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 Ca²⁺ 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 [Ca²⁺] _i by using a fluorescent Na⁺ indicator (SBFI-AM or Na-Green) or a Ca²⁺ indicator (Fura 2-AM) in freshly isolated cardiomyocytes. We examined both [Na⁺] _i and [Ca²⁺] _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 [Ca²⁺] _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⁺/Ca²⁺ 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 [Ca²⁺] _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.