Suresh Govindan

Myocardial Infarction-induced N-terminal Fragment of Cardiac Myosin-binding Protein C (cMyBP-C) Impairs Myofilament Function in Human Myocardium

Background: Myocardial infarction (MI) leads to proteolytic cleavage of cMyBP-C (hC0C1f) and decreased contractility. Results: hC0C1f can incorporate into the human cardiac sarcomere, depressing force generation and increasing tension cost. Conclusion: Interaction between hC0C1f and both actin and α-tropomyosin causes disruption of intact cMyBP-C function. Significance: Proteolytic cleavage of cMyBP-C is sufficient to cause contractile dysfunction following MI. Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca2+ transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 μm) and long (2.3 μm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca2+ sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca2+ sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.

Cardiac Myosin-binding Protein C and Troponin-I Phosphorylation Independently Modulate Myofilament Length-dependent Activation

Background: Myofilament length-dependent activation (LDA) is modulated by phosphorylation. Results: Myosin binding protein C (MyBP-C) constitutes the dominant molecular mechanism underlying phosphorylation, with a minor and independent contribution of cardiac troponin-I (cTnI). Conclusion: MyBP-C, and to a lesser extent cTnI, both contribute to enhance LDA. Significance: Novel insights into the molecular basis LDA and its regulation by phosphorylation. β-Adrenergic stimulation in heart leads to increased contractility and lusitropy via activation of protein kinase A (PKA). In the cardiac sarcomere, both cardiac myosin binding protein C (cMyBP-C) and troponin-I (cTnI) are prominent myofilament targets of PKA. Treatment of permeabilized myocardium with PKA induces enhanced myofilament length-dependent activation (LDA), the cellular basis of the Frank-Starling cardiac regulatory mechanism. It is not known, however, which of these targets mediates the altered LDA and to what extent. Here, we employed two genetic mouse models in which the three PKA sites in cMyBP-C were replaced with either phospho-mimic (DDD) or phospho-null (AAA) residues. AAA- or DDD-permeabilized myocytes (n = 12–17) were exchanged (∼93%) for recombinant cTnI in which the two PKA sites were mutated to either phospho-mimic (DD) or phospho-null (AA) residues. Force-[Ca2+] relationships were determined at two sarcomere lengths (SL = 1.9 μm and SL = 2.3 μm). Data were fit to a modified Hill equation for each individual cell preparation at each SL. LDA was indexed as ΔEC50, the difference in [Ca2+] required to achieve 50% force activation at the two SLs. We found that PKA-mediated phosphorylation of cMyBP-C and cTnI each independently contribute to enhance myofilament length-dependent activation properties of the cardiac sarcomere, with relative contributions of ∼67 and ∼33% for cMyBP-C for cTnI, respectively. We conclude that β-adrenergic stimulation enhances the Frank-Starling regulatory mechanism predominantly via cMyBP-C PKA-mediated phosphorylation. We speculate that this molecular mechanism enhances cross-bridge formation at long SL while accelerating cross-bridge detachment and relaxation at short SLs.

Cardiac Myosin Binding Protein-C Plays No Regulatory Role in Skeletal Muscle Structure and Function

Myosin binding protein-C (MyBP-C) exists in three major isoforms: slow skeletal, fast skeletal, and cardiac. While cardiac MyBP-C (cMyBP-C) expression is restricted to the heart in the adult, it is transiently expressed in neonatal stages of some skeletal muscles. However, it is unclear whether this expression is necessary for the proper development and function of skeletal muscle. Our aim was to determine whether the absence of cMyBP-C alters the structure, function, or MyBP-C isoform expression in adult skeletal muscle using a cMyBP-C null mouse model (cMyBP-C(t/t)). Slow MyBP-C was expressed in both slow and fast skeletal muscles, whereas fast MyBP-C was mostly restricted to fast skeletal muscles. Expression of these isoforms was unaffected in skeletal muscle from cMyBP-C(t/t) mice. Slow and fast skeletal muscles in cMyBP-C(t/t) mice showed no histological or ultrastructural changes in comparison to the wild-type control. In addition, slow muscle twitch, tetanus tension, and susceptibility to injury were all similar to the wild-type controls. Interestingly, fMyBP-C expression was significantly increased in the cMyBP-C(t/t) hearts undergoing severe dilated cardiomyopathy, though this does not seem to prevent dysfunction. Additionally, expression of both slow and fast isoforms was increased in myopathic skeletal muscles. Our data demonstrate that i) MyBP-C isoforms are differentially regulated in both cardiac and skeletal muscles, ii) cMyBP-C is dispensable for the development of skeletal muscle with no functional or structural consequences in the adult myocyte, and iii) skeletal isoforms can transcomplement in the heart in the absence of cMyBP-C.

