Intracellular Levels of Na+ and TTX-sensitive Na+ Channel Current in Diabetic Rat Ventricular Cardiomyocytes

Abstract

Intracellular Na(+) ([Na(+)](i)) is an important modulator of excitation-contraction coupling via regulating Ca(2+) efflux/influx, and no investigation has been so far performed in diabetic rat heart. Here, we examined whether any change of [Na(+)](i) in paced cardiomyocytes could contribute to functional alterations during diabetes. Slowing down in depolarization phase of the action potential, small but significant decrease in its amplitude with a slight depolarized resting membrane potential was traced in live cardiomyocytes from diabetic rat, being parallel with a decreased TTX-sensitive Na(+) channel current (I(Na)) density. We recorded either [Na(+)](i) or [Ca(2+)](i) by using a fluorescent Na(+) indicator (SBFI-AM or Na-Green) or a Ca(2+) indicator (Fura 2-AM) in freshly isolated cardiomyocytes. We examined both [Na(+)](i) and [Ca(2+)](i) at rest, and also [Na(+)](i) during pacing with electrical field stimulation in a range of 0.2-2.0 Hz stimulation frequency. In order to test the possible contribution of Na(+)/H(+) exchanger (NHE) to [Na(+)](i), we examined the free cytoplasmic [H(+)](i) changes from time course of [H(+)](i) recovery in cardiomyocytes loaded with SNARF1-AM by using ammonium prepulse method. Our data showed that [Na(+)](i) in resting cells from either diabetic or control group was not significantly different, whereas the increase in [Na(+)](i) was significantly smaller in paced diabetic cardiomyocytes compared to that of the controls. However, resting [Ca(2+)](i) in diabetic cardiomyocytes was significantly higher than that of the controls. Here, a lower basal pH(i) in diabetics compared with the controls correlates also with a slightly higher but not significantly different NHE activity and consequently a similar Na(+) loading rate at resting state with a leftward shift in pH sensitivity of NHE-dependent H(+)-flux. NHE protein level remained unchanged, while protein levels of Na(+)/K(+) ATPase and Na(+)/Ca(2+) exchanger were decreased in the diabetic cardiomyocytes. Taken together, the present data indicate that depressed I(Na) plays an important role in altered electrical activity with less Na(+) influx during contraction, and an increased [Ca(2+)](i) load in these cells seems to be independent of [Na(+)](i). The data with insulin treatment suggest further a recent balance between Na(+) influx and efflux proteins associated with the [Na(+)](i), particularly during diabetes.