EHD3-dependent endosome pathway regulates cardiac membrane excitability and physiology.
BACKGROUND
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.


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.
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.
Jerry
J
Curran
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.).
Michael A
MA
Makara
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.).
Sean C
SC
Little
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.).
Hassan
H
Musa
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.).
Bin
B
Liu
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.).
Xiangqiong
X
Wu
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.).
Iuliia
I
Polina
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.).
Joseph S
JS
Alecusan
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.).
Patrick
P
Wright
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.).
Jingdong
J
Li
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.).
George E
GE
Billman
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.).
Penelope A
PA
Boyden
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.).
Sandor
S
Gyorke
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.).
Hamid
H
Band
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.).
Thomas J
TJ
Hund
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 J
PJ
Mohler
D000818:Animals; D017487:Ankyrins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000502:physiology; D002352:Carrier Proteins / Q000502:physiology*; D011992:Endosomes / Q000502:physiology*; D006321:Heart / Q000502:physiology*; D006339:Heart Rate; D051379:Mice; D032383:Myocytes, Cardiac / Q000502:physiology; D013318:Stroke Volume
C094681:Ank3 protein, mouse; D017487:Ankyrins; C514350:CACNA1C protein, mouse; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; C470993:Ehd3 protein, mouse; D002118:Calcium
Ehd3 protein; ankyrins; cell biology; electrophysiology; ion channels; mice; protein transport