Altered transarcolemmal Ca transport modifies the myofibrillar ultrastructure and protein metabolism in cultured adult ventricular cardiomyocytes

The present study was designed to investigate how prolonged (24-72 h) expos ure to modifiers of Ca transarcolemmal transport affects the myofibrillar s tructure, protein turnover and content of myofibrillar proteins in adult gu inea pig cardiomyocytes maintained beating synchronously in long-term cultu res. First we established the functional responses (the contractile activit y and [Ca](i) transients) of the cultured myocytes to acute exposures to se veral drugs used in this study. The ultrastructural characteristics of thes e cultures under the various treatments were determined using immunohistoch emistry and confocal scanning laser microscopy, and their biochemical prope rties were evaluated using analysis of total cellular protein content, myof ibrillar protein content and SDS-PAGE electrophoretic examination. We compa red the effects of 24, 36 and 72 h-long exposures to the various specific C a-flux modifiers. Increased Ca influx via Ca-L-channel agonist (Bay K 8644) or via the reversed-mode of the Na/Ca exchanger (veratrine) did not alter the myofibrillar structure or the specific protein profiles or proteosynthe sis. However, when cytosolic Ca was increased by three different types of i nhibitors of Ca extrusion from the cells via Na/Ca exchange, (Na-free solut ion, 5 mM NiCl2 and 10(-6) M ouabain), very significant changes in all inve stigated parameters occurred almost immediately. Twenty-four h-long exposur e to Na-free did not affect significantly the total cellular protein (TCP), but the protein synthesis was decreased by 87% and the total myofibrillar protein (TMP) content was decreased by 38%. The myofibrils were heavily fra gmented. Similarly, 24 h-long exposure to 5 mM NiCl2 did not affect the TCP , but it reduced protein synthesis by about 90% and decreased the total myo fibrillar protein content by 30%. These effects were even more pronounced a t 72 h of exposure and they were accompanied with a complete disassembly of myofilaments. Exposure to 10(-6) M ouabain over 72 h resulted in > 80% inh ibition of protein synthesis, a 45% decrease in TCP content and a 53% in TM P content. In contrast, 10(-7) M ouabain did not produce any such changes. The changes produced by the Na/Ca-exchange inhibitors were accompanied by o nly minor changes in DNA content, indicating that the myocytes remained via ble. Moreover, these effects were not due to the associated contractile arr est, since exposure to Ca-L-channel antagonists (5-20 mu M nifedipine or 10 mu M verapamil) produced only very minor changes in the myofibrillar struc ture and in protein profiles. Our data demonstrate that short-term (up to 72 h) increased Ca influx or co ntractile arrest of well-interconnected, spontaneously beating adult cardio myocytes does not affect their ultrastructural characteristics or their myo fibrillar protein turnover greatly, while any situations leading to Ca accu mulation (via inhibition of Na/Ca exchange) affect cardiomyocyte function a nd ultrastructure almost immediately. These data are in sharp contrast to t hose previously reported from immature, neonatal myocytes.