LQTS mutation N1325S in cardiac sodium channel gene SCN5A causes cardiomyocyte apoptosis, cardiac fibrosis and contractile dysfunction in mice - PubMed (original) (raw)

LQTS mutation N1325S in cardiac sodium channel gene SCN5A causes cardiomyocyte apoptosis, cardiac fibrosis and contractile dysfunction in mice

Teng Zhang et al. Int J Cardiol. 2011.

Abstract

Objective: Mutations in the cardiac sodium channel gene SCN5A cause long QT syndrome (LQTS). We previously generated an LQTS mouse model (TG-NS) that overexpresses the LQTS mutation N1325S in SCN5A. The TG-NS mice manifested the clinical features of LQTS including spontaneous VT, syncope and sudden death. However, the long-term prognosis of LQTS on the structure of the heart has not been investigated in this or any other LQTS models and human patients.

Methods and results: Impaired systolic function and reduced left ventricular fractional shortening were detected by echocardiography, morphological and histological examination in two lines of adult mutant transgenic mice. Histological and TUNEL analyses of heart sections revealed fibrosis lesions and increased apoptosis in an age-dependent manner. Cardiomyocyte apoptosis was associated with the increased activation of caspases 3 and 9 in TG-NS hearts. Western blot analysis showed a significantly increased expression of the key Ca(2+) handling proteins L-type Ca(2+) channel, RYR2 and NCX in TG-NS hearts. Increased apoptosis and an altered expression of Ca(2+) handling proteins could be detected as early as 3months of age when echocardiography showed little or no alterations in TG-NS mice.

Conclusions: Our findings revealed for the first time that the LQTS mutation N1325S in SCN5A causes cardiac fibrosis and contractile dysfunction in mice, possibly through cellular mechanisms involving aberrant cardiomyocyte apoptosis. Therefore, we provide the experimental evidence supporting the notion that some LQTS patients have an increased risk of structural and functional cardiac damage in a prolonged disease course.

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Conflict of interest statement

Conflict of interest: All of the authors report no conflicts.

Figures

Figure 1

Abnormal cardiac structure in TG-NS mice. The gross morphology of the whole heart (first row) and H&E staining of transverse heart sections (second row; horizontal bars indicate 200 µm) revealed cardiac enlargement and ventricular dilatation in 6–10-month old TG-NSL12 and TG-NSL3, but not in TG-WTL10 or NT mice. Representative echocardiographic M-mode images for each of the corresponding hearts are illustrated in the third row.

Figure 2

Histological analysis of the lung. (A) Significant increases in the lung weight/body weight ratio were observed in both lines of TG-NS mice (TG-NS-L12, n=12; TG-NS-L3, n=11), compared with TG-WTL10 (n=10) or NT (n=19) mice (*P<0.01). The heart/body weight ratios of 6–10-month old TG-NSL12, TG-NSL3, TG-WTL10, and NT mice were not different. (B) Pulmonary congestion and edema were observed in both lines of TG-NS mice at 6–10 months of age. The alveolar capillaries of the lung were distended and filled with erythrocytes. In some areas, the alveolar space was filled with fluids. Normal alveolar structures were shown as in TG-WTL10 or NT mice. n=3; Scale bar (bottom right panel) = 20 µm.

Figure 3

Significant increase of development of cardiac fibrosis in both lines of TG-NS mice. (A) Representative stained sections from 4 groups of mice (n=5 mice/group) indicates fibrosis (light blue color) in both lines of TG-NS mice, but not in TG-WTL10 and NT mice. (B) Fibrotic tissue was greater in 6–10-month old TG-L12 and TG-L3 mice than in TG-WTL10 or NT mice (bar graph), by Masson’s trichrome staining of heart sections. Scale bar (bottom right panel) = 20 µm. “**” Denotes statistically significant difference from TG-WTL10 (P<0.0001).

