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.
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.
| Basheer, Wassim A; Fu, Ying; Shimura, Daisuke et al. (2018) Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia/reperfusion injury. JCI Insight 3: |
| Epifantseva, Irina; Shaw, Robin M (2018) Intracellular trafficking pathways of Cx43 gap junction channels. Biochim Biophys Acta Biomembr 1860:40-47 |
| Fu, Ying; Shaw, Robin M (2017) CASAAV Technology to Examine Regulators of Heart Failure: Cause or Effect. Circ Res 120:1846-1848 |
| Hong, TingTing; Shaw, Robin M (2017) Cardiac T-Tubule Microanatomy and Function. Physiol Rev 97:227-252 |
| Fu, Ying; Zhang, Shan-Shan; Xiao, Shaohua et al. (2017) Cx43 Isoform GJA1-20k Promotes Microtubule Dependent Mitochondrial Transport. Front Physiol 8:905 |
| Basheer, Wassim A; Xiao, Shaohua; Epifantseva, Irina et al. (2017) GJA1-20k Arranges Actin to Guide Cx43 Delivery to Cardiac Intercalated Discs. Circ Res 121:1069-1080 |
| 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 |
| Basheer, Wassim; Shaw, Robin (2016) The ""tail"" of Connexin43: An unexpected journey from alternative translation to trafficking. Biochim Biophys Acta 1863:1848-56 |
| 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 |
| Fu, Ying; Xiao, Shaohua; Hong, TingTing et al. (2015) Cytoskeleton regulation of ion channels. Circulation 131:689-91 |
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