A hypertrophic cardiomyopathy-associated MYBPC3 mutation common in populations of South Asian descent causes contractile dysfunction

Background: A 25-base pair deletion in the MYBPC3 gene is the most prevalent hypertrophic cardiomyopathy-associated mutation among South Asian populations. Results: Expression of mutant protein (cMyBP-CC10mut) in cultured adult rat cardiomyocytes led to improper localization and contractile dysfunction. Conclusion: cMyBP-CC10mut expression is sufficient to cause contractile dysfunction. Significance: The study sheds light on the etiology of this ethnic-specific hypertrophic cardiomyopathy mutation. Hypertrophic cardiomyopathy (HCM) results from mutations in genes encoding sarcomeric proteins, most often MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). A recently discovered HCM-associated 25-base pair deletion in MYBPC3 is inherited in millions worldwide. Although this mutation causes changes in the C10 domain of cMyBP-C (cMyBP-CC10mut), which binds to the light meromyosin (LMM) region of the myosin heavy chain, the underlying molecular mechanism causing HCM is unknown. In this study, adenoviral expression of cMyBP-CC10mut in cultured adult rat cardiomyocytes was used to investigate protein localization and evaluate contractile function and Ca2+ transients, compared with wild-type cMyBP-C expression (cMyBP-CWT) and controls. Forty-eight hours after infection, 44% of cMyBP-CWT and 36% of cMyBP-CC10mut protein levels were determined in total lysates, confirming equal expression. Immunofluorescence experiments showed little or no localization of cMyBP-CC10mut to the C-zone, whereas cMyBP-CWT mostly showed C-zone staining, suggesting that cMyBP-CC10mut could not properly integrate in the C-zone of the sarcomere. Subcellular fractionation confirmed that most cMyBP-CC10mut resided in the soluble fraction, with reduced presence in the myofilament fraction. Also, cMyBP-CC10mut displayed significantly reduced fractional shortening, sarcomere shortening, and relaxation velocities, apparently caused by defects in sarcomere function, because Ca2+ transients were unaffected. Co-sedimentation and protein cross-linking assays confirmed that C10mut causes the loss of C10 domain interaction with myosin LMM. Protein homology modeling studies showed significant structural perturbation in cMyBP-CC10mut, providing a potential structural basis for the alteration in its mode of interaction with myosin LMM. Therefore, expression of cMyBP-CC10mut protein is sufficient to cause contractile dysfunction in vitro.

Oxidative Stress in Dilated Cardiomyopathy Caused by MYBPC3 Mutation

Cardiomyopathies can result from mutations in genes encoding sarcomere proteins including MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). However, whether oxidative stress is augmented due to contractile dysfunction and cardiomyocyte damage in MYBPC3-mutated cardiomyopathies has not been elucidated. To determine whether oxidative stress markers were elevated in MYBPC3-mutated cardiomyopathies, a previously characterized 3-month-old mouse model of dilated cardiomyopathy (DCM) expressing a homozygous MYBPC3 mutation (cMyBP-C(t/t)) was used, compared to wild-type (WT) mice. Echocardiography confirmed decreased percentage of fractional shortening in DCM versus WT hearts. Histopathological analysis indicated a significant increase in myocardial disarray and fibrosis while the second harmonic generation imaging revealed disorganized sarcomeric structure and myocyte damage in DCM hearts when compared to WT hearts. Intriguingly, DCM mouse heart homogenates had decreased glutathione (GSH/GSSG) ratio and increased protein carbonyl and lipid malondialdehyde content compared to WT heart homogenates, consistent with elevated oxidative stress. Importantly, a similar result was observed in human cardiomyopathy heart homogenate samples. These results were further supported by reduced signals for mitochondrial semiquinone radicals and Fe-S clusters in DCM mouse hearts measured using electron paramagnetic resonance spectroscopy. In conclusion, we demonstrate elevated oxidative stress in MYPBC3-mutated DCM mice, which may exacerbate the development of heart failure.

