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.


Mitochondrial Isolation
Mice were euthanized by cervical dislocation, and the hearts were very quickly removed. Ventricles' tissues were minced on ice in isolation buffer A (in mM: 50 Tris, 1 EDTA, 70 saccharose, 210 mannitol, pH 7.4). They were cut roughly and homogenized in a glass-glass potter on ice. The ground material was then subjected to differential centrifugations at 4 °C. The first centrifugation was carried out at 1300 g for 3 min, allowing the cellular debris to be pushed. The supernatant was then recovered and centrifuged at 10000 g for 10 min, causing the sedimentation of the mitochondria. The pellet obtained was then resuspended in buffer B (same composition as buffer A without EDTA), and a final centrifugation was carried out at 10000 g for 10 min. The final pellet was then re-suspended in 100 μl of buffer B, and the mitochondrial proteins were finally quantified by Bradford assay.
Following a 2 min pre-incubation period, 20 nM of CaCl2 were added every 2 min until an increase in Ca 2+ -green fluorescence was detected, indicating mPTP opening. Experiments were performed in the presence and absence of puromycin (200 µM). The effect of the acute and the 30 min pre-treatment with puromycin were studied.

Probe multilabeling
Cells were loaded at 37°C 10 min before the end of the reperfusion phase, with DilC1(5), MitoSOX Red or MitoTracker Deep Red as previously described [46].

Arterial pressure and heart rate monitoring
Heart rate was measured using three 30G electrodes disposed subcutaneously at the anterior and posterior limbs of the animal. Data were retrieved via the iox2 software (Emka®) in a continuous mode.
Puromycin pretreated mice were injected IV under anesthesia with the 0.8 mg/kg dose while control mice were injected with the same volume of saline solution 0.9 %.
Blood pressure was determined non-invasively at the animal's tail (Coda, KentScientific®). After IV injection of the 0.8 mg/kg of puromycin, mice were kept in contention in a cylinder provided for this purpose and positioned on a heating pad at 37 °C, while the cuff and the sensor are positioned at the base of the tail of the animal. The measurements are made every 5 min and the data are recorded directly by computer.
Habituation sessions were performed once or twice a day for several days upstream measurement day to reduce animal stress due to the restraint.