Intercalated Disc-Associated Protein, mXin-alpha, influences surface expression of ITO currents in ventricular myocytes
Fu-Chi Chan1, Chiao-Pei Cheng1,2, Kuo-Ho Wu2, Yao-Chang Chen3, Chih-Hsiung Hsu4, Elisabeth A. Gustafson-Wagner5, Jenny Li-Chun Lin5, Qinchuan Wang5, Jim Jung-Ching Lin5, Cheng-I Lin1
1
Institute of Physiology, National Defense Medical Center, Taipei, Taiwan, ROC, 2Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, ROC, 3Department of Biomedical Engineering, National Defense Medical Center, Taipei, Taiwan, ROC, 4Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC, 5Department of Biology, University of Iowa, Iowa City, IA, U.S.A.
TABLE OF CONTENTS
- 1. Abstract
- 2. Introduction
- 3. Materials and methods
- 3.1. Animals
- 3.2. Isolation of cardiomyocytes
- 3.3. Cellular electrophysiological study
- 3.4. Measurement of intracellular calcium concentration
- 3.5. Northern blot analysis and quantitative RT-PCR (qRT-PCR)
- 3.6. Membrane preparation and Western blot analysis
- 3.7. Yeast two hybrid interaction assay
- 3.8. Data analysis
- 4. Results
- 4.1. Prolonged action potential duration (APD) and higher incidence of early after-depolarization (EAD) were associated with both juvenile and adult mXina-null ventricular myocytes
- 4.2. Depressed transient outward currents (ITO) were detected in both juvenile and adult mXina-null ventricular myocytes
- 4.3. A small but significant reduction in the delayed rectifier outward K+ currents (IK) was observed in juvenile but not adult mXina-null ventricular myocytes
- 4.4. No changes in the inward rectifier K+ currents (IK1) and the L-type Ca2+ currents (ICa,L) were detected in juvenile mXina-null ventricular myocytes, but both current densities were consistently reduced in adult myocytes
- 4.5. Intracellular Ca2+ concentration was significantly decreased in both juvenile and adult mXina-null cardiomyocytes
- 4.6. The KChIP2 message was significantly decreased in adult mXina-deficient hearts
- 4.7. The membrane-associated KChIP2 and filamin proteins were reduced in juvenile mXina-null hearts
- 4.8. mXina interacted with KChIP2 and filamin
- 5. Discussion
- 5.1. Reductions in ITO and IK of juvenile mXina-null cardiomyocytes were primarily responsible for the prolonged APD and higher incidence of EAD.
- 5.2. Reduced ITO and prolonged APD did not cause cardiac hypertrophy in juvenile mXina-null hearts
- 5.3. Molecular mechanisms underlying the depressed ITO in mXina-null cardiomyocytes
- 6. Acknowledgements
- 7. References
1. ABSTRACT
Mouse Xin-alpha (mXin-alpha) encodes a Xin repeat-containing, actin-binding protein localized to the intercalated disc (ICD). Ablation of mXin-alpha progressively leads to disrupted ICD structure, cardiac hypertrophy and cardiomyopathy with conduction defects during adulthood. Such conduction defects could be due to ICD structural defects and/or cell electrophysiological property changes. Here, we showed that despite the normal ICD structure, juvenile mXina-null cardiomyocytes (from 3~4-week-old mice) exhibited a significant reduction in the transient outward K+ current (ITO), similar to adult mutant cells. Juvenile but not adult mutant cardiomyocytes also had a significant reduction in the delayed rectifier K+ current. In contrast, the mutant adult ventricular myocytes had a significant reduction in the inward rectifier K+ current (IK1) on hyperpolarization. These together could account for the prolongation of action potential duration (APD) and the ease of developing early afterdepolarization observed in juvenile mXin-alpha-null cells. Interestingly, juvenile mXin-alpha-null cardiomyocytes had a notable decrease in the amplitude of intracellular Ca2+ transient and no change in the L-type Ca2+ current, suggesting that the prolonged APD did not promote an increase in intracellular Ca2+ for cardiac hypertrophy. Juvenile mXin-alpha-null ventricles had reduced levels of membrane-associated Kv channel interacting protein 2, an auxiliary subunit of ITO, and filamin, an actin cross-linking protein. We further showed that mXin-alpha interacted with both proteins, providing a novel mechanism for ITO surface expression.
