Myosin Binding protein-C Phosphorylation Research Articles

Effects of Aging and Fatigue on Human Skeletal Muscle Cellular Contractile Function

Aging is associated with a reduction in skeletal muscle mass and function, known as sarcopenia. While reductions in contractile force are typically explained by loss in muscle cellular cross sectional area (CSA) loss of contractile power during high velocity contractions exceed reductions in force and cannot be solely explained by loss of muscle size and are observed at the single fiber within the same myosin heavy chain isoform (MHC). These phenomenon may be explained by myosin binding protein C (MyBP-C), which is known to modulate contractile force and velocity in a phosphorylation-dependent manner in both cardiac and skeletal muscle. Few studies have investigated the inherent contractile performance of single fibers between old and young adults, and none have explored the effects of altered MyBP-C phosphorylation on single fiber function. Therefore, the PURPOSE of this study was to investigate the effects of age and fatigue-induced MyBP-C phosphorylation on single fiber contractile performance. We HYPOTHESIZED that isometric tension will be enhanced in older adults but velocity and power will be reduced, and fatigue will enhance velocity and power in single fibers. To test this hypothesis, we recruited 4 young (18-35) and 3 older (65-80) adult males to perform a single bout of unilateral knee extensions at 30% maximal voluntary isometric contraction (MVIC) until task failure. Immediately following, a bilateral biopsy of the vastus lateralis (VL) muscle was performed on the fatigued and non-fatigued control limbs. Tissue was either flash frozen for western blot or prepared for mechanical analysis of single fiber tension (Tmax), velocity (Vmax), and power (Pmax) under optimal intracellular metabolic conditions. Our analyses were limited to MHC IIA due to differences in fiber type distribution between the groups. Our RESULTS showed that neither age nor fatigue had an effect on contractile performance in MHC IIA fibers. However, independent analysis of fatigue within age groups suggest trends towards an increase in velocity (p=0.055) in young (MHC IIA) and reduction in tension (p=0.085) in old (MHC I) adult fibers. We CONCLUDE that fatigue may modulate contractile function in the single fiber in an age- and fiber-type dependent manner. Future studies will be aimed at collecting multiple fiber types and investigating the role of biological sex. Wu Tsai Human Performance Alliance. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Roles of cMyBP-C phosphorylation on cardiac contractile dysfunction in db/db mice

Type 2 diabetes mellitus (T2DM) is a metabolic disease and comorbidity associated with several conditions, including cardiac dysfunction leading to heart failure with preserved ejection fraction (HFpEF), in turn resulting in T2DM-induced cardiomyopathy (T2DM-CM). However, the molecular mechanisms underlying the development of T2DM-CM are poorly understood. It is hypothesized that molecular alterations in myopathic genes induced by diabetes promote the development of HFpEF, whereas cardiac myosin inhibitors can rescue the resultant T2DM-mediated cardiomyopathy. To test this hypothesis, a Leptin receptor-deficient db/db homozygous (Lepr db/db) mouse model was used to define the pathogenesis of T2DM-CM. Echocardiographic studies at 4 and 6 months revealed that Lepr db/db hearts started developing cardiac dysfunction by four months, and left ventricular hypertrophy with diastolic dysfunction was evident at 6 months. RNA-seq data analysis, followed by functional enrichment, revealed the differential regulation of genes related to cardiac dysfunction in Lepr db/db heart tissues. Strikingly, the level of cardiac myosin binding protein-C phosphorylation was significantly increased in Lepr db/db mouse hearts. Finally, using isolated skinned papillary muscles and freshly isolated cardiomyocytes, CAMZYOS® (mavacamten, MYK-461), a prescription heart medicine used for symptomatic obstructive hypertrophic cardiomyopathy treatment, was tested for its ability to rescue T2DM-CM. Compared with controls, MYK-461 significantly reduced force generation in papillary muscle fibers and cardiomyocyte contractility in the db/db group. This line of evidence shows that 1) T2DM-CM is associated with hyperphosphorylation of cardiac myosin binding protein-C and 2) MYK-461 significantly lessened disease progression in vitro, suggesting its promise as a treatment for HFpEF.

Single-molecule imaging reveals how mavacamten and PKA modulate ATP turnover in skeletal muscle myofibrils.

