Cardiac Myosin Binding protein-C Phosphorylation Research Articles

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.

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.

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.

CMyBP-C phosphorylation modulates the time-dependent slowing of unloaded shortening in murine skinned myocardium.

In myocardium, phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) is thought to modulate the cooperative activation of the thin filament by binding to myosin and/or actin, thereby regulating the probability of cross-bridge binding to actin. At low levels of Ca2+ activation, unloaded shortening velocity (Vo) in permeabilized cardiac muscle is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The velocities in these phases scale with the level of activation, culminating in a single high-velocity phase (Vmax) at saturating Ca2+. To test the idea that cMyBP-C phosphorylation contributes to the activation dependence of Vo, we measured Vo before and following treatment with protein kinase A (PKA) in skinned trabecula isolated from mice expressing either wild-type cMyBP-C (tWT), nonphosphorylatable cMyBP-C (t3SA), or phosphomimetic cMyBP-C (t3SD). During maximal Ca2+ activation, Vmax was monophasic and not significantly different between the three groups. Although biphasic shortening was observed in all three groups at half-maximal activation under control conditions, the high- and low-velocity phases were faster in the t3SD myocardium compared with values obtained in either tWT or t3SA myocardium. Treatment with PKA significantly accelerated both the high- and low-velocity phases in tWT myocardium but had no effect on Vo in either the t3SD or t3SA myocardium. These results can be explained in terms of a model in which the level of cMyBP-C phosphorylation modulates the extent and rate of cooperative spread of myosin binding to actin.

CaMKII activity contributes to homeometric autoregulation of the heart: A novel mechanism for the Anrep effect.

The Anrep effect represents the alteration of left ventricular (LV) contractility to acutely enhanced afterload in a few seconds, thereby preserving stroke volume (SV) at constant preload. As a result of the missing preload stretch in our model, the Anrep effect differs from the slow force response and has a different mechanism. The Anrep effect demonstrated two different phases. First, the sudden increased afterload was momentary equilibrated by the enhanced LV contractility as a result of higher power strokes of strongly-bound myosin cross-bridges. Second, the slightly delayed recovery of SV is perhaps dependent on Ca2+ /calmodulin-dependent protein kinase II activation caused by oxidation and myofilament phosphorylation (cardiac myosin-binding protein-C, myosin light chain 2), maximizing the recruitment of available strongly-bound myosin cross-bridges. Short-lived oxidative stress might present a new facet of subcellular signalling with respect to cardiovascular regulation. Relevance for human physiology was demonstrated by echocardiography disclosing the Anrep effect in humans during handgrip exercise. The present study investigated whether oxidative stress and Ca2+ /calmodulin-dependent protein kinase II (CaMKII) activity are involved in triggering the Anrep effect. LV pressure-volume (PV) analyses of isolated, preload controlled working hearts were performed at two afterload levels (60 and 100mmHg) in C57BL/6N wild-type (WT) and CaMKII-double knockout mice (DKOCaMKII ). In snap-frozen WT hearts, force-pCa relationship, H2 O2 generation, CaMKII oxidation and phosphorylation of myofilament and Ca2+ handling proteins were assessed. Acutely raised afterload showed significantly increased wall stress, H2 O2 generation and LV contractility in the PV diagram with an initial decrease and recovery of stroke volume, whereas end-diastolic pressure and volume, as well as heart rate, remained constant. Afterload induced increase in LV contractility was blunted in DKOCaMKII -hearts. Force development of single WT cardiomyocytes was greater with elevated afterload at submaximal Ca2+ concentration and associated with increases in CaMKII oxidation and phosphorylation of cardiac-myosin binding protein-C, myosin light chain and Ca2+ handling proteins. CaMKII activity is involved in the regulation of the Anrep effect and associates with stimulation of oxidative stress, presumably starting a cascade of CaMKII oxidation with downstream phosphorylation of myofilament and Ca2+ handling proteins. These mechanisms improve LV inotropy and preserve stroke volume within few seconds.

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<0.0001; S282P r=-0.868, p<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.

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.

