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Ca2+ ionophores are not suitable for inducing mPTP opening in murine isolated adult cardiac myocytes.

ABSTRACT: Opening of the mitochondrial permeability transition pore (mPTP) plays a major role in cell death during cardiac ischaemia-reperfusion. Adult isolated rodent cardiomyocytes are valuable cells to study the effect of drugs targeting mPTP. This study investigated whether the use of Ca2+ ionophores (A23187, ionomycin and ETH129) represent a reliable model to study inhibition of mPTP opening in cardiomyocytes. We monitored mPTP opening using the calcein/cobalt fluorescence technique in adult rat and wild type or cyclophilin D (CypD) knock-out mice cardiomyocytes. Cells were either treated with Ca2+ ionophores or subjected to hypoxia followed by reoxygenation. The ionophores induced mPTP-dependent swelling in isolated mitochondria. A23187, but not ionomycin, induced a decrease in calcein fluorescence. This loss could not be inhibited by CypD deletion and was explained by a direct interaction between A23187 and cobalt. ETH129 caused calcein loss, mitochondrial depolarization and cell death but CypD deletion did not alleviate these effects. In the hypoxia-reoxygenation model, CypD deletion delayed both mPTP opening and cell death occurring at the time of reoxygenation. Thus, Ca2+ ionophores are not suitable to induce CypD-dependent mPTP opening in adult murine cardiomyocytes. Hypoxia-reoxygenation conditions appear therefore as the most reliable model to investigate mPTP opening in these cells.

SUBMITTER: Panel M

PROVIDER: S-EPMC5487341 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Ca<sup>2+</sup> ionophores are not suitable for inducing mPTP opening in murine isolated adult cardiac myocytes.

Panel Mathieu M Ghaleh Bijan B Morin Didier D

Scientific reports 20170627 1

Opening of the mitochondrial permeability transition pore (mPTP) plays a major role in cell death during cardiac ischaemia-reperfusion. Adult isolated rodent cardiomyocytes are valuable cells to study the effect of drugs targeting mPTP. This study investigated whether the use of Ca<sup>2+</sup> ionophores (A23187, ionomycin and ETH129) represent a reliable model to study inhibition of mPTP opening in cardiomyocytes. We monitored mPTP opening using the calcein/cobalt fluorescence technique in adu ...[more]

PMID: 28655872

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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.

Project description:Background The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib is associated with a higher incidence of cardiotoxic side effects including heart failure (HF). Objectives Ibrutinib is capable of inhibiting PI3K/Akt signaling in neonatal rat ventricular cardiomyocytes when stimulated with insulin-like growth factor 1 (IGF-1). We therefore hypothesized that Ibrutinib might disrupt IGF-1-mediated activation of intracellular Ca handling in adult mouse cardiomyocytes by inhibiting PI3K/Akt signaling. Methods Isolated ventricular myocytes (C57BL6/J) were exposed to IGF-1 at 10 nmol/L in the presence or absence of Ibrutinib (1 µmol/L) or Acalabrutinib (10 µmol/L; cell culture for 24 ± 2 h). Intracellular Ca handling was measured by epifluorescence (Fura-2 AM) and confocal microscopy (Fluo-4 AM). Ruptured-patch whole-cell voltage-clamp was used to measure ICa. Levels of key cardiac Ca handling proteins were investigated by immunoblots. Results IGF-1 significantly increased Ca transient amplitudes by ∼83% as compared to vehicle treated control cells. This was associated with unaffected diastolic Ca, enhanced SR Ca loading and increased ICa. Co-treatment with Ibrutinib attenuated both the IGF-1-mediated increase in SR Ca content and in ICa. IGF-1 treated cardiomyocytes had significantly increased levels of pS473Akt/Akt and SERCA2a expression as compared to cells concomitantly treated with IGF-1 and Ibrutinib. SR Ca release (as assessed by Ca spark frequency) was unaffected by either treatment. In order to test for potential off-target effects, second generation BTK inhibitor Acalabrutinib with greater BTK selectivity and lower cardiovascular toxicity was tested for IGF1-mediated activation of intracellular Ca handling. Acalabrutinib induced similar effects on Ca handling in IGF-1 treated cultured myocytes as Ibrutinib in regard to decreased Ca transient amplitude and slowed Ca transient decay, hence implying a functional class effect of BTK inhibitors in cardiac myocytes. Conclusions Inhibition of BTK by Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in adult ventricular mouse myocytes in the face of disrupted Akt signaling and absent SERCA2a upregulation.

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Ca2+ ionophores are not suitable for inducing mPTP opening in murine isolated adult cardiac myocytes.

Publications

Ca<sup>2+</sup> ionophores are not suitable for inducing mPTP opening in murine isolated adult cardiac myocytes.

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