Procurement and preservation of neonatal porcine cardiac tissue.

The porcine and human heart are remarkably similar in cardiac physiology and biochemistry. Translational research involving the porcine biomedical model is becoming increasingly applicable for the study of human cardiac function in health and disease. Presently, few protocols exist for collecting experimentally viable cardiac tissue from large animal models, particularly during neonatal maturation.

The W792R HCM missense mutation in the C6 domain of cardiac myosin binding protein-C increases contractility in neonatal mouse myocardium.

Missense mutations in cardiac myosin binding protein C (cMyBP-C) are known to cause hypertrophic cardiomyopathy (HCM). The W792R mutation in the C6 domain of cMyBP-C causes severe, early onset HCM in humans, yet its impact on the function of cMyBP-C and the mechanism through which it causes disease remain unknown. To fully characterize the effect of the W792R mutation on cardiac morphology and function in vivo, we generated a murine knock-in model.

Cooperative mechanisms underlie differences in myocardial contractile dynamics between large and small mammals.

Ca2+ binding to troponin C (TnC) and myosin cross-bridge binding to actin act in a synergistic cooperative manner to modulate myocardial contraction and relaxation. The responsiveness of the myocardial thin filament to the activating effects of Ca2+ and myosin cross-bridge binding has been well-characterized in small mammals (e.g.

RLC phosphorylation amplifies Ca2+ sensitivity of force in myocardium from cMyBP-C knockout mice.

Hypertrophic cardiomyopathy (HCM) is the leading genetic cause of heart disease. The heart comprises several proteins that work together to properly facilitate force production and pump blood throughout the body. Cardiac myosin binding protein-C (cMyBP-C) is a thick-filament protein, and mutations in cMyBP-C are frequently linked with clinical cases of HCM.

Cardiac MyBP-C phosphorylation regulates the Frank-Starling relationship in murine hearts.

The Frank-Starling relationship establishes that elevated end-diastolic volume progressively increases ventricular pressure and stroke volume in healthy hearts. The relationship is modulated by a number of physiological inputs and is often depressed in human heart failure. Emerging evidence suggests that cardiac myosin-binding protein-C (cMyBP-C) contributes to the Frank-Starling relationship.

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+.

Deletion of Enigma Homologue from the Z-disc slows tension development kinetics in mouse myocardium.

Enigma Homologue (ENH) is a component of the Z-disc, a structure that anchors actin filaments in the contractile unit of muscle, the sarcomere. Cardiac-specific ablation of ENH protein expression causes contractile dysfunction that ultimately culminates in dilated cardiomyopathy. However, whether ENH is involved in the regulation of myocardial contractility is unknown.

Recovery of left ventricular function following in vivo reexpression of cardiac myosin binding protein C.

The loss of cardiac myosin binding protein C (cMyBP-C) results in left ventricular dilation, cardiac hypertrophy, and impaired ventricular function in both constitutive and conditional cMyBP-C knockout ( null) mice. It remains unclear whether the structural and functional phenotypes expressed in the null mouse are reversible, which is an important question, since reduced expression of cMyBP-C is an important cause of hypertrophic cardiomyopathy in humans. To investigate this question, we generated a cardiac-specific transgenic mouse model using a Tet-Off inducible system to permit the controlled expression of WT cMyBP-C on the null background.

Acting on an impulse (or two): Advantages of high-frequency tetanic onset in skeletal muscle.

Moss et al. highlight why high-frequency bursts at the onset of tetany increase force development in fast-twitch skeletal muscle fibers.

Phosphorylation of cardiac Myosin-binding protein-C is a critical mediator of diastolic function.

Background: Heart failure (HF) with preserved ejection fraction (HFpEF) accounts for ≈50% of all cases of HF and currently has no effective treatment. Diastolic dysfunction underlies HFpEF; therefore, elucidation of the mechanisms that mediate relaxation can provide new potential targets for treatment. Cardiac myosin-binding protein-C (cMyBP-C) is a thick filament protein that modulates cross-bridge cycling rates via alterations in its phosphorylation status.

Cardiac MyBP-C regulates the rate and force of contraction in mammalian myocardium.

Cardiac myosin-binding protein-C (cMyBP-C) is a thick filament-associated protein that seems to contribute to the regulation of cardiac contraction through interactions with either myosin or actin or both. Several studies over the past several years have suggested that the interactions of cardiac myosin-binding protein-C with its binding partners vary with its phosphorylation state, binding predominantly to myosin when dephosphorylated and to actin when it is phosphorylated by protein kinase A or other kinases. Here, we summarize evidence suggesting that phosphorylation of cardiac myosin binding protein-C is a key regulator of the kinetics and amplitude of cardiac contraction during β-adrenergic stimulation and increased stimulus frequency.

Myosin binding protein-C phosphorylation is the principal mediator of protein kinase A effects on thick filament structure in myocardium.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a regulator of pump function in healthy hearts. However, the mechanisms of regulation by cAMP-dependent protein kinase (PKA)-mediated cMyBP-C phosphorylation have not been completely dissociated from other myofilament substrates for PKA, especially cardiac troponin I (cTnI). We have used synchrotron X-ray diffraction in skinned trabeculae to elucidate the roles of cMyBP-C and cTnI phosphorylation in myocardial inotropy and lusitropy.

Dissociation of structural and functional phenotypes in cardiac myosin-binding protein C conditional knockout mice.

