Cardiac Myofilament Function Research Articles

Cardiac myosin binding protein-C phosphorylation as a function of multiple protein kinase and phosphatase activities

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-C’s multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2 A (PP1 and PP2A), and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and various protein kinases known to phosphorylate cMyBP-C. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by potein kinase A. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results suggest non-redundant roles for PP1 and PP2A under both physiological and heart failure conditions, and emphasize the importance of phosphatases for cMyBP-C regulation.

Dystrophic cardiomyopathy: role of the cardiac myofilaments.

Dystrophic cardiomyopathy arises from mutations in the dystrophin gene. Dystrophin forms part of the dystrophin glycoprotein complex and is postulated to act as a membrane stabilizer, protecting the sarcolemma from contraction-induced damage. Duchenne muscular dystrophy (DMD) is the most severe dystrophinopathy, caused by a total absence of dystrophin. Patients with DMD present with progressive skeletal muscle weakness and, because of treatment advances, a cardiac component of the disease (i.e., dystrophic cardiomyopathy) has been unmasked later in disease progression. The role that myofilaments play in dystrophic cardiomyopathy is largely unknown and, as such, this study aimed to address cardiac myofilament function in a mouse model of muscular dystrophy. To assess the effects of DMD on myofilament function, isolated permeabilized cardiomyocytes of wild-type (WT) littermates and Dmdmdx-4cv mice were attached between a force transducer and motor and subjected to contractile assays. Maximal tension and rates of force development (indexed by the rate constant, k tr) were similar between WT and Dmdmdx-4cv cardiac myocyte preparations. Interestingly, Dmdmdx-4cv cardiac myocytes exhibited greater sarcomere length dependence of peak power output compared to WT myocyte preparations. These results suggest dystrophin mitigates length dependence of activation and, in the absence of dystrophin, augmented sarcomere length dependence of myocyte contractility may accelerate ventricular myocyte contraction-induced damage and contribute to dystrophic cardiomyopathy. Next, we assessed if mavacamten, a small molecule modulator of thick filament activation, would mitigate contractile properties observed in Dmdmdx-4cv permeabilized cardiac myocyte preparations. Mavacamten decreased maximal tension and k tr in both WT and Dmdmdx-4cv cardiac myocytes, while also normalizing the length dependence of peak power between WT and Dmdmdx-4cv cardiac myocyte preparations. These results highlight potential benefits of mavacamten (i.e., reduced contractility while maintaining exquisite sarcomere length dependence of power output) as a treatment for dystrophic cardiomyopathy associated with DMD.

Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays

The large unmet demand for new heart failure therapeutics is widely acknowledged. Over the last decades the contractile myofilaments themselves have emerged as an attractive target for the development of new therapeutics for both systolic and diastolic heart failure. However, the clinical use of myofilament-directed drugs has been limited, and further progress has been hampered by incomplete understanding of myofilament function on the molecular level and screening technologies for small molecules that accurately reproduce this function in vitro. In this study we have designed, validated and characterized new high throughput screening platforms for small molecule effectors targeting the interactions between the troponin C and troponin I subunits of the cardiac troponin complex. Fluorescence polarization-based assays were used to screen commercially available compound libraries, and hits were validated using secondary screens and orthogonal assays. Hit compound-troponin interactions were characterized using isothermal titration calorimetry and NMR spectroscopy. We identified NS5806 as novel calcium sensitizer that stabilizes active troponin. In good agreement, NS5806 greatly increased the calcium sensitivity and maximal isometric force of demembranated human donor myocardium. Our results suggest that sarcomeric protein-directed screening platforms are suitable for the development of compounds that modulate cardiac myofilament function.