Figure 4

Evaluation of cardiomyocyte apoptosis. (A) Representative images of heart sections using the TUNEL assay. Brown nuclei are TUNEL positive and apoptotic cells (arrows). i, NT; ii, TG-NSL12; iii and iv, TUNEL stained images counterstained with H&E for NT and TG-NSL12, respectively; v, a representative image of a TUNEL-positive nucleus undergoing condensation of nuclear chromatin (arrow); vi, image of TG-NSL12 with staining for both TUNEL and a monoclonal anti-α-actinin antibody, a probe specific for cardiomyocytes. Scale bar = 20 µm. (B) The fraction of TUNEL-positive nuclei in hearts of 6–10-month old TG-NSL12 (n=10) and TG-NSL3 (n=6) mice was significantly greater than in hearts of TG-WTL10 (n=10), and NT (n=17) mice. (** P<0.0001 vs TG-WT; * P<0.05 vs NT). (C) The number of TUNEL-positive nuclei in hearts of TG-NSL12 mice increased with age. (*P<0.05; NS means no significant difference; n=4 for 2–3 months, n=6 for 4–6 months, n=6 for 8–10 months, n=5 for more than 10 months group).

Figure 5

Assessment of caspase activation in heart tissue. (A) Increased caspase-3 activation was detected in hearts from TG-NSL12 (n=17) compared to those from TG-WTL10 (n=9) and NT (n=17) mice. **_P_=2.6 × 10−9 vs NT. (B) No significant difference was detected in caspase-8 activation amongst the three heart groups. (C) Increased caspase-9 activation was detected in TG-NSL12 hearts (n=10) compared to TG-WTL10 (n=12) and NT (n=11).age, 3–5 months old; *P<0.05.vs NT.

Figure 6

Expression of NCX-1 (A), RyR2 (B), SERCA2 (C) and L-type Ca2+ channel (D) in hearts from TG-NS and NT mice at 3–5 months of age. Representative Western blots are shown at left panels and summarized data for the Western blot densitometrical analysis are at right panels. Cardiac expression of NCX-1 (A), RyR2 (B) and L-type Ca2+ (D) was significantly increased in TG-NSL3 and TG-NSL12 mice, but not in TG-WTL10 mice or NT mice. Expression of SERCA2 (C) was not significantly different amongst the four groups of mice. (TG-NSL12 and TG-NSL3, n = 7, respectively; TG-WTL10 and NT, n = 8, respectively). **P<0.001; *P<0.005.

References

1. Wang Q, Shen J, Li Z, Timothy K, Vincent GM, Priori SG, Schwartz PJ, Keating MT. Cardiac sodium channel mutations in patients with long QT syndrome, an inherited cardiac arrhythmia. Hum Mol Genet. 1995;4:1603–1607. - PubMed
1. Wang Q, Shen J, Splawski I, Atkinson D, Li Z, Robinson JL, Moss AJ, Towbin JA, Keating MT. SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome. Cell. 1995;80:805–811. - PubMed
1. Yong SL, Ni Y, Zhang T, Tester DJ, Ackerman MJ, Wang QK. Characterization of the cardiac sodium channel SCN5A mutation, N1325S, in single murine ventricular myocytes. Biochem Biophys Res Commun. 2007;352:378–383. - PMC - PubMed
1. Tian XL, Yong SL, Wan X, Wu L, Chung MK, Tchou PJ, Rosenbaum DS, Van Wagoner DR, Kirsch GE, Wang Q. Mechanisms by which SCN5A mutation N1325S causes cardiac arrhythmias and sudden death in vivo. Cardiovasc Res. 2004;61:256–267. - PMC - PubMed
1. Tian XL, Cheng Y, Zhang T, Liao ML, Yong SL, Wang QK. Optical mapping of ventricular arrhythmias in LQTS mice with SCN5A mutation N1325S. Biochem Biophys Res Commun. 2007;352:879–883. - PMC - PubMed

LQTS mutation N1325S in cardiac sodium channel gene SCN5A causes cardiomyocyte apoptosis, cardiac fibrosis and contractile dysfunction in mice - PubMed (original) (raw)