Myosin heavy chain synthesis is independently regulated in hypertrophy and atrophy of isolated adult cardiac myocytes.

Hypertrophy of isolated adult feline cardiac muscle cells may be induced in culture by either alpha- or beta-adrenergic agonists. However, it has been shown previously that each of these agonists activate different subsets of immediate-early response genes and have different effects on expression of "fetal" protein isoforms and stimulation of protein synthesis. Moreover, in adult feline heart cells, beta-adrenergic agonists, such as isoproterenol, activate sustained synchronous beating and sarcomeric reorganization while alpha-adrenergic agonists, such as phenylephrine, do not. The objective of the present study was to determine whether these differences in proximal signaling events converged in a common signal pathway during activation of contractile protein synthesis. By direct comparisons of actin and myosin heavy chain (HC) synthesis and accumulation following isoproterenol and phenylephrine, it was determined that both agonists stimulate a coordinated accumulation of these proteins during cardiomyocyte growth. However, each agonist stimulated a very different program of contractile protein synthesis. During phenylephrine-induced hypertrophy, actin and myosin HC syntheses were rapidly and coordinately activated and continuously maintained at rates 10-25% greater than untreated cultures. The pattern of myosin HC synthesis following isoproterenol was very much more complex with periods during which it was as much as 40% greater or 25% less than in control cultures. Furthermore, there was no correlation between rates of actin and myosin HC synthesis following isoproterenol. It was concluded that actin and myosin HC syntheses and accumulation were regulated independently and in a very different manner following isoproterenol or phenylephrine. Since protein accumulation was not correlated with synthesis rates during development of hypertrophy, it was also concluded that post-translational mechanisms played a significant role in the maintenance of contractile protein stoichiometry during beta-adrenergic/beating-induced hypertrophy. Myosin HC synthesis also appeared to be independently regulated during cardiomyocyte atrophy induced by the calcium channel blocker nifedipine. Unlike the case in hypertrophy, however, protein balance was not maintained in nifedipine, and the depression of myosin HC synthesis and loss of myosin HC content were much greater than in the case of other contractile proteins.

Regulation of hypertrophy and atrophy in cultured adult heart cells.

Mechanical loading and alpha-adrenergic receptor stimulation have both been shown to induce hypertrophy in isolated neonatal heart cells. The present study examined the effects of adrenergic hormones and contractile activity on the hypertrophic response in isolated adult feline cardiomyocytes maintained for more than 14 days in insulin- and serum-supplemented medium. Measurements of the hypertrophic response included cell size, total protein content, myosin heavy chain content, and the time course of activation of increased protein synthesis. Reactivation of the "fetal" gene program was evaluated by secretion of atrial natriuretic factor (ANF) into the medium. Significant myocyte hypertrophy was induced in both quiescent myocytes treated with alpha 1-adrenergic agonists and in beating myocytes treated with beta-adrenergic agonists. However, there were both quantitative and qualitative differences in the response to each type of stimulation. alpha-Adrenergic agonists promoted an increase in cell size, protein content, and ANF secretion but not myofibrillar reorganization, which was observed only in beating myocytes. In contrast to results reported for neonatal heart cells, determinants of hypertrophy in beating myocytes exceeded those in nonbeating alpha 1-adrenergic agonist-treated heart cells in every parameter examined. In addition, in the case of both beating and alpha-adrenergic stimulation, there were marked time-dependent variations in rates of protein synthesis over the interval of 4 hours to 7 days of treatment with each type of stimulus. Differences were also encountered in correlations between rates of protein synthesis and protein accumulation over this interval. The effect of beating was particularly important both to the reorganization of myofibrillar structure and the metabolism of myosin heavy chain. In cultures in which beating was inhibited with the calcium channel antagonist nifedipine, the loss of myosin heavy chain was significantly greater than that of total protein.

Cultured adult cardiac myocytes maintain protein synthetic capacity of intact adult hearts.

