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

Abstract

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.

Keywords: calcium; ischemia; myocytes; reactive oxygen species; stretch; ventricle.

Figure 1

Effect of axial stretch on the occurrence of calcium sparks in single quiescent mouse ventricular myocytes exposed to either normal Tyrode (A) or simulated ischemia (B). Fluorescence surface plots are derived from a line of pixels across the cell, providing a temporal depiction of the relative Fluo-4 fluorescence (F_REL) before, during, and after 15 s of stretch (dashed red lines). Movies of the cells are provided as Supplemental Videos 1, 2.

Figure 2

Effect of duration of exposure of quiescent mouse ventricular myocytes to normal Tyrode (A,B) or simulated ischemia solution (C,D) on calcium (Ca²⁺) spark rate before (Baseline), during (Stretch), and after stretch (Release) (A,C), as well as the change in Ca²⁺ spark rate with stretch [Δ Ca²⁺ Spark Rate; (B,D)] over the 30 min measurement window. Cells were subjected to stretch at a single time point and values from all cells stretched within 5 min intervals were averaged (presented as mean ± SEM). N indicates the number of mice; n indicates the number of cells. *p < 0.01 by post-hoc Dunn's multiple comparisons test.

Figure 3

Effect of stretch of quiescent mouse ventricular myocytes on calcium (Ca²⁺) spark rate before (Baseline) and during stretch (Stretch; A) and the change in Ca²⁺ spark rate with stretch (Δ Ca²⁺ Spark Rate; B) averaged across all cells exposed to either normal Tyrode or simulated ischemia solution (presented as mean ± SEM). *p < 0.0001 by Wilcoxon paired test; ^#p < 0.0001 by Mann-Whitney unpaired test. N indicates the number of mice; n indicates the number of cells.

Figure 4

Effect of stretch of quiescent mouse ventricular myocytes on the rate of reactive oxygen species (ROS) production (quantified as the slope of the change in 2′,7′-dichlorofluorescein diacetate, DCF, fluorescence over time) before (Baseline) and during stretch (Stretch; A) and the change in ROS production with stretch (Δ ROS Production; B) averaged across all cells exposed to either normal Tyrode or simulated ischemia solution (presented as mean ± SEM). *p < 0.001 by two-tailed, paired Student's t-test; ^#p < 0.05 and ^##p < 0.01, by two-tailed, unpaired Student's t-test. N indicates the number of mice; n indicates the number of cells.