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Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart
Abstract:
Changes in cytosolic free calcium concentration during myocardial ischemia were measured by 19F NMR in 5FBAPTA-loaded perfused rat hearts. The hearts were perfused with Krebs-Henseleit buffer containing 5 microM of the acetoxymethyl ester of 5FBAPTA, which was hydrolyzed by cytosolic esterases to achieve cytosolic concentrations of 5FBAPTA of 0.12 to 0.65 mM. Cytosolic free calcium concentrations were calculated as the product of the ratio of peak areas for bound and free 5FBAPTA in the NMR spectra and the dissociation constant (708 nM). The basal cytosolic calcium concentration, measured in potassium or magnesium arrested hearts, was 252 nM, and the time-average calcium concentration in beating hearts was 630 nM. Following the onset of total ischemia, there was no immediate substantial change in cytosolic calcium despite a rapid decline in creatine phosphate and ATP and a marked increase in inorganic phosphate as monitored by 31P NMR, but by 10 minutes, there was a substantial increase in free calcium concentration. The ratio of peak areas of bound and free 5FBAPTA returned to the preischemic value during reperfusion, and there was no detectable loss of 5FBAPTA from the heart. Creatine phosphate was also restored to its preischemic level during reperfusion. These results indicate that cytosolic free calcium increases during ischemia and is not immediately associated with lethal injury. This increase in cytosolic calcium may activate degradative enzymes that eventually could compromise myocyte viability.
Insights
Cytosolic free calcium increases during myocardial ischemia, measured by 19F NMR. This rise in calcium, observed after 10 minutes of ischemia, may activate enzymes compromising heart cell viability.
Area of Science:
- Cardiovascular Physiology
- Cellular Biology
- Biochemistry
Background:
- Myocardial ischemia leads to significant metabolic changes.
- Understanding calcium handling during ischemia is crucial for cell survival.
Purpose of the Study:
- To measure cytosolic free calcium concentration changes during myocardial ischemia using 19F NMR.
- To investigate the relationship between ischemia, calcium levels, and metabolic changes.
Main Methods:
- Perfused rat hearts loaded with 5-carboxyfluorescein-based BAPTA (5FBAPTA) acetoxymethyl ester.
- 19F and 31P Nuclear Magnetic Resonance (NMR) spectroscopy to monitor calcium and metabolites.
- Calculation of cytosolic free calcium based on 5FBAPTA binding ratios and dissociation constant.
Main Results:
- Basal cytosolic calcium was 252 nM; time-average in beating hearts was 630 nM.
- No immediate change in cytosolic calcium during early ischemia, despite metabolic shifts.
- A substantial increase in free calcium occurred by 10 minutes of ischemia.
- Calcium levels and creatine phosphate normalized upon reperfusion without 5FBAPTA loss.
Conclusions:
- Cytosolic free calcium concentration increases during myocardial ischemia.
- This increase is not immediately linked to lethal injury but may activate degradative enzymes.
- The findings provide insights into mechanisms of myocyte injury during ischemic events.