Structural Measurements within the M-Domain Reveal Unique Details of Cardiac Myosin Binding Protein-C Phosphorylation

We have used molecular dynamics (MD) simulations in atomic-level detail to resolve the structural changes associated with cardiac myosin binding protein-C (cMyBP-C) phosphorylation by protein kinase A (PKA), focusing on the N-terminal phosphorylation motif (pm/M-domain). The results are supported by spectroscopic measurements, and have implications for cMyBP-C's role in myocardial contraction, with particular relevance to -adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. PKA phosphorylation in the pm mediates the affinity of cMyBP-C interactions with actin and the myosin-neck that modulate cardiac muscle contractility. Our spectroscopic studies using time-resolved FRET (TR-FRET) had previously detected distance distributions between probe pairs engineered at sites of cMyBP-C domains C0 through C2. Phosphorylation reduces the level of molecular disorder and the distribution of N-terminal inter-domain distances become more compact (e.g., C1-to-C2). Here, we gain further insight from 20 independent microsecond-long MD simulations of the intrinsically disordered pm. We modeled the motif structure (residues His255-Lys356) and simulated the unphosphorylated and phosphorylated (Ser- 273, 282, and 302) characteristics of molecular disorder, secondary and tertiary structure, intra-motif distance measurements, surface accessibility, and predictions of binding probability. A large structural change in the disordered region of pm was revealed, whereby phosphorylation unmasks a surface of positively-charged residues on an α-helix within the ordered region of pm. Distance measurements between residues in the disordered N-terminal portion of the motif to the helix residues of the motif C-terminus changed proximity to one another by as much as 2 nm upon phosphorylation. These findings help to elucidate cMyBP-C structural dynamics, providing new molecular insight into the modulatory role of cMyBP-C in force development. This work was supported by NIH grants to DDT (R01 AR032961) and to BC (R00 HL122397) and to LME-F from the American Heart Association (12SDG12060656).

Phosphoregulation of Cardiac Inotropy via Myosin Binding Protein-C During Increased Pacing Frequency or β1-Adrenergic Stimulation.

Mammalian hearts exhibit positive inotropic responses to β-adrenergic stimulation as a consequence of protein kinase A-mediated phosphorylation or as a result of increased beat frequency (the Bowditch effect). Several membrane and myofibrillar proteins are phosphorylated under these conditions, but the relative contributions of these to increased contractility are not known. Phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) by protein kinase A accelerates the kinetics of force development in permeabilized heart muscle, but its role in vivo is unknown. Such understanding is important because adrenergic responsiveness of the heart and the Bowditch effect are both depressed in heart failure. The roles of cMyBP-C phosphorylation were studied using mice in which either WT or nonphosphorylatable forms of cMyBP-C [ser273ala, ser282ala, ser302ala: cMyBP-C(t3SA)] were expressed at similar levels on a cMyBP-C null background. Force and [Ca(2+)]in measurements in isolated papillary muscles showed that the increased force and twitch kinetics because increased pacing or β1-adrenergic stimulation were nearly absent in cMyBP-C(t3SA) myocardium, even though [Ca(2+)]in transients under each condition were similar to WT. Biochemical measurements confirmed that protein kinase A phosphorylated ser273, ser282, and ser302 in WT cMyBP-C. In contrast, CaMKIIδ, which is activated by increased pacing, phosphorylated ser302 principally, ser282 to a lesser degree, and ser273 not at all. Phosphorylation of cMyBP-C increases the force and kinetics of twitches in living cardiac muscle. Further, cMyBP-C is a principal mediator of increased contractility observed with β-adrenergic stimulation or increased pacing because of protein kinase A and CaMKIIδ phosphorylations of cMyB-C.

The contribution of cardiac myosin binding protein-c Ser282 phosphorylation to the rate of force generation and in vivo cardiac contractility.

