Recent studies indicate that critically ill patients have profound respiratory muscle weakness. Infection (e.g. pneumonia) is common in these patients and likely an important contributor to the genesis of diaphragmatic weakness. The precise mechanisms by which infection induces respiratory muscle weakness remain, however, unclear. The purpose of the present proposal is to examine these issues in detail, testing our central hypothesis that caspase activation plays a critical role in initiating diaphragm dysfunction in infection. This theory will be evaluated in three groups of studies carried out in mice and C2C12 cells: Objective I studies will test the hypothesis that caspase activation mediates reductions in diaphragm and limb muscle force generation in acute infection/inflammation. Two models of infection/inflammation will be studied (endotoxin injection and pseudomonas pneumonia) in both wild type and transgenic animals with altered caspase pathway genes. Fluorogenic and Western techniques will be used to assess caspase activation indices. Both the effect of specific caspase inhibitors and the responses of caspase pathway gene knockout transgenics will be used to determine which caspase pathways mediate force reductions. Objective II will test the hypothesis that activated caspase alters force via effects on three cellular targets, i.e. specific contractile proteins, sarcolemmal membrane support proteins, and the calpain proteolytic system. These studies will use proteomic and single fiber techniques to identify specific contractile protein cleavage patterns, assays to assess cell integrity, and a variety of calpain activity assays to assess the downstream effects of caspase activation. Objective III will examine the upstream pathways of diaphragmatic caspase activation and test the specific hypotheses that both superoxide and nitric oxide radicals regulate inflammatory mediator induced caspase activation in muscle cells. These experiments will use both transgenic animal models and C2C12 cells to assess the effect of genetic and chemical inhibition of superoxide/nitric oxide on caspase responses. ? ?

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MOSS-D (14))
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Harabin, Andrea L
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University of Kentucky
Internal Medicine/Medicine
Schools of Medicine
United States
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Supinski, Gerald S; Alimov, Alexander P; Wang, Lin et al. (2016) Calcium-dependent phospholipase A2 modulates infection-induced diaphragm dysfunction. Am J Physiol Lung Cell Mol Physiol 310:L975-84
Supinski, Gerald S; Alimov, Alexander P; Wang, Lin et al. (2015) Neutral sphingomyelinase 2 is required for cytokine-induced skeletal muscle calpain activation. Am J Physiol Lung Cell Mol Physiol 309:L614-24
Supinski, Gerald S; Callahan, Leigh A (2015) How Important is Diaphragm Function as a Determinant of Outcomes for MICU Patients in Respiratory Failure? Physiology (Bethesda) 30:336-7
Supinski, Gerald S; Wang, Lin; Song, Xiao-Hong et al. (2014) Muscle-specific calpastatin overexpression prevents diaphragm weakness in cecal ligation puncture-induced sepsis. J Appl Physiol (1985) 117:921-9
Callahan, Leigh A; Supinski, Gerald S (2014) Hyperglycemia-induced diaphragm weakness is mediated by oxidative stress. Crit Care 18:R88
Supinski, Gerald S; Callahan, Leigh A (2014) ?-hydroxy-?-methylbutyrate (HMB) prevents sepsis-induced diaphragm dysfunction in mice. Respir Physiol Neurobiol 196:63-8
Supinski, Gerald S; Callahan, Leigh Ann (2010) Calpain activation contributes to endotoxin-induced diaphragmatic dysfunction. Am J Respir Cell Mol Biol 42:80-7
Callahan, Leigh Ann; Supinski, Gerald S (2010) Diaphragm weakness and mechanical ventilation--what's the critical issue? Crit Care 14:187
Supinski, G S; Vanags, J; Callahan, L A (2009) Effect of proteasome inhibitors on endotoxin-induced diaphragm dysfunction. Am J Physiol Lung Cell Mol Physiol 296:L994-L1001
Supinski, G S; Murphy, M P; Callahan, L A (2009) MitoQ administration prevents endotoxin-induced cardiac dysfunction. Am J Physiol Regul Integr Comp Physiol 297:R1095-102

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