Abstract-The role of cardiac myosin binding protein-C (cMyBP-C) phosphorylation in cardiac physiology or pathophysiology is unclear. To investigate the status of cMyBP-C phosphorylation in vivo, we determined its phosphorylation state in stressed and unstressed mouse hearts. cMyBP-C phosphorylation is significantly decreased during the development of heart failure or pathologic hypertrophy. We then generated transgenic (TG) mice in which the phosphorylation sites of cMyBP-C were changed to nonphosphorylatable alanines (MyBP-C AllPϪ ). A TG line showing Ϸ40% replacement with MyBP-C AllPϪ showed no changes in morbidity or mortality but displayed depressed cardiac contractility, altered sarcomeric structure and upregulation of transcripts associated with a hypertrophic response. To explore the effect of complete replacement of endogenous cMyBP-C with MyBP-C AllPϪ , the mice were bred into the MyBP-C (t/t) background, in which less than 10% of normal levels of a truncated MyBP-C are present. Although MyBP-C AllPϪ was incorporated into the sarcomere and expressed at normal levels, the mutant protein could not rescue the MyBP-C (t/t) phenotype. The mice developed significant cardiac hypertrophy with myofibrillar disarray and fibrosis, similar to what was observed in the MyBP-C (t/t) animals. In contrast, when the MyBP-C (t/t) mice were bred to a TG line expressing normal MyBP-C (MyBP-C WT ), the MyBP-C (t/t) phenotype was rescued. These data suggest that cMyBP-C phosphorylation is essential for normal cardiac function. Key Words: mouse Ⅲ mouse mutants Ⅲ muscle Ⅲ muscle contraction Ⅲ myocardial contractility U nderstanding the structure/function relations for cardiac myosin binding protein-C (cMyBP-C) is clinically relevant, as cMyBP-C mutations are a widely recognized cause of familial hypertrophic cardiomyopathy. 1 Various cMyBP-C transgenic (TG) and gene-targeted mouse models have demonstrated the importance of the protein for long-term integrity of sarcomeric structure and for maintaining normal cardiac contractility. 2,3 Functional insight can be gained from appreciating the crucial structural differences between cMyBP-C and the skeletal isoform. Only the cardiac isoform contains an extra immunoglobulin domain at the N terminus (C0), an insertion of 28 residues within the C5 domain, and three phosphorylation sites (Ser273, that are substrates for cAMP-dependent protein kinase A (PKA), Ca 2ϩ -calmodulin-activated kinase and protein kinase C.In vivo, PKA-mediated phosphorylation of cMyBP-C is linked to modulation of cardiac contraction. 4 On adrenergic stimulation, PKA phosphorylates Ser273, -282, and -302, whereas protein kinase C phosphorylates only Ser273 and -302. 5 These residues, located near the N terminus of the protein, are of particular interest, as this region binds to the S2 segment of the myosin heavy chain (MHC), 6,7 which is close to the lever arm domain of myosin. It has been hypothesized that cMyBP-C/MHC interactions are dynamically regulated by the phosphorylation/dephosphorylation of cMyBP-C. 8...

Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during ␤-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser 23 /Ser 24 ) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation.Recent studies have demonstrated that changes in the phosphorylation states of key cardiac regulatory proteins can have dramatic effects on normal cardiac function (1, 2). Phosphorylation of cardiac troponin I (cTnI), 1 a thin filament regulatory protein, may be particularly important in modulating cardiac function. cTnI, together with cardiac troponin T (cTnT) and cardiac troponin C (cTnC), form the troponin complex. The protein binds to both cTnC and actin and is a critical component in activating contraction as it serves as the Ca 2ϩ -sensing apparatus. Within the cardiac isoform's amino-terminal extension, serines are present at residues 23 and 24 (Ser 23 /Ser 24 ), which serve as substrates for protein kinase A (PKA), which is activated in response to ␤-adrenergic stimulation of the heart (3). Several investigations report that PKA-mediated phosphorylation of cTnI results in a reduction in myofilament Ca 2ϩ sensitivity (4), an increase in cross-bridge cycling (5), and increased binding of cTnI to the thin filament. Cardiac TnI is also a substrate for protein kinase C (PKC) phosphorylation at Ser 43 /Ser 45 and Thr 144 (position 143 in the human protein) (6). However, the substrate specificity of these sites is not absolute, since PKC can phosphorylate the PKA sites (7-9). Whereas PKA-mediated phosphorylation is th...

Background-The biochemical differences between the 2 mammalian cardiac myosin heavy chains (MHCs), ␣-MHC and ␤-MHC, are well described, but the physiological consequences of basal isoform expression and isoform shifts in response to altered cardiac load are not clearly understood. Mature human ventricle contains primarily the ␤-MHC isoform. However, the ␣-MHC isoform can be detected in healthy human ventricle and appears to be significantly downregulated in failing hearts. The unique biochemical properties of the ␣-MHC isoform might offer functional advantages in a failing heart that is expressing only the ␤-MHC isoform. This hypothesis cannot be tested in mice or rats because both species express ␣-MHC as the predominant isoform. Methods and Results-To test the effects of persistent ␣-MHC expression on the background of ␤-MHC, we made transgenic (TG) rabbits that expressed rabbit ␣-MHC cDNA in the ventricle so that the endogenous myosin was partially replaced by the transgenically encoded species. Molecular, histological, and functional analyses showed no significant baseline effects in the TG rabbits compared with nontransgenic (NTG) littermates. To determine whether ␣-MHC expression afforded any advantages to stressed myocardium, a cohort of TG and NTG rabbits was subjected to rapid ventricular pacing. Although both the TG and NTG rabbits developed dilated cardiomyopathy, the TG rabbits had a higher shortening fraction, less septal thinning, and more normal ϮdP/dt than paced NTG rabbits. Conclusions-Transgenic expression of ␣-MHC does not have any apparent detrimental effects under basal conditions and is cardioprotective in experimental tachycardia-induced cardiomyopathy.

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2−/−) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2−/− mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2−/− mice. Small-angle X-ray diffraction of C2−/− EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2−/− EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2−/− mice. Finally, C2−/− muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.