Calpain And Caspase-3 Participate In Regulatory Crosstalk During Disuse Muscle Atrophy

Abstract

Diaphragmatic weakness is a well-documented response to prolonged mechanical ventilation (MV). The activation of major proteolytic systems (e.g. calpains, caspase-3, and ubiquitin-proteasome) occurs in the diaphragm during prolonged MV and these proteases contribute to MV-induced diaphragmatic atrophy and contractile dysfunction. Specifically, calpain and caspase-3 are proteases capable of degrading both cytoskeletal proteins and actomyosin complexes. Furthermore, independent inhibition of either calpain or caspase-3 activation prevents diaphragmatic atrophy and weakness. These findings suggest that a regulatory crosstalk exists between calpain and caspase-3, whereby calpain can activate caspase-3 and vice versa, caspase-3 regulates calpain activity. PURPOSE: We tested the hypothesis that during prolonged MV, selective inhibition of calpain will prevent diaphragmatic calpain-caspase-3 crosstalk, resulting in diminished activation of caspase-3. We also predicted that selective caspase-3 inhibition would likewise prevent MV-induced calpain activation in the diaphragm. METHODS: Sprague-Dawley rats were randomly divided into 4 groups: 1) control, 2) 12 hr MV, 3) 12 hr MV with a selective caspase-3 inhibitor and 4) 12 hr MV with a selective calpain inhibitor. RESULTS: Compared to control, MV resulted in calpain and caspase-3 activation in the diaphragm accompanied by diaphragmatic myofiber atrophy in type I, type IIA, and type IIX/IIB fibers. Independent inhibition of either calpain or caspase-3 prevented this MV-induced decrease in cross-sectional area. Pharmacological inhibition of calpain prevented MV-induced activation of diaphragmatic caspase-3 and inhibition of caspase-3 prevented activation of diaphragmatic calpain. Further, calpain inhibition also prevented cleavage of Bid to tBid, an upstream signal for caspase-3 activation. Also, caspase-3 inhibition prevented the MV-induced degradation of the endogenous calpain inhibitor, calpastatin. CONCLUSION: Collectively, these results indicate that MV-induced diaphragmatic atrophy is dependent upon the activation of calpain and caspase-3. Importantly, these findings indicate calpain inhibition prevents the activation of caspase-3 and vice versa, caspase-3 inhibition prevents the activation of calpain. These findings support the concept that calpain and caspase-3 participate in regulatory crosstalk in skeletal muscle during disuse muscle atrophy. Supported by the NIH RO1 HL087839 awarded to SKP