Dissecting the mechanisms by which a pathogenic variant in cardiac TnT tail domain leads to disturbed myofilament performance.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and high rate of sudden cardiac arrest at juvenile age. cTnT-I79N is located in the cTnT N-terminal (TNT1) loop region and is well-known for its pathological and prognostic relevance. Recent structural study revealed that I79 is part of hydrophobic interface between TNT1 loop and actin which stabilizes the OFF state of the cardiac thin filament. Given the importance of understanding: i) the role of TNT1 loop region on the regulation of the cardiac thin filament, and ii) the underlying mechanisms of cTnT-I79N-linked pathogenesis; we sought to investigate the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased passive force and passive sinusoidal stiffness, which could be due in part to an increase in force-generating cross-bridges during diastole. Tg-I79N also displayed increased myofilament Ca2+-sensitivity, shorter myofilament lattice spacing, and slower cross-bridge kinetics. These findings can be attributed to the destabilization of cardiac thin filament's inhibitory property, and the increase in the overall number of cross-bridges during Ca2+ activation. Finally, we show that cTnT-I79N dysregulates SRX↔DRX equilibrium, which can be explained by the excessive binding of myosin heads to the dysregulated thin filament that causes the SRX↔DRX equilibrium to shift towards the ON state. Altogether, biochemical and functional measurements, and structural data points to the following mechanism: cTnT-I79N weakens the interaction of the TNT1 loop with the actin filament, which in turn destabilizes the OFF state of the cardiac thin filament.

The HCM I79N pathogenic variant in cardiac TNT induces thick filament malfunction and myofilament lattice rearrangement

Missense variant Ile79Asn in human cardiac troponin T (HcTnT-I79N) has been associated with familial hypertrophic cardiomyopathy (HCM), arrhythmia and sudden cardiac death. Little is known about effects of this pathogenic variant on cardiac myofilament function and structure. To fill this gap, cardiac tissues were harvested from non-transgenic (NTg) control mice and transgenic mice bearing HcTnT-I79N. Left ventricular papillary muscle bundles were permeabilized and mounted for mechanical measurements.

Optimal cutting temperature medium embedding and cryostat sectioning are valid for cardiac myofilament function assessment.

To maximize data obtainment from valuable cardiac tissue, we hypothesized that myocardium fixed in optimal cutting temperature (OCT) medium for histology could also be used to investigate the function of myofilament proteins in situ. We compared tissue prepared via conventional liquid nitrogen (LN) snap freezing with tissue fixed in OCT and then sectioned in fiber-parallel orientation. We found that actin-myosin Ca2+ sensitivity, activation rate by Ca2+, cooperativity along the thin filament, as well as cross-bridge cycling rate were unaffected by OCT storage and could reliably be interpreted after sectioning. Absolute values in maximum force generation per cross-sectional area, as well as passive strain, are difficult to investigate after sectioning, as myofibrillar continuity along the preparation cannot be guaranteed. We have shown that myocardial tissue stored in OCT and sectioned before analysis is available for functional analysis, a valuable means of maximizing usage of precious cardiac biopsies.NEW & NOTEWORTHY Myocardial tissue in optimal cutting temperature (OCT) fixation and cryostat sectioning was tested as a means of storing and preparing tissue for myofilament function analysis in relation to conventional liquid nitrogen freezing and dissection. Actomyosin interaction, Ca2+ force activation, and passive compliance were tested. The study concluded that OCT storage and cryostat sectioning do not interfere with the actomyosin cross-bridge dynamics or Ca2+ activation but that absolute tension values suffer and may not be investigated by this method.

Bisphenol S rapidly depresses heart function through estrogen receptor-\u03b2 and decreases phospholamban phosphorylation in a sex-dependent manner

The health effects of the endocrine disruptor Bisphenol A (BPA) led to its partial replacement with Bisphenol S (BPS) in several products including food containers, toys, and thermal paper receipts. The acute effects of BPS on myocardial contractility are unknown. We perfused mouse hearts from both sexes for 15 min with physiologically relevant doses of BPS or BPA. In females BPS (1 nM) decreased left ventricular systolic pressure by 5 min, whereas BPA (1 nM) effects were delayed to 10 min. BPS effects in male mice were attenuated. In both sexes ER-β antagonism abolished the effects of BPS. Cardiac myofilament function was not impacted by BPS or BPA in either sex, although there were sex-dependent differences in troponin I phosphorylation. BPS increased phospholamban phosphorylation at S16 only in female hearts, whereas BPA reduced phosphorylation in both sexes. BPA decreased phospholamban phosphorylation at T17 in both sexes while BPS caused dephosphorylation only in females. This is the first study to compare sex differences in the acute myocardial response to physiologically relevant levels of BPS and BPA, and demonstrates a rapid ability of both to depress heart function. This study raises concerns about the safety of BPS as a replacement for BPA.