Muscle contraction is controlled at two levels: the thin and the thick filaments. The latter level of control involves three states of myosin heads: active, disordered relaxed (DRX), and super-relaxed (SRX), the distribution of which controls the number of myosins available to interact with actin. How these are controlled is still uncertain. Using fluorescently labeled ATP, we were able to spatially assign the activity of individual myosins within the sarcomere. We observed that SRX comprises 53% of all heads in the C-zone compared with 35% and 44% in the P- and D-zones, respectively. The recently FDA-approved hypertrophic cardiomyopathy drug, mavacamten (mava), significantly decreased DRX, favoring SRX in both the C- and D-zones at 60% and 63%, respectively. Since thick filament regulation is in part regulated by the myosin-binding protein-C (MyBP-C), we also studied PKA phosphorylation. This had the opposite effect as mava, specifically in the C-zone where it decreased SRX to 34%, favoring DRX. These results directly show that excess concentrations of mava do increase SRX, but the effect is limited across the sarcomere, suggesting mava is less effective on skeletal muscle. In addition, we show that PKA directly affects the contractile machinery of skeletal muscle leading to the liberation of repressed heads. Since the effect is focused on the C-zone, this suggests it is likely through MyBP-C phosphorylation, although our data suggest that a further reserve of myosins remain that are not accessible to PKA treatment.

Mapping the binding interaction of MyBP-C and myosin in hypertrophic cardiomyopathy

Cardiac myosin binding protein-C (MyBP-C) accounts for about half of hypertrophic cardiomyopathy (HCM) mutations, and in the events where another sarcomeric protein is mutated, MyBP-C phosphorylation is frequently altered. MyBP-C is thought to regulate normal cardiac contractility through its interactions with actin and myosin, and this regulation is dependent on phosphorylation. Efforts in elucidating the details of MyBP-C interactions with myosin and actin have been limited due to low-throughput and labor-intensive assays.

Abstract 14317: Left Ventricular Free Wall Adaptations in Heart Failure With Preserved Ejection Fraction: Insights From a Murine Model

Introduction: Heart failure with preserved ejection fraction has heterogeneous causes. Understanding the cause of diastolic dysfunction can help with pairing correct treatment. Hypothesis: We hypothesize that one can elucidate the underlying cause using echocardiography. Methods: We performed echocardiography on hearts of cardiac myosin binding protein-C phosphorylation deficient mouse t3SA vs. its control tWT at 3 months of age with both sexes (n=6 for each group). Left ventricular free wall (LVFW) specimens were harvested and biaxially stretched along circumferential and longitudinal directions, followed by picrosirius red staining. Fiber architecture was quantified by analyzing histology images. Results: t3SA hearts demonstrated hypertrophy (see Fig. 1a-b for representative mice) and diastolic dysfunction (slowed e’ and increased E/e’ ratio; see Table 1). t3SA LVFW specimens showed increased longitudinal stiffness (p<0.05 for strains >0.25; Fig. 1c-d). Consistent with this stiffening, t3SA specimens exhibited pronounced fiber orientation towards the longitudinal direction (see Fig. 1e-f for representative mice). The smaller e’+S showed that t3SA mice had slowed cross-bridge cycling rates. Biomechanical analyses indicated that LVFW myocardial adaptations contributed to diastolic dysfunction. Conclusions: Echocardiography indices can be used to noninvasively infer underlying remodeling events contributing to diastolic dysfunction facilitating remodeling-targeted therapy selection.

NAD + Redox Imbalance Exacerbates Diabetic Cardiomyopathy via Enhanced Troponin I Phosphorylation, Impaired Energetics and Prolonged Cardiac Relaxation

