Pregnancy and oestrogen regulate sinoatrial node calcium homeostasis and accelerate pacemaking.

Aims: During pregnancy, there is a significant increase in heart rate (HR) potentially associated with an increased risk of arrhythmias or exacerbation of pre-existing cardiac conditions endangering both mother and foetus. Calcium homeostasis plays an important role in regulating automaticity of the sinoatrial node (SAN); however, its contribution to the accelerated HR during pregnancy remains unknown. Methods and results: Using murine SAN cells, we showed that pregnancy increased L-type Ca2+ current (ICaL) and CaV1.3 mRNA expression, whereas T-type Ca2+ current (ICaT) and its underlying channel were unchanged. Analysis of SAN intra-cellular Ca2+ oscillations showed that the rate of spontaneous Ca2+ transients was significantly higher in pregnant mice along with a higher mRNA expression of ryanodine receptor. Assessment of supra-ventricular arrhythmias using programmed electrical stimulation protocols on anaesthetized mice revealed higher susceptibility in pregnancy. Of note, the modifications associated with pregnancy were reversible following delivery. Furthermore, chronic administration of 17ß-estradiol (E2) to nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM), control mice, oestrogen-receptor-ß knockout (ERKOß) but not ERKOα mice, accelerated cardiac automaticity, recapitulating the pregnancy phenotype in both mouse and human SAN cell models. Conclusion: Together, these results indicate that pregnancy considerably alters intra-cellular Ca2+ homeostasis sustaining faster HR during pregnancy. Importantly, these changes were dependent on an oestrogen receptor α (ERα) mechanism that resulted in increased ICaL and spontaneous Ca2+ release from the sarcoplasmic reticulum, highlighting a novel role for oestrogen in regulating HR.

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.

Characterization of a Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Model for the Study of Variant Pathogenicity: Validation of a KCNJ2 Mutation.

BACKGROUND: Long-QT syndrome is a potentially fatal condition for which 30% of patients are without a genetically confirmed diagnosis. Rapid identification of causal mutations is thus a priority to avoid at-risk situations that can lead to fatal cardiac events. Massively parallel sequencing technologies are useful for the identification of sequence variants; however, electrophysiological testing of newly identified variants is crucial to demonstrate causality. Long-QT syndrome could, therefore, benefit from having a standardized platform for functional characterization of candidate variants in the physiological context of human cardiomyocytes. METHODS AND RESULTS: Using a variant in Kir2.1 (Gly52Val) revealed by whole-exome sequencing in a patient presenting with symptoms of long-QT syndrome as a proof of principle, we demonstrated that commercially available human induced pluripotent stem cell-derived cardiomyocytes are a powerful model for screening variants involved in genetic cardiac diseases. Immunohistochemistry experiments and whole-cell current recordings in human embryonic kidney cells expressing the wild-type or the mutant Kir2.1 demonstrated that Kir2.1-52V alters channel cellular trafficking and fails to form a functional channel. Using human induced pluripotent stem cell-derived cardiomyocytes, we not only confirmed these results but also further demonstrated that Kir2.1-52V is associated with a dramatic prolongation of action potential duration with evidence of arrhythmic activity, parameters which could not have been studied using human embryonic kidney cells. CONCLUSIONS: Our study confirms the pathogenicity of Kir2.1-52V in 1 patient with long-QT syndrome and also supports the use of isogenic human induced pluripotent stem cell-derived cardiomyocytes as a physiologically relevant model for the screening of variants of unknown function.

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.

Estrogen regulation of the transient outward K(+) current involves estrogen receptor α in mouse heart.

BACKGROUND AND OBJECTIVE: We have previously shown that androgens upregulate cardiac K(+) channels and shorten repolarization. However, the effects that estrogens (E2) and estrogen receptors (ER) might have on the various repolarizing K(+) currents and underlying ion channels remain incompletely understood. Accordingly, our objective was to verify whether and how E2 and its ERs subtypes influence these K(+) currents. METHODS AND RESULTS: In order to examine the influence of E2 and ERs on K(+) currents we drastically lowered the E2 level through ovariectomy (OVX; 74% reduction vs CTL) and in parallel, we used female mice lacking either ERα (ERαKO) or ERß (ERßKO). In OVX mice, results showed a specific increase of 35% in the density of the Ca(2+)-independent transient outward K(+) current (Ito) compared to CTL. Western blots showed increase in Kv4.2 and Kv4.3 sarcolemmal protein expression while qPCR revealed higher mRNA expression of only Kv4.3 in OVX mice. This upregulation of Ito was correlated with a shorter ventricular action potential duration and QTc interval. In ERαKO but not ERßKO mice, the mRNA of Kv4.3 was selectively increased. Furthermore, when ventricular myocytes obtained from ERαKO and ERßKO were cultured in the presence of E2, results showed that E2 reduced Ito density only in ERßKO myocytes confirming the repressive role of E2-ERα in regulating Ito. CONCLUSION: Altogether, these results suggest that E2 negatively regulates the density of Ito through ERα, this highlights a potential role for this female hormone and its α-subtype receptor in modulating cardiac electrical activity.

