Intracellular calcium mobilization in cardiomyocytes of rats with experimental diabetes
Creators: name: Yu, Zhen
Publisher: name: University of British Columbia
Year: 2008

description: It is well known that alterations in intracellular calcium, [Ca2]i, mobilization in the heart are among the major causes of cardiac dysfunction. To study the mechanism(s) involved in the development of diabetic cardio myopathy, [Ca2]i (determined by fluorescence microscopy and Ca-indicators) and contraction of the cardiomyocytes (measured by a video-edge detector system) were studied in rats with experimental diabetes induced by streptozotocin (STZ). Ryanodine receptor (the calcium release channel) binding and sarcolemmal Na-Ca exchange activity were also investigated. Cardiomyocytes were successfully isolated from rat hearts of diabetics and age-matched controls. The cell contractile function, determined by percentage of cell shortening, rate of shortening and relengthening (±dlidt), and response to isoproterenol stimulation were significantly depressed in diabetic cells. These reflect similar features presented in intact diabetic heart and cardiac tissues. The sarcoplasmic reticulum (SR) Ca2+ content assessed by rapid cooling contracture (RCC), caffeine contracture (CC), and caffeine-induced [Ca2li transient was markedly reduced. Insulin treatment reversed depressed RCCs and caffeine-induced [Ca2li transients, and also reversed the diabetes-induced depression of peak -d[Ca21i/dt values which presumably are indications of depressed function of Na-Ca exchange. Two binding sites for ryanodine were found in cardiac homogenates. The number of sites for high affinity and low capacity binding (Bmax) were less in diabetic heart than in controls, while the binding affinity (Kid) was unchanged. Alteration in SR calcium release channels may be involved in the SR dysfunction observed in diabetes. Measurement of Na-Ca exchange activity in diabetic hearts showed a decreased Vmax suggesting a reduced number of exchangers and a decreased Km, an index of apparent affinity for calcium. These changes may occur as a result of the diabetes-induced membrane environmental change and/or adaptation. In quiescent myocytes isoproterenol (1 pM) induced a sustained [Ca2+]; decrease which was blocked by timolol (0.1 pM) and thapsigargin (1 pM) suggesting the SR Ca-ATPase was involved in this fl-adrenergic stimulated [Ca24]i response. In electrically-paced myocytes, isoproterenol caused a dose dependent increase of[Ca2÷]i. The maximum response was depressed in diabetic cells. 8-bromo-cAMPcaused a similar but less marked increase in [Ca2+]i compared with isoproterenol. The maximum response to 8-bromo-cAMP was also decreased in diabetic cells. The data are in good agreement with the 11-adrenoceptor deficiency observed in diabetic hearts, and also suggest that post-receptor alterations occur in diabetes. Paced diabetic myocytes showed a decreased maximum response but an increased sensitivity to ouabain stimulation. This suggests that diabetic cells are less tolerant to ouabain, which may be related to the depressed Na-pump activity and increased [Na]i in diabetic hearts. ATP and KCI both caused an enhanced increase in [Ca2]i in diabetic myocytes (paced and quiescent, respectively). The exact mechanisms involved in the action of ATP is unknown although, like KCl, it activates the L-type Ca-channels. By using different blockers, [Ca2-f]1 transients induced by KCl could be separated into different components mediated by the L-type Ca-channel, Na-Ca exchange and SR release. An enhanced response to these agents might be related to the enhanced and altered Ca-channel activity reported in diabetic hearts. In summary, diabetic cardiomyocytes provide a valuable model for studying cellular function of the diabetic heart. Myocytes from diabetic hearts retain the features of cardiac dysfunction seen in intact diabetic rat hearts. There appear to be multiple changes in the control of [Ca2]i in diabetic cardiomyopathy. The cardiac SR calcium content was reduced, which may consequently depress cardiac contraction. The [Ca2+ji mobilization is altered in diabetes, notably a decreased response to isoproterenol and an enhanced sensitivity to ouabain. These observations provide some useful information about diabetic cardiomyopathy.