This stretch induced increase in [Ca2+]i may be associated with stretch activated arrhythmias in the heart. The effects of streptomycin are consistent with its reported inhibitory action on stretch activated channels.

SUMMARYUntil recently the investigation of length-dependent effects in cardiac muscle was restricted to multicellular preparations. We describe our experimental set-up which for the first time, in single cardiac myocytes, permits the effects of changes in cell length on auxotonic contractions (measured by carbon fibre transducers) to be simultaneously recorded with the effects on membrane potential and/or changes in intracellular calcium concentration (using indo-1 AM, acetoxylmethyl form). Consistent with previous findings (in experiments at 20-25°C and 025 Hz) we report that following a stretch there was an increase in passive tension and contraction. A stretch which increased sarcomere length by approximately 3 % had no significant effect on resting membrane potential or action potential amplitude. There was, however, a significant decrease in the action potential duration (P < 0-01, n = 8). No significant change in the amplitude of the intracellular calcium transient was seen following a stretch but a reduction in its duration was observed (P < 0-025, n = 11). Our observations on intracellular calcium transients are consistent with the hypothesis that, in mechanically loaded preparations, their time course is more dependent on changes in tension than changes in length.

C‐protein is a major component of muscle thick filaments whose function is unknown. We have examined for the first time the role of the regulatory binding domain of C‐protein in modulating contraction and intracellular Ca2+ concentration ([Ca2+]i) in intact cardiac myocytes. Rat ventricular myocytes were reversibly permeabilised with the pore‐forming toxin streptolysin O. Myosin S2 (which binds to the regulatory domain of C‐protein) was introduced into cells during permeabilisation to compete with the endogenous C‐protein‐thick filament interaction. Introduction of S2 into myocytes increased contractility by ∼30%, significantly lengthened the time to peak of the contraction and the time to half‐relaxation, but had no effect on [Ca2+]i transient amplitude. Our data are consistent with increased myofilament Ca2+ sensitivity when there is reduced binding of C‐protein to myosin near the head‐tail junction. We propose that the effects of introducing S2 into intact cardiac cells can be equated with the consequences of selectively phosphorylating C‐protein in vivo, and that the regulation of contraction by C‐protein is mediated by the effects of crossbridge cycling on the Ca2+ affinity of troponin C.

Cardiac hypertrophy is associated with a dramatic change in the gene expression profile of cardiac myocytes. Many genes important during development of the fetal heart but repressed in the adult tissue are reexpressed, resulting in gross physiological changes that lead to arrhythmias, cardiac failure, and sudden death. One transcription factor thought to be important in repressing the expression of fetal genes in the adult heart is the transcriptional repressor REST (repressor element 1-silencing transcription factor). Although REST has been shown to repress several fetal cardiac genes and inhibition of REST function is sufficient to induce cardiac hypertrophy, the molecular mechanisms employed in this repression are not known. Here we show that continued REST expression prevents increases in the levels of the BNP (Nppb) and ANP (Nppa) genes, encoding brain and atrial natriuretic peptides, in adult rat ventricular myocytes in response to endothelin-1 and that inhibition of REST results in increased expression of these genes in H9c2 cells. Increased expression of Nppb and Nppa correlates with increased histone H4 acetylation and histone H3 lysine 4 methylation of promoter-proximal regions of these genes. Furthermore, using deletions of individual REST repression domains, we show that the combined activities of two domains of REST are required to efficiently repress transcription of the Nppb gene; however, a single repression domain is sufficient to repress the Nppa gene. These data provide some of the first insights into the molecular mechanism that may be important for the changes in gene expression profile seen in cardiac hypertrophy.The repressor element 1-silencing transcription factor (REST) was originally identified as an important transcription factor regulating the expression of neuron-specific genes (12, 53) but has since been shown to be a key transcriptional regulator in heart development (28) and vascular smooth muscle growth (11). Disruption of REST function by expression of a dominant-negative form specifically in the heart results in cardiomyopathy, arrhythmias, and sudden death (28). These effects are thought to result from the reexpression of fetal cardiac genes and have led to the proposition that REST represses the fetal cardiac gene program in the adult heart (28). In vascular smooth muscle, loss of REST has been implicated in neointimal hyperplasia, and inhibition of REST results in increased smooth muscle proliferation (11). Several genes that are repressed by REST in myocytes have been identified, including the genes encoding the brain and atrial natriuretic peptides (Nppb and Nppa, encoding BNP and ANP, respectively), ␣-skeletal actin (Acta1), potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channels 2 and 4 (Hcn2 and Hcn4), and voltage-gated calcium channel subunit alpha Cav3.2 (Cacna1h) (26, 43). Levels of BNP and ANP are particularly important, since increased levels of these peptides in circulation are clinical indicators of the severity of hypertrophy (8,29,38,61)....