Intracellular acidosis decreases the occurrence of spontaneous Ca(2+i) sparks in rat ventricular myocytes (Balnave & Vaughan-Jones, 2000). Under conditions of Ca(2+i) overload, spark amplitude can increase, triggering arrhythmogenic spontaneous Ca2+ waves. At 22 °C, such waves are suppressed by intracellular acidosis, provided sarcolemmal acid-extrusion proteins are also inactive (Vaughan-Jones & Balnave, 2001). We have now examined the pHi sensitivity of spontaneous waves at 37 °C.
Ventricular myocytes, isolated from rat (humanely killed by cervical dislocation), AM-loaded with Fluo-3, and Ca(2+i)-overloaded by raising [Ca2+]o to 7.5 mM, were confocally imaged (Leica DM IRBE X 100 1.4 NA objective) in line-scan mode (100 µM line scanned at 450 Hz, excitation at 515 nm). pHi was reduced by superfusion with 40 or 80 mM acetate. Data, collected during the second minute of acetate superfusion, were normalized to control measurements recorded over 1 min before acetate addition. All results are expressed as means ± S.E.M. P values given were calculated with Student’s paired t test.
At 37 °C, reducing pHi from 7.32 ± 0.02 (n = 15) to 6.82 ± 0.03 (n = 8) (measured in parallel experiments with intracellular carboxy SNARF-1) increased wave frequency by 341 ± 47 % (n = 9, P < 0.05). In comparison, at 22 °C a 0.74 pH unit decrease in pHi increased spontaneous wave frequency (P < 0.05) by a smaller amount (210 ± 36% n = 10) (Vaughan-Jones & Balnave, 2001). At both temperatures in the presence of 30 µM cariporide (a selective cardiac Na+-H+ exchange (NHE-1) inhibitor) intracellular acidosis almost entirely suppressed wave formation.
At 37 °C decreasing pHi from 7.32 to 6.72 (decrease of 0.6 pH units) enhanced average wave velocity from 75 µm s-1 to 120 µm s-1, i.e. by 150 ± 3 % (n = 9, P < 0.05). In the presence of 30 µM cariporide, there was still an increase in velocity (130 ± 15% n = 3). At 22 °C reducing pHi by 0.74 pH units caused an even greater increase in velocity (176 ± 3% n = 10, P < 0.05), and this was again unaffected (n = 7) by inhibiting NHE-1.
In conclusion, during Ca(2+i) overload a fall of pHi increases the frequency of spontaneous Ca2+ waves, an effect attenuated by moderate hypothermia. In the presence of cariporide, however, acidosis suppresses waves at both 22 °C and 37 °C. Intracellular acidosis increases the velocity of Ca2+ waves, an effect enhanced by hypothermia. This increase, however, is not influenced by cariporide and is therefore independent of NHE activity. The effect of intracellular pH on spontaneous Ca2+ waves thus depends on at least two mechanisms, one of which relies on the activity of sarcolemmal Na+-H+ exchange.
This work was funded by the BHF and Wellcome Trust. We thank Dr H. W. Kleeman of Aventis, for kindly providing cariporide.