Diabetes is a major risk factor of heart failure. Diabetes and heart failure have been linked to NAD redox imbalance, whose role in the progression of diabetic cardiomyopathy (DCM) is far from established. Streptozotocin (STZ)-induced diabetes in C57BL/6 mice for 16 weeks promoted declines in systolic and diastolic function and lowered cardiac NAD/NADH ratio. We employed cardiac-specific Ndufs4-KO mice (cKO), which exhibit lowered cardiac NAD/NADH ratio with normal baseline function and energetics, to determine the role of NAD redox imbalance in DCM. Plasma metabolomic analysis showed similar diabetic stress in diabetic control and cKO mice. Systolic (fractional shortening) and diastolic (E’/A’ ratio; isovolumic relaxation time, IVRT) dysfunctions were further exacerbated in diabetic cKO mice in both male and female cohorts. Collagen levels and transcript analyses of fibrosis and extracellular matrix-dependent pathways did not show changes in diabetic cKO hearts, suggesting that the exacerbated cardiac dysfunction was likely due to cardiomyocyte dysfunction. We next analyzed protein acetylation-dependent pathways to account for the exacerbated decline of function in diabetic cKO hearts. NAD redox imbalance in diabetic cKO hearts promoted protein acetylation, including SOD2 acetylation (SOD2-K68Ac). This SOD2 acetylation inhibits its antioxidant function and we observed elevated protein oxidation levels in diabetic cKO hearts. To gain further insights how NAD redox imbalance may regulate cardiomyocyte function, phosphorylation levels of myosin binding protein C (MyBPC) and troponin I (TnI) were examined. MyBPC phosphorylation at S282 is suppressed in failing hearts and remained unchanged in diabetic cKO hearts. TnI phosphorylation at S150 (TnI-S150Pi) increases myofilament calcium sensitivity and prolongs calcium dissociation, while TnI-S23/24Pi decreases calcium sensitivity and accelerates calcium dissociation. In diabetic cKO hearts, levels of TnI-S150Pi were elevated while TnI-S23/24Pi remained unchanged. TnI-S150Pi is sensitive to AMPK, the master energy sensor, and we found that ATP levels decreased, and AMP/ATP ratio increased in diabetic cKO hearts. Elevation of cardiac NAD levels by nicotinamide phosphoribosyltransferase (NAMPT) normalized NAD redox balance and alleviated systolic and diastolic dysfunction. Importantly, elevated TnI-S150Pi, exacerbated diastolic dysfunction and prolonged IVRT in diabetic cKO hearts were all restored by NAMPT over-expression. The data collectively suggest that NAD redox imbalance promotes impairments in cellular energetics and diastolic function via enhancing TnI-S150Pi, and is a critical mediator of the progression of DCM.

Cardiac myosin binding protein-C phosphorylation accelerates β-cardiac myosin detachment rate in mouse myocardium.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that influences sarcomere stiffness and modulates cardiac contraction-relaxation through its phosphorylation. Phosphorylation of cMyBP-C and ablation of cMyBP-C have been shown to increase the rate of MgADP release in the acto-myosin cross-bridge cycle in the intact sarcomere. The influence of cMyBP-C on Pi-dependent myosin kinetics has not yet been examined. We investigated the effect of cMyBP-C, and its phosphorylation, on myosin kinetics in demembranated papillary muscle strips bearing the β-cardiac myosin isoform from nontransgenic and homozygous transgenic mice lacking cMyBP-C. We used quick stretch and stochastic length-perturbation analysis to characterize rates of myosin detachment and force development over 0-12 mM Pi and at maximal (pCa 4.8) and near-half maximal (pCa 5.75) Ca2+ activation. Protein kinase A (PKA) treatment was applied to half the strips to probe the effect of cMyBP-C phosphorylation on Pi sensitivity of myosin kinetics. Increasing Pi increased myosin cross-bridge detachment rate similarly for muscles with and without cMyBP-C, although these rates were higher in muscle without cMyBP-C. Treating myocardial strips with PKA accelerated detachment rate when cMyBP-C was present over all Pi, but not when cMyBP-C was absent. The rate of force development increased with Pi in all muscles. However, Pi sensitivity of the rate force development was reduced when cMyBP-C was present versus absent, suggesting that cMyBP-C inhibits Pi-dependent reversal of the power stroke or stabilizes cross-bridge attachment to enhance the probability of completing the power stroke. These results support a functional role for cMyBP-C in slowing myosin detachment rate, possibly through a direct interaction with myosin or by altering strain-dependent myosin detachment via cMyBP-C-dependent stiffness of the thick filament and myofilament lattice. PKA treatment reduces the role for cMyBP-C to slow myosin detachment and thus effectively accelerates β-myosin detachment in the intact myofilament lattice.NEW & NOTEWORTHY Length perturbation analysis was used to demonstrate that β-cardiac myosin characteristic rates of detachment and recruitment in the intact myofilament lattice are accelerated by Pi, phosphorylation of cMyBP-C, and the absence of cMyBP-C. The results suggest that cMyBP-C normally slows myosin detachment, including Pi-dependent detachment, and that this inhibition is released with phosphorylation or absence of cMyBP-C.