Molecular Screen Identifies Cardiac Myosin–Binding Protein-C as a Protein Kinase G-Iα Substrate

Background—Pharmacological activation of cGMP-dependent protein kinase G I (PKGI) has emerged as a therapeutic strategy for humans with heart failure. However, PKG-activating drugs have been limited by hypotension arising from PKG-induced vasodilation. PKGI&agr; antiremodeling substrates specific to the myocardium might provide targets to circumvent this limitation, but currently remain poorly understood. Methods and Results—We performed a screen for myocardial proteins interacting with the PKGI&agr; leucine zipper (LZ)–binding domain to identify myocardial-specific PKGI antiremodeling substrates. Our screen identified cardiac myosin–binding protein-C (cMyBP-C), a cardiac myocyte–specific protein, which has been demonstrated to inhibit cardiac remodeling in the phosphorylated state, and when mutated leads to hypertrophic cardiomyopathy in humans. GST pulldowns and precipitations with cGMP-conjugated beads confirmed the PKGI&agr;–cMyBP-C interaction in myocardial lysates. In vitro studies demonstrated that purified PKGI&agr; phosphorylates the cMyBP-C M-domain at Ser-273, Ser-282, and Ser-302. cGMP induced cMyBP-C phosphorylation at these residues in COS cells transfected with PKGI&agr;, but not in cells transfected with LZ mutant PKGI&agr;, containing mutations to disrupt LZ substrate binding. In mice subjected to left ventricular pressure overload, PKGI activation with sildenafil increased cMyBP-C phosphorylation at Ser-273 compared with untreated mice. cGMP also induced cMyBP-C phosphorylation in isolated cardiac myocytes. Conclusions—Taken together, these data support that PKGI&agr; and cMyBP-C interact in the heart and that cMyBP-C is an anti remodeling PKGI&agr; kinase substrate. This study provides the first identification of a myocardial-specific PKGI&agr; LZ-dependent antiremodeling substrate and supports further exploration of PKGI&agr; myocardial LZ substrates as potential therapeutic targets for heart failure.

Alterations in Multi‐Scale Cardiac Architecture in Association With Phosphorylation of Myosin Binding Protein‐C

Background The geometric organization of myocytes in the ventricular wall comprises the structural underpinnings of cardiac mechanical function. Cardiac myosin binding protein‐C (MYBPC3) is a sarcomeric protein, for which phosphorylation modulates myofilament binding, sarcomere morphology, and myocyte alignment in the ventricular wall. To elucidate the mechanisms by which MYBPC3 phospho‐regulation affects cardiac tissue organization, we studied ventricular myoarchitecture using generalized Q‐space imaging (GQI). GQI assessed geometric phenotype in excised hearts that had undergone transgenic (TG) modification of phospho‐regulatory serine sites to nonphosphorylatable alanines (MYBPC3AllP−/(t/t)) or phospho‐mimetic aspartic acids (MYBPC3AllP+/(t/t)). Methods and Results Myoarchitecture in the wild‐type (MYBPC3WT) left‐ventricle (LV) varied with transmural position, with helix angles ranging from −90/+90 degrees and contiguous circular orientation from the LV mid‐myocardium to the right ventricle (RV). Whereas MYBPC3AllP+/(t/t) hearts were not architecturally distinct from MYBPC3WT, MYBPC3AllP−/(t/t) hearts demonstrated a significant reduction in LV transmural helicity. Null MYBPC3(t/t) hearts, as constituted by a truncated MYBPC3 protein, demonstrated global architectural disarray and loss in helicity. Electron microscopy was performed to correlate the observed macroscopic architectural changes with sarcomere ultrastructure and demonstrated that impaired phosphorylation of MYBPC3 resulted in modifications of the sarcomere aspect ratio and shear angle. The mechanical effect of helicity loss was assessed through a geometric model relating cardiac work to ejection fraction, confirming the mechanical impairments observed with echocardiography. Conclusions We conclude that phosphorylation of MYBPC3 contributes to the genesis of ventricular wall geometry, linking myofilament biology with multiscale cardiac mechanics and myoarchitecture.

