710;]]CTS incorporates normally into the cardiac sarcomere and results in complete rescue of the cMyBP-Ct/t phenotype, compared control groups. Moreover, echocardiography and in vivo hemodynamic studies revealed that cMyBP-C[[Unable to Display Character: &amp;#8710;]]CTS:t/t mice have normal cardiac function, similar to control mice. Altogether, these data suggest that cMyBP-C[[Unable to Display Character: &amp;#8710;]]CTS is benign. Compared to cMyBP-CWT, cMyBP-C[[Unable to Display Character: &amp;#8710;]]CTS protein is protected from calpain-mediated degradation. Finally, we determined that ablation of the CTS reduced infarct size and preserved cMyBP-C stability and cardiac function during I-R injury. Conclusion: Our data suggest that preserving cMyBP-C stability by thwarting calpain-mediated proteolysis protects cardiac structure and function from I-R injury, as a supportive therapy.