Ischemia Enhances the Acute Stretch-Induced Increase in Calcium Spark Rate in Ventricular Myocytes

Introduction: In ventricular myocytes, spontaneous release of calcium (Ca²⁺) from the sarcoplasmic reticulum via ryanodine receptors ("Ca²⁺ sparks") is acutely increased by stretch, due to a stretch-induced increase of reactive oxygen species (ROS). In acute regional ischemia there is stretch of ischemic tissue, along with an increase in Ca²⁺ spark rate and ROS production, each of which has been implicated in arrhythmogenesis. Yet, whether there is an impact of ischemia on the stretch-induced increase in Ca²⁺ sparks and ROS has not been investigated. We hypothesized that ischemia would enhance the increase of Ca²⁺ sparks and ROS that occurs with stretch. Methods: Isolated ventricular myocytes from mice (male, C57BL/6J) were loaded with fluorescent dye to detect Ca²⁺ sparks (4.6 μM Fluo-4, 10 min) or ROS (1 μM DCF, 20 min), exposed to normal Tyrode (NT) or simulated ischemia (SI) solution (hyperkalemia [15 mM potassium], acidosis [6.5 pH], and metabolic inhibition [1 mM sodium cyanide, 20 mM 2-deoxyglucose]), and subjected to sustained stretch by the carbon fiber technique (~10% increase in sarcomere length, 15 s). Ca²⁺ spark rate and rate of ROS production were measured by confocal microscopy. Results: Baseline Ca²⁺ spark rate was greater in SI (2.54 ± 0.11 sparks·s^-1·100 μm^-2; n = 103 cells, N = 10 mice) than NT (0.29 ± 0.05 sparks·s^-1·100 μm^-2; n = 33 cells, N = 9 mice; p < 0.0001). Stretch resulted in an acute increase in Ca²⁺ spark rate in both SI (3.03 ± 0.13 sparks·s^-1·100 μm^-2; p < 0.0001) and NT (0.49 ± 0.07 sparks·s^-1·100 μm^-2; p < 0.0001), with the increase in SI being greater than NT (+0.49 ± 0.04 vs. +0.20 ± 0.04 sparks·s^-1·100 μm^-2; p < 0.0001). Baseline rate of ROS production was also greater in SI (1.01 ± 0.01 normalized slope; n = 11, N = 8 mice) than NT (0.98 ± 0.01 normalized slope; n = 12, N = 4 mice; p < 0.05), but there was an acute increase with stretch only in SI (+12.5 ± 2.6%; p < 0.001). Conclusion: Ischemia enhances the stretch-induced increase of Ca²⁺ sparks in ventricular myocytes, with an associated enhancement of stretch-induced ROS production. This effect may be important for premature excitation and/or in the development of an arrhythmogenic substrate in acute regional ischemia.