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CYP2J2 overexpression protects against arrhythmia susceptibility in cardiac hypertrophy - PubMed

  • ️Tue Jan 01 2013

CYP2J2 overexpression protects against arrhythmia susceptibility in cardiac hypertrophy

Christina Westphal et al. PLoS One. 2013.

Abstract

Maladaptive cardiac hypertrophy predisposes one to arrhythmia and sudden death. Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) promote anti-inflammatory and antiapoptotic mechanisms, and are involved in the regulation of cardiac Ca(2+)-, K(+)- and Na(+)-channels. To test the hypothesis that enhanced cardiac EET biosynthesis counteracts hypertrophy-induced electrical remodeling, male transgenic mice with cardiomyocyte-specific overexpression of the human epoxygenase CYP2J2 (CYP2J2-TG) and wildtype littermates (WT) were subjected to chronic pressure overload (transverse aortic constriction, TAC) or β-adrenergic stimulation (isoproterenol infusion, ISO). TAC caused progressive mortality that was higher in WT (42% over 8 weeks after TAC), compared to CYP2J2-TG mice (6%). In vivo electrophysiological studies, 4 weeks after TAC, revealed high ventricular tachyarrhythmia inducibility in WT (47% of the stimulation protocols), but not in CYP2J2-TG mice (0%). CYP2J2 overexpression also enhanced ventricular refractoriness and protected against TAC-induced QRS prolongation and delocalization of left ventricular connexin-43. ISO for 14 days induced high vulnerability for atrial fibrillation in WT mice (54%) that was reduced in CYP-TG mice (17%). CYP2J2 overexpression also protected against ISO-induced reduction of atrial refractoriness and development of atrial fibrosis. In contrast to these profound effects on electrical remodeling, CYP2J2 overexpression only moderately reduced TAC-induced cardiac hypertrophy and did not affect the hypertrophic response to β-adrenergic stimulation. These results demonstrate that enhanced cardiac EET biosynthesis protects against electrical remodeling, ventricular tachyarrhythmia, and atrial fibrillation susceptibility during maladaptive cardiac hypertrophy.

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Conflict of interest statement

Competing Interests: Please note that W.-H. Schunck is a PLOS ONE editorial board member (academic editor). This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Chronic pressure overload-induced mortality and cardiac hypertrophy.

(A) The survival rate was significantly higher in CYP2J2-TG (15 out of 16 animals survived over 8 weeks after TAC operation) compared with WT mice (11/19); Log rank-test ‡p<0.05. None of the sham operated WT (n = 17) or CYP2J2-TG mice (n = 11) died over the same period. (B) TAC-induced left ventricular hypertrophy was gradually ameliorated in CYP2J2-TG compared with WT mice. The difference was significant 8 weeks after TAC (13.1±0.8 vs. 15.1±0.8 mg/mm in 15 CYP2J2-TG vs. 11 WT mice). (C) Myocyte area significantly increased in both WT and CYP2J2-TG mice. (D) Systolic function was significantly decreased in both animal groups two weeks after TAC compared to the sham controls. Eight weeks after TAC, CYP2J2-TG mice (n = 15) showed significantly higher EF values (20.4±2.8 vs. 10.7±1.9%) than WT littermates (n = 11). Results represent mean±SEM; ANOVA, Post-Hoc Tukey; *p<0.05 vs. WT+Sham; †p<0.05 vs. CYP+Sham; ‡p<0.05 vs. WT+TAC.

Figure 2
Figure 2. Arrhythmia susceptibility after pressure overload-induced cardiac hypertrophy.

(A) Representative original tracings showing the induction of ventricular tachyarrhythmia by programmed electrical stimulation in WT mice 4 weeks after TAC (upper panel) and the resistance of TAC operated CYP2J2-TG mice under the same conditions (lower panel). (B) Ventricular arrhythmia inducibility significantly increased in WT mice after TAC (n = 5) compared with the sham control (n = 7). Arrhythmias were not inducible in any of the CYP2J2-TG mice both after sham (n = 5) and TAC operation (n = 6). Each animal was subjected to three protocols of programmed electrical stimulation and statistical evaluation was performed as described in Materials and Methods. (C) The severity of ventricular tachyarrhythmias scored according to the length of induced episodes (number of consecutive ventricular extrasystoles; VES) increased in WT mice after TAC compared with the sham control, whereas neither non-sustained nor sustained arrhythmias were inducible in corresponding CYP2J2-TG mice. (D) Analysis of arrhythmia inducibility in Langendorff preparations of isolated perfused hearts (n = 4 per group). Comparison of the vehicle treated groups confirmed the contrasting vulnerabilities of hypertrophied WT and CYP2J2-TG hearts after TAC. Perfusion with the mitochondrial KATP-channel opener diazoxide (100 µM, 20 min prior to programmed electrical stimulation) reduced the arrhythmia inducibility of WT-TAC hearts to the levels of hearts isolated from sham WT mice as well as CYP2J2-TG mice after TAC. Pretreatment with the EET antagonist 14,15-EEZE-mSi (48.5 µM for 20 min) reversed the protection of hypertrophied CYP2J2-TG hearts towards arrhythmia inducibility. Results represent mean±SEM; ANOVA, Post-Hoc Tukey; *p<0.05 vs. WT-Sham (vehicle); ‡p<0.05 vs. WT-TAC (vehicle); # p<0.05 vs. CYP-TAC (vehicle).

