Global and Local Effects of Intracellular pH on Ca2+ Waves in Rat Ventricular Myocytes

Abstract

Arrhythmogenic Ca2+ signalling may be influenced by alterations in intracellular pH (pHi), as found in myocardial ischaemia. Here we characterise the effects of altering pHi on Ca2+ waves in rat isolated ventricular myocytes.Ca2+ waves were induced by raising [Ca2+]o, and imaged confocally using fluo-3. Parallel measurements of pHi were made using cSNARF-1. pHi changes were induced by superfusion of acetate (20-80mM) or trimethylamine (20mM).Reducing pHi acutely decreased (to 16±6% of control) then increased (to 360±62% by 60s) wave frequency. Inhibiting the sarcolemmal Na+/H+ exchanger (NHE) with 30μM 5-(N,N-dimethyl)amiloride suppressed this secondary increase. A linear relationship was found between pHi and wave velocity: intracellular acidosis increased velocity up to 140±3%. Acidosis also slowed wave decay and increased diastolic Ca2+, while alkalosis decreased diastolic Ca2+.A stable longitudinal pHi gradient (pHi 6.6-7.3) was generated in single myocytes by microperfusing half of the cell with normal Tyrode and half with 80mM acetate, using a double-barrelled micropipette. With NHE inhibited, a decrease in wave frequency was seen in the acidic microdomain and an increase in the more alkaline microdomain. Conversely, wave velocity was increased in acidic and decreased in alkaline microdomains. Thus, wave properties map onto a spatial gradient of pHi.Wave velocity may be influenced by multiple pH-dependent components. Computational modelling of Ca2+ waves predicts that, at steady-state, sarco/endoplasmic reticulum Ca2+ ATPase inhibition decreases wave velocity, due to a fall in sarcoplasmic reticulum Ca2+ content (Swietach et al, 2010). However, decreasing the kon of Ca2+ buffers predicted an increase in wave velocity, suggesting the effect of pHi changes on velocity is multifactorial.We conclude that pHi heterogeneity locally regulates Ca2+ waves, suggesting that it may contribute to the heterogeneity of Ca2+ signalling observed in myocardial ischaemia.