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The amount of work the heart can perform during ejection is governed by the inherent contractile properties of individual myocytes. One way to alter contractile properties is to alter contractile proteins such as myosin heavy chain (MyHC), which is known to demonstrate isoform plasticity in response to disease states. The purpose of this study was to examine myocyte functionality over the complete range of MyHC expression in heart, from 100% [alpha]-MyHC to 100% [beta]-MyHC, using euthyroid and hypothyroid rats. Peak power output in skinned cardiac myocytes decreased as a nearly linear function of [beta]-MyHC expression during maximal ([r.sup.2] = 0.85, n = 44 myocyte preparations) and submaximal ([r.sup.2] = 0.82, n = 31 myocyte preparations) [Ca.sup.2+] activation. To determine whether single myocyte function translated to the level of the whole heart, power output was measured in working heart preparations expressing varied ratios of MyHC. Left ventricular power output of isolated working heart preparations also decreased as a linear function of increasing [beta]-MyHC expression ([r.sup.2] = 0.82, n = 34 myocyte preparations). These results demonstrate that power output is highly dependent on MyHC expression in single myocytes, and this translates to the performance of working left ventricles. myosin heavy chain; myocardium; myocardial contractility; cardiac muscle

The purpose of this study was to examine the role of myosin heavy chain (MHC) in determining loaded shortening velocities and power output in cardiac myocytes. Cardiac myocytes were obtained from euthyroid rats that expressed [Alpha]-MHC or from thyroidectomized rats that expressed [Beta]-MHC. Skinned myocytes were attached to a force transducer and a position motor, and isotonic shortening velocities were measured at several loads during steady-state maximal [Ca.sup.2+] activation ([P.sub.pCa4.5]). MHC expression was determined after mechanical measurements using SDS-PAGE. Both [Alpha]-MHC and [Beta]-MHC myocytes generated similar maximal [Ca.sup.2+]-activated force, but [Alpha]-MHC myocytes shortened faster at all loads and generated ~170% greater peak normalized power output. Additionally, the curvature of force-velocity relationships was less, and therefore the relative load optimal for power output ([F.sub.opt]) was greater in [Alpha]-MHC myocytes. [F.sub.opt] was 0.31 [+ or -] 0.03 [P.sub.pCa4.5] and 0.20 [+ or -] 0.06 [P.sub.pCa4.5] for [Alpha]-MHC and [Beta]-MHC myocytes, respectively. These results indicate that MHC expression is a primary determinant of the shape of force-velocity relationships, velocity of loaded shortening, and overall power output-generating capacity of individual cardiac myocytes. cardiac muscle contraction; sarcomere proteins

Key points β-Adrenergic stimulation is an important control mechanism, matching cardiac output to venous return during increased metabolic demand., β-Adrenergic signalling leads to protein kinase A (PKA) phosphorylation of myofilament proteins cardiac troponin I (cTnI), cardiac myosin binding protein-C (cMyBP-C) and titin, but their specific effects on the sarcomeric length (SL) dependence of contraction - which underlies the Frank-Starling Law of the Heart - is debated., Recombinant cTnI phosphomimetics were exchanged into cardiac muscle to isolate the effects of cTnI from those of cMyBP-C/titin phosphorylation on SL-dependent force-Ca2+ relations and sarcomeric structure., Results suggest cTnI or cMyBP-C/titin phosphorylation, separately or together, eliminate the SL dependence of Ca2+ sensitivity of force, but not maximal force. The reduction occurs particularly at long SL, suggesting effects on thin filament access and crossbridge recruitment., The net effect of PKA phosphorylation is to blunt SL dependence of force at submaximal [Ca2+] to maintain elevated systolic function., Abstract Protein kinase A (PKA) phosphorylation of myofibrillar proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility. In myofilaments PKA targets troponin I (cTnI), myosin binding protein-C (cMyBP-C) and titin. We studied how this affects the sarcomere length (SL) dependence of force- pCa relations in demembranated cardiac muscle. To distinguish cTnI from cMyBP-C/titin phosphorylation effects on the force- pCa relationship, endogenous troponin (Tn) was exchanged in rat ventricular trabeculae with either wild-type (WT) Tn, non-phosphorylatable cTnI (S23/24A) Tn or phosphomimetic cTnI (S23/24D) Tn. PKA cannot phosphorylate either cTnI S23/24 variant, leaving cMyBP-C/titin as PKA targets. Force was measured at 2.3 and 2.0 μm SL. Decreasing SL reduced maximal force ( Fmax) and Ca2+ sensitivity of force ( pCa50) similarly with WT and S23/24A trabeculae. PKA treatment of WT and S23/24A trabeculae reduced pCa50 at 2.3 but not at 2.0 μm SL, thus eliminating the SL dependence of pCa50. In contrast, S23/24D trabeculae reduced pCa50 at both SL values, primarily at 2.3 μm, also eliminating SL dependence of pCa50. Subsequent PKA treatment moderately reduced pCa50 at both SLs. At each SL, Fmax was unaffected by either Tn exchange and/or PKA treatment. Low-angle X-ray diffraction was performed to determine whether pCa50 shifts were associated with changes in myofilament spacing ( d1,0) or thick-thin filament interaction. PKA increased d1,0 slightly under all conditions. The ratios of the integrated intensities of the equatorial X-ray reflections ( I1,1/ I1,0) indicate that PKA treatment increased crossbridge proximity to thin filaments under all conditions. The results suggest that phosphorylation by PKA of either cTnI or cMyBP-C/titin independently reduces the pCa50 preferentially at long SL, possibly through reduced availability of thin filament binding sites (cTnI) or altered crossbridge recruitment (cMyBP-C/titin). Preferential reduction of pCa50 at long SL may not reduce cardiac output during periods of high metabolic demand because of increased intracellular Ca2+ during β-adrenergic stimulation. [ABSTRACT FROM AUTHOR]

