Cardiovascular disease remains the primary cause of death and disability in the United States. Death is most commonly a direct result of ventricular arrhythmia which, in turn, is the consequence of alternations in ion channel function, affecting action potentials and cell-cell communication. Altered ion channel function in failing hearts also limits the heart's contractility, further worsening heart failure and the vicious cycle towards arrhythmia and death. Individual ion channels have half-lives that are on the order of hours, and a major cause of altered ion channel function in failing hearts is altered trafficking. Altered trafficking implies that the channels are not delivered to the correct subdomain on the cardiomyocyte plasma membrane (forward trafficking), or are removed incorrectly (reverse trafficking or internalization). Altered forward and reverse trafficking are components of most cardiac disorders. However the mechanisms of forward and reverse trafficking of membrane proteins, especially of cardiac ion channels, remain poorly understood. My laboratory explores the basic mechanisms of cardiac ion channel trafficking. In this competitive renewal, we will identify three novel pathways with strong preliminary data for each. The channels of interest are the Connexin43 gap junction channel responsible for cell-cell communication, and the L-type Calcium Channel (alpha subunit Cav1.2), which is responsible for triggering the beat-to- beat intracellular calcium transient which initiates and helps determine the strength of each cardiac contraction. Each of the three pathways explored is a separate specific aim of this proposal.
Aim #1 is to determine if alternative translation is a mechanism of Cx43 auto regulation of forward traffic in health and disease. We have evidence in human heart that as many as six in frame AUG sequences initiate translation within the Cx43 (GJA1) exon. Our data indicate the truncated Cx43 isoforms are necessary at least for Cx43 exit from the endoplasmic reticulum and Golgi apparatus.
Aim #2 is to determine how the actin cytoskeleton contributes to directed channel delivery. Our studies indicate that the majority of intracellular Cx43 is moving slowly or not moving at all, due to association with actin. We have developed a novel microfluidics based cell patterning system to quantify actin dynamics and its contribution to ion channel trafficking and are finding that actin is important to working with microtubules in providing specificity of Cx43 and Cav1.2 delivery.
Aim #3 is to elucidate the post-translation cascade by which Cx43 is internalized from the plasma membrane. We have evidence that serine 373 phosphorylation on the Cx43 C-terminus needs to precede serine 368 phopsphorylation. Our new data will explore mechanism of phosphorylation dependent internalization and whether the products of alternative translation are involved.

Public Health Relevance

The proposed research is relevant to NIH because it will introduce new targets for therapeutic interventions capable of limiting malignant arrhythmias and progression of heart failure in millions of Americans. Sudden cardiac death and heart failure are major syndromes in the United States. A better molecular understanding of how cardiomyocyte are regulated and how, in failing hearts, they progressively lose their electrical and mechanical function is a critical step to developing new medical solutions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL094414-09
Application #
9172659
Study Section
Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
Program Officer
Lathrop, David A
Project Start
2008-12-01
Project End
2018-10-31
Budget Start
2016-11-01
Budget End
2017-10-31
Support Year
9
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Cedars-Sinai Medical Center
Department
Type
DUNS #
075307785
City
Los Angeles
State
CA
Country
United States
Zip Code
90048
Fu, Ying; Zhang, Shan-Shan; Xiao, Shaohua et al. (2017) Cx43 Isoform GJA1-20k Promotes Microtubule Dependent Mitochondrial Transport. Front Physiol 8:905
Levitan, Bryana M; Manning, Janet R; Withers, Catherine N et al. (2016) Rad-deletion Phenocopies Tonic Sympathetic Stimulation of the Heart. J Cardiovasc Transl Res 9:432-444
Basheer, Wassim; Shaw, Robin (2016) The ""tail"" of Connexin43: An unexpected journey from alternative translation to trafficking. Biochim Biophys Acta 1863:1848-56
Basheer, Wassim A; Shaw, Robin M (2016) Connexin 43 and CaV1.2 Ion Channel Trafficking in Healthy and Diseased Myocardium. Circ Arrhythm Electrophysiol 9:e001357
Fu, Ying; Xiao, Shaohua; Hong, TingTing et al. (2015) Cytoskeleton regulation of ion channels. Circulation 131:689-91
Xiao, Shaohua; Shaw, Robin M (2015) Cardiomyocyte protein trafficking: Relevance to heart disease and opportunities for therapeutic intervention. Trends Cardiovasc Med 25:379-89
Smyth, James W; Zhang, Shan-Shan; Sanchez, Jose M et al. (2014) A 14-3-3 mode-1 binding motif initiates gap junction internalization during acute cardiac ischemia. Traffic 15:684-99
Schumacher-Bass, Sarah M; Vesely, Eileen D; Zhang, Lian et al. (2014) Role for myosin-V motor proteins in the selective delivery of Kv channel isoforms to the membrane surface of cardiac myocytes. Circ Res 114:982-92
Hong, TingTing; Yang, Huanghe; Zhang, Shan-Shan et al. (2014) Cardiac BIN1 folds T-tubule membrane, controlling ion flux and limiting arrhythmia. Nat Med 20:624-32
Zhang, Shan-Shan; Hong, SoonGweon; Kléber, André G et al. (2014) A micropatterning approach for imaging dynamic Cx43 trafficking to cell-cell borders. FEBS Lett 588:1439-45

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