Age, Sex and Overall Health, Measured As Frailty, Modify Myofilament Proteins in Hearts From Naturally Aging Mice

We investigated effects of age, sex and frailty on contractions, calcium transients and myofilament proteins to determine if maladaptive changes associated with aging were sex-specific and modified by frailty. Ventricular myocytes and myofilaments were isolated from middle-aged (~12 mos) and older (~24 mos) mice. Frailty was assessed with a non-invasive frailty index. Calcium transients declined and slowed with age in both sexes, but contractions were largely unaffected. Actomyosin Mg-ATPase activity increased with age in females but not males; this could maintain contractions with smaller calcium transients in females. Phosphorylation of myosin-binding protein C (MyBP-C), desmin, tropomyosin and myosin light chain-1 (MLC-1) increased with age in males, but only MyBP-C and troponin-T increased in females. Enhanced phosphorylation of MyBP-C and MLC-1 could preserve contractions in aging. Interestingly, the age-related decline in Hill coefficients (r = −0.816; p = 0.002) and increase in phosphorylation of desmin (r = 0.735; p = 0.010), tropomyosin (r = 0.779; p = 0.005) and MLC-1 (r = 0.817; p = 0.022) were graded by the level of frailty in males but not females. In these ways, cardiac remodeling at cellular and subcellular levels is graded by overall health in aging males. Such changes may contribute to heart diseases in frail older males, whereas females may be resistant to these effects of frailty.

Roles of NAD Metabolism in Diabetic Cardiomyopathy

Diabetes is a major risk factor of heart failure. Diabetes and heart failure have been linked to altered NAD metabolism, whose roles in the progression of diabetic cardiomyopathy (DC) are far from established. We induced diabetes in wild type C57BL6N mice (WT) by streptozotocin (STZ) injections. WT mice with chronic diabetic stress (fasting glucose ~600 mg/dl) for 16 weeks had gradual declines in systolic and diastolic function and lowered NAD/NADH ratio, while acute STZ treatment (1‐day) did not affect cardiac function and NAD pool. To determine whether NAD redox imbalance promotes DC, we employed cardiac‐specific Ndufs4‐KO mice (cKO), which exhibit lowered cardiac NAD/NADH ratio without overt dysfunction. While insulin depletion and hyperglycemia were similar in diabetic control and cKO mice stressed by the same diabetogenic protocol, systolic and diastolic dysfunctions were accelerated in diabetic cKO mice. We next determined how NAD metabolism is altered to accelerate DC. In addition to the NAD redox balance, NAD metabolism also involves metabolites and enzymes in the NAD consumption and synthesis pathways, coordinating the homeostasis of NAD. Targeted analyses of transcripts and metabolites involved in these pathways were performed using qPCR and quantitative LC‐MS/MS analyses. Of 22 genes measured, we identified that Nmrk mRNA levels were up‐regulated in diabetic cKO hearts, compared to diabetic control hearts. Of thirteen cardiac NAD metabolite levels surveyed, product metabolites of NMRK enzymatic reaction (i.e. NMN or NAMN) showed decreases in levels in diabetic cKO hearts. The transcript and metabolite data suggest a role of NMRK in DC. Histologically, cardiac fibrosis levels of diabetic control and cKO hearts were slightly elevated compared to non‐diabetic controls, but they were not different. The results suggest that the accelerated decline of cardiac function in diabetic cKO hearts is not be due to altered extracellular matrix environment. We next analyzed acetylation‐dependent pathways to account for the accelerated decline of function in diabetic cKO hearts. NAD redox imbalance in diabetic cKO hearts promoted protein acetylation, including SOD2 acetylation (SOD2‐K68Ac). SOD2 acetylation inhibits its antioxidant function and we observed elevated protein oxidation levels in diabetic cKO hearts. To gain further insights how NAD redox imbalance may regulate cardiomyocyte function, phosphorylation levels of myosin binding protein C (MyBPC) and troponin I (TnI) were examined. In diabetic cKO hearts, TnI phosphorylation was elevated while MyBPC phosphorylation remained unchanged, suggesting a regulation of cardiomyocyte contraction/relaxation by NAD‐dependent mechanisms. To normalize NAD redox imbalance, we elevated NAD levels in diabetic cKO mice with cardiac‐specific NAMPT overexpression. Cardiac NAMPT expression slowed the accelerated decline of systolic and diastolic functions in diabetic cKO hearts. Our results support that altered NAD metabolism is a critical determinant for the progression of DC and warrants further investigations.Support or Funding Information1. Scientist Development Grant, AHA2. Seed Funding, Presbyterian Health Foundation of Oklahoma City 1.

Pressure Overload Is Associated With Low Levels of Troponin I and Myosin Binding Protein C Phosphorylation in the Hearts of Patients With Aortic Stenosis.

