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Cardiac remodeling and atrial fibrillation in transgenic mice overexpressing junctin¹

CHANG-SOO HONG, MYEONG-CHAN CHO^*, YONG-GEUN KWAK, CHANG-HO SONG, YOUNG-HOON LEE, JUNG SU LIM, YUNHEE KIM KWON, SOO-WAN CHAE and DO HAN KIM²

Department of Life Science, Kwangju Institute of Science and Technology (K-JIST), Kwangju, Korea;
^* Department of Cardiology, College of Medicine, Chungbuk National University, Cheongju, Korea;
Department of Pharmacology, Institute of Cardiovascular Research, and
Department of Anatomy, Chonbuk National University Medical School, Chonju, Korea; and
Department of Biology, Kyunghee University, Seoul, Korea

²Correspondence: Department of Life Science, Kwangju Institute of Science and Technology (K-JIST), 1 Oryong-dong, Puk-gu, Kwangju, 500–712, Korea. E-mail: dhkim{at}kjist.ac.kr

SPECIFIC AIMS

The present study was conducted to investigate the functional role of junctin in heart by generating transgenic (TG) mice with cardiac overexpression of junctin in order to provide insight into the role of junctin in the development and function of heart.

PRINCIPAL FINDINGS

1. Cardiac overexpression of junctin induced heart enlargement, fibrosis, and ultrastructural changes
Hearts from 6- to 8-wk-old TG mice overexpressing dog junctin (24–29 folds) showed dramatic enlargements of ventricles and atria compared to wild-type (WT) litters (Fig. 1 A). Heart-to-body weight ratio increased twofold. Dissection of the TG heart revealed significant remodeling compared with the WT (Fig. 1B ). The right ventricular and atrial cavities were markedly dilated. Intra-atrial thrombus was found in both atria, which might be due to a blood clot formation caused by the development of atrial fibrillation. To examine whether fibrosis exists in the TG hearts, trichrome staining was performed (Fig. 1C-F) . The atrial myocardium of TG mice was replaced substantially with collagen fibers (shown in blue-green) (Fig. 1D ). The ventricular wall of TG mice showed mild collagen deposition (Fig. 1F ).

View larger version (91K): [in this window] [in a new window]   Figure 1. Phenotype changes in the heart of TG mice. A) Gross morphology of WT and TG mouse hearts. B) Longitudinal section of WT and TG mouse hearts. Both bi-atrial and biventricular enlargements were seen in TG mice. C–F) Trichrome staining of the heart in the WT and TG mice. Atria (C, D) and ventricles (E, F) consisted of endocardium (arrow), myocardium (M), and epicardium (arrowhead) in WT (C, E) and TG mice (D, F). Cardiomyocytes and collagen fibers were stained with red and blue-green, respectively. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle. Scale bar: 50 µm. G–J) Sections of WT and TG ventricles were immunostained with anti-junctin (G, H) or anti-triadin (I, J) antibodies. Scale bar: 5 µm.

To compare subcellular localization of junctin in TG and WT cardiomyocytes, immunocytochemistry was performed (Fig. 1G-J ). The localization of junctin was not significantly changed in TG cells (Fig. 2 H) and that of triadin was unaltered in TG cells (Fig. 2J ). Transmission electron microscopy revealed ultrastructural alterations such as fragmented nuclei and indistinct sarcomeres in junctin TG atria. To examine any possible alterations in the T-SR junction area in junctin TG heart, high-resolution transmission electron microscopy was performed. In TG heart, all junctional SR cisternae facing the T-tubule showed narrow and dense lumen, probably due to highly clustered content of calsequestrin. The dense appearance of junctional cisternae of SR in TG heart might be caused by increased expression of junctin in the junctional SR membrane, which may recruit calsequestrin to junctional SR lumen.

View larger version (28K): [in this window] [in a new window]   Figure 2. Electrophysiological changes in TG hearts. A, B) ICa in WT and TG ventricular cardiomyocytes. A) Typical whole cell L-type Ca2+ currents recorded in WT (n=26) and TG (n=20) cells. Shown are traces of currents recorded from a holding potential of -50 mV to the indicated test potentials. B) Peak current-voltage relationships obtained from these cells. The peak currents were normalized to cell capacitance. C–F) Action potentials in ventricular and atrial muscles of WT and junctin TG mice. C) Typical representations of action potentials in WT and junctin TG ventricular muscle. D) Averaged values of action potential durations at 70% repolarization (APD70) of ventricular muscle isolated from WT (n=16) and junctin TG mice (n=7). E) Typical representations of action potentials in WT and junctin TG atrial muscle. F) Averaged values of APD70 of atrial muscle isolated from WT (n=16) and junctin TG mice (n=12). *P < 0.05 vs. WT.