Cardiac myosin binding protein-C phosphorylation plays an important role in modulating cardiac muscle function and accelerating contraction. It has been proposed that Ser282 phosphorylation may serve as a critical molecular switch that regulates the phosphorylation of neighbouring Ser273 and Ser302 residues, and thereby govern myofilament contractile acceleration in response to protein kinase A (PKA). Therefore, to determine the regulatory roles of Ser282 we generated a transgenic (TG) mouse model expressing cardiac myosin binding protein-C with a non-phosphorylatable Ser282 (i.e. serine to alanine substitution, TG(S282A)). Myofibrils isolated from TG(S282A) hearts displayed robust PKA-mediated phosphorylation of Ser273 and Ser302, and the increase in phosphorylation was identical to TG wild-type (TG(WT)) controls. No signs of pathological cardiac hypertrophy were detected in TG(S282A) hearts by either histological examination of cardiac sections or echocardiography. Baseline fractional shortening, ejection fraction, isovolumic relaxation time, rate of pressure development and rate of relaxation (τ) were unaltered in TG(S282A) mice. However, the increase in cardiac contractility as well as the acceleration of pressure development observed in response to β-adrenergic stimulation was attenuated in TG(S282A) mice. In agreement with our in vivo data, in vitro force measurements revealed that PKA-mediated acceleration of cross-bridge kinetics in TG(S282A) myocardium was significantly attenuated compared to TG(WT) myocardium. Taken together, our data suggest that while Ser282 phosphorylation does not regulate the phosphorylation of neighbouring Ser residues and basal cardiac function, full acceleration of cross-bridge kinetics and left ventricular pressure development cannot be achieved in its absence.

Cardiac Myosin Binding Protein-C Phosphorylation and Sarcomere Function

Rationale: Cardiac myosin binding protein-C (cMyBP-C) has three phosphorylatable serines at N-terminus (Ser-273, Ser-282, and Ser-302) and phosphorylation states of these serines may alter the thick filament structure and function. Phosphorylation of these serines in cMyBP-C may also alter sarcomere length-dependent modulation of contractile force development (LDA), which would be expected to modulate the Frank-Starling Law of the Heart.Objective: To analyze length dependent activation in cMyBP-C for nonphosphorylated and phosphomimetic states of serines 273, 282, 302 and to study the contribution of cMyBP-C in regulating cardiac contraction at different sarcomere lengths.Methods & Results: We studied the force-Ca2+ relationship in skinned myocytes isolated from (a) nonphosphorylated alanine transgenic mice (cMyBP-CAAA), (b) phosphomimetic aspartic acid transgenic mice (cMyBP-CDDD), and normal cMyBP-CWT mice at 2.0 and 2.3 μm sarcomere length (SL).At short SL, Ca2+ sensitivity was similar in all groups. However, Ca2+ sensitivity was increased in the cMyBP-CAAA group compared to either the WT or the cMyBP-CDDD group. Similar results were obtained for maximum force development. As a consequence, LDA was blunted (∼40%) in cMyBP-CAAA myocardium. There were no differences in the level of cooperativety as indexed by the Hill coefficient in any group.Conclusion: Phosphorylated cMyBP-C has been shown to contribute to regulation of cardiac sarcomere function via modulation of the cMyBP-C-actin interaction as well as the disposition of the cross-bridges in relation to the thin filament. Moreover removal of cMyBP-C results in blunted LDA, and a cardiac dysfunction that can be prevented by cMyBP-CDDD but not cMyBP-CAAA. Our data showed that lack of cMyBP-C phosphorylation results in blunted LDA, similar to that found previously in the absence cMyBP-C. We conclude that cMyBP-C phosphorylation modulates myofilament length dependent activation, possibly via modulation of the cMyBP-C interaction with actin.

The extent of cardiac myosin binding protein-C phosphorylation modulates actomyosin function in a graded manner

Cardiac myosin binding protein-C (cMyBP-C), a sarcomeric protein with 11 domains, C0-C10, binds to the myosin rod via its C-terminus, while its N-terminus binds regions of the myosin head and actin. These N-terminal interactions can be attenuated by phosphorylation of serines in the C1-C2 motif linker. Within the sarcomere, cMyBP-C exists in a range of phosphorylation states, which may affect its ability to regulate actomyosin motion generation. To examine the functional importance of partial phosphorylation, we bacterially expressed N-terminal fragments of cMyBP-C (domains C0-C3) with three of its phosphorylatable serines (S273, S282, and S302) mutated in combinations to either aspartic acids or alanines, mimicking phosphorylation and dephosphorylation respectively. The effect of these C0-C3 constructs on actomyosin motility was characterized in both the unloaded in vitro motility assay and in the load-clamped laser trap assay where force:velocity (F:V) relations were obtained. In the motility assay, phosphomimetic replacement (i.e. aspartic acid) reduced the slowing of actin velocity observed in the presence of C0-C3 in proportion to the total number phosphomimetic replacements. Under load, C0-C3 depressed the F:V relationship without any effect on maximal force. Phosphomimetic replacement reversed the depression of F:V by C0-C3 in a graded manner with respect to the total number of replacements. Interestingly, the effect of C0-C3 on F:V was well fitted by a model that assumed C0-C3 acts as an effective viscous load against which myosin must operate. This study suggests that increasing phosphorylation of cMyBP-C incrementally reduces its modulation of actomyosin motion generation providing a tunable mechanism to regulate cardiac function.

