Jerry Curran1, Michael A Makara2, Sean C Little2, Hassan Musa2, Bin Liu2, Xiangqiong Wu2, Iuliia Polina2, Joseph S Alecusan2, Patrick Wright2, Jingdong Li2, George E Billman2, Penelope A Boyden2, Sandor Gyorke2, Hamid Band2, Thomas J Hund2, Peter J Mohler2. 1. From The Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus (J.C., M.A.M., S.C.L., H.M., B.L., X.W., I.P., J.S.A., P.W., J.L., G.E.B., S.G., T.J.H., P.J.M.); Departments of Internal Medicine (P.J.M.) and Physiology and Cell Biology (J.C., M.A.M., S.C.L., H.M., B.L., P.W., G.E.B., S.G., P.J.M.), The Ohio State University, Columbus; Department of Biomedical Engineering, The Ohio State University College of Engineering, Columbus (T.J.H.); Department of Pharmacology and Center for Molecular Therapeutics, Columbia University Medical Center, New York, NY (P.A.B.); and The Eppley Institute and UNMC-Eppley Cancer Center, University of Nebraska Medical Center, Omaha (H.B.). peter.mohler@osumc.edu jerry.curran@osumc.edu. 2. From The Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus (J.C., M.A.M., S.C.L., H.M., B.L., X.W., I.P., J.S.A., P.W., J.L., G.E.B., S.G., T.J.H., P.J.M.); Departments of Internal Medicine (P.J.M.) and Physiology and Cell Biology (J.C., M.A.M., S.C.L., H.M., B.L., P.W., G.E.B., S.G., P.J.M.), The Ohio State University, Columbus; Department of Biomedical Engineering, The Ohio State University College of Engineering, Columbus (T.J.H.); Department of Pharmacology and Center for Molecular Therapeutics, Columbia University Medical Center, New York, NY (P.A.B.); and The Eppley Institute and UNMC-Eppley Cancer Center, University of Nebraska Medical Center, Omaha (H.B.).
Abstract
RATIONALE: Cardiac function is dependent on the coordinate activities of membrane ion channels, transporters, pumps, and hormone receptors to tune the membrane electrochemical gradient dynamically in response to acute and chronic stress. Although our knowledge of membrane proteins has rapidly advanced during the past decade, our understanding of the subcellular pathways governing the trafficking and localization of integral membrane proteins is limited and essentially unstudied in vivo. In the heart, to our knowledge, there are no in vivo mechanistic studies that directly link endosome-based machinery with cardiac physiology. OBJECTIVE: To define the in vivo roles of endosome-based cellular machinery for cardiac membrane protein trafficking, myocyte excitability, and cardiac physiology. METHODS AND RESULTS: We identify the endosome-based Eps15 homology domain 3 (EHD3) pathway as essential for cardiac physiology. EHD3-deficient hearts display structural and functional defects including bradycardia and rate variability, conduction block, and blunted response to adrenergic stimulation. Mechanistically, EHD3 is critical for membrane protein trafficking, because EHD3-deficient myocytes display reduced expression/localization of Na/Ca exchanger and L-type Ca channel type 1.2 with a parallel reduction in Na/Ca exchanger-mediated membrane current and Cav1.2-mediated membrane current. Functionally, EHD3-deficient myocytes show increased sarcoplasmic reticulum [Ca], increased spark frequency, and reduced expression/localization of ankyrin-B, a binding partner for EHD3 and Na/Ca exchanger. Finally, we show that in vivo EHD3-deficient defects are attributable to cardiac-specific roles of EHD3 because mice with cardiac-selective EHD3 deficiency demonstrate both structural and electric phenotypes. CONCLUSIONS: These data provide new insight into the critical role of endosome-based pathways in membrane protein targeting and cardiac physiology. EHD3 is a critical component of protein trafficking in heart and is essential for the proper membrane targeting of select cellular proteins that maintain excitability.
RATIONALE: Cardiac function is dependent on the coordinate activities of membrane ion channels, transporters, pumps, and hormone receptors to tune the membrane electrochemical gradient dynamically in response to acute and chronic stress. Although our knowledge of membrane proteins has rapidly advanced during the past decade, our understanding of the subcellular pathways governing the trafficking and localization of integral membrane proteins is limited and essentially unstudied in vivo. In the heart, to our knowledge, there are no in vivo mechanistic studies that directly link endosome-based machinery with cardiac physiology. OBJECTIVE: To define the in vivo roles of endosome-based cellular machinery for cardiac membrane protein trafficking, myocyte excitability, and cardiac physiology. METHODS AND RESULTS: We identify the endosome-based Eps15 homology domain 3 (EHD3) pathway as essential for cardiac physiology. EHD3-deficient hearts display structural and functional defects including bradycardia and rate variability, conduction block, and blunted response to adrenergic stimulation. Mechanistically, EHD3 is critical for membrane protein trafficking, because EHD3-deficient myocytes display reduced expression/localization of Na/Ca exchanger and L-type Ca channel type 1.2 with a parallel reduction in Na/Ca exchanger-mediated membrane current and Cav1.2-mediated membrane current. Functionally, EHD3-deficient myocytes show increased sarcoplasmic reticulum [Ca], increased spark frequency, and reduced expression/localization of ankyrin-B, a binding partner for EHD3 and Na/Ca exchanger. Finally, we show that in vivo EHD3-deficient defects are attributable to cardiac-specific roles of EHD3 because mice with cardiac-selective EHD3 deficiency demonstrate both structural and electric phenotypes. CONCLUSIONS: These data provide new insight into the critical role of endosome-based pathways in membrane protein targeting and cardiac physiology. EHD3 is a critical component of protein trafficking in heart and is essential for the proper membrane targeting of select cellular proteins that maintain excitability.
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