In previous studies of septal heart muscle from HCM patients with hypertrophic obstructive cardiomyopathy (HOCM, LVOT gradient 50–120 mmHg) we found that the level of phosphorylation of troponin I (TnI) and myosin binding protein C (MyBP-C) was extremely low yet samples from hearts with HCM or DCM mutations that did not have pressure overload were similar to donor heart controls. We therefore investigated heart muscle samples taken from patients undergoing valve replacement for aortic stenosis, since they have pressure overload that is unrelated to inherited cardiomyopathy. Thirteen muscle samples from septum and from free wall were analyzed (LVOT gradients 30–100 mmHg) The levels of TnI and MyBP-C phosphorylation were determined in muscle myofibrils by separating phosphospecies using phosphate affinity SDS-PAGE and detecting with TnI and MyBP-C specific antibodies. TnI was predominantly monophosphorylated and total phosphorylation was 0.85 ± 0.03 molsPi/mol TnI. This phosphorylation level was significantly different (p < 0.0001) from both donor heart TnI (1.6 ± 0.06 molsPi/mol TnI) and HOCM heart TnI (0.19 ± 0.04 molsPi/mol TnI). MyBP-C is phosphorylated at up to four sites. In donor heart the 4P and 3P species predominate but in the pressure overload samples the 4P species was much reduced and 3P and 1P species predominated. Total phosphorylation was 2.0 ± 0.2 molsPi/mol MyBP-C (n = 8) compared with 3.4 ± 0.07 (n = 21) in donor heart and 1.1 ± 0.1 (n = 10) in HOCM heart. We conclude that pressure overload may be associated with substantial dephosphorylation of troponin I and MyBP-C.

Altered Thick and Thin Filament Structural Dynamics in Mouse Myocardium Due to Ablation and Phosphorylation of Myosin Binding Protein-C

Cardiac Myosin Binding Protein-C (cMyBP-C) is an important regulator of myocardial contraction and cross-bridge disposition; however, its contributions to thick and thin filament structural dynamics is unknown. Therefore, we performed time-resolved fluorescence (TR-F) experiments to quantify lifetime changes of environmentally-sensitive probes introduced in myofibrils isolated from ventricles of wild-type (WT) and cMyBP-C null (cMyBP-C-/-) mice. Mutant ventricular regulatory light chain (RLC V105C) labeled with CPM or cardiac troponin C (cTnC T53C) labeled with IANBD were exchanged in skinned myofibrils to replace the endogenous proteins. ATPase measurements demonstrated that the exchanges did not affect contractile function. TR-F measurements revealed differences in lifetimes of both RLC and cTnC between WT and cMyBP-C-/- myofibrils. PKA-mediated phosphorylation of myofibrils also affected lifetimes with greater effects observed in WT as compared to cMyBP-C-/-. For the RLC probe, the differences in lifetimes due to PKA phosphorylation were greatest with addition of myosin inhibitors: blebbistatin or vanadate. These results are consistent with the idea that cMyBP-C plays a role in stabilizing myosin on the thick filament and promoting the super-relaxed state, whereas its absence or phosphorylation leads to increased molecular disorder. For the cTnC probe, PKA treatment of myofibrils reduced the Ca2+-sensitivity of lifetime in both WT and cMyBP-C-/-. These results suggest unique phosphorylation-dependent roles for cMyBP-C and cardiac troponin I (cTnI) on thin filament structural dynamics. Our findings demonstrate that cMyBP-C and cTnI contribute to phosphorylation-dependent effects on thick and thin filament structural dynamics to influence force development. These TR-F probes can also be useful for screening of compounds that alter cMyBP-C interactions with the myofilaments.

Abstract 906: Age-Dependent Modifications in Cardiac Myofilament Proteins are Graded by Overall Health, Measured as Frailty, in Aging Male and Female C57BL/6 Mice

Previously, we showed that the age-dependent decline in ventricular function in male mice is proportional to overall health, as quantified with a frailty index (FI) tool. This tool is based on the accumulation of deficits in health across many systems, but not the cardiovascular system per se . Here we investigated whether frailty grades age-dependent modifications in myofilaments in young and aged mice of both sexes. Myofilaments were isolated from ventricles of adult (12-13 mos; n=5 males, 5 females) and aged (22-23 mos; n=6 males, 5 females) mice. FI scores were obtained from all mice. Actomyosin Mg-ATPase activity as a function of increasing calcium concentration and phosphorylation of major myofilament proteins were quantified and compared between groups. Results showed that myofilament calcium sensitivity (EC 50 values) was similar regardless of age or sex and was not affected by FI score. Maximal actomyosin Mg-ATPase activity increased with age in females, but this was not linked to frailty. Hill coefficients declined with age in males only and were lower than females in the older group (p&lt;0.05). However when all mice were examined, Hill coefficients declined as frailty increased (r=0.68, p=0.001). Total phosphorylation levels of myosin-binding protein C (MyBP-C), desmin, tropomyosin and essential myosin light chain (ELC) increased with age, but only in males. Interestingly, phosphorylation of MyBP-C (r=0.49; p=0.03), desmin (r=0.65; p=0.003), tropomyosin (r=0.62; p=0.004) and ELC (r=0.71; p=0.001) were highly correlated with overall health (FI scores). By contrast there was no link between frailty and phosphorylation of actin, troponin T and troponin I. These findings suggest that poor overall health, quantified in an FI, predicts changes in the myofilaments across the life course in both sexes. This may contribute to changes in cardiac contractile function in frail older adults.

