Céline Fiset

Functional properties of K + currents in adult mouse ventricular myocytes

Although the K+ currents expressed in hearts of adult mice have been studied extensively, detailed information concerning their relative sizes and biophysical properties in ventricle and atrium is lacking. Here we describe and validate pharmacological and biophysical methods that can be used to isolate the three main time‐ and voltage‐dependent outward K+ currents which modulate action potential repolarization. A Ca2+‐independent transient outward K+ current, Ito, can be separated from total outward current using an ‘inactivating prepulse’. The rapidly activating, slowly inactivating delayed rectifier K+ current, IKur, can be isolated using submillimolar concentrations of 4‐aminopyridine (4‐AP). The remaining K+ current, Iss, can be obtained by combining these two procedures: (i) inactivating Ito and (ii) eliminating IKur by application of low concentration of 4‐AP. Iss activates relatively slowly and shows very little inactivation, even during depolarizations lasting several seconds. Our findings also show that the rate of reactivation of Ito is more than 20‐fold faster than that of IKur. These results demonstrate that the outward K+ currents in mouse ventricles can be separated based on their distinct time and voltage dependence, and different sensitivities to 4‐AP. Data obtained at both 22 and 32°C demonstrate that although the duration of the inactivating prepulse has to be adapted for the recording temperature, this approach for separation of K+ current components is also valid at more physiological temperatures. To demonstrate that these methods also allow separation of these K+ currents in other cell types, we have applied this same approach to myocytes from mouse atria. Molecular approaches have been used to compare the expression levels of different K+ channels in mouse atrium and ventricle. These findings provide new insights into the functional roles of IKur, Ito and Iss during action potential repolarization.

Effect of androgen deficiency on mouse ventricular repolarization

We previously demonstrated that female mouse ventricles have longer action potential durations (APDs) than males. This delayed repolarization results from a lower current density of the ultrarapid delayed rectifier K+ current (IK,ur) and a lower expression level of its underlying K+ channel (Kv1.5). To evaluate whether this sex difference could be attributable to the action of male sex hormones, we studied the effect of androgen deficiency on ventricular repolarization. We compared cardiac electrophysiological properties in castrated (orchiectomized; ORC) and control (CTL) male mice. Q‐Tc intervals as well as APDs measured at 20 %, 50 % and 90 % of repolarization were all significantly longer in ORC than in CTL. The current density of IK,ur was significantly lower in ORC than in CTL (at +50 mV, ORC: 29 ± 4 pA pF−1, n= 25; CTL: 48 ± 5 pA pF−1, n= 17; P= 0.006). In contrast, all the other K+ currents present in mouse ventricular myocytes were comparable between ORC and CTL. Moreover, results of Western blot analysis showed a lower expression level of Kv1.5 protein in ORC but no difference between the two groups for the other K+ channels studied. This study demonstrates that androgen deficiency leads to a reduction in the density of IK,ur and Kv1.5 in mouse ventricle, and consequently, to prolongation of APD and Q‐Tc interval. In conclusion, these findings strongly suggest that male sex hormones contribute to the sex difference that we previously reported in cardiac repolarization in adult mouse heart.

Sex Differences in Cardiac Electrophysiology and Clinical Arrhythmias: Epidemiology, Therapeutics, and Mechanisms

Sex differences in cardiac electrophysiological properties and arrhythmias are evident in epidemiologic and investigative studies as well as in daily patient care. At the supraventricular level, women are at increased risk of sick sinus syndrome and atrioventricular (AV) node re-entrant tachycardia, whereas men manifest more AV block and accessory pathway-mediated arrhythmias. At the ventricular level, women are generally at higher risk of long QT-associated arrhythmias, whereas men are more likely to present with early repolarization, idiopathic ventricular fibrillation, and Brugada syndromes. Great advances have been made in unraveling the fundamental mechanisms underlying sex differences in ventricular arrhythmias, particularly those associated with abnormal repolarization. Conversely, the basis for male-predominant arrhythmia risk in structural heart disease and differences in supraventricular arrhythmia susceptibility are poorly understood. Beyond biological differences, arrhythmia occurrence and patient care decisions are also influenced by gender-related factors. This article reviews the current knowledge regarding the nature and underlying mechanisms of sex differences in basic cardiac electrophysiology and clinical arrhythmias.

Effect of pressure overload-induced hypertrophy on the expression and localization of p38 MAP kinase isoforms in the mouse heart.

p38 mitogen-activated protein kinases (MAPKs) are serine/threonine specific protein kinases that respond to cellular stress and regulate a broad range of cellular activities. There are four major isoforms of p38 MAPK: alpha, beta, gamma, and delta. To date, the prominent isoform in heart has been thought to be p38alpha. We examined the expression of each p38 isoform at both the mRNA and protein level in murine heart. mRNA for all four p38 isoforms was detected. p38gamma and p38delta were expressed at protein levels comparable to p38alpha and 38beta, respectively. In the early phase of pressure-overload hypertrophy (1-7 days after constriction of the transverse aorta), the abundance of p38beta, p38gamma and p38delta mRNA increased; however, no corresponding changes were detected at the protein level. Confocal immunofluorescence studies revealed p38alpha and p38gamma in both the cytoplasm and nucleus. In the established phase of hypertrophy induced by chronic pressure overload (7-28 days after constriction of the transverse aorta), p38gamma immunoreactivity accumulated in the nucleus whereas the distribution of p38alpha remained unaffected. Hence, both p38alpha and p38gamma are prominent p38 isoforms in heart and p38gamma may play a role in mediating the changes in gene expression associated with cardiac remodeling during pressure-overload hypertrophy.

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.

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.

Reduction of ventricular sodium current in a mouse model of HIV.

Effect of HIV on Cardiac Sodium Current. Introduction: Cardiac arrhythmias have been reported in AIDS patients. Arrhythmias can arise from alterations in ventricular Na+ channel function. However, it is unknown whether HIV affects cardiac Na+ channel function. Therefore, the purpose of this study was to characterize the effect of HIV on ventricular Na+ current (INa) in a transgenic model of HIV (CD4C/HIV mice), which exhibit a severe AIDS‐like disease.

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

Background: Cytokines are important inflammatory mediators that can affect cardiac function. Results: Treatment with IL-1β but not TNFα reduced the density of L-type Ca2+ current. Conclusion: Mechanisms underlying IL-1β effects implicate oxidative stress and PKCϵ signaling to alter L-type Ca2+ current. Significance: Elucidating the cytokine signaling pathways are important in understanding their role in heart disease. 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-1β (IL-1β). 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 Ca2+ 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-1β 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-1β for 24 h. Voltage-clamp recordings showed that TNFα had no effect on ICaL, whereas IL-1β decreased the current density by 36%. Although both IL-1β- and TNFα-treated myocytes showed significant increase in reactive oxidative species (ROS), Western blot experiments revealed that only IL-1β 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-1β on ICaL. The reduction of ICaL by IL-1β was also seen in cultured adult ventricular myocytes. Overall, chronic IL-1β treatment decreased ICaL density in cardiomyocytes. These effects implicated ROS signaling and PKCϵ activation. These findings could contribute to explain the role of IL-1β in the development of arrhythmia and heart failure.

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.

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity.

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca2+ channel complexes. CaVα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type CaV1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant CaVα2δ1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the CaVα2δ1-mediated increase in the peak current density and voltage-dependent gating of CaV1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored CaVα2δ1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of CaVα2δ1 as well as the CaVα2δ1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of CaVα2δ1, and furthermore that N-glycosylation of CaVα2δ1 is essential to produce functional L-type Ca2+ channels.