Figure 3
Figure 3. Chronic pressure overload-induced alterations in Cx43 localization.

(A) Representative immunofluorescence staining of left ventricular cryosections prepared from WT and CYP2J2-TG mice 4 weeks after TAC surgery. The sections were co-stained for detecting Cx43 (green fluorescent signal) and N-cadherin (red). Cx43 and N-cadherin are colocalized (yellow) to the intercalated disks (indicated by white arrows). This normal Cx43 localization was largely preserved in CYP2J2-TG mice, whereas WT mice featured TAC- induced redistribution of Cx43 to the cytoplasm and lateral borders of the cardiomyocytes (pink arrows). Nuclei were stained with DAPI (blue). Scale bar: 50 µm. (B) Quantitative analysis of Cx43 and N-cadherin colocalization. Results represent mean±SEM based on the analysis of 5 sections per heart and 4–6 animals per group; ANOVA, Post-Hoc Tukey; *p<0.05 vs. WT-Sham; ‡p<0.05 vs. WT-TAC.

Figure 4
Figure 4. Induction of cardiac hypertrophy by chronic β-adrenergic stimulation.

(A) Two weeks of chronic ISO infusion significantly increased the heart weight to tibia length-ratio in WT and CYP2J2-TG mice (n = 7 per group) compared with the vehicle controls (n = 7 and 5). The hypertrophic response was not different in CYP2J2-TG compared to WT mice. (B) Systolic function was not significantly altered upon chronic ISO infusion as indicated by preserved EF values compared to the respective vehicle controls. EF was slightly but significantly higher in CYP2J2-TG than WT mice two weeks after chronic ISO stimulation. Results represent mean±SEM; ANOVA, Post-Hoc Tukey; *p<0.05 vs. WT+Vehicle; †p<0.05 vs. CYP+Vehicle; ‡p<0.05 vs. WT+ISO.

Figure 5
Figure 5. Arrhythmia susceptibility after chronic β-adrenergic stimulation-induced cardiac hypertrophy.

(A) Representative original tracings showing the induction of atrial fibrillation by programmed electrical stimulation in WT mice 2 weeks after chronic ISO infusion (upper panel) and the resistance of CYP2J2-TG mice treated in the same manner (lower panel). (B) Atrial fibrillation inducibility significantly increased in WT mice after chronic ISO infusion (n = 9) compared with the vehicle control (n = 8) and was significantly higher than in CYP2J2-TG mice (n = 7 and n = 8 for the vehicle and ISO groups). (C) The relative percentage of inducible sustained atrial fibrillation was markedly higher in WT compared with CYP2J2-TG after chronic ISO infusion. For statistical evaluation of arrhythmia inducibilities and severity scoring compare Fig. 2. Results represent mean±SEM; ANOVA, Post-Hoc Tukey; *p<0.05 vs. WT+Vehicle; †p<0.05 vs. CYP+Vehicle; ‡p<0.05 vs. WT+ISO.

Figure 6
Figure 6. Effect of chronic β-adrenergic stimulation on the expression of markers of fibrosis in WT and CYP2J2-TG mice.

RNA isolated from atrial tissue 2 weeks after vehicle or ISO infusion was reverse transcribed and analyzed by quantitative Taqman- or SYBR-PCR for the expression of Col1 (A), Col3 (B) and fibronectin (C). WT+Vehicle: n = 8; CYP+Vehicle n = 5; WT+ISO: n = 10; CYP+ISO: n = 8. ANOVA, Post-Hoc Tukey; *p<0.05 vs. WT+vehicle †p<0.05 vs. CYP+vehicle; ‡p<0.05 vs. WT+ISO.

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