Cardiac Myosin Binding Protein-C Phosphorylation, Contractile Function and Cardioprotection

Cardiac myosin binding protein-C (cMyBP-C) is a sarcomeric thick filament assembly protein with regulatory functions in the heart. The cMyBP-C protein differs from the skeletal isoform in that it has a small insertion near the carboxyl terminus that contains 3 phosphorylatable serines, Ser-273, −282 and −302. While the precise functional correlates of cMyBP-C phosphorylation remain obscure, we do know that cMyBP-C is targeted by multiple kinases, such as PKA, PKC, RSK, PKD, CaMKII and PKG, suggesting that it plays a vital role in cardiac signaling. We previously reported that cMyBP-C phosphorylation is essential for normal heart function and that Ser-282 phosphorylation is critical for the subsequent phosphorylation of Ser-302 and normal cardiac function. However, the role of Ser-282 cMyBP-C phosphorylation in cardiac function, particularly as it affects contractile properties and sarcomere organization, is unclear. Therefore, to better understand the mechanisms and significance of cMyBP-C phosphorylation, we established several transgenic mouse models to determine the necessity and sufficiency of Ser-282 phosphorylation for normal cardiac function. Our findings suggested that cMyBP-C phosphorylation at Ser-282 is essential for normal cardiac function and that dephosphorylation at this site accelerates cMyBP-C degradation and cleavage of a 40 kDa fragment. During MI, we showed that cMyBP-C is extensively fragmented when dephosphorylated and that such fragmentation correlates well with contractile dysfunction and heart failure. Meanwhile, we also established that the release of cMyBP-C in the blood post-MI could be a potential diagnostic biomarker for MI. Overall, these studies show that Ser-282 phosphorylation is a critical determinant of Ser-302 phosphorylation and that cMyBP-C dephosphorylation accelerates its degradation and release into the circulation. In conclusion, we provide strong evidence that cMyBP-C phosphorylation directly affects the heart's contractile properties, sarcomere organization and cardioprotection.

A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function

Cardiac myosin-binding protein-C (cMyBP-C) phosphorylation at Ser-273, Ser-282, and Ser-302 regulates myocardial contractility. In vitro and in vivo experiments suggest the nonequivalence of these sites and the potential importance of Ser-282 phosphorylation in modulating the protein's overall phosphorylation and myocardial function. To determine whether complete cMyBP-C phosphorylation is dependent on Ser-282 phosphorylation and to define its role in myocardial function. We hypothesized that Ser-282 regulates Ser-302 phosphorylation and cardiac function during β-adrenergic stimulation. Using recombinant human C1-M-C2 peptides in vitro, we determined that protein kinase A can phosphorylate Ser-273, Ser-282, and Ser-302. Protein kinase C can also phosphorylate Ser-273 and Ser-302. In contrast, Ca(2+)-calmodulin-activated kinase II targets Ser-302 but can also target Ser-282 at nonphysiological calcium concentrations. Strikingly, Ser-302 phosphorylation by Ca(2+)-calmodulin-activated kinase II was abolished by ablating the ability of Ser-282 to be phosphorylated via alanine substitution. To determine the functional roles of the sites in vivo, three transgenic lines, which expressed cMyBP-C containing either Ser-273-Ala-282-Ser-302 (cMyBP-C(SAS)), Ala-273-Asp-282-Ala-302 (cMyBP-C(ADA)), or Asp-273-Ala-282-Asp-302 (cMyBP-C(DAD)), were generated. Mutant protein was completely substituted for endogenous cMyBP-C by breeding each mouse line into a cMyBP-C null (t/t) background. Serine-to-alanine substitutions were used to ablate the abilities of the residues to be phosphorylated, whereas serine-to-aspartate substitutions were used to mimic the charged state conferred by phosphorylation. Compared to control nontransgenic mice, as well as transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-C(SAS(t/t)), cMyBP-C(ADA(t/t)), and cMyBP-C(DAD(t/t)) mice showed no increases in morbidity and mortality and partially rescued the cMyBP-C((t/t)) phenotype. The loss of cMyBP-C phosphorylation at Ser-282 led to an altered β-adrenergic response. In vivo hemodynamic studies revealed that contractility was unaffected but that cMyBP-C(SAS(t/t)) hearts showed decreased diastolic function at baseline. However, the normal increases in cardiac function (increased contractility/relaxation) as a result of infusion of β-agonist was significantly decreased in all of the mutants, suggesting that competency for phosphorylation at multiple sites in cMyBP-C is a prerequisite for normal β-adrenergic responsiveness. Ser-282 has a unique regulatory role in that its phosphorylation is critical for the subsequent phosphorylation of Ser-302. However, each residue plays a role in regulating the contractile response to β-agonist stimulation.

Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle

A unique feature of MyBP-C in cardiac muscle is that it has multiple phosphorylation sites. MyBP-C phosphorylation, predominantly by PKA, plays an essential role in modulating contractility as part of the cellular response to β-adrenergic stimulation. In vitro studies indicate MyBP-C can be phosphorylated at Serine 273, 282, 302 and 307 (mouse sequence) but little is known about the level of MyBP-C phosphorylation or the sites phosphorylated in heart muscle. Since current methodologies are limited in specificity and are not quantitative we have investigated the use of phosphate affinity SDS-PAGE together with a total anti MyBP-C antibody and a range of phosphorylation site-specific antibodies for the main sites (Ser-273, -282 and -302). With these newly developed methods we have been able to make a detailed quantitative analysis of MyBP-C phosphorylation in heart tissue in situ. We have found that MyBP-C is highly phosphorylated in non-failing human (donor) heart or mouse heart; tris and tetra-phosphorylated species predominate and less than 10% of MyBP-C is unphosphorylated (0, 9.3 ± 1%: 1P, 13.4 ± 2.7%: 2P, 10.5 ± 3.3%: 3P, 28.7 ± 3.7%: 4P, 36.4 ± 2.7%, n = 21). Total phosphorylation was 2.7 ± 0.07 molPi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C was unphosphorylated. Total phosphorylation levels were 23% of normal in failing heart myofibrils (0, 60.1 ± 2.8%: 1P, 27.8 ± 2.8%: 2P, 4.8 ± 2.0%: 3P, 3.7 ± 1.2%: 4P, 2.8 ± 1.3%, n = 19) and 39% of normal in myectomy samples. The site-specific antibodies showed a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser-273, Ser-282 and Ser-302 were all present in the 4P band of MyBP-C but none of them were significant in the 1P band, indicating that there must be at least one other site of MyBP-C phosphorylation in human heart. The pattern of phosphorylation at the three sites was not random, but indicated positive and negative interactions between the three sites. Phosphorylation at Ser-282 was not proportional to the number of sites available. The 2P band contained 302 but not 273; the 3P band contained 273 but not 302.

Hypothesis Testing

In most biomedical research, investigators hypothesize about the relationships of various factors, collect data to test those relationships, and try to draw conclusions about those relationships from the data collected. In many cases, investigators test relationships by comparing the average level of a factor between 2 groups or between 1 group and a standard reference. This framework is as true for understanding the basic role of cardiac myosin binding protein-C phosphorylation in cardiac physiology1 as it is for evaluating non–high-density lipoprotein cholesterol (HDL-C) as a predictor of myocardial infarction in large groups of individuals.2 In this article we describe hypothesis testing, which is the process of drawing conclusions on the basis of statistical testing of collected data, and the specific approach used to test means (or average levels of a collected data element). These concepts are covered in detail in many statistical textbooks at various levels, including Pagano and Gauvreau,3 Zar,4 and Kleinbaum et al.5 The purpose of statistical inference is to draw conclusions about a population on the basis of data obtained from a sample of that population. Hypothesis testing is the process used to evaluate the strength of evidence from the sample and provides a framework for making determinations related to the population, ie, it provides a method for understanding how reliably one can extrapolate observed findings in a sample under study to the larger population from which the sample was drawn. The investigator formulates a specific hypothesis, evaluates data from the sample, and uses these data to decide whether they support the specific hypothesis. The first step in testing hypotheses is the transformation of the research question into a null hypothesis, H, and an alternative hypothesis, HA.6 The null and alternative hypotheses are concise statements, usually in …