Overexpression of type 1 angiotensin II receptors impairs excitation-contraction coupling in the mouse heart.

Transgenic mice that overexpress human type 1 angiotensin II receptor (AT(1)R) in the heart develop cardiac hypertrophy. Previously, we have shown that in 6-mo AT(1)R mice, which exhibit significant cardiac remodeling, fractional shortening is decreased. However, it is not clear whether altered contractility is attributable to AT(1)R overexpression or is secondary to cardiac hypertrophy/remodeling. Thus the present study characterized the effects of AT(1)R overexpression on ventricular L-type Ca(2+) currents (I(CaL)), cell shortening, and Ca(2+) handling in 50-day and 6-mo-old male AT(1)R mice. Echocardiography showed there was no evidence of cardiac hypertrophy in 50-day AT(1)R mice but that fractional shortening was decreased. Cellular experiments showed that cell shortening, I(CaL), and Ca(v)1.2 mRNA expression were significantly reduced in 50-day and 6-mo-old AT(1)R mice compared with controls. In addition, Ca(2+) transients and caffeine-induced Ca(2+) transients were reduced whereas the time to 90% Ca(2+) transient decay was prolonged in both age groups of AT(1)R mice. Western blot analysis revealed that sarcoplasmic reticulum Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger protein expression was significantly decreased in 50-day and 6-mo AT(1)R mice. Overall, the data show that cardiac contractility and the mechanisms that underlie excitation-contraction coupling are altered in AT(1)R mice. Furthermore, since the alterations in contractility occur before the development of cardiac hypertrophy, it is likely that these changes are attributable to the increased activity of the renin-angiotensin system brought about by AT(1)R overexpression. Thus it is possible that AT(1)R blockade may help maintain cardiac contractility in individuals with heart disease.

Cardiac-specific overexpression of the human type 1 angiotensin II receptor causes delayed repolarization.

AIMS: Mice with cardiac-specific overexpression of human angiotensin II type 1 receptor (AT1R) undergo cardiac remodelling and die prematurely of sudden death. Since excessive QT prolongation is a major risk factor for ventricular arrhythmias and sudden death, we hypothesize that chronic stimulation of AT1R might contribute to sudden death by promoting delayed repolarization and ventricular arrhythmias. METHODS: In the present study, a detailed analysis of ventricular repolarization parameters was undertaken in AT1R mice. RESULTS: Measurement of K+ currents in ventricular myocytes isolated from 6-8 months AT1R male mice revealed a significant reduction of the Ca2+-independent transient outward (I(to)), the ultra-rapid delayed rectifier (I Kur)), and the inward rectifier (I K1) K+ currents compared with littermate controls (CTL). The expression of the underlying K+ channels was also decreased in AT1R ventricles. Moreover, reactivation of I(to) was slower in AT1R mice. Consistent with these findings, AT1R mice presented a longer action potential duration (APD90, CTL: 19.0 +/- 1.8 ms; AT1R: 39.1 +/- 4.7 ms, P = 0.0001) and QTc interval (CTL: 53.6 +/- 1.5 ms, AT1R: 64.2 +/- 1.4 ms, P = 0.0005). In addition, spontaneous ventricular arrhythmias were reported in the AT1R mice. Importantly, the increased incidence of arrhythmia and the repolarization defects also occurred in much younger AT1R mice that do not present signs of hypertrophy, confirming that these arrhythmogenic changes are not secondary to cardiac remodelling. CONCLUSION: These results strongly suggest that chronic stimulation of AT1R directly leads to an increased incidence of cardiac arrhythmia associated with delayed repolarization.

Angiotensin II Overstimulation Leads to an Increased Susceptibility to Dilated Cardiomyopathy and Higher Mortality in Female Mice.

Heart failure (HF) is associated with high mortality and affects men and women differently. The underlying mechanisms for these sex-related differences remain largely unexplored. Accordingly, using mice with cardiac-specific overexpression of the angiotensin II (ANGII) type 1 receptor (AT1R), we explored male-female differences in the manifestations of hypertrophy and HF. AT1R mice of both sexes feature electrical and Ca2+ handling alterations, systolic dysfunction, hypertrophy and develop HF. However, females had much higher mortality (21.0%) rate than males (5.5%). In females, AT1R stimulation leads to more pronounced eccentric hypertrophy (larger increase in LV mass/body weight ratio [+31%], in cell length [+27%], in LV internal end-diastolic [LVIDd, +34%] and systolic [LVIDs, +67%] diameter) and dilation (larger decrease in LV posterior wall thickness, +17%) than males. In addition, in female AT1R mice the cytosolic Ca2+ extrusion mechanisms were more severely compromised and were associated with a specific increased in Ca2+ sparks (by 187%) and evidence of SR Ca2+ leak. Altogether, these results suggest that female AT1R mice have more severe eccentric hypertrophy, dysfunction and compromised Ca2+ dynamics. These findings indicate that females are more susceptible to the adverse effects of AT1R stimulation than males favouring the development of HF and increased mortality.

Sex and strain differences in adult mouse cardiac repolarization: importance of androgens.

