The time courses of changes in pH(i) and cytosolic calcium were compared in isolated perfused rabbit hearts with the use of the calcium-sensitive fluorescent indicator indo-1 and the pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cell-permeant forms of these indicators were loaded into myocytes by arterial infusion or by direct infusion into the extravascular space. Indo-1 fluorescence was recorded from the epicardial surface of the left ventricle at an excitation wavelength of 360 nm and emission wavelengths of 400 and 550 nm. BCECF fluorescence was recorded at an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Calibration procedures were developed for each indicator that allowed [Ca2+](i) and pH(i) to be quantified during ischemia. Global ischemia decreased contractility and caused a rapid increase in both the systolic and end-diastolic levels of the calcium transients. Ninety seconds of ischemia increased peak systolic [Ca2+](i) from 609 ± 29 to 1,341±159 nM, while end-diastolic [Ca2+](i) increased from 315±25 to 553±52 nM. The observed increase in diastolic [Ca2+](i) was shown not to arise from indo-1-loaded endothelial cells. The initial increase in [Ca2+](i) was followed by a gradual decline and then a secondary rise occurring between 5 and 15 minutes of ischemia. In contrast, ischemia caused a monotonic decrease in pH(i) from a baseline of 7.03±0.06 to 6.83±0.02 after 2 minutes, 6.32±0.1 after 10 minutes, and 6.11±0.04 after 15 minutes. Perfusion of hearts with acidified (hypercarbic) saline increased the systolic and diastolic levels of the calcium transients, but only when pH(i) fell below a threshold value, which was more acidic than values achieved during the first 2 minutes of ischemia (6.83±0.03). Lesser degrees of acidification caused a decrease in contractility but did not affect the calcium transients. Effects of pH(i) on the calcium transients were not due to altered calcium sensitivity of indo-1. These results suggest that cytosolic acidification may contribute to the increase in [Ca2+](i) during the first 15 minutes of global ischemia, but the [Ca2+](i) increase during the first 2 minutes is mediated by other factors.
- Calcium transient
- Intracellular pH
ASJC Scopus subject areas
- Cardiology and Cardiovascular Medicine