Diminished function and expression of the cardiac Na+-Ca2+ exchanger in diabetic rats: implication in Ca2+ overload

Abstract

1. The present work was carried out in order to determine whether a decrease in cardiac Na+-Ca2+ exchanger (NCX) activity observed in diabetes is caused by a reduction in NCX protein and mRNA levels and to elucidate the significance of this decrease in alterations in [Ca2+]i homeostasis in diabetic cardiomyocytes. 2. The NCX current was significantly reduced in ventricular myocytes freshly isolated from streptozotocin-induced diabetic rat hearts, and its current density was about 55 % of age-matched controls. 3. Diabetes resulted in a 30 % decrease in cardiac protein and mRNA levels of NCX1, a NCX isoform which is expressed at high levels in the heart. 4. The reduced NCX current and the decreased protein and mRNA levels of NCX1 in diabetes were prevented by insulin therapy. 5. Although both diastolic and peak systolic [Ca2+]i were not different between the two groups of myocytes, increasing external Ca2+ concentration to high levels greatly elevated diastolic [Ca2+]i in diabetic myocytes. Inhibition of NCX by reduction in extracellular Na+ by 50 % could produce a marked rise in diastolic [Ca2+]i in control myocytes in response to high Ca2+, as seen in diabetic myocytes. However, cyclopiazonic acid, an inhibitor of sarcoplasmic reticulum Ca2+ pump ATPase, did not modify the high Ca2+-induced changes in diastolic [Ca2+]i in either control or diabetic myocytes. 6. Only in papillary muscles from diabetic rats did the addition of high Ca2+ cause a marked rise in resting tension signifying a partial contracture that was possibly due to an increase in diastolic [Ca2+]i. 7. In conclusion, the diminished NCX function in diabetic myocytes shown in this study results in part from the decreased levels of cardiac NCX protein and mRNA. We suggest that this impaired NCX function may play an important role in alterations in Ca2+ handling when [Ca2+]i rises to pathological levels.

Figure 1. Assessment of the NCX current in control and diabetic rat ventricular myocytes

A and B represent the I–V curves obtained at an external Ca²⁺ concentration of 0 mM (a) and 5 mM (b) in control and diabetic myocytes, respectively. The ordinate shows the current density. The difference current between the I–V curves at 0 and 5 mM Ca²⁺ indicates a net NCX current. In C, the difference currents in control and diabetic myocytes are shown. Note that the net NCX current is apparently smaller in diabetic cells than in control. D, bar graph comparing the NCX current density in control, diabetic and insulin-treated diabetic myocytes. The net NCX current was estimated as a difference current between the currents at +40 mV under external Ca²⁺ concentrations of 0 and 5 mM. Values are the result of dividing the net NCX current by the capacitance of each cell, and represent means ±s.e.m. of 4–6 cells from at least 3 rats. *P < 0.05vs. the value with diabetes. There was no significant difference between control and insulin-treated diabetic groups.

Figure 2. Immunoblot analysis of NCX1 in ventricular myocardium from control and diabetic rats

A, representative Western blot indicating clearly a decrease in the single protein band with an apparent molecular mass of 120–140 kDa in diabetes. B, bar graph comparing the immunostained band for NCX1 in the two groups of ventricular myocardium. Densitometric results are expressed as a percentage of the band obtained with control in each of the experiments. Bars are the means ±s.e.m. of 5 experiments. ***P < 0.001.

Figure 3. Northern blot analysis of the expression of NCX1 mRNA in ventricular myocardium from control, diabetic and insulin-treated diabetic rats

A, representative autograph of Northern blot analysis of NCX1 mRNA and β-actin mRNA expression. The blots had been hybridized sequentially using probes for NCX1 and β-actin. The positive signal localized at the molecular size appropriate for the specific mRNA with minimal background. The standard size is indicated as number of bases (b). While there appeared to be no relative change in the abundance of mRNA for β-actin, the relative content of NCX1 mRNA appeared to be decreased in diabetes. B, bar graph showing quantification of the steady-state levels of NCX1 mRNA in total myocardial RNA from control, diabetic and insulin-treated diabetic rats. The quantitative analysis was made using a Fujix BAS 2000. The steady-state levels of NCX1 mRNA are normalized to those of β-actin mRNA. Bars are means ±s.e.m. of 5 experiments. ***P < 0.001vs. the value with diabetes.

Figure 5. Changes in diastolic and peak systolic [Ca²⁺]_i in response to different external Ca²⁺ concentrations in control and diabetic rat ventricular myocytes

The [Ca²⁺]_i transients were elicited by a field stimulation of 0.5 Hz. The values of diastolic and peak systolic [Ca²⁺]_i when external Ca²⁺ concentration is increased stepwise from 1.3 mM to 2.6, 3.9 and 5.2 mM are shown. Data represent means ±s.e.m. of 5 cells from at least 3 rats. n.s., not significant. ***P < 0.001.

Figure 4. Changes in the [Ca²⁺]_i transients in response to different external Ca²⁺ concentrations in control and diabetic rat ventricular myocytes

After loading with indo-1 AM, myocytes were field stimulated at 0.5 Hz. Myocytes were exposed to Tyrode solution containing different concentrations of Ca²⁺ during the period indicated by the horizontal bars. Note that the increases in diastolic [Ca²⁺]_i in response to increasing external Ca²⁺ concentration from 1.3 mM to 2.6, 3.9 and 5.2 mM were apparently more marked in diabetic cells than in controls.

Figure 6. The influence of a reduction in external Na⁺ concentration on the change in the [Ca²⁺]_i transients in response to increasing external Ca²⁺ concentration in control and diabetic rat ventricular myocytes

After loading with indo-1 AM, myocytes were field stimulated with 0.5 Hz in normal Tyrode solution. The external Na⁺ concentration was then reduced by 50 % by replacing NaCl with equimolar choline chloride. Note that the increase in diastolic [Ca²⁺]_i was apparently the same in the two groups of myocytes when increasing the Ca²⁺ concentration from 1.3 to 5.2 mM.

Figure 7. The effect of the addition of high Ca²⁺ concentration on force of contraction in left ventricular papillary muscles isolated from control and diabetic rats

Muscles were electrically stimulated at 1 Hz. A, in control muscle, the addition of 10 mM CaCl₂ caused an increase in the peak amplitude of force of contraction without any change in resting tension. B, in diabetic muscle, a marked rise in resting tension was elicited by 10 mM CaCl₂.