Usefulness of Released Cardiac Myosin Binding Protein-C as a Predictor of Cardiovascular Events

Cardiac myosin binding protein-C (cMyBP-C) is a heart muscle-specific thick filament protein. Elevated level of serum cMyBP-C is an indicator of early myocardial infarction (MI), but its value as a predictor of future cardiovascular disease is unknown. Based on the presence of significant amount of cMyBP-C in the serum of previous study subjects independent of MI, we hypothesized that circulating cMyBP-C is a sensitive indicator of ongoing cardiovascular stress and disease. To test this hypothesis, 75 men and 83 women of similar ages were recruited for a prospective study. They underwent exercise stress echocardiography to provide pre- and poststress blood samples for subsequent determination of serum cMyBP-C levels. The subjects were followed for 1 to 1.5 years. Exercise stress increased serum cMyBP-C in all subjects. Twenty-seven primary events (such as death, MI, revascularization, invasive cardiovascular procedure, or cardiovascular-related hospitalization) and 7 critical events (CE; such as death, MI, stroke, or pulmonary embolism) occurred. After adjusting for sex and cardiovascular risk factors with multivariate Cox regression, a 96% sensitive prestress cMyBP-C threshold carried a hazard ratio of 8.1 with p = 0.041 for primary events. Most subjects (6 of 7) who had CE showed normal ejection fraction on echocardiography. Pre-stress cMyBP-C demonstrated area under receiver operating curve of 0.91 and multivariate Cox regression hazard ratio of 13.8 (p = 0.000472) for CE. Thus, basal cMyBP-C levels reflected susceptibility for a variety of cardiovascular diseases. Together with its high sensitivity, cMyBP-C holds potential as a screening biomarker for the existence of severe cardiovascular diseases.

ID: 136: N-TERMINAL REGION OF CARDIAC MYOSIN BINDING PROTEIN-C IS NECESSARY FOR CARDIAC FUNCTION

Rationale Cardiac myosin binding protein-C (cMyBP-C) is a trans-filament protein that has been shown to regulate cardiac function via its amino terminal (N′) region. In vitro studies have suggested the importance of the first 271 N′-residues of cMyBP-C (C0-C1f region) in slowing actin filament sliding over myosin to regulate cross-bridge cycling kinetics within the cardiac sarcomere. However, the role and necessity of the C0-C1f region of cMyBP-C in regulating contractile and cardiac function in vivo have not been elucidated. Hypothesis The N′-C0-C1f region of cMyBP-C is critical for proper cardiac function in vivo. Methods and Results Transgenic mice with approximately 95% expression of a mutant truncated cMyBP-C missing the N′-C0-C1f region (cMyBP-C110 kDa), compared to endogenous cMyBP-C, were generated and characterized at 3-months of age. cMyBP-C110 kDa hearts had significantly elevated heart weight/body weight ratio, fibrosis, nuclear area and collagen content compared to hearts from non-transgenic (NTG) littermates. Electron microscopic analysis revealed normal sarcomere structure in cMyBP-C110 kDa hearts but with apparently weaker cMyBP-C stripes. Furthermore, the ability of cMyBP-C to slow actin-filament sliding within the C-zone of native thick filaments isolated from NTG hearts was lost on thick filaments from cMyBP-C110 kDa hearts. Short axis M-mode echocardiography revealed a significant increase in left ventricular (LV) internal diameter during diastole in cMyBP-C110 kDa hearts. Importantly, cMyBP-C110 kDa hearts displayed a significant reduction in fractional shortening compared to hearts from NTG mice. We further observed a decrease in the thickness of the LV interventricular septum and free wall during systole in cMyBP-C110 kDa hearts. Strain analysis using images acquired from ECG-Gated Kilohertz Visualization identified a significant deficit in global longitudinal strain in cMyBP-C110 kDa hearts compared to NTG hearts. Consistent with cardiac hypertrophy, we observed a significant increase in the expression of the hypertrophic genes MYH7 and NPPA by real-time PCR analysis. As expected, the expression levels of the MYBPC3 gene were significantly elevated in cMyBP-C110 kDa hearts compared to NTG hearts. Surprisingly, our Western blot analyses revealed no significant difference in total cMyBP-C levels between NTG and cMyBP-C110 kDa heart homogenates. However, intriguingly, we observed a significant elevation in cMyBP-C phosphorylation at Ser-273, Ser-282, and Ser-302, sites important for cMyBP-Cs regulation of actomyosin interaction, in cMyBP-C110 kDa heart homogenates compared to those from NTG mice. Conclusion The N′-C0-C1f region of cMyBP-C is essential for maintaining normal cardiac morphology and function in vivo and loss of this region promotes contractile dysfunction both at the molecular and tissue level.