ABSTRACT
AIMS: Prior to reperfusion, Calpains remain inactive due to the acidic pH and elevated ionic strength in the ischemic myocardium; but Calpain is activated during myocardial reperfusion. The underlying mechanism of Calpain activation in the ischemia-reperfusion (I/R) is yet to be determined. Therefore, the present study aims to investigate the mechanism of Calpain in I/R-induced mice. MAIN METHODS: In order to detect the function of Calpain and the NLRP3/ASC/Caspase-1 axis in cardiomyocyte pyroptosis, endoplasmic reticulum (ER) stress and myocardial function, the cardiomyocytes were treated with hypoxia-reoxygenation (H/R), and NLRP3 were silenced, Calpain was overexpressed and Caspase-1 inhibitors were used to determine cardiomyocyte pyroptosis. The results obtained from the cell experiments were then verified with an animal experiment in I/R mice. KEY FINDINGS: There was an overexpression in Calpain, ASC, NLRP3, GRP78 and C/EBP homologous protein (CHOP) in cardiomyocytes following H/R. A significant increase was witnessed in lactic acid dehydrogenase (LDH) activity, cardiomyocyte pyroptosis rate, Calpain activity, reactive oxygen species (ROS) concentration, as well as activation of ER stress in cardiomyocytes after H/R. However, opposing results were observed in H/R cardiomyocytes that received siRNA Calpain, siRNA NLRP3 or Caspase-1 inhibitor treatment. Overall, the results obtained from the animal experiment were consistent with the results from the cell experiment. SIGNIFICANCE: The silencing of Calpain suppresses the activation of the NLRP3/ASC/Caspase-1 axis, thus inhibiting ER stress in mice and improving myocardial dysfunction induced by I/R, providing a novel therapeutic pathway for I/R.
Subject(s)
Amino Acid Transport System y+/antagonists & inhibitors , Calpain/antagonists & inhibitors , Caspase 1/chemistry , Endoplasmic Reticulum Stress , Myocardial Reperfusion Injury/prevention & control , Myocytes, Cardiac/pathology , NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors , Amino Acid Transport System y+/genetics , Amino Acid Transport System y+/metabolism , Animals , Calpain/genetics , Calpain/metabolism , Caspase 1/genetics , Caspase 1/metabolism , Cells, Cultured , Endoplasmic Reticulum Chaperone BiP , Inflammasomes , Male , Mice , Mice, Inbred C57BL , Myocardial Reperfusion Injury/etiology , Myocardial Reperfusion Injury/pathology , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , RNA, Small Interfering/geneticsABSTRACT
OBJECTIVE: To investigate the possible mechanism of lycopene on protecting against hypoxia/reoxygenation (H/R)-injury. METHODS: Primary cultured cardiomyocytes, isolated from neonatal mouse, were divided into three groups randomly: control group (C) ; H/R group(4 h H followed by 8 h R); lycopene+H/R group(L+H/R), in which the cardiomyocytes were pretreated with lycopene for 4 h before H/R. The survival of cardiomyocytes was counted. Apoptotic cells were detected by TUNEL assays. The release of cytochrome c from mitochondrial matrix into the cytosol, the activity of caspase-3, intracellular ROS levels and the activity of calpain were also determined in these groups respectively at the same time. RESULTS: The pretreatment of cardiomyocytes with lycopene significantly improved the survival of cardiomyocytes [C: (89.84 ± 5.15)%, H/R: (63.59 ± 5.11)%, L+H/R: (79.25 ± 1.48)%, P < 0.05] and reduced the extent of apoptosis [C: ( 10.37 ± 1.25)%, H/R: (32.03 ± 4.79)%, L+H/R: (22.57 ± 3.22)%, P < 0.05], significantly reduced caspase-3 activation [C: (2.61 ± 0.19), H/R: (5.82 ± 0.92), L+H/R: (3.74 ± 0.64) pNA pmol/µg protein, P < 0.05]. To further study the mechanism underlying the benefits of lycopene, interactions between lycopene and calpain activation were examined. Lycopene pretreatment of cardiomyocytes suppressed the activation of calpain(C:272.33 ± 300.46, H/R: 1156.00 ± 212.02, L+H/R: 607.33 ± 166.23, P < 0.05) by reducing the H/R induced increased intracellular ROS levels [C: 100%, H/R: (239.79 ± 27.27)%, L+H/R: (188.19 ± 17.63)%, P < 0.05]. CONCLUSION: Lycopene may protect against hypoxia/reoxygenation-induced injury by preventing calpain activation.
