Effects of acidosis on resting cytosolic and mitochondrial Ca²⁺ in mammalian myocardium

Acidosis increases resting cytosolic [Ca²⁺], (Ca_i) of myocardial prepa-rations; 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 ester (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 equilibrated with 5% CO₂ to a buffer equilibrated with 20% CO₂. Electrical stimulation at either 0.5 or 1.5 Hz had no effect on pH_i in 5% CO₂, nor did it affect the magnitude of pH_i decrease in response to hypercarbic 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-1 fluorescence with MnCl₂. In quiescent intact myocytes bathed in 1.5 mM [Ca²⁺], hypercarbia increased Ca_i from 130 ± 5 to 221 ± 13 nM. However, when acidosis was effected in electrically stimulated myocytes, diastolic Ca_i increased more than resting Ca_i in quiescent myocytes, and during pacing at 1.5 Hz diastolic 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 affected either by sarcoplasmic reticulum (SR) Ca²⁺ depletion with ryanodine 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 μM ruthenium red in order to block the mitochondrial uniporter. In mitochondrial suspensions loaded either with BCECF/AM or indo-1/ AM, acidosis produced by lactic acid addition decreased both intra- and extramitochondrial pH and increased Ca_m. Studies of mitochondrial suspensions bathed in indo-1/free acid - containing solution showed an increase in extramitochondrial Ca²⁺ after the addition of lactic acid. Thus, in quiescent myocytes, cytoplasmic and intramitochondrial buffers, rather than transsarcolemmal Ca²⁺ influx or SR Ca²⁺ release, are the likely Ca²⁺ sources for the increase in Ca_i and Ca_m, respectively; additionally, Ca²⁺ efflux from the mitochondria may contribute to the raise in Ca_i. In contrast, in response to acidosis, diastolic Ca_i in electrically stimulated myocytes increases more than resting Ca_i in quiescent cells; this suggests that during pacing, net cell Ca²⁺ gain contributes to enhance diastolic Ca_i.