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Interaction of background Ca²⁺ influx, sarcoplasmic reticulum threshold and heart failure in determining propensity for Ca²⁺ waves in sheep heart.

ABSTRACT: Ventricular arrhythmias can cause death in heart failure (HF). A trigger is the occurrence of Ca²⁺ waves which activate a Na⁺ -Ca²⁺ exchange (NCX) current, leading to delayed after-depolarisations and triggered action potentials. Waves arise when sarcoplasmic reticulum (SR) Ca²⁺ content reaches a threshold and are commonly induced experimentally by raising external Ca²⁺ , although the mechanism by which this causes waves is unclear and was the focus of this study. Intracellular Ca²⁺ was measured in voltage-clamped ventricular myocytes from both control sheep and those subjected to rapid pacing to produce HF. Threshold SR Ca²⁺ content was determined by applying caffeine (10 mM) following a wave and integrating wave and caffeine-induced NCX currents. Raising external Ca²⁺ induced waves in a greater proportion of HF cells than control. The associated increase of SR Ca²⁺ content was smaller in HF due to a lower threshold. Raising external Ca²⁺ had no effect on total influx via the L-type Ca²⁺ current, I_Ca-L , and increased efflux on NCX. Analysis of sarcolemmal fluxes revealed substantial background Ca²⁺ entry which sustains Ca²⁺ efflux during waves in the steady state. Wave frequency and background Ca²⁺ entry were decreased by Gd³⁺ or the TRPC6 inhibitor BI 749327. These agents also blocked Mn²⁺ entry. Inhibiting connexin hemi-channels, TRPC1/4/5, L-type channels or NCX had no effect on background entry. In conclusion, raising external Ca²⁺ induces waves via a background Ca²⁺ influx through TRPC6 channels. The greater propensity to waves in HF results from increased background entry and decreased threshold SR content. KEY POINTS: Heart failure is a pro-arrhythmic state and arrhythmias are a major cause of death. At the cellular level, Ca²⁺ waves resulting in delayed after-depolarisations are a key trigger of arrhythmias. Ca²⁺ waves arise when the sarcoplasmic reticulum (SR) becomes overloaded with Ca²⁺ . We investigate the mechanism by which raising external Ca²⁺ causes waves, and how this is modified in heart failure. We demonstrate that a novel sarcolemmal background Ca²⁺ influx via the TRPC6 channel is responsible for SR Ca²⁺ overload and Ca²⁺ waves. The increased propensity for Ca²⁺ waves in heart failure results from an increase of background influx, and a lower threshold SR content. The results of the present study highlight a novel mechanism by which Ca²⁺ waves may arise in heart failure, providing a basis for future work and novel therapeutic targets.

SUBMITTER: Hutchings DC

PROVIDER: S-EPMC9310721 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Excessive cardiac fibrosis, characterized by increased collagen-rich extracellular matrix (ECM) deposition, is a major predisposing factor for mechanical and electrical dysfunction in heart failure (HF). The human ventricular fibroblast (hVF) remodeling mechanisms that cause excessive collagen deposition in HF are unclear, although reports suggest a role for intracellular free Ca2+ in fibrosis. Therefore, we determined the association of differences in cellular Ca2+ dynamics and collagen secretion/deposition between hVFs from failing and normal (control) hearts. Histology of left ventricle sections (Masson trichrome) confirmed excessive fibrosis in HF versus normal. In vitro, hVFs from HF showed increased secretion/deposition of soluble collagen in 48 h of culture compared with control [85.9±7.4 µg/106 cells vs 58.5±8.8 µg/106 cells, P<0.05; (Sircol™ assay)]. However, collagen gene expressions (COL1A1 and COL1A2; RT-PCR) were not different. Ca2+ imaging (fluo-3) of isolated hVFs showed no difference in the thapsigargin-induced intracellular Ca2+ release capacity (control 16±1.4% vs HF 17±1.1%); however, Ca2+ influx via store-operated Ca2+ entry/Ca2+ release-activated channels (SOCE/CRAC) was significantly (P≤0.05) greater in HF-hVFs (47±3%) compared with non-failing (35±5%). Immunoblotting for ICRAC channel components showed increased ORAI1 expression in HF-hVFs compared with normal without any difference in STIM1 expression. The Pearson's correlation coefficient for co-localization of STIM1/ORAI1 was significantly (P<0.01) greater in HF (0.5±0.01) than control (0.4±0.01) hVFs. The increase in collagen secretion of HF versus control hVFs was eliminated by incubation of hVFs with YM58483 (10 µM), a selective ICRAC inhibitor, for 48 h (66.78±5.87 µg/106 cells vs 55.81±7.09 µg/106 cells, P=0.27). In conclusion, hVFs from HF have increased collagen secretion capacity versus non-failing hearts and this is related to increase in Ca2+ entry via SOCE and enhanced expression of ORAI, the pore-forming subunit. Therapeutic inhibition of SOCE may reduce the progression of cardiac fibrosis/HF.

Project description:Introduction: Safranal, which endows saffron its unique aroma, causes vasodilatation and has a hypotensive effect in animal studies, but the mechanisms of these effects are unknown. In this study, we investigated the mechanisms of safranal vasodilation. Methods: Isolated rat endothelium-intact or -denuded aortic rings were precontracted with phenylephrine and then relaxed with safranal. To further assess the involvement of nitric oxide, prostaglandins, guanylate cyclase, and phospholipase A2 in safranal-induced vasodilation, aortic rings were preincubated with L-NAME, indomethacin, methylene blue, or quinacrine, respectively, then precontracted with phenylephrine, and safranal concentration-response curves were established. To explore the effects of safranal on Ca2+ influx, phenylephrine and CaCl2 concentration-response curves were established in the presence of safranal. Furthermore, the effect of safranal on aortic rings in the presence of ouabain, a Na+-K+ ATPase inhibitor, was studied to explore the contribution of Na+/Ca2+ exchanger to this vasodilation. Results: Safranal caused vasodilation in endothelium-intact and endothelium-denuded aortic rings. The vasodilation was not eliminated by pretreatment with L-NAME, indomethacin, methylene blue, or quinacrine, indicating the lack of a role for NO/cGMP. Safranal significantly inhibited the maximum contractions induced by phenylephrine, or by CaCl2 in Ca2+-free depolarizing buffer. Safranal also relaxed contractions induced by ouabain, but pretreatment with safranal totally abolished the development of ouabain contractions. Discussion/Conclusion: Inhibition of Na+-K+ ATPase by ouabain leads to the accumulation of Na+ intracellularly, forcing the Na+/Ca2+ exchanger to work in reverse mode, thus causing a contraction. Inhibition of the development of this contraction by preincubation with safranal indicates that safranal inhibited the Na+/Ca2+ exchanger. We conclude that safranal vasodilation is mediated by the inhibition of calcium influx from extracellular space through L-type Ca2+ channels and by the inhibition of the Na+/Ca2+ exchanger.

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Interaction of background Ca²⁺ influx, sarcoplasmic reticulum threshold and heart failure in determining propensity for Ca²⁺ waves in sheep heart.

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Dataset Information

Interaction of background Ca2+ influx, sarcoplasmic reticulum threshold and heart failure in determining propensity for Ca2+ waves in sheep heart.

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OmicsDI is part of the ELIXIR infrastructure

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Interaction of background Ca²⁺ influx, sarcoplasmic reticulum threshold and heart failure in determining propensity for Ca²⁺ waves in sheep heart.