Background: Cardiac myosin-binding protein C (cMyBP-C) is a sarcomeric protein that dynamically regulates thick-filament structure and function. In constitutive cMyBP-C knockout (cMyBP-C(-/-)) mice, loss of cMyBP-C has been linked to left ventricular dilation, cardiac hypertrophy, and systolic and diastolic dysfunction, although the pathogenesis of these phenotypes remains unclear.

Methods And Results: We generated cMyBP-C conditional knockout (cMyBP-C-cKO) mice expressing floxed cMyBP-C alleles and a tamoxifen-inducible Cre-recombinase fused to 2 mutated estrogen receptors to study the onset and progression of structural and functional phenotypes caused by the loss of cMyBP-C.

Transgenic overexpression of γ-cytoplasmic actin protects against eccentric contraction-induced force loss in mdx mice.

Background: γ-cytoplasmic (γ-cyto) actin levels are elevated in dystrophin-deficient mdx mouse skeletal muscle. The purpose of this study was to determine whether further elevation of γ-cyto actin levels improve or exacerbate the dystrophic phenotype of mdx mice.

Methods: We transgenically overexpressed γ-cyto actin, specifically in skeletal muscle of mdx mice (mdx-TG), and compared skeletal muscle pathology and force-generating capacity between mdx and mdx-TG mice at different ages.

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development.

Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca(2+) sensitivity of force (pCa(50)), PKA treatment has been shown to decrease pCa(50), presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca(2+)-independent force and maximum Ca(2+)-activated force, whereas PKA treatment has no effect on either force.

Context-dependent functional substitution of alpha-skeletal actin by gamma-cytoplasmic actin.

We generated transgenic mice that overexpressed gamma-(cyto) actin 2000-fold above wild-type levels in skeletal muscle. gamma-(cyto) actin comprised 40% of total actin in transgenic skeletal muscle, with a concomitant 40% decrease in alpha-actin. Surprisingly, transgenic muscle was histologically and ultrastructurally identical to wild-type muscle despite near-stoichiometric incorporation of gamma-(cyto) actin into sarcomeric thin filaments.

Overexpression of TEAD-1 in transgenic mouse striated muscles produces a slower skeletal muscle contractile phenotype.

TEA domain (TEAD) transcription factors serve important functional roles during embryonic development and in striated muscle gene expression. Our previous work has implicated a role for TEAD-1 in the fast-to-slow fiber-type transition in response to mechanical overload. To investigate whether TEAD-1 is a modulator of slow muscle gene expression in vivo, we developed transgenic mice expressing hemagglutinin (HA)-tagged TEAD-1 under the control of the muscle creatine kinase promoter.

Protein kinase A-mediated phosphorylation of cMyBP-C increases proximity of myosin heads to actin in resting myocardium.

Protein kinase A-mediated (PKA) phosphorylation of cardiac myosin binding protein C (cMyBP-C) accelerates the kinetics of cross-bridge cycling and may relieve the tether-like constraint of myosin heads imposed by cMyBP-C. We favor a mechanism in which cMyBP-C modulates cross-bridge cycling kinetics by regulating the proximity and interaction of myosin and actin. To test this idea, we used synchrotron low-angle x-ray diffraction to measure interthick filament lattice spacing and the equatorial intensity ratio, I(11)/I(10), in skinned trabeculae isolated from wild-type and cMyBP-C null (cMyBP-C(-/-)) mice.

Destabilization of the dystrophin-glycoprotein complex without functional deficits in alpha-dystrobrevin null muscle.

Alpha-dystrobrevin is a component of the dystrophin-glycoprotein complex (DGC) and is thought to have both structural and signaling roles in skeletal muscle. Mice deficient for alpha-dystrobrevin (adbn(-/-)) exhibit extensive myofiber degeneration and neuromuscular junction abnormalities. However, the biochemical stability of the DGC and the functional performance of adbn(-/-) muscle have not been characterized.

Mutation that dramatically alters rat titin isoform expression and cardiomyocyte passive tension.

Titin is a very large alternatively spliced protein that performs multiple functions in heart and skeletal muscles. A rat strain is described with an autosomal dominant mutation that alters the isoform expression of titin. While wild type animals go through a developmental program where the 3.

Radial displacement of myosin cross-bridges in mouse myocardium due to ablation of myosin binding protein-C.

Myosin binding protein-C (cMyBP-C) is a thick filament accessory protein, which in cardiac muscle functions to regulate the kinetics of cross-bridge interaction with actin; however, the underlying mechanism is not yet understood. To explore the structural basis for cMyBP-C function, we used synchrotron low-angle X-ray diffraction to measure interfilament lattice spacing and the equatorial intensity ratio, I(11)/I(10), in skinned myocardial preparations isolated from wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)). In relaxed myocardium, ablation of cMyBP-C appeared to result in radial displacement of cross-bridges away from the thick filaments, as there was a significant increase ( approximately 30%) in the I(11)/I(10) ratio for cMyBP-C(-/-) (0.

Cytoplasmic gamma-actin is not required for skeletal muscle development but its absence leads to a progressive myopathy.

Nonmuscle gamma(cyto)-actin is expressed at very low levels in skeletal muscle but uniquely localizes to costameres, the cytoskeletal networks that couple peripheral myofibrils to the sarcolemma. We generated and analyzed skeletal muscle-specific gamma(cyto)-actin knockout (Actg1-msKO) mice. Although muscle development proceeded normally, Actg1-msKO mice presented with overt muscle weakness accompanied by a progressive pattern of muscle fiber necrosis/regeneration.