Previous studies have shown that the rates of protein synthesis observed in embryonic and neonatal heart cells in culture are as much as nine times greater than the rates of synthesis observed in the intact adult heart either in situ or in isolated perfusion studies. This study addressed whether adult cardiomyocytes in long-term culture maintain the protein synthetic capacity of the adult myocardium or, rather, whether the protein synthetic capacity expands or falls as adult cardiac myocytes progress in culture. Protein synthesis was evaluated in isolated adult feline cardiomyocytes maintained in serum and insulin-supplemented medium for up to 53 days in vitro. With the use of both pulse- and saturation-labeling techniques it was determined that the rate of protein synthesis in adult cardiomyocytes was maintained at a level very close to that observed in the intact heart for over 1 mo in culture. Saturation-labeling studies indicate a fractional rate of protein synthesis at 6.1%/day and an absolute synthesis rate of 1,300 nmol leucine incorporated.g protein-1.h-1. Pulse-labeling studies revealed an initial increase in protein synthesis rates during adaptation to culture and a further increase after activation of beating and cellular hypertrophy.

Hypertrophy of isolated adult feline heart cells following beta-adrenergic-induced beating.

Catecholamine-induced beating and myocardial hypertrophy were evaluated in isolated adult feline cardiomyocytes maintained in culture for up to 30 days. Adult feline cardiomyocytes were used in this study because they displayed several unique characteristics that facilitated assessment of factors regulating cardiomyocyte hypertrophy in vitro. These characteristics included the following. 1) A single heart provides a high yield of 20-40 x 10(6) calcium-tolerant rod-shaped myocytes. 2) In culture, isolated adult feline cardiomyocytes maintain a stable population of differentiated myocytes that could be maintained without the dramatic loss of cell number, DNA content, or cell structure seen in adult rat cardiomyocyte cultures. 3) Cultured feline cardiomyocytes remained quiescent in culture unless appropriately stimulated to begin beating. 4) Sustained regular beating activity could be readily initiated up to 3 wk in culture by addition of 1 x 10(-5) M isoproterenol, other beta-adrenergic agonists, or agents known to elevate adenosine 3',5'-cyclic monophosphate. Beating could be maintained indefinitely in the presence of isoproterenol, but ceased upon removal of isoproterenol from the medium. Initiation of beating in 7-day-old cultures resulted in a profound restructuring of cardiomyocyte morphology compared with quiescent cultures. Beating heart cells were 66% larger with increased protein content, and they had significantly greater development of striated myofibrillar structure than quiescent myocytes at the same age in culture. We conclude that maintenance of an organized myofibrillar structure in cultured adult cardiac myocytes requires activation of intrinsic beating. Cardiomyocyte hypertrophy also develops following beta-adrenergic activation of beating, but it is unclear whether beating per se is required for inducing hypertrophy in isolated adult cardiomyocytes in vitro.

Halothane anesthesia decreases human monocyte hydrogen peroxide generation. Protection of monocytes by activation with gamma interferon.

In an effort to determine the impact of halothane anesthesia on certain human cell-mediated immune functions, normal, purified human monocytes and lymphocytes were exposed to halothane in vitro at varying concentrations for up to 8 hours. Subsequently, these human effector cells were analyzed for their ability to function in several cell-mediated immunologic assays. Natural killer cell activity against K-562 was unaffected by halothane in most of the donors tested. Similarly, the ability of purified monocytes to inhibit MBL-2 tumor cell growth was unchanged. Halothane appeared to decrease the proliferative response of lymphocytes to phytohemagglutinin (PHA) in approximately 50% of the normal donors tested. In contrast, the ability of monocytes to lyse antibody-coated red cell targets (ADCC) was unaffected by even maximal exposure to halothane. Of interest was the finding that human monocytes exposed to as low as 2% halothane anesthesia for 4 hours displayed a dramatic down-regulation of hydrogen peroxide (H2O2) release. Since it is known that hydrogen peroxide and other incompletely reduced forms of oxygen secreted by monocytes can play a major role in the antimicrobial, antitumor, and inflammatory functions of these cells, this finding may help explain the enhanced susceptibility of post-operative patients to infections.