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Persistent increases in Ca2+ influx through Cav1.2 shortens action potential and causes Ca2+ overload-induced afterdepolarizations and arrhythmias

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Abstract

Persistent elevation of Ca2+ influx due to prolongation of the action potential (AP), chronic activation of the β-adrenergic system and molecular remodeling occurs in stressed and diseased hearts. Increases in Ca2+ influx are usually linked to prolonged myocyte action potentials and arrhythmias. However, the contribution of chronic enhancement of Cav1.2 activity on cardiac electrical remodeling and arrhythmogenicity has not been completely defined and is the subject of this study. Chronically increased Cav1.2 activity was produced with a cardiac specific, inducible double transgenic (DTG) mouse system overexpressing the β2a subunit of Cav (Cavβ2a). DTG myocytes had increased L-type Ca2+ current (ICa-L), myocyte shortening, and Ca2+ transients. DTG mice had enhanced cardiac performance, but died suddenly and prematurely. Telemetric electrocardiograms revealed shortened QT intervals in DTG mice. The action potential duration (APD) was shortened in DTG myocytes due to significant increases of potassium currents and channel abundance. However, shortened AP in DTG myocytes did not fully limit excess Ca2+ influx and increased the peak and tail ICa-L. Enhanced ICa promoted sarcoplasmic reticulum (SR) Ca2+ overload, diastolic Ca2+ sparks and waves, and increased NCX activity, causing increased occurrence of early and delayed afterdepolarizations (EADs and DADs) that may contribute to premature ventricular beats and ventricular tachycardia. AV blocks that could be related to fibrosis of the AV node were also observed. Our study suggests that increasing ICa-L does not necessarily result in AP prolongation but causes SR Ca2+ overload and fibrosis of AV node and myocardium to induce cellular arrhythmogenicity, arrhythmias, and conduction abnormalities.

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Abbreviations

4-AP:

4-Aminopyridine

AP:

Action potential

APD:

Action potential duration

CaMK II:

Ca2+/Calmodulin-dependent kinase II

Cavβ2a:

The β2a splicing variant of the β2 subunit of the L-type Ca2+ channel

DAD:

Delayed afterdepolarization

DTG:

Double transgenic

EAD:

Early afterdepolarization

ECG:

Electrocardiography

ECHO:

Echocardiography

FDHM:

Full duration at half maximum

FWHM:

Full width at half maximum

ICa-L :

L-type Ca2+ current

LTCC or Cav1.2:

The L-type Ca2+ channel

NCX:

Na+/Ca2+ Exchange

NFAT:

Nuclear factor of activated T-cells

PKA:

Protein kinase A

QTc:

Corrected QT interval

SQTS:

Short QT syndrome

RyR2:

Ryanodine receptor type 2

SR:

Sarcoplasmic reticulum

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Author notes

    Authors and Affiliations

    1. Daping Hospital, The Third Military Medical University, Chongqing, China

      Xiaoying Zhang & Xiongwen Chen

    2. Cardiovascular Research Center and Department of Physiology, Temple University School of Medicine, Philadelphia, PA, 19140, USA

      Xiaoying Zhang, Xiaojie Ai, Mingxin Tang, Christopher Szeto, Yingxin Li, Ying Li, Hongyu Zhang & Xiongwen Chen

    3. Cincinnati Children’s Hospital Medical Center, Department of Pediatrics, University of Cincinnati, Cincinnati, OH, 45229, USA

      Hiroyuki Nakayama & Jeffery D. Molkentin

    4. Department of Internal Medicine, University of Iowa, Iowa City, IA, 52242, USA

      Biyi Chen & Yuping Xie

    5. College of Medicine, University of Central Florida, Orlando, FL, 32827, USA

      David M. Harris

    6. School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China

      Xiaojie Ai

    7. The Second Artillery General Hospital, Beijing, 100088, China

      Ying Li

    8. MedThink SciCom, Raleigh, NC, 27609, USA

      Andrea D. Eckhart

    9. Center for Translational Medicine and Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA, USA

      Walter J. Koch

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    1. Xiaoying Zhang
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    Correspondence to Xiongwen Chen.