Calcium sensitivity of the force-pCa relationship depends strongly on sarcomere length (SL) in cardiac muscle and is considered to be the cellular basis of the Frank-Starling law of the heart. SL dependence may involve changes in myofilament lattice spacing and/or myosin crossbridge orientation to increase probability of binding to actin at longer SLs. We used the L48Q cardiac troponin C (cTnC) variant, which has enhanced Ca(2+) binding affinity, to test the hypotheses that the intrinsic properties of cTnC are important in determining 1) thin filament binding site availability and responsiveness to crossbridge activation and 2) SL dependence of force in cardiac muscle. Trabeculae containing L48Q cTnC-cTn lost SL dependence of the Ca(2+) sensitivity of force. This occurred despite maintaining the typical SL-dependent changes in maximal force (F(max)). Osmotic compression of preparations at SL 2.0 μm with 3% dextran increased F(max) but not pCa(50) in L48Q cTnC-cTn exchanged trabeculae, whereas wild-type (WT)-cTnC-cTn exchanged trabeculae exhibited increases in both F(max) and pCa(50). Furthermore, crossbridge inhibition with 2,3-butanedione monoxime at SL 2.3 μm decreased F(max) and pCa(50) in WT cTnC-cTn trabeculae to levels measured at SL 2.0 μm, whereas only F(max) was decreased with L48Q cTnC-cTn. Overall, these results suggest that L48Q cTnC confers reduced crossbridge dependence of thin filament activation in cardiac muscle and that changes in the Ca(2+) sensitivity of force in response to changes in SL are at least partially dependent on properties of thin filament troponin.

Heart failure is a leading cause of morbidity and mortality in Western society. The cardiovascular transcription factor CHF1/Hey2 has been linked to experimental heart failure in mice, but the mechanisms by which it regulates myocardial function remain incompletely understood. The objective of this study was to determine how CHF1/Hey2 affects development of heart failure through examination of contractility in a myocardial knockout mouse model. We generated myocardial-specific knockout mice. At baseline, cardiac function was normal, but, after aortic banding, the conditional knockout mice demonstrated a greater increase in ventricular weight-to-body weight ratio compared with control mice (5.526 vs. 4.664 mg/g) and a significantly decreased ejection fraction (47.8 vs. 72.0% control). Isolated cardiac myocytes from these mice showed decreased calcium transients and fractional shortening after electrical stimulation. To determine the molecular basis for these alterations in excitation-contraction coupling, we first measured total sarcoplasmic reticulum calcium stores and calcium-dependent force generation in isolated muscle fibers, which were normal, suggesting a defect in calcium cycling. Analysis of gene expression demonstrated normal expression of most genes known to be involved in myocardial calcium cycling, with the exception of the ryanodine receptor binding protein FKBP12.6, which was expressed at increased levels in the conditional knockout hearts. Treatment of the isolated knockout myocytes with FK506, which inhibits the association of FKBP12.6 with the ryanodine receptor, restored contractile function. These findings demonstrate that conditional deletion of CHF1/Hey2 in the myocardium leads to abnormalities in calcium handling mediated by FKBP12.6 that predispose to pressure overload-induced heart failure.