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Cardiac injury is a common pathological change frequently accompanied by diabetes mellitus. Recently, some evidence indicated that calcium-sensing receptor (CaSR) expressed in the cardiac tissue. However, the functional role of CaSR in diabetic cardiac injury remains unclear. The present study was designed to investigate the relationship between CaSR activation and diabetes-induced cardiac injury. Diabetic model was successfully established by administration of streptozotocin (STZ) in vivo, and cardiomyocyte injury was simulated by 25.5 mM glucose in vitro. Apoptotic rate, intracellular calcium concentration ([Ca²⁺](i)) and the expression of Bcl-2, Bax, extracellular signal-regulated protein kinase (ERK), c-Jun NH₂-terminal protein kinase (JNK), and p38 were examined. We demonstrated a significant increase in left ventricular end-diastolic pressure (LVEDP) as well as decrease in maximum rate of left ventricular pressure rise and fall (±dp/dtmax), and left ventricular systolic pressure (LVSP), apoptosis of cardiomyocytes was also observed by TUNEL staining. In vitro, 25.5 mM glucose-induced apoptosis was detected by flow cytometry in neonatal rat cardiomyocytes. Further results showed that 25.5 mM glucose significantly increased [Ca²⁺](i), up-regulated the expression of Bax, P-ERK and P-JNK, and suppressed Bcl-2 expression. However, the above deleterious changes were further confirmed when co-treatment with CaSR agonist GdCl₃ (300 µM). But the effects of GdCl₃ were attenuated by 10 µM NPS-2390, a specific CaSR inhibitor. When CaSR was silence by siRNA transfection, the effects of high glucose were inhibited. These results suggest that CaSR activation could lead to the apoptosis of cardiomyocytes in diabetic cardiac injury through the induction of calcium overload, the activation of the mitochondrial, and mitogen-activated protein kinase pathway.

The M(3) subtype of muscarinic acetylcholine receptors (M(3)-mAChR) plays a protective role in myocardial ischemia and microRNAs (miRNAs) participate in many cardiac pathophysiological processes, including ischemia-induced cardiac injury. However, the role of miRNAs in M(3)-mAChR mediated cardioprotection remains unexplored. The present study was designed to identify miRNAs that are involved in cardioprotective effects of M(3)-mAChR against myocardial ischemia and elucidate the underlying mechanisms. We established rat model of myocardial ischemia and performed miRNA microarray analysis to identify miRNAs involved in the cardioprotection of M(3)-mAChR. In H9c2 cells, the viability, intracellular free Ca(2+) concentration ([Ca(2+)]i), intracellular reactive oxygen species (ROS), miR-376b-5p expression level, brain derived neurophic factor (BDNF) and nuclear factor kappa-B (NF-κB) levels were measured. Our results demonstrated that M(3)-mAChR protected myocardial ischemia injury. Microarray analysis and qRT-PCR revealed that miR-376b-5p was significantly up-regulated in ischemic heart tissue and the M(3)-mAChRs agonist choline reversed its up-regulation. In vitro, miR-376b-5p promoted H(2)O(2)-induced H9c2 cell injuries measured by cells viability, [Ca(2+)]i and ROS. Western blot and luciferase assay identified BDNF as a direct target of miR-376b-5p. M(3)-mAChR activated NF-κB and thereby inhibited miR-376b-5p expression. Our data show that a novel M(3)-mAChR/NF-κB/miR-376b-5p/BDNF axis plays an important role in modulating cardioprotection. MiR-376b-5p promotes myocardial ischemia injury possibly by inhibiting BDNF expression and M(3)-mAChR provides cardioprotection at least partially mediated by the downregulation of miR-376b-5p through NF-κB. These findings provide new insight into the potential mechanism by which M(3)-mAChR provides cardioprotection against myocardial ischemia injury.