EFFECTS OF ACIDOSIS ON RESTING CYTOSOLIC AND MITOCHONDRIAL CA2+ IN MAMMALIAN MYOCARDIUM

Acidosis increases resting cytosolic [Ca2+], (Ca(i)) of myocardial pre parations; however, neither the Ca2+ sources for the increase in Ca(i) nor the effect of acidosis on mitochondrial free [Ca2+], (Ca(m)) have been characterized. In this study cytosolic pH (pH(i)) was monitored in adult rat left ventricular myocytes loaded with the acetoxymethyl e ster (AM form) of SNARF-1. A stable decrease in the pH(i) of 0.52 +/- 0.05 U (n = 16) was obtained by switching from a bicarbonate buffer eq uilibrated with 5% CO2 to a buffer equilibrated with 20% CO2. Electric al stimulation at either 0.5 or 1.5 Hz had no effect on pH(i) in 5% CO 2, nor did it affect the magnitude of pH(i) decrease in response to hy percarbic acidosis. Ca(i) was measured in myocytes loaded with indo-1/ free acid and Ca(m) was monitored in cells loaded with indo-1/AM after quenching cytosolic indo-I fluorescence with MnCl2. In quiescent inta ct myocytes bathed in 1.5 mM [Ca2+], hypercarbia increased Ca(i) from 130 +/- 5 to 221 +/- 13 nM. However, when acidosis was effected in ele ctrically stimulated myocytes, diastolic Ca(i) increased more than res ting Ca(i) in quiescent myocytes, and during pacing at 1.5 Hz diastoli c Ca(i) was higher (285 +/- 17 nM) than at 0.5 Hz (245 +/- 18 nM; P < 0.05). The magnitude of Ca(i) increase in quiescent myocytes was not a ffected either by sarcoplasmic reticulum (SR) Ca2+ depletion with ryan odine or by SR Ca2+ depletion and concomitant superfusion with a Ca2+- free buffer. In unstimulated intact myocytes hypercarbia increased Ca( m) from 95 +/- 12 to 147 +/- 19 nM and this response was not modified either by ryanodine and a Ca2+-free buffer or by 50 muM ruthenium red in order to block the mitochondrial uniporter. In mitochondrial suspen sions loaded either with BCECF/AM or indo-1/AM, acidosis produced by l actic acid addition decreased both intra- and extramitochondrial pH an d increased Ca(m). Studies of mitochondrial suspensions bathed in indo -1/free acid-containing solution showed an increase in extramitochondr ial Ca2+ after the addition of lactic acid. Thus, in quiescent myocyte s, cytoplasmic and intramitochondrial buffers, rather than transsarcol emmal Ca2+ influx or SR Ca2+ release, are the likely Ca2+ sources for the increase in Ca(i) and Ca(m), respectively; additionally, Ca2+ effl ux from the mitochondria may contribute to the raise in Ca(i). In cont rast, in response to acidosis, diastolic Ca(i) in electrically stimula ted myocytes increases more than resting Ca(i) in quiescent cells; thi s suggests that during pacing, net cell Ca2+ gain contributes to enhan ce diastolic Ca(i).