Abstract 900: Troponin I Tyrosine 26 Phosphorylation Accelerates i n vivo Myocardial Relaxation

Heart failure results in depressed cardiac systolic contraction and diastolic relaxation, both of which limit the heart’s ability to pump blood. Currently there is no therapy to accelerate the detrimental slowed relaxation in heart failure. A fundamental mechanism of the heart to modulate cardiac relaxation is through serine/threonine kinase-mediated phosphorylation of the contractile regulatory protein troponin I (TnI). Non-receptor tyrosine-specific kinases are expressed in the heart and their activity is altered in disease; yet, the role of tyrosine kinase-mediated phosphorylation to directly modulate cardiac relaxation has not been described. TnI is phosphorylated at tyrosine 26 (Tyr26) in the heart and we previously demonstrated TnI Tyr26 phosphorylation accelerates myofilament deactivation. TnI Tyr26 phosphorylation is therefore the first tyrosine phosphorylation identified to directly modulate cardiac myofilament function, however the effects of tyrosine phosphorylation on in vivo function of the heart are unknown. To determine the effect of tyrosine phosphorylation on heart function, we generated a TnI Tyr26 phosphorylation (Tg Y26E) mouse. At the muscle level, myofibrils from Tg Y26E mice exhibit accelerated myofibril relaxation. Echocardiography and pressure-volume hemodynamic measurements demonstrate Tg Y26E mice have enhanced diastolic function evident in accelerated myocardial relaxation, without depressed systolic function or altered morphology. As a whole these data support the phosphorylation of TnI at Tyr26 as a novel tyrosine signaling mechanism to accelerate in vivo diastolic function without depressing systolic contraction.

Chronic high-dose testosterone treatment: impact on rat cardiac contractile biology.

Androgen therapy provides cardiovascular benefits for hypogonadism. However, myocardial hypertrophy, fibrosis, and infarction have been reported in testosterone or androgenic anabolic steroid abuse. Therefore, better understanding of the factors leading to adverse results of androgen abuse is needed. The aim of the present study was to examine the impact of high dose of androgen treatment on cardiac biology, and whether exposure duration modulates this response. Male rats were treated with 10 mg/kg testosterone, three times a week, for either 4 or 12 weeks; vehicle injections served as controls. Four weeks of testosterone treatment induced an increase in ventricular wall thickness, indicative of concentric hypertrophy, as well as increased ejection fraction; in contrast, both parameters were blunted following 12 weeks of high‐dose testosterone treatment. Cardiac myocyte contractile parameters were assessed in isolated electrically stimulated myocytes (sarcomere and intracellular calcium dynamics), and in chemically permeabilized isolated myocardium (myofilament force development and tension‐cost). High‐dose testosterone treatment for 4 weeks was associated with increased myocyte contractile parameters, while 12 weeks treatment induced significant depression of these parameters, mirroring the cardiac pump function results. In conclusion, chronic administration of high‐dose testosterone initially induces increased cardiac function. However, this initial beneficial impact is followed by significant depression of cardiac pump function, myocyte contractility, and cardiac myofilament function. Our results indicate that chronic high‐testosterone usage is of limited use and may, instead, induce significant cardiac dysfunction.

Scaffold Proteins Regulating Extracellular Regulated Kinase Function in Cardiac Hypertrophy and Disease.

The mitogen activated protein kinase (MAPK)-extracellular regulated kinase 1/2 (ERK1/2) pathway is a central downstream signaling pathway that is activated in cardiac muscle cells during mechanical and agonist-mediated hypertrophy. Studies in genetic mouse models deficient in ERK-associated MAPK components pathway have further reinforced a direct role for this pathway in stress-induced cardiac hypertrophy and disease. However, more recent studies have highlighted that these signaling pathways may exert their regulatory functions in a more compartmentalized manner in cardiac muscle. Emerging data has uncovered specific MAPK scaffolding proteins that tether MAPK/ERK signaling specifically at the sarcomere and plasma membrane in cardiac muscle and show that deficiencies in these scaffolding proteins alter ERK activity and phosphorylation, which are then critical in altering the cardiac myocyte response to stress-induced hypertrophy and disease progression. In this review, we provide insights on ERK-associated scaffolding proteins regulating cardiac myofilament function and their impact on cardiac hypertrophy and disease.