Abstract 621: Cardiac Myosin Binding Protein-C Phosphorylation in Ser-273 and Ser-282 is Critical to Maintain Cardiac Function During Aging

Background/hypothesis: Heart failure (HF) afflicts 6.2M Americans as of 2019 and its prevalence is expected to increase due to a growing aging population. HF progression involves depression in contractility and relaxation, which are strongly controlled by phosphorylation of cardiac myosin binding protein-C (cMyBP-C). We hypothesize that phosphorylated cMyBP-C supports normal heart function during aging. Methods: We aged mouse models of cMyBP-C de-phosphorylation mimetic (t3SA: S273A/S282A/S302A mutations), phosphorylation mimetic (t3SD: S273D/S282D/S302D mutations) and WT equivalent expressed onto cMyBP-C(-/-) background (tWT). Results: Western blotting (N=16, 2-24 months) showed that cMyBP-C phosphorylation decreases with aging at S273 and S282, showing strong Pearson correlations with age (see figure; S273P r= -0.931, p&lt;0.0001; S282P r=-0.868, p&lt;0.00001). Simultaneous force and [Ca 2+ ] i measurements on intact papillary muscles showed that phosphorylation-mimetic t3SD has greatest dFR=(-dF/dt)min/(+dF/dt)max, signifying better lusitropy with similar calcium decay rate constant (k Ca ) as others. Echocardiography at 3, 12, 15, 18 months showed that t3SD has preserved ejection fraction, faster peak myocardial relaxation velocity e’ and preserved posterior ventricular wall thickness with aging. Moreover, t3SD demonstrated superior 600-days survival than others (t3SD 87.5% n=96; tWT 69.3% n=122; t3SA 53.2% n=97; overall LogRank p=0.0002244). Conclusions: De-phosphorylation of cMyBP-C at S273 and S282 contributes to worsening of cardiac function with aging. Conversely, phosphorylated cMyBP-C mitigates aging-related cardiac dysfunction.

Site-specific phosphorylation of myosin binding protein-C coordinates thin and thick filament activation in cardiac muscle

The heart's response to varying demands of the body is regulated by signaling pathways that activate protein kinases which phosphorylate sarcomeric proteins. Although phosphorylation of cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator of myocardial contractility, little is known about its mechanism of action. Here, we used protein kinase A (PKA) and Cε (PKCε), as well as ribosomal S6 kinase II (RSK2), which have different specificities for cMyBP-C's multiple phosphorylation sites, to show that individual sites are not independent, and that phosphorylation of cMyBP-C is controlled by positive and negative regulatory coupling between those sites. PKA phosphorylation of cMyBP-C's N terminus on 3 conserved serine residues is hierarchical and antagonizes phosphorylation by PKCε, and vice versa. In contrast, RSK2 phosphorylation of cMyBP-C accelerates PKA phosphorylation. We used cMyBP-C's regulatory N-terminal domains in defined phosphorylation states for protein-protein interaction studies with isolated cardiac native thin filaments and the S2 domain of cardiac myosin to show that site-specific phosphorylation of this region of cMyBP-C controls its interaction with both the actin-containing thin and myosin-containing thick filaments. We also used fluorescence probes on the myosin-associated regulatory light chain in the thick filaments and on troponin C in the thin filaments to monitor structural changes in the myofilaments of intact heart muscle cells associated with activation of myocardial contraction by the N-terminal region of cMyBP-C in its different phosphorylation states. Our results suggest that cMyBP-C acts as a sarcomeric integrator of multiple signaling pathways that determines downstream physiological function.

