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

Abstract

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.

Mechano-Electric Coupling and Arrhythmogenic Current Generation in a Computational Model of Coupled Myocytes

“…Late diastolic stretches, such as stretch pattern S, have been observed in various pathological conditions such as ischemia or heart failure (Neves et al, 2016 ) and isometric conditions are often used in experimental set-ups to immobilize the cells for optical mapping. In pathological conditions, such as ischemia, stretch is assumed to enhance calcium spark probability (Cameron et al, 2020 ), which may trigger calcium waves and DADs originating from these sites (Boyden et al, 2003 ; Boyden and ter Keurs, 2005 ; ter Keurs and Boyden, 2007 ). In addition to calcium waves, spontaneous contractile waves have been related to pro-arrhythmic effects in arrangements of weaker damaged regions in-series with normal muscle segments.…”

Mechano-arrhythmogenicity is enhanced during late repolarisation in ischemia and driven by a TRPA1-, calcium-, and reactive oxygen species-dependent mechanism

“…Next, we assessed whether Ca 2+ release via RyR may be involved in the observed arrhythmogenic role of cytosolic Ca 2+ (like X-ROS, stretch-induced Ca 2+ -sparks are also enhanced by ischemia). 22 To test this, RyR in ischemic cells were stabilised in their closed state with dantrolene, however, this had no effect on arrhythmia incidence with stretch in diastole or the VP (Fig. 5a).…”

“…4b). As fluorophore photoactivation is associated with the generation of ROS, 26 which is increased in ischemia, 22 ROS might also play a role in mechano-arrhythmogenicity in ischemic cells. To test this, we pre-incubated ischemic cells with NAC to chelate intracellular ROS and found that arrhythmia incidence in the VP decreased compared to ischemia alone (0.8 vs 6.8%; p<0.0005), with no effect in diastole (Fig.…”

“…4c). Further, as we have shown that X-ROS production is enhanced in ischemia, 22 we tested the effect of the NOX2 inhibitor DPI to block X-ROS production in ischemic cells. DPI also reduced arrhythmia incidence in the VP (2.4 vs 6.8%; p<0.05) with no diastolic effect (Fig.…”

“…Stretch is known to increase NADPH oxidase-dependent ROS production (X-ROS) 20 and subsequent sarcoplasmic Ca 2+ release events via ryanodine receptors (RyR). 21 As these mechanically-induced effects are enhanced in ischemia, 22 regional stretch may lead to localised increases in ROS and cytosolic Ca 2+ , resulting in arrhythmic activity. 23 The goal of this study was to investigate cellular mechanisms of ischemic mechanoarrhythmogenicity and the importance of the VP.…”