The Cardiac Troponin T Mutant Missing the N-Terminal Extension Causes Dose-Dependent Effects on Cardiac Function and Remodeling in Transgenic Mice

The N-terminal extension (NTE; residues 42-73) of mouse cardiac troponin T (TnT) desensitizes cardiac myofilaments to Ca2+ by stabilizing thin filaments in the blocked-state. We arrived at this conclusion using detergent-skinned muscle from transgenic (TG) mouse hearts that expressed 54% of chimeric TnT (residues 1-73 of mouse cardiac TnT were replaced by residues 1-41 of mouse fast skeletal TnT). Here, we extended our investigation to include higher dose effects of the modified TnT on cardiac myofilament function/phenotype using detergent-skinned fiber studies and echocardiography measurements in two different TG mouse lines (TG-55 and TG-64 that expressed 55% and 64% of chimeric TnT, respectively). Both TG-55 and TG-64 mice showed a similar increase in myofilament Ca2+ sensitivity at sarcomere lengths (SL) of 1.9 and 2.3 μm. However, Ca2+-activated maximal tension increased significantly only in TG-64 mice at either SL. There was a progressive decrease in the overall heart size and heart-to-body weight ratios in both TG-55 and TG-64 mice. Left ventricular diastolic functional parameters (isovolumic relaxation time and E-wave deceleration time) showed a graded increase in TG-55 and TG-64 mice; however, such effects were only significant in TG-64 mice, suggesting impaired relaxation. Systolic functional parameters (stroke volume, ejection fraction and fractional shortening) were unaffected in TG-55 mice, but significantly decreased in TG-64 mice. Thus, higher levels of chimeric TnT (64%) depressed both diastolic and systolic function significantly in TG-64 mice. We will discuss the link between the effects of the modified N-terminus of TnT on cardiac myofilament function and the resultant pathological remodeling of the heart. Our findings have pathological relevance because a growing number of disease-related mutations are found both in and near the NTE of cardiac TnT.

N‐terminal region of cardiac myosin binding protein‐C impairs myofilament function

Cardiac myosin binding protein‐C (cMyBP‐C) is a thick filament protein that plays a critical role in the regulation of sarcomere structure and contractility. Previously, we demonstrated that the N‐terminal fragment of cMyBP‐C (C0‐C1f), released during ischemia‐reperfusion (I‐R) injury, is cytotoxic and significantly impairs contractile function. However, the mechanisms in which C0‐C1f impairs cardiomyocyte contractile function remain unknown. The hypothesis of this study is that C0‐C1f depresses cardiac myofilament function. The molecular mass of C0‐C1f was analyzed by top‐down mass spectrometry. The observed molecular weights were 30974.72 Da and 31989.20 Da for mouse and human species respectively. Permeabilized left ventricular (LV) muscle strips were incubated with purified C0‐C1f. Myofilament function was characterized by measuring active tension‐[Ca2+] relationships to determine changes in maximum tension development (tmax) and Ca2+ sensitivity. Tension development and ATP consumption were simultaneously measured to determine tension‐cost over a range of [Ca2+]. C0‐C1f significantly reduced myofilament Ca2+ sensitivity and tmax, increased tension‐cost, and increased the rate of tension redevelopment (ktr). These data contribute to the understanding of contractile dysfunction following I‐R injury and provide a novel therapeutic target for the treatment of heart failure.