Cardiac myosin binding protein-C phosphorylation regulates the super-relaxed state of myosin.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) accelerates cardiac contractility. However, the mechanisms by which cMyBP-C phosphorylation increases contractile kinetics have not been fully elucidated. In this study, we tested the hypothesis that phosphorylation of cMyBP-C releases myosin heads from the inhibited super-relaxed state (SRX), thereby determining the fraction of myosin available for contraction. Mice with various alanine (A) or aspartic acid (D) substitutions of the three main phosphorylatable serines of cMyBP-C (serines 273, 282, and 302) were used to address the association between cMyBP-C phosphorylation and SRX. Single-nucleotide turnover in skinned ventricular preparations demonstrated that phosphomimetic cMyBP-C destabilized SRX, whereas phospho-ablated cMyBP-C had a stabilizing effect on SRX. Strikingly, phosphorylation at serine 282 site was found to play a critical role in regulating the SRX. Treatment of WT preparations with protein kinase A (PKA) reduced the SRX, whereas, in nonphosphorylatable cMyBP-C preparations, PKA had no detectable effect. Mice with stable SRX exhibited reduced force production. Phosphomimetic cMyBP-C with reduced SRX exhibited increased rates of tension redevelopment and reduced binding to myosin. We also used recombinant myosin subfragment-2 to disrupt the endogenous interaction between cMyBP-C and myosin and observed a significant reduction in the population of SRX myosin. This peptide also increased force generation and rate of tension redevelopment in skinned fibers. Taken together, this study demonstrates that the phosphorylation-dependent interaction between cMyBP-C and myosin is a determinant of the fraction of myosin available for contraction. Furthermore, the binding between cMyBP-C and myosin may be targeted to improve contractile function.

MYBPC3 truncation mutations enhance actomyosin contractile mechanics in human hypertrophic cardiomyopathy

RationaleTruncation mutations in the MYBPC3 gene, encoding for cardiac myosin-binding protein C (MyBP-C), are the leading cause of hypertrophic cardiomyopathy (HCM). Whole heart, fiber and molecular studies demonstrate that MyBP-C is a potent modulator of cardiac contractility, but how these mutations contribute to HCM is unresolved. ObjectivesTo readdress whether MYBPC3 truncation mutations result in loss of MyBP-C content and/or the expression of truncated MyBP-C from the mutant allele and determine how these mutations effect myofilament sliding in human myocardium. Methods and resultsSeptal wall tissue samples were obtained from HCM patients undergoing myectomy (n = 18) and donor controls (n = 8). The HCM samples contained 40% less MyBP-C and reduced levels of MyBP-C phosphorylation, when compared to the donor control samples using quantitative mass spectrometry. These differences occurred in the absence of changes in the stoichiometry of other myofilament proteins or production of truncated MyBP-C from the mutant MYBPC3 allele. The functional impact of MYBPC3 truncation mutations on myofilament sliding was determined using a total internal reflection microscopy (TIRFM) single particle assay. Myosin-thick filaments containing their native complement of MyBP-C, and actin-thin filaments decorated with the troponin/tropomyosin calcium regulatory proteins, were isolated from a subgroup of the HCM (n = 4) and donor (n = 5) heart samples. The maximal sliding velocity of native thin filaments was enhanced within the C-zones of the native thick filaments isolated from the HCM samples, when compared to velocity within the C-zones of thick filaments isolated from the donor samples. Analytical modeling demonstrated that the 40% reduction in MyBP-C content was sufficient to enhance the myofilament sliding velocity, as observed in the TIRFM assay. ConclusionsHCM-causing MYBPC3 truncation mutations result in a loss of MyBP-C content that enhances maximal myofilament sliding velocities, only where MyBP-C is localized within the C-zone. These findings support therapeutic rationale for restoring normal levels of MyBP-C and/or dampening maximal contractile velocities for the treatment of human HCM.

Structural and functional effects of myosin-binding protein-C phosphorylation in heart muscle are not mimicked by serine-to-aspartate substitutions

Myosin-binding protein-C (cMyBP-C) is a key regulator of contractility in heart muscle, and its regulatory function is controlled in turn by phosphorylation of multiple serines in its m-domain. The structural and functional effects of m-domain phosphorylation have often been inferred from those of the corresponding serine-to-aspartate (Ser–Asp) substitutions, in both in vivo and in vitro studies. Here, using a combination of in vitro binding assays and in situ structural and functional assays in ventricular trabeculae of rat heart and the expressed C1mC2 region of cMyBP-C, containing the m-domain flanked by domains C1 and C2, we tested whether these substitutions do in fact mimic the effects of phosphorylation. In situ changes in thin and thick filament structure were determined from changes in polarized fluorescence from bifunctional probes attached to troponin C or myosin regulatory light chain, respectively. We show that both the action of exogenous C1mC2 to activate contraction in the absence of calcium and the accompanying change in thin filament structure are abolished by tris-phosphorylation of the m-domain, but unaffected by the corresponding Ser–Asp substitutions. The latter produced an intermediate change in thick filament structure. Both tris-phosphorylation and Ser–Asp substitutions abolished the interaction between C1mC2 and myosin sub-fragment 2 (myosin S2) in vitro, but yielded different effects on thin filament binding. These results suggest that some previous inferences from the effects of Ser–Asp substitutions in cMyBP-C should be reconsidered and that the distinct effects of tris-phosphorylation and Ser–Asp substitutions on cMyBP-C may provide a useful basis for future studies.

