Direct, autocrine and paracrine effects of cyclic stretch on growth of myocytes and fibroblasts isolated from neonatal rat ventricles.

Several studies have demonstrated that static stretch of cardiomyocytes induces cardiomyocyte hypertrophy. We investigated the effects of cyclic stretch, a more physiological stimulus, on protein synthesis and DNA synthesis of rat ventricular cardiomyocytes and cardiofibroblasts. Further-more, we investigated whether these effects are caused by autocrine mechanisms. In addition, we studied the paracrine influences of stretched cardiofibroblasts on cardiomyocyte growth. Short-term cyclic stretch (0-24 h) of cardiomyocytes induced a growth response indicative of cardiomyocyte hypertrophy, given the fact that increased rates of protein synthesis and DNA synthesis were accompanied by an elevated release of atrial natriuretic peptide into the culture medium. In cardiofibroblasts, short-term cyclic stretch also induced a growth response as indicated by an increased rate of protein synthesis and DNA synthesis. Furthermore, incubation of stationary cardiofibroblasts with conditioned medium derived from stretched cardiofibroblasts revealed an autocrine effect of stretch as illustrated by an increased rate of protein synthesis in stationary cardiofibroblasts. In analogy, there was an autocrine effect of stretch on stationary cardiomyocytes incubated with conditioned medium derived from stretched cardiomyocytes. Moreover, we observed a paracrine effect of the conditioned medium derived from stretched cardiofibroblasts on stationary cardiomyocytes. Thus, short-term cyclic stretch of cardiomyocytes and cardiofibroblasts induces growth responses that are the result of direct, autocrine, and paracrine effects. These autocrine/paracrine effects of stretch are most probably due to release of factors from stretched cells.

Role of calcium-activated neutral protease (calpain) in cell death in cultured neonatal rat cardiomyocytes during metabolic inhibition.

Calcium-activated neutral protease (CANP), also known as calpain, has been implicated in the development of cell death in ischemic hearts. CANP is thought to be activated by the calcium overload that develops during ischemia. We studied the involvement of CANP in cell death in cultured neonatal rat cardiomyocytes during metabolic inhibition (5 mmol/L NaCN + 10 mmol/L 2-deoxyglucose). First, we isolated CANP using ion exchange and affinity chromatography. Then the efficacy of the CANP inhibitors calpain I inhibitor, leupeptin, and E64 to inhibit isolated CANP activity was tested with the use of fluorescently labeled beta-casein as a substrate. The IC50 for the inhibitors was between 2.1 and 56 mumol/L. Uptake of the inhibitors by intact cells was assessed with the use of 99mTc-radiolabeled inhibitors. The calculated intracellular inhibitor concentrations were sufficiently high to yield substantial inhibition of intracellular CANP activity. Intracellular CANP activity was measured directly with the use of the cell-permeant fluorogenic CANP-specific substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl-coumarin. During metabolic inhibition, intracellular CANP activity was increased compared with control incubation. The time course of CANP activation was compatible with that of the rise in [Ca2+]i, as measured by fura 2 and digital imaging fluorescence microscopy. Calpain I inhibitor and leupeptin inhibited intracellular CANP activity both during metabolic inhibition and control incubation, whereas E64 did not. Despite their substantial inhibition of intracellular CANP activity, calpain I inhibitor and leupeptin did not attenuate cell death during metabolic inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic inhibition of cardiomyocytes causes an increase in sarcolemmal fluidity which may be due to loss of cellular cholesterol.

We examined whether metabolic inhibition (5 mM NaCN + 10 mM 2-deoxyglucose) affects sarcolemmal fluidity in cultured neonatal cardiomyocytes. As a measure of sarcolemmal fluidity we determined the fluorescence steady-state anisotropy (rss, which is reciprocally related to membrane fluidity) of cardiomyocytes labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, p-toluenesulfonate. During metabolic inhibition, membrane fluidity increased progressively: after 30 min rss had fallen by 6.7 +/- 1.2% (mean +/- SE; n = 9; P < 0.05) compared to baseline values, and after 90 min by 14.5 +/- 3.5% (P < 0.05; n = 5). Beyond 90 min rss did not decrease any further. During control incubations (without metabolic inhibition), no significant changes in rss were observed. During metabolic inhibition cellular free cholesterol content declined: after 30 min free cholesterol content had decreased by 12.2 +/- 3.1% (P < 0.02; n = 4), compared to baseline values, and after 90 min by 31.1 +/- 8.3% (P < 0.02; n = 4). We conclude that metabolic inhibition induces an increase in sarcolemmal fluidity, which may be caused by a decrease in sarcolemmal free cholesterol content.

Release of alpha hydroxybutyrate from neonatal rat heart cell cultures exposed to anoxia and reoxygenation: comparison with impairment of structure and function of damaged cardiac cells.

Spontaneously beating monolayer cultures of neonatal rat heart cells first exposed to depletion of oxygen and metabolic substrates for 1 to 7 h (anoxia). Subsequently the cultures were resupplied with oxygen and substrates (reoxygenation). The release of alpha-hydroxybutyrate dehydrogenase (HBDH) from the cells, the extent of necrosis, and the changes in spontaneous contractile activity were measured. HBDH release was observed to start after 1 h of anoxia and to increase to 84% of intracellular HBDH activity after 7 h of anoxia. Reoxygenation of anoxic heart cells is associated with accelerated HBDH release (oxygen paradox). The activity of HBDH released by cardiac cells, the number of necrotic cells and the impairment of beating capacity of the cells depend on the duration of the anoxic period in a similar way. This study demonstrates that cardiac cell death can be assessed quantitatively and reliably by the measurement of the activity of HBDH released by the cells.