Rapid effects of stretched myocardial and vascular cells on gene expression of neonatal rat cardiomyocytes with emphasis on autocrine and paracrine mechanisms.

Passive stretch of the heart has a direct effect on cardiomyocytes and other cell types including cardiac fibroblasts, endothelial cells, and vascular smooth muscle cells (VSMCs). Cardiomyocytes are targets for the action of peptide growth factors found in myocardium, suggesting an autocrine or paracrine model of the hypertrophic process. In this study we examined stretch-dependent cellular communication between cardiomyocytes, cardiac fibroblasts, endothelial cells, and VSMCs. Stationary cardiomyocytes were incubated with stretch-conditioned medium (CM0-CM60) derived from stretched (for 0-60 min) cardiomyocytes, cardiac fibroblasts, endothelial cells, and VSMCs. The expression levels of protooncogenes (as c-fos, c-jun, and fra-1) were measured, and as an indication of a hypertrophic response the expression of atrial natriuretic peptide (ANP) was measured. Stationary cardiomyocytes that have been incubated for 30 min with CM from stretched (for 0-60 min) cardiomyocytes, cardiac fibroblasts, endothelial cells, and VSMCs showed distinct gene expression patterns that were time-dependent and cell-type specific. In stationary cardiomyocytes, CM derived from stretched cardiomyocytes caused decreased c-fos and fra-1 expression by 37 and 20%, respectively (CM30), elevated c-jun expression by 20% (CM45-CM60), and increased ANP expression by 106% (CM45). CM derived from stretched cardiac fibroblasts caused increased c-fos expression by 41% (CM60), no significant changes in c-jun expression, and increased fra-1 and ANP expression by 39 and 20%, respectively (CM45). CM derived from stretched VSMCs induced an initial decrease in c-fos expression followed by an increase of 13% (CM45) and induced increased c-jun, fra-1, and ANP expression by 39, 24, and 22%, respectively. CM15-CM60 derived from stretched endothelial cells caused decreased c-fos, c-jun and fra-1 expression by 20, 25, and 25%, respectively, and increased ANP expression by 18%. Our data indicate that gene expression of cardiomyocytes in stretched myocardium is regulated by mediators released by cardiomyocytes, cardiac fibroblasts, endothelial cells, and VSMCs. This observation emphasizes the involvement of nonmyocyte cells in the early stages of cardiomyocyte hypertrophy caused by cardiac stretch.

The effect of sarcolemmal cholesterol content on the tolerance to anoxia in cardiomyocyte cultures.

To determine whether the sarcolemmal-free cholesterol content influences the tolerance to anoxia in cultured neonatal rat cardiomyocytes, we modulated the free cholesterol content of the cultures, and determined the time course of anoxia-induced cell death. Incubation for 5 h with liposomes having a free cholesterol to phospholipid molar ratio of 2, 0.5 and 0, resulted in a change of cellular free cholesterol content by +54.7 +/- 5.8% (P < 0.001), by -5.2 +/- 6.3% (n.s.), and by -22.1 +/- 4.9% (P < 0.01), respectively, when compared to cultures incubated without liposomes (control). In cells which were loaded with cholesterol, it was verified that the extra cholesterol was transferred predominantly to the sarcolemma, which was associated with a decrease in sarcolemmal fluidity. In cells which underwent cholesterol reduction it was verified that free cholesterol was retracted selectively from the sarcolemma, associated with an increase in sarcolemmal fluidity. Cellular enrichment with free cholesterol caused a delay of anoxia-induced release of lactate dehydrogenase (LDH): the time at which half-maximal release of LDH was reached (LDH50%) occurred later by 35.5 +/- 3.4 min (P < 0.001) compared to anoxic control cultures. Cholesterol reduction diminished the tolerance to anoxia: LDH50% occurred earlier by 36.4 +/- 7.8 min (P < 0.01), compared to anoxic control cultures. Cultures which were incubated with liposomes having a free cholesterol to phospholipid molar ratio of 0.5 had unchanged sarcolemmal free cholesterol content, unchanged sarcolemmal fluidity, and an unchanged LDH50% during anoxia in comparison to control cultures, excluding any effect of the incubation with liposomes per se. The present study indicates that the sarcolemmal free cholesterol content of cardiomyocytes is a determinant of their tolerance to anoxia.