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    Funding sources

    This study was supported by NIH and HMMI grants to JDM, NIH HL088243 and AHA 0730347N to XC.

    Additional information

    X. Zhang and X. Ai contributed equally to this study.

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    Supplementary material 1 (DOCX 33 kb)

    395_2015_523_MOESM2_ESM.pdf

    Supplemental Figure 1: Cavβ2a DTG mouse model. Cavβ2a single transgenic (STG) mice interbreed with ttA single transgenic mice to produce double transgenic mice. Offspring were screened for genotypes with the primers as shown. Four genotypes were produced in the offspring and only double transgenic (DTG) mice off doxycycline (DOX) were used as the experimental group. Mice with other genotypes were used as control mice (PDF 52 kb)

    395_2015_523_MOESM3_ESM.pdf

    Supplemental Figure 2: Similar properties of Ca2+ sparks were observed in control and DTG VMs. No differences in Amplitudes (A), full width at half maximum (FWHM) (B) and full duration at half maximum (FDHM) (C) of Ca2+ sparks were detected in control and DTG VMs (PDF 47 kb)

    395_2015_523_MOESM4_ESM.pdf

    Supplemental Figure 3: Increased amplitudes but similar time to peak of Ca2+ transients in DTG VMs at pacing frequencies of 1Hz (A and B) and 3Hz (C and D). *: p<0.05, **: p<0.01 control vs. DTG, student t-test (PDF 50 kb)

    395_2015_523_MOESM5_ESM.pdf

    Supplemental Figure 4: Bradycardia in a DTG mouse before its death. A mouse died during telemetric recording of ECG with bradycardia before death (PDF 111 kb)

    395_2015_523_MOESM6_ESM.pdf

    Supplemental Figure 5: APDs and K+ currents in Cav1.2α1c heterozygous knockout mice. (A) Action potentials recorded from one c57/bl6 control VM and one α1c +/- (α1c heterozygous knockout) VM. (B) & (C) Action potential durations (B) and maximal phase 1 decay rates of action potentials (C) in control and α1c +/- myocytes. (D)-(E) amplitudes of total K+ currents, sustained 4-AP-insensitive K+ currents and 4-AP-sensitive K+ currents at different test potentials in control and α1c +/- VMs. *: p<0.05, &: p<0.01, #: p<0.001, αlc +/- vs. control. “n” cells from “N” hearts were studied (PDF 99 kb)

    395_2015_523_MOESM7_ESM.pdf

    Supplemental Figure 6: K+ currents in myocytes from Cavβ2a high expression mice with heart failure. Amplitudes of total K+ currents (A), sustained 4-AP insensitive K+ currents (B) and 4-AP sensitive K+ currents (C) at different test potentials in control, LE DTG and HE DTG (with HF) myocytes. *: p<0.05, &: p<0.01, #: p<0.001, Cavβ2a HE vs. control; %: p<0.01; $: p<0.0001, Cavβ2a HE vs. Cavβ2a LE. “n” cells from “N” hearts were studied. (PDF 67 kb)

    395_2015_523_MOESM8_ESM.pdf

    Supplemental Figure 7: Whole-blot images of KChIP2 (A) and Kv4.3 (B). (A). The KChIP2 antibody from NeuroMab detected multiple strong bands in the high molecular weight region (>50 kDa region). However, in the region for molecular weights lower than 50 kDa, there is only one strong band corresponding to 32-35kD of KChIP2. (B). The Kv4.3 antibody from NeuroMab detected only one major band between 75–100 kDa, corresponding to Kv4.3. (PDF 410 kb)

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    Zhang, X., Ai, X., Nakayama, H. et al. Persistent increases in Ca2+ influx through Cav1.2 shortens action potential and causes Ca2+ overload-induced afterdepolarizations and arrhythmias. Basic Res Cardiol 111, 4 (2016). https://doi.org/10.1007/s00395-015-0523-4

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