Previous studies indicated that Ca 2+ /calmodulin-dependent kinase II (CaMKII), a kinase involved in the modulation of ryanodine receptor activity, activates Ca 2+-regulated protease μ-calpain to promote myocardial ischemia/ reperfusion injury. This study was performed to explore the underlying mechanisms in CaMKII-induced calpain activation to better understand heart injury. To examine the Ca 2+ paradox and ischemia/reperfusion injury, isolated rat hearts were subjected to a Ca 2+-free solution for 3 min, or left coronary artery occlusion for 40 min, prior to restoration of normal perfusion. Blockade of trans-sarcoplasmic reticulum Ca 2+ flux using ryanodine and thapsigargin failed to prevent Ca 2+ paradox-induced heart injury. In contrast, the Ca 2+ paradox increased CaMKII auto-phosphorylation at Thr 287 , while the CaMKII inhibitor KN-62 and the Na + /Ca 2+ exchanger inhibitor KB-R7943 alleviated heart injury and calpain activity. Intriguingly, the binding of μ-calpain large subunit calpain-1 (CAPN1) to phospho-CaMKII was blunted by both inhibitors. Thus, a Ca 2+ leak via the ryanodine receptor is not an essential element in CaMKII-elicited calpain activation. In hearts receiving vector injection, ischemia/reperfusion caused elevated calpain activity and α-fodrin degradation, along with membrane integrity damage, similar to the effects noted in control hearts. Importantly, all these alterations were diminished with delivery of adeno-associated virus expressing mutant CaMKIIδC T287A. Ischemia/reperfusion increased CaMKII auto-phosphorylation and binding of CAPN1 to phospho-CaMKII, and facilitated the translocation of phospho-CaMKII and CAPN1 to the plasma membrane, all of which were reversed by injecting CaMKII mutant. Furthermore, the relocation capacity and the interaction of CaMKII with CAPN1 appeared to be dependent upon CaMKII autophosphorylation, as its mutant delivery increased the level of CaMKII, but did not increase membrane content of CaMKII and CAPN1, or their interactions. Together, CaMKII/calpain interaction represents a new avenue for mediating myocardial ischemia/ reperfusion injury, and CaMKII likely serves as both a kinase and a carrier, thereby promoting calpain membrane translocation and activation.

BackgroundThe molecular pattern of severe burn-induced acute lung injury, characterized by cell structure damage and leukocyte infiltration, remains unknown. This study aimed to determine whether calpain, a protease involved in both processes, mediates severe burn-induced acute lung injury.MethodsRats received full-thickness scald burns covering 30% of the total body surface area, followed by instant fluid resuscitation. MDL28170 (Tocris Bioscience), an inhibitor of calpain, was given intravenously 1 h before or after the scald burn. The histological score, wet/dry weight ratio, and caspase-3 activity were examined to evaluate the degree of lung damage. Calpain activity and its source were detected by an assay kit and immunofluorescence staining. The proteolysis of membrane skeleton proteins α-fodrin and ankyrin-B, which are substrates of calpain, was measured by Western blot.ResultsTime-course studies showed that tissue damage reached a peak between 1 and 6 h post-scald burn and gradually diminished at 24 h. More importantly, calpain activity reached peak levels at 1 h and was maintained until 24 h, paralleled by lung damage to some extent. Western blot showed that the levels of the proteolyzed forms of α-fodrin and ankyrin-B correlated well with the degree of damage. MDL28170 at a dose of 3 mg/kg b. w. given 1 h before burn injury not only antagonized the increase in calpain activity but also ameliorated scald burn-induced lung injury, including the degradation of α-fodrin and ankyrin-B. Immunofluorescence images revealed calpain 1 and CD45 double-positive cells in the lung tissue of rats exposed to scald burn injury, suggesting that leukocytes were a dominant source of calpain. Furthermore, this change was blocked by MDL28170. Finally, MDL28170 given at 1 h post-scald burn injury significantly ameliorated the wet/dry weight ratio compared with burn injury alone.ConclusionsCalpain, a product of infiltrating leukocytes, is a mediator of scald burn-induced acute lung injury that involves enhancement of inflammation and proteolysis of membrane skeleton proteins. Its late effects warrant further study.

Stress cardiomyopathy is a major clinical complication after severe burn. Multiple upstream initiators have been identified; however, the downstream targets are not fully understood. This study assessed the role of the plasma membrane in this process and its relationship with the protease μ-calpain and tumor necrosis factor-alpha (TNF-α). Here, third-degree burn injury of approximately 40% of the total body surface area was established in rats. Plasma levels of LDH and cTnI and cardiac cell apoptosis increased at 0.5 h post burn, reached a peak at 6 h, and gradually declined at 24 h. This effect correlated well with not only the disruption of cytoskeletal proteins, including dystrophin and ankyrin-B, but also with the activation of μ-calpain, as indicated by the cleaved fragments of α-spectrin and membrane recruitment of the catalytic subunit CAPN1. More importantly, these alterations were diminished by blocking calpain activity with MDL28170. Burn injury markedly increased the cellular uptake of Evans blue, indicating membrane integrity disruption, and this effect was also reversed by MDL28170. Compared with those in the control group, cardiac cells in the burn plasma-treated group were more prone to damage, as indicated by a marked decrease in cell viability and increases in LDH release and apoptosis. Of note, these alterations were mitigated by CAPN1 siRNA. Moreover, after neutralizing TNF-α with rhTNFR:Fc, calpain activity was blocked, and heart function was improved. In conclusion, we identified μ-calpain as a trigger for severe burn-induced membrane disruption in the heart and provided evidence for the application of rhTNFR:Fc to inhibit calpain for cardioprotection.

No abstract