Elevated rates of force development and MgATP binding in F764L and S532P myosin mutations causing dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a disease characterized by dilation of the ventricular chambers and reduced contractile function. We examined the contractile performance of chemically-skinned ventricular strips from two heterozygous murine models of DCM-causing missense mutations of myosin, F764L/+ and S532P/+, in an α-myosin heavy chain (MyHC) background. In Ca2+-activated skinned myocardial strips, the maximum developed tension in F764L/+ was only ~50% that of litter-mate controls (+/+). The F764L/+ also exhibited significantly reduced rigor stiffness, loaded shortening velocity and power output. Corresponding indices for S532P/+ strips were not different from controls. Manipulation of MgATP concentration in conjunction with measures of viscoelasticity, which provides estimates of myosin detachment rate 2πc, allowed us to probe the molecular basis of changes in crossbridge kinetics that occur with the myosin mutations. By examining the response of detachment rate to varying MgATP we found the rate of MgADP release was unaffected by the myosin mutations. However, MgATP binding rate was higher in the DCM groups compared to controls (422±109mM−1·s−1 in F764L/+, 483±74mM−1·s−1 in S532P/+ and 303±18mM−1·s−1 in +/+). In addition, the rate constant of force development, 2πb, was significantly higher in DCM groups compared to controls (at 5mM MgATP: 36.9±4.9s−1 in F764L/+, 32.9±4.5s−1 in S532P/+ and 18.2±1.7s−1 in +/+). These results suggest that elevated rates of force development and MgATP binding are features of cardiac myofilament function that underlie the development of DCM.

Rapid Changes in Cardiac Myofilament Function following the Acute Activation of Estrogen Receptor-Alpha

Estrogens have well-recognized and complex cardiovascular effects, including altering myocardial contractility through changes in myofilament function. The presence of multiple estrogen receptor (ER) isoforms in the heart may explain some discrepant findings about the cardiac effects of estrogens. Most studies examining the impact of estrogens on the heart have focused on chronic changes in estrogen levels, and have not investigated rapid, non-genomic pathways. The first objective of this study was to determine how acute activation of ERα impacts cardiac myofilaments. Nongenomic myocardial estrogen signaling is associated with the activation of a variety of signaling pathways. p38 MAPK has been implicated in acute ER signaling in the heart, and is known to affect myofilament function. Thus, the second objective of this study was to determine if acute ERα activation mediates its myofilament effects through p38 MAPK recruitment. Hearts from female C57Bl/6 mice were perfused with the ERα agonist PPT and myofilaments isolated. Activation of ERα depressed actomyosin MgATPase activity and decreased myofilament calcium sensitivity. Inhibition of p38 MAPK attenuated the myofilament effects of ERα activation. ERα stimulation did not affect global myofilament protein phosphorylation, but troponin I phosphorylation at the putative PKA phosphorylation sites was decreased. Changes in myofilament activation did not translate into alterations in whole heart function. The present study provides evidence supporting rapid, non-genomic changes in cardiac myofilament function following acute ERα stimulation mediated by the p38 MAPK pathway.

Protein kinase D increases maximal Ca2+-activated tension of cardiomyocyte contraction by phosphorylation of cMyBP-C-Ser315

Cardiac myosin-binding protein C (cMyBP-C) is involved in the regulation of cardiac myofilament contraction. Recent evidence showed that protein kinase D (PKD) is one of the kinases that phosphorylate cMyBP-C. However, the mechanism by which PKD-induced cMyBP-C phosphorylation affects cardiac contractile responses is not known. Using immunoprecipitation, we showed that, in contracting cardiomyocytes, PKD binds to cMyBP-C and phosphorylates it at Ser(315). The effect of PKD-mediated phosphorylation of cMyBP-C on cardiac myofilament function was investigated in permeabilized ventricular myocytes, isolated from wild-type (WT) and from cMyBP-C knockout (KO) mice, incubated in the presence of full-length active PKD. In WT myocytes, PKD increased both myofilament Ca(2+) sensitivity (pCa(50)) and maximal Ca(2+)-activated tension of contraction (T(max)). In cMyBP-C KO skinned myocytes, PKD increased pCa(50) but did not alter T(max). This suggests that cMyBP-C is not involved in PKD-mediated sensitization of myofilaments to Ca(2+) but is essential for PKD-induced increase in T(max). Furthermore, the phosphorylation of both PKD-Ser(916) and cMyBP-C-Ser(315) was contraction frequency-dependent, suggesting that PKD-mediated cMyBP-C phosphorylation is operational primarily during periods of increased contractile activity. Thus, during high contraction frequency, PKD facilitates contraction of cardiomyocytes by increasing Ca(2+) sensitivity and by an increased T(max) through phosphorylation of cMyBP-C.