2. Cardiac overexpression of junctin induced bradycardia, atrial fibrillation, and cardiac remodeling
TG mice had a significantly lower heart rate than WT litters and limb-lead electrocardiographic analysis of TG mice revealed signs of atrial fibrillation. According to 2-dimensional echocardiographic analyses, the junctin TG mice showed enlarged left ventricle, markedly dilated right atrium and right ventricle. M-mode tracing demonstrated impaired systolic function and dilated right ventricle with paradoxical interventricular septal motion probably due to increased right ventricular cavity in the TG mice.

3. Cardiac overexpression of junctin induced alteration of expression levels of several E-C coupling proteins
The expression level of junctin in TG hearts was 29-fold higher than in WT hearts (line #129), whereas those of endogenous mouse junctin and triadin were significantly lower in TG vs. WT hearts. The expression level of RyR decreased slightly whereas that of DHPR increased by 2.4-fold. Expression levels of calsequestrin, calreticulin, FKBP12, phospholamban, SERCA2a, and Na⁺-Ca²⁺ exchanger did not change significantly. To examine whether junctin overexpression alters the characteristics of RyR, ryanodine binding to whole homogenates was measured at various [³H]ryanodine concentrations The densities of RyR, as determined by B_max of ryanodine to the receptor, were significantly lower in TG hearts than in WT heart (0.751±0.014 vs. 0.979±0.032 pmol/mg protein, n=4, P<0.05).

4. Cardiac overexpression of junctin induced increased L-type Ca²⁺ currents (I_Ca)
In light of evidence that the expression level of DHPR increased in the TG mice, the characteristics of I_Ca currents in ventricular myocytes were examined (Fig. 2A ). Figure 2B shows typical current-voltage relationships of I_Ca for WT and TG cardiomyocytes. The current-voltage relationships were similar between the two groups, but the current density of peak I_Ca was significantly greater in TG cardiomyocytes.

5. Cardiac overexpression of junctin induced prolongation of action potential duration
To test whether overexpression of junctin could affect the general electrophysiological characteristics of the heart, characteristics of the action potential in WT and TG ventricular and atrial muscles were examined (Fig. 2C, E ). In TG hearts, the resting membrane potential was not changed, but action potential duration at 70% repolarization (APD₇₀) was significantly increased compared to WT in both ventricular and atrial muscles (Fig. 2D ).

CONCLUSION AND SIGNIFICANCE

Cardiac junctin is localized in the junctional SR and appears to interact with calsequestrin and RyR. However, the functional consequences of the interaction in heart have not been fully elucidated. The junctin TG mice in the present study have shown various morphological and physiological alterations. The most outstanding phenotype changes would be the substantial bi-atrial and biventricular enlargements and the occurrence of the atrial fibrillation. Those phenotype changes could be caused by perturbations of the intracellular Ca²⁺ homeostasis due to the overexpression of junctin and consequent changes in protein expression of the other E-C coupling proteins such as DHPR (Fig. 3 ). Our TG model may help clarify the relationship between the altered expression levels of cardiac membrane proteins and the pathogenesis of hearts, especially when related to conduction abnormalities.

View larger version (34K): [in this window] [in a new window]   Figure 3. Schematic diagram of possible mechanisms to explain the phenotype changes due to the overexpression of junctin in heart. Increased L-type Ca2+ current leads to the prolongation of action potential duration and induction of bradycardia. Such impaired conduction may induce cardiac remodeling.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0908fje; to cite this article, use FASEB J. (June 21, 2002) 10.1096/fj.01-0908fje

This Article Full Text (PDF) All Versions of this Article: 16/10/1310 01-0908fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by HONG, C.-S. Articles by KIM, D. H. Search for Related Content PubMed PubMed Citation Articles by HONG, C.-S. Articles by KIM, D. H.