Stephanie M Cossette1, Vijesh J Bhute1, Xiaoping Bao1, Leanne M Harmann1, Mark A Horswill1, Indranil Sinha1, Adam Gastonguay1, Shabnam Pooya1, Michelle Bordas1, Suresh N Kumar1, Shama P Mirza1, Sean P Palecek1, Jennifer L Strande1, Ramani Ramchandran2. 1. From the Department of Pediatrics (S.M.C., A.G., S. Pooya, M.B., R.R.), OBGYN, Developmental Vascular Biology Program, Children's Research Institute (R.R.), Division of Cardiovascular Medicine, Cardiovascular Center, Clinical and Translational Science Institute (L.M.H.), Division of Cardiovascular Medicine, Department of Cell Biology, Neurobiology and Anatomy, Cardiovascular Center, Clinical and Translational Science Institute (J.L.S.), and Division of Pediatric Pathology, Department of Pathology (S.N.K.), Medical College of Wisconsin, Milwaukee; Department of Chemical and Biological Engineering (V.J.B., X.B., S. Palecek), Morgridge Institute for Research (M.A.H.), University of Wisconsin-Madison; Marginalen Bank, Stockholm, Sweden (I.S.); Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee (S.P.M.). 2. From the Department of Pediatrics (S.M.C., A.G., S. Pooya, M.B., R.R.), OBGYN, Developmental Vascular Biology Program, Children's Research Institute (R.R.), Division of Cardiovascular Medicine, Cardiovascular Center, Clinical and Translational Science Institute (L.M.H.), Division of Cardiovascular Medicine, Department of Cell Biology, Neurobiology and Anatomy, Cardiovascular Center, Clinical and Translational Science Institute (J.L.S.), and Division of Pediatric Pathology, Department of Pathology (S.N.K.), Medical College of Wisconsin, Milwaukee; Department of Chemical and Biological Engineering (V.J.B., X.B., S. Palecek), Morgridge Institute for Research (M.A.H.), University of Wisconsin-Madison; Marginalen Bank, Stockholm, Sweden (I.S.); Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee (S.P.M.). rramchan@mcw.edu.
Abstract
BACKGROUND: Cardiac metabolism is critical for the functioning of the heart, and disturbance in this homeostasis is likely to influence cardiac disorders or cardiomyopathy. Our laboratory has previously shown that SNRK (sucrose nonfermenting related kinase) enzyme, which belongs to the AMPK (adenosine monophosphate-activated kinase) family, was essential for cardiac metabolism in mammals. Snrk global homozygous knockout (KO) mice die at postnatal day 0, and conditional deletion of Snrk in cardiomyocytes (Snrk cmcKO) leads to cardiac failure and death by 8 to 10 months. METHODS AND RESULTS: We performed additional cardiac functional studies using echocardiography and identified further cardiac functional deficits in Snrk cmcKO mice. Nuclear magnetic resonance-based metabolomics analysis identified key metabolic pathway deficits in SNRK knockdown cardiomyocytes in vitro. Specifically, metabolites involved in lipid metabolism and oxidative phosphorylation are altered, and perturbations in these pathways can result in cardiac function deficits and heart failure. A phosphopeptide-based proteomic screen identified ROCK (Rho-associated kinase) as a putative substrate for SNRK, and mass spec-based fragment analysis confirmed key amino acid residues on ROCK that are phosphorylated by SNRK. Western blot analysis on heart lysates from Snrk cmcKO adult mice and SNRK knockdown cardiomyocytes showed increased ROCK activity. In addition, in vivo inhibition of ROCK partially rescued the in vivo Snrk cmcKO cardiac function deficits. CONCLUSIONS: Collectively, our data suggest that SNRK in cardiomyocytes is responsible for maintaining cardiac metabolic homeostasis, which is mediated in part by ROCK, and alteration of this homeostasis influences cardiac function in the adult heart.
BACKGROUND: Cardiac metabolism is critical for the functioning of the heart, and disturbance in this homeostasis is likely to influence cardiac disorders or cardiomyopathy. Our laboratory has previously shown that SNRK (sucrose nonfermenting related kinase) enzyme, which belongs to the AMPK (adenosine monophosphate-activated kinase) family, was essential for cardiac metabolism in mammals. Snrk global homozygous knockout (KO) mice die at postnatal day 0, and conditional deletion of Snrk in cardiomyocytes (Snrk cmcKO) leads to cardiac failure and death by 8 to 10 months. METHODS AND RESULTS: We performed additional cardiac functional studies using echocardiography and identified further cardiac functional deficits in Snrk cmcKO mice. Nuclear magnetic resonance-based metabolomics analysis identified key metabolic pathway deficits in SNRK knockdown cardiomyocytes in vitro. Specifically, metabolites involved in lipid metabolism and oxidative phosphorylation are altered, and perturbations in these pathways can result in cardiac function deficits and heart failure. A phosphopeptide-based proteomic screen identified ROCK (Rho-associated kinase) as a putative substrate for SNRK, and mass spec-based fragment analysis confirmed key amino acid residues on ROCK that are phosphorylated by SNRK. Western blot analysis on heart lysates from Snrk cmcKO adult mice and SNRK knockdown cardiomyocytes showed increased ROCK activity. In addition, in vivo inhibition of ROCK partially rescued the in vivo Snrk cmcKO cardiac function deficits. CONCLUSIONS: Collectively, our data suggest that SNRK in cardiomyocytes is responsible for maintaining cardiac metabolic homeostasis, which is mediated in part by ROCK, and alteration of this homeostasis influences cardiac function in the adult heart.
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