Interleukin-1ß reduces L-type Ca2+ current through protein kinase Cϵ activation in mouse heart.

Inflammation is now widely recognized as a key component of heart disease. Patients suffering from arrhythmias and heart failure have increased levels of tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß). Evidence suggests that these cytokines are important mediators of cardiac remodeling; however, their effects on ion channels and arrhythmogenesis remain incompletely understood. The L-type Ca(2+) current (ICaL) is a major determinant of the plateau phase of cardiac action potential and has a critical excitation-contraction coupling role. Thus, altering its properties could have detrimental effects on cardiac electrical and contractile functions. Accordingly, the objective of this study was to elucidate the effect of TNFα and IL-1ß on ICaL, while exploring the underlying regulatory mechanisms. Neonatal mouse ventricular myocytes were treated with a pathophysiological concentration (30 pg/ml) of TNFα and IL-1ß for 24 h. Voltage-clamp recordings showed that TNFα had no effect on ICaL, whereas IL-1ß decreased the current density by 36%. Although both IL-1ß- and TNFα-treated myocytes showed significant increase in reactive oxidative species (ROS), Western blot experiments revealed that only IL-1ß increased PKCϵ membrane translocation. The antioxidant N-acetyl-L-cysteine normalized ROS levels and restored ICaL density. Furthermore, the PKCϵ translocation inhibitor ϵ-V1-2 blocked the effect of IL-1ß on ICaL. The reduction of ICaL by IL-1ß was also seen in cultured adult ventricular myocytes. Overall, chronic IL-1ß treatment decreased ICaL density in cardiomyocytes. These effects implicated ROS signaling and PKCϵ activation. These findings could contribute to explain the role of IL-1ß in the development of arrhythmia and heart failure.

Upregulation of the hyperpolarization-activated current increases pacemaker activity of the sinoatrial node and heart rate during pregnancy in mice.

BACKGROUND: Pregnancy is associated with a faster heart rate (HR), which is a risk factor for arrhythmias. However, the underlying mechanisms for this increased HR are poorly understood. Therefore, this study was performed to gain mechanistic insight into the pregnancy-induced increase in HR. METHODS AND RESULTS: Using surface ECG we observed that pregnant (P) mice have faster HR (531±14 beats per minute [bpm]) compared with nonpregnant (NP) mice (470±27 bpm; P<0.03). Results obtained with Langendorff-perfused hearts showed that this difference persisted in the absence of autonomic nervous innervation (NP, 327±16 bpm; P, 385±18 bpm; P<0.02). Spontaneous action potentials of sinoatrial node cells from pregnant mice exhibited higher automaticity (NP, 292±13 bpm; P, 330±12 bpm; P=0.047) and steeper diastolic depolarization (NP, 0.20±0.03 V/s; P, 0.40±0.06 V/s; P=0.004). Pregnancy increased the density of the hyperpolarization-activated current (If) (at -90mV: NP, -15.2±1.0 pA/pF; P, -28.6±2.9 pA/pF; P=0.0002) in sinoatrial node cells. Voltage dependence of the If activation curve and the intracellular cAMP levels were unchanged in sinoatrial node cells of pregnant mice. However, there was a significant increase in HCN2 channel protein expression with no change in HCN4 expression. Maximal depolarizing shift of the If activation curve induced by isoproterenol was attenuated in pregnancy. This reduced response to isoproterenol may be attributable to the lower cAMP sensitivity of HCN2 isoform compared with that of HCN4. CONCLUSIONS: This study shows that an increase in If current density contributes to the acceleration of sinoatrial node automaticity and explains, in part, the higher HR observed in pregnancy.