Rapamycin (sirolimus) protects against hypoxic damage in primary heart cultures via Na⁺/Ca²⁺ exchanger activation

Aims: Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian targeting of rapamycin (mTOR) signaling, and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients. Rapamycin confers preconditioning-like protection against ischemic-reperfusion injury in isolated mouse heart cultures. Our aim was to further define the role of rapamycin in intracellular Ca²⁺ homeostasis and to investigate the mechanism by which rapamycin protects cardiomyocytes from hypoxic damage. Main methods: We demonstrate here that rapamycin protects rat heart cultures from hypoxic-reoxygenation (H/R) damage, as revealed by assays of lactate dehydrogenase (LDH) and creatine kinase (CK) leakage to the medium, by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) measurements, and desmin immunostaining. As a result of hypoxia, intracellular calcium levels ([Ca²⁺]_i) were elevated. However, treatment of heart cultures with rapamycin during hypoxia attenuated the increase of [Ca²⁺]_i. Rapamycin also attenuated ⁴⁵Ca²⁺ uptake into the sarcoplasmic reticulum (SR) of skinned heart cultures in a dose- and time-dependent manner. KB-R7943, which inhibits the "reverse" mode of Na⁺/Ca²⁺ exchanger (NCX), protected heart cultures from H/R damage with or without the addition of rapamycin. Rapamycin decreased [Ca²⁺]_i following its elevation by extracellular Ca²⁺ ([Ca²⁺] _o) influx, thapsigargin treatment, or depolarization with KCl. Key findings: We suggest that rapamycin induces cardioprotection against hypoxic/reoxygenation damage in primary heart cultures by stimulating NCX to extrude Ca²⁺ outside the cardiomyocytes. Significance: According to our findings, rapamycin preserves Ca²⁺ homeostasis and prevents Ca²⁺ overload via extrusion of Ca²⁺ surplus outside the sarcolemma, thereby protecting the cells from hypoxic stress.