OBJECTIVE: Gender differences in mouse cardiac repolarization have been reported to be due to the stimulatory action of androgens on the ultrarapid delayed rectifier K(+) current (I(Kur)) and its underlying Kv1.5 channel. To confirm the regulation of ventricular repolarization by androgens, the present study compared two strains of mice (CD-1 and C57BL/6) that present different androgen levels. METHODS AND RESULTS: Measurement of testosterone levels in different strains of mice (CD-1, C57BL/6, C3H and FVB) revealed that male C57BL/6 mice had very low levels of testosterone, whereas males of the other strains displayed normal testosterone levels. Furthermore, whole-cell voltage clamp recordings in isolated ventricular myocytes showed that the current density of I(Kur) in male C57BL/6 mice was similar to that in female mice but smaller with respect to male CD-1 mice. Androgen replacement in male C57BL/6 mice as well as in castrated male CD-1 mice shortened ventricular repolarization, increased I(Kur) current density, and increased expression of Kv1.5 channels. CONCLUSION: Strain and gender differences observed in mouse cardiac repolarization can be explained by different androgen levels. As a consequence, androgens are major regulatory factors in cardiac repolarization and special attention should be paid to the hormonal status of the animal when studying hormonal regulation of cardiac repolarization.

Reduction in Na(+) current by angiotensin II is mediated by PKCα in mouse and human-induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Ventricular arrhythmias and sudden cardiac deaths are among the leading causes of mortality in patients with heart failure, and the underlying mechanisms remain incompletely understood. Chronic elevation of angiotensin II (ANGII) is known to be one of the main contributors to heart failure. OBJECTIVE: We tested whether ANGII can alter ventricular conduction and Na(+) current using transgenic mice with cardiomyocyte-restricted overexpression of ANGII type 1 receptor (AT1R). METHODS: We used surface electrocardiograms along with current- and voltage-clamp techniques to characterize the electrophysiological properties of AT1R mice while the underlying regulatory mechanisms were explored using reverse transcription/quantitative polymerase chain reaction, Western blots, and immunofluorescence techniques. RESULTS: Electrophysiological data indicated that chronic AT1R activation in ventricular myocytes caused a 60% reduction in Na(+) current density that slowed the maximal velocity of the action potential upstroke, leading to a prolongation of the QRS complex. These changes occur independently of cardiac hypertrophy, suggesting a direct role for ANGII/AT1R in slowing ventricular conduction. Western blots demonstrated a selective increase in sarcolemmal protein kinase Cα (PKCα) in AT1R mice, indicating PKCα activation. Furthermore, immunofluorescence analysis showed reorganization of PKCα expression to sarcolemma and colocalization with NaV1.5 in AT1R myocytes. The involvement of PKCα in regulating Na(+) current was subsequently demonstrated in human-induced pluripotent stem cell-derived cardiomyocytes where ANGII treatment reduced Na(+) current density. Concomitant treatment with αV5-3, a PKCα translocation inhibitor peptide, blocked the ANGII effect. CONCLUSION: Overall, this study suggests that in mouse and human cardiomyocytes, PKCα is an important mediator of the ANGII-induced reduction in Na(+) current and may contribute to ventricular arrhythmias.

Electrical remodeling in a transgenic mouse model of alpha1B-adrenergic receptor overexpression.

Cardiac-specific overexpression of wild-type alpha(1B)-adrenergic receptors (alpha(1B)-AR) in mice predisposes to dilated cardiomyopathy and sudden death. Although alpha-adrenergic stimulation is thought to contribute to induction of arrhythmias in heart failure, the electrophysiological consequences of chronic alpha(1)-adrenergic activation have not been clearly defined. Thus we characterized ventricular repolarization and monitored incidence of spontaneous arrhythmias in end-stage heart failure alpha(1B)-AR mice (9-12 mo) and younger alpha(1B)-AR mice (2-3 mo) that do not present signs of heart failure. Compared with aged-matched controls, the corrected QT interval was 34% longer in the 9- to 12-mo alpha(1B)-AR mice, and the action potential durations were also significantly prolonged in these mice. These changes were associated with a decrease in the density of the outward K(+) currents, Ca(2+)-independent transient, ultrarapid delayed rectifier, and steady state (at +30 mV, reduction of 68, 64, and 41%, respectively), and underlying K(+) channel expression. Electrocardiogram (ECG) recordings revealed that older alpha(1B)-AR mice exhibited spontaneous ventricular arrhythmias. The alterations in repolarization can contribute to these rhythm abnormalities and are likely caused by chronic alpha(1B)-AR activity. Additional data obtained in 2- to 3-mo alpha(1B)-AR mice clearly showed that electrical remodeling was already observed in younger transgenic animals. However, it appeared to be slightly less pronounced than in older mice. These results suggest that there are two waves of remodeling: one due to chronic alpha(1B)-AR activity, and a second due to heart failure. Taken together, these data provide strong evidence for a pathological role of chronic alpha(1B)-AR activity in the development of repolarization defects and ventricular arrhythmias.