MYBP-C Phosphorylation Accelerates Sarcomere Shortening as Thin Filaments Slide through the C-Zone

Myosin Binding Protein-C (MyBP-C) is a 140-150 kDa thick filament protein located in the middle third of each sarcomere, i.e., the C-zone, and is likely a key regulator of contraction. Phosphorylation of MyBP-C occurs at 4 residues within its N-terminus and PKA phosphorylation of MyBP-C in hearts is associated with faster cardiac muscle twitch dynamics (Tong et. al. Circ. Res. 2008). Here we investigated potential mechanisms by which MyBP-C phosphorylation regulates myofilament function. We utilized slow-twitch skeletal muscle fibers since PKA phosphorylates slow skeletal (ss)MyBP-C but not (ss)troponin I. Permeabilized rat slow-twitch fibers were mounted between a force transducer and motor, and calcium activated to elicit ∼30% maximal force. We measured rates of force development and transient force overshoots following a slack re-stretch maneuver and monitored sarcomere shortening during load-clamps. Following PKA, force development rates increased ∼40% and there was a near-doubling in the magnitude of transient force overshoot. We also observed faster shortening velocities at all loads following PKA treatment; this may arise from (i) faster cross-bridge cycling rates, (ii) greater number of cross-bridges during load clamps, and/or (iii) attenuation of internal drag imposed by binding of MyBP-C to actin. To address these mechanisms, we quantified sarcomere length shortening as thin filaments traversed into the C-zone. Before PKA, there tended to be a slowdown as sarcomeres shortened from ∼3.05 to 2.90 µm. After PKA, sarcomeres shortened a considerably greater distance during similar load clamps and, at times, shortening even accelerated as thin filaments slid through the C-zone. Overall these results suggest MyBP-C phosphorylation speeds sarcomere shortening and power output by a combination of faster cross-bridge cycling kinetics, recruitment of cross-bridges, and reduction in drag forces as thin filaments slide through the C-zone.

Muscle Lim Protein and myosin binding protein C form a complex regulating muscle differentiation

Muscle Lim Protein (MLP) is a protein with multiple functional roles in striated muscle physiology and pathophysiology. Herein, we demonstrate that MLP directly binds to slow, fast, and cardiac myosin-binding protein C (MyBP-C) during myogenesis, as shown by yeast two-hybrid and a range of protein-protein interaction assays. The minimal interacting domains involve MLP inter-LIM and MyBP-C [C4]. The interaction is sensitive to cytosolic Ca2+ concentrations changes and to MyBP-C phosphorylation by PKA or CaMKII. Confocal microscopy of differentiating myoblasts showed MLP and MyBP-C colocalization during myoblast differentiation. Suppression of the complex formation with recombinant MyBP-C [C4] peptide overexpression, inhibited myoblast differentiation by 65%. Suppression of both MLP and MyBP-C expression in myoblasts by siRNA revealed negative synergistic effects on differentiation. The MLP/MyBP-C complex modulates the actin activated myosin II ATPase activity in vitro, which could interfere with sarcomerogenesis and myofilaments assembly during differentiation. Our data demonstrate a critical role of the MLP/MyBP-C complex during early myoblast differentiation. Its absence in muscles with mutations or aberrant expression of MLP or MyBP-C could be directly implicated in the development of cardiac and skeletal myopathies.

The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations.

Hypertrophic cardiomyopathy (HCM) is primarily caused by mutations in β-cardiac myosin and myosin-binding protein-C (MyBP-C). Changes in the contractile parameters of myosin measured so far do not explain the clinical hypercontractility caused by such mutations. We propose that hypercontractility is due to an increase in the number of myosin heads (S1) that are accessible for force production. In support of this hypothesis, we demonstrate myosin tail (S2)-dependent functional regulation of actin-activated human β-cardiac myosin ATPase. In addition, we show that both S2 and MyBP-C bind to S1 and that phosphorylation of either S1 or MyBP-C weakens these interactions. Importantly, the S1-S2 interaction is also weakened by four myosin HCM-causing mutations but not by two other mutations. To explain these experimental results, we propose a working structural model involving multiple interactions, including those with myosin's own S2 and MyBP-C, that hold myosin in a sequestered state.