Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis.
Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcA/cytochrome-c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome-c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification.
Malhotra
Ashwani
A
Division of Nephrology and Hypertension, Department of Medicine, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA. malhotas@umdnj.edu
Vashistha
Himanshu
H
Yadav
Virendra S
VS
Dube
Michael G
MG
Kalra
Satya P
SP
Abdellatif
Maha
M
Meggs
Leonard G
LG
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D009202:Cardiomyopathies / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D053148:Caspase 3 / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D004195:Disease Models, Animal; D015316:Genetic Therapy / Q000379:methods*; D006943:Hyperglycemia / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D020738:Leptin / Q000235:genetics / Q000378:metabolism; D008297:Male; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008810:Mice, Inbred C57BL; D016688:Mice, Inbred NOD; D008929:Mitochondria, Heart / Q000201:enzymology; D009154:Mutation; D032383:Myocytes, Cardiac / Q000201:enzymology* / Q000473:pathology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013482:Superoxide Dismutase / Q000378:metabolism; D014161:Transduction, Genetic; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C494828:Bax protein, rat; D020738:Leptin; D017382:Reactive Oxygen Species; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D002374:Catalase; D013482:Superoxide Dismutase; C505410:Casp3 protein, rat; D053148:Caspase 3