Protein kinase D increases maximal Ca2+ activated tension of cardiomyocyte contraction by selective phosphorylation of cMyBP‐C‐Ser315

Cardiac myosin binding protein-C (cMyBP-C) regulates cardiac myofilament contraction. Recent evidence showed that Protein kinase-D (PKD) is a kinase that phosphorylates cMyBP-C. However, the mechanism by which PKD-induced cMyBP-C phosphorylation mediates cardiac contractile responses is not known yet. Using immunoprecipitation, PKD was shown to bind to cMyBP-C and to phosphorylate it at Ser315 in contracting cardiomyocytes. The effect of PKD-mediated phosphorylation of cMyBP-C on cardiac myofilament function was investigated in permeabilized ventricular myocytes isolated from wild-type (WT) and cMyBP-C knock-out (KO) mice. In WT myocytes, PKD increased both myofilament Ca2+ sensitivity (pCa50) and maximal Ca2+-activated tension of contraction (Tmax). The PKD-induced increase of Tmax and sub-maximal active tension was not observed in cMyBP-C KO myocytes, which proofs that cMyBP-C is involved in the PKD-mediated increase of submaximal activation and Tmax. Furthermore, phosphorylation of both PKD-Ser916 and cMyBP-C-Ser315 is contraction frequency-dependent, indicating that PKD-mediated cMyBP-C phosphorylation is operational especially during periods of increased contractile activity. We propose that during increased contraction, PKD is a key factor to match energy metabolism and cardiac mechanics. Grant Funding Source: NWO (Dutch foundation for scientific research)

Novel Role for p90 Ribosomal S6 Kinase in the Regulation of Cardiac Myofilament Phosphorylation

In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.

H2O2 alters rat cardiac sarcomere function and protein phosphorylation through redox signaling

ROS, such as H(2)O(2), are a component of pathological conditions in many organ systems and have been reported to be elevated in cardiac pathophysiology. The experiments presented here test the hypothesis that H(2)O(2) induces alterations in cardiac myofilament function by the posttranslational modification of sarcomeric proteins indirectly through PKC signaling. In vitro assessment of actomyosin Mg(2+)-ATPase activity of myofibrillar fractions showed blunted relative ATP consumption in the relaxed state (pCa 8.0) in response to treatment with 0.5 mM H(2)O(2) before myofilament isolation. The effect was attributable to downstream "redox signaling," inasmuch as the direct application of H(2)O(2) to isolated myofibrils did not alter Mg(2+)-ATPase activity. Ca(2+)-ATPase activity, which was used as a measure of myofibrillar myosin function, was unaffected by H(2)O(2). Functional experiments using rat cardiac trabeculae treated with 0.5 or 5 mM H(2)O(2) followed by detergent extraction of membranes demonstrated increased Ca(2+) sensitivity of force production, a faster rate of force redevelopment, and (for 5 mM) decreased maximum tension. Biochemical analysis of myocardial samples treated with 0.5 mM H(2)O(2) demonstrated increased phosphorylation of two sarcomeric proteins: cardiac troponin I and myosin-binding protein-C. These changes were eliminated by a general PKC inhibitor. However, H(2)O(2) and the general PKC activator PMA induced different phosphorylation patterns in cardiomyocytes in which PKC-delta was elevated by viral infection. These data provide evidence that PKC-dependent redox signaling affects the function of cardiac myofilaments and indicate modification of specific proteins through this signaling mechanism.

Distinct Sarcomeric Substrates Are Responsible for Protein Kinase D-mediated Regulation of Cardiac Myofilament Ca2+ Sensitivity and Cross-bridge Cycling

Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser(22)/Ser(23), reduces myofilament Ca(2+) sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser(22)/Ser(23) remains to be established. To determine the role of cTnI phosphorylation at Ser(22)/Ser(23) in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser(22)/Ser(23) are substituted by nonphosphorylatable Ala (cTnI-Ala(2)). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser(22)/Ser(23) and decreased the Ca(2+) sensitivity of force. In contrast, PKD had no effect on the Ca(2+) sensitivity of force in myocardium from cTnI-Ala(2) mice, in which Ser(22)/Ser(23) were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala(2) mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser(273), Ser(282), and Ser(302), and revealed that PKD phosphorylates only Ser(302). Furthermore, PKD phosphorylated Ser(302) selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala(2) mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca(2+) sensitivity through cTnI phosphorylation at Ser(22)/Ser(23) but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser(302), which may mediate the latter effect.