Intracellular Ca2+ modulation during short exposure to ischemia-mimetic factors in isolated rat ventricular myocytes

Abstract

We investigated the effects of different ischemia-mimetic factors on intracellular Ca2+ concentration ([Ca2+]i). Ventricular myocytes were isolated from adult Wistar rats, and [Ca2+]i was measured using fluorescent indicator fluo-4 AM by confocal microscopy. Intracellular pH was measured using c5-(and-6)-carboxy SNARF-1 AM, a dual emission pH-sensitive ionophore. Myocytes were exposed to hypoxia, extracellular acidosis (pH(o) 6.8), Na-lactate (10 mM), or to combination of those factors for 25 min. Monitoring of [Ca2+]i using fluo-4 AM fluorescent indicator revealed that [Ca2+]i accumulation increased immediately after exposing the cells to Na-lactate and extracellular acidosis, but not during cell exposure to moderate ischemia. Increase in [Ca2+]i during Na-lactate exposure decreased to control levels at the end of exposure period at extracellular pH 7.4, but not at pH 6.8. When combined, Na-lactate and acidosis had an additive effect on [Ca2+]i increase. After removal of solutions, [Ca2+]i continued to rise only when acidosis, hypoxia, and Na-lactate were applied together. Analysis of intracellular pH revealed that treatment of cells by Na-lactate and acidosis caused intracellular acidification, while short ischemia did not significantly change intracellular pH. Our experiments suggest that increase in [Ca2+]i during short hypoxia does not occur if pH(i) does not fall, while extracellular acidosis is required for sustained rise in [Ca2+]i induced by Na-lactate. Comparing to the effect of Na-lactate, extracellular acidosis induced slower [Ca2+]i elevation, accompanied with slower decrease in intracellular pH. These multiple effects of hypoxia, extracellular acidosis, and Na-lactate are likely to cause [Ca2+]i accumulation after the hypoxic stress.

Figure 1

Intracellular Ca²⁺ changes in response to hypoxia and acidosis. A) Summarized recordings of relative fluo-4 fluorescence in cardiomyocytes exposed to glucose-free Tyrode solution with decreased O₂ concentration (25 mmHg) for 25 min and in cells which were perfused with normoxic Tyrode solution (Time control). B) Time course of the relative fluo-4 fluorescence change in cells exposed to normoxic Tyrode solution with decreased pH to 6.8. *P<0.05 versus time control, n=12 from 3 rats, error bars represent SD.

Figure 2

The effect of Na-lactate and hypoxia with Na-lactate and extracellular acidosis on intracellular Ca²⁺. A) Fluorescence measurements of intracellular pH in cardiomyocytes exposed to 10 mM Na-lactate (pH 7.4) for 25 min. B) Relative fluo-4 fluorescence intensity during hypoxia together with Na-lactate (10 mM) and extracellular acidosis (pH 6.8). *P<0.05 versus time control, n=12 from 3 rats, error bars represent SD.

Figure 3

Changes in intracellular Ca²⁺ in response to hypoxia, Na-lactate, extracellular acidosis, and combination of those factors. Graph shows summarized data for relative fluo-4 fluorescence of cardiomyocytes exposed to different ischemia-mimetic factors. *P<0.05 versus time control, #P<0.05 versus pH 6.8 or Na-lactate, n=10 from 3 rats, error bars represent SD.

Figure 4

Changes in pH_i in response to hypoxia, Na-lactate, extracellular acidosis, and combination of those factors. Figure shows representative examples of pHi changes of cardiomyocytes following exposure to different ischemia-mimetic factors. *P<0.05 versus time control, #P<0.05 versus pH 6.8 or Na-lactate, n=12 from 5 rats, error bars represent SD.