In ventricular myocytes, spontaneous release of calcium (Ca 2+) from the sarcoplasmic reticulum via ryanodine receptors ("Ca 2+ 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 Ca 2+ 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 Ca 2+ sparks and ROS has not been investigated. We hypothesized that ischemia would enhance the increase of Ca 2+ sparks and ROS that occurs with stretch. Methods: Isolated ventricular myocytes from mice (male, C57BL/6J) were loaded with fluorescent dye to detect Ca 2+ 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). Ca 2+ spark rate and rate of ROS production were measured by confocal microscopy. Results: Baseline Ca 2+ 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 Ca 2+ 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 Ca 2+ 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.
Myocardial stretch physiologically activates NADPH oxidase 2 (NOX2) to increase reactive oxygen species (ROS) production. Although physiological low‐level ROS are known to be important as signalling molecules, the role of stretch‐induced ROS in the intact myocardium remains unclear. To address this, we investigated the effects of stretch‐induced ROS on myocardial cellular contractility and calcium transients in C57BL/6J and NOX2−/− mice. Axial stretch was applied to the isolated cardiomyocytes using a pair of carbon fibres attached to both cell ends to evaluate stretch‐induced modulation in the time course of the contraction curve and calcium transient, as well as to evaluate maximum cellular elastance, an index of cellular contractility, which is obtained from the end‐systolic force–length relationship. In NOX2−/− mice, the peak calcium transient was not altered by stretch, as that in wild‐type mice, but the lack of stretch‐induced ROS delayed the rise of calcium transients and reduced contractility. Our mathematical modelling studies suggest that the augmented activation of ryanodine receptors by stretch‐induced ROS causes a rapid and large increase in the calcium release flux, resulting in a faster rise in the calcium transient. The slight increase in the magnitude of calcium transients is offset by a decrease in sarcoplasmic reticulum calcium content as a result of ROS‐induced calcium leakage, but the faster rise in calcium transients still maintains higher contractility. In conclusion, a physiological role of stretch‐induced ROS is to increase contractility to counteract a given preload, that is, it contributes to the Frank–Starling law of the heart. imageKey points Myocardial stretch increases the production of reactive oxygen species by NADPH oxidase 2. We used NADPH oxidase 2 knockout mice to elucidate the physiological role of stretch‐induced reactive oxygen species in the heart. We showed that stretch‐induced reactive oxygen species modulate the rising phase of calcium transients and increase myocardial contractility. A mathematical model simulation study demonstrated that rapid activation of ryanodine receptors by reactive oxygen species is important for increased contractility. This response is advantageous for the myocardium, which must contract against a given preload.
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