Acute Induction of Translocon-Mediated Ca2+ Leak Protects Cardiomyocytes Against Ischemia/Reperfusion Injury.

During myocardial infarction, dysregulation of Ca2+ homeostasis between the reticulum, mitochondria, and cytosol occurs in cardiomyocytes and leads to cell death. Ca2+ leak channels are thought to be key regulators of the reticular Ca2+ homeostasis and cell survival. The present study aimed to determine whether a particular reticular Ca2+ leak channel, the translocon, also known as translocation channel, could be a relevant target against ischemia/reperfusion-mediated heart injury. To achieve this objective, we first used an intramyocardial adenoviral strategy to express biosensors in order to assess Ca2+ variations in freshly isolated adult mouse cardiomyocytes to show that translocon is a functional reticular Ca2+ leak channel. Interestingly, translocon activation by puromycin mobilized a ryanodine receptor (RyR)-independent reticular Ca2+ pool and did not affect the excitation-concentration coupling. Second, puromycin pretreatment decreased mitochondrial Ca2+ content and slowed down the mitochondrial permeability transition pore (mPTP) opening and the rate of cytosolic Ca2+ increase during hypoxia. Finally, this translocon pre-activation also protected cardiomyocytes after in vitro hypoxia reoxygenation and reduced infarct size in mice submitted to in vivo ischemia-reperfusion. Altogether, our report emphasizes the role of translocon in cardioprotection and highlights a new paradigm in cardioprotection by functionally uncoupling the RyR-dependent Ca2+ stores and translocon-dependent Ca2+ stores.

An innovative sequence of hypoxia-reoxygenation on adult mouse cardiomyocytes in suspension to perform multilabeling analysis by flow cytometry.

Cardiovascular diseases remain the leading cause of death worldwide. Although major therapeutic progress has been made during the past decades, a better understanding of the underlying mechanisms will certainly help to improve patient's prognosis. In vitro models, particularly adult mouse cardiomyocytes, have been largely used; however, their fragility and large size are major obstacles to the use of flow cytometry. Conventional techniques, such as cell imaging, require the use of large numbers of animals and are time consuming. Here, we described a new, simple, and rapid one-day protocol using living adult mouse cardiomyocytes in suspension exposed to hypoxia-reoxygenation that allows a multilabeling analysis by flow cytometry. Several parameters can be measured by fluorescent probes labeling to assess cell viability (propidium iodide, calcein-AM, and Sytox Green), mitochondrial membrane potential [DilC1(5) and TMRM], reactive oxygen species production (MitoSOX Red), and mitochondrial mass (MitoTracker Deep Red). We address the robustness and sensitivity of our model using a cardioprotective agent, cyclosporine A. Overall, our new experimental set-up offers a high-speed quantitative multilabeling analysis of adult mouse cardiomyocytes exposed to hypoxia-reoxygenation. Our model might be interesting to investigate other cellular stresses (oxidative and inflammation) or to perform pharmacological screening.