Sex-Based Mechanisms of Cardiac Development and Function: Applications for Induced-Pluripotent Stem Cell Derived-Cardiomyocytes.

Males and females exhibit intrinsic differences in the structure and function of the heart, while the prevalence and severity of cardiovascular disease vary in the two sexes. However, the mechanisms of this sex-based dimorphism are yet to be elucidated. Sex chromosomes and sex hormones are the main contributors to sex-based differences in cardiac physiology and pathophysiology. In recent years, the advances in induced pluripotent stem cell-derived cardiac models and multi-omic approaches have enabled a more comprehensive understanding of the sex-specific differences in the human heart. Here, we provide an overview of the roles of these two factors throughout cardiac development and explore the sex hormone signaling pathways involved. We will also discuss how the employment of stem cell-based cardiac models and single-cell RNA sequencing help us further investigate sex differences in healthy and diseased hearts.

Rapamycin treatment unmasks a sex-specific pattern of scar expansion of the infarcted rat heart: The relationship between mTOR and KATP channel.

Inhibition of the mammalian target of rapamycin (mTOR) with the macrolide rapamycin or pharmacological suppression of KATP channel opening translated to scar expansion of the myocardial infarcted (MI) adult female rodent heart. The present study tested the hypotheses that rapamycin-mediated scar expansion was sex-specific and that mTOR signaling directly influenced KATP channel subunit expression/activity. Scar size was significantly larger in post-MI male rats as compared to the previous data reported in post-MI female rats. The reported scar expansion of rapamycin-treated post-MI female rats was not observed following the administration of the macrolide to post-MI male rats. Protein levels of the KATP channel subunits Kir6.2 and SUR2A and phosphorylation of the serine2448 residue of mTOR were similar in the normal heart of adult male and female rats. By contrast, greater tuberin inactivation characterized by the increased phosphorylation of the threonine1462 residue and reduced raptor protein levels were identified in the normal heart of adult female rats. Rapamycin pretreatment of phorbol 12,13-dibutyrate (PDBu)-treated neonatal rat ventricular cardiomyocytes (NNVMs) suppressed hypertrophy, inhibited p70S6K phosphorylation, and attenuated SUR2A protein upregulation. In the presence of low ATP levels, KATP channel activity detected in untreated NNVMs was significantly attenuated in PDBu-induced hypertrophied NNVMs via a rapamycin-independent pathway. Thus, rapamycin administration to post-MI rats unmasked a sex-specific pattern of scar expansion and mTOR signaling in PDBu-induced hypertrophied NNVMs significantly increased SUR2A protein levels. However, the biological advantage associated with SUR2A protein upregulation was partially offset by an mTOR-independent pathway that attenuated KATP channel activity in PDBu-induced hypertrophied NNVMs.

Programmable self-regulated molecular buffers for precise sustained drug delivery.

Unlike artificial nanosystems, biological systems are ideally engineered to respond to their environment. As such, natural molecular buffers ensure precise and quantitative delivery of specific molecules through self-regulated mechanisms based on Le Chatelier's principle. Here, we apply this principle to design self-regulated nucleic acid molecular buffers for the chemotherapeutic drug doxorubicin and the antimalarial agent quinine. We show that these aptamer-based buffers can be programmed to maintain any specific desired concentration of free drug both in vitro and in vivo and enable the optimization of the chemical stability, partition coefficient, pharmacokinetics and biodistribution of the drug. These programmable buffers can be built from any polymer and should improve patient therapeutic outcome by enhancing drug activity and minimizing adverse effects and dosage frequency.

Connexin Lateralization Contributes to Male Susceptibility to Atrial Fibrillation.

Men have a higher risk of developing atrial fibrillation (AF) than women, though the reason for this is unknown. Here, we compared atrial electrical and structural properties in male and female mice and explored the contribution of sex hormones. Cellular electrophysiological studies revealed that action potential configuration, Na+ and K+ currents were similar in atrial myocytes from male and female mice (4-5 months). Immunofluorescence showed that male atrial myocytes had more lateralization of connexins 40 (63 ± 4%) and 43 (66 ± 4%) than females (Cx40: 45 ± 4%, p = 0.006; Cx43: 44 ± 4%, p = 0.002), with no difference in mRNA expression. Atrial mass was significantly higher in males. Atrial myocyte dimensions were also larger in males. Atrial fibrosis was low and similar between sexes. Orchiectomy (ORC) abolished sex differences in AF susceptibility (M: 65%; ORC: 38%, p = 0.050) by reducing connexin lateralization and myocyte dimensions. Ovariectomy (OVX) did not influence AF susceptibility (F: 42%; OVX: 33%). This study shows that prior to the development of age-related remodeling, male mice have more connexin lateralization and larger atria and atrial myocyte than females. Orchiectomy reduced AF susceptibility in males by decreasing connexin lateralization and atrial myocyte size, supporting a role for androgens. These sex differences in AF substrates may contribute to male predisposition to AF.

Higher Na+-Ca2+ Exchanger Function and Triggered Activity Contribute to Male Predisposition to Atrial Fibrillation.

Male sex is one of the most important risk factors of atrial fibrillation (AF), with the incidence in men being almost double that in women. However, the reasons for this sex difference are unknown. Accordingly, in this study, we sought to determine whether there are sex differences in intracellular Ca2+ homeostasis in mouse atrial myocytes that might help explain male predisposition to AF. AF susceptibility was assessed in male (M) and female (F) mice (4-5 months old) using programmed electrical stimulation (EPS) protocols. Males were 50% more likely to develop AF. The Ca2+ transient amplitude was 28% higher in male atrial myocytes. Spontaneous systolic and diastolic Ca2+ releases, which are known sources of triggered activity, were significantly more frequent in males than females. The time to 90% decay of Ca2+ transient was faster in males. Males had 54% higher Na+-Ca2+ exchanger (NCX1) current density, and its expression was also more abundant. L-type Ca2+ current (ICaL) was recorded with and without BAPTA, a Ca2+ chelator. ICaL density was lower in males only in the absence of BAPTA, suggesting stronger Ca2+-dependent inactivation in males. CaV1.2 expression was similar between sexes. This study reports major sex differences in Ca2+ homeostasis in mouse atria, with larger Ca2+ transients and enhanced NCX1 function and expression in males resulting in more spontaneous Ca2+ releases. These sex differences may contribute to male susceptibility to AF by promoting triggered activity.

Atrial Electrical Remodeling in Mice With Cardiac-Specific Overexpression of Angiotensin II Type 1 Receptor.

Background Elevated angiotensin II levels are thought to play an important role in atrial electrical and structural remodeling associated with atrial fibrillation. However, the mechanisms by which this remodeling occurs are still unclear. Accordingly, we explored the effects of angiotensin II on atrial remodeling using transgenic mice overexpressing angiotensin II type 1 receptor (AT1R) specifically in cardiomyocytes. Methods and Results Voltage-clamp techniques, surface ECG, programmed electrical stimulations along with quantitative polymerase chain reaction, Western blot, and Picrosirius red staining were used to compare the atrial phenotype of AT1R mice and their controls at 50 days and 6 months. Atrial cell capacitance and fibrosis were increased only in AT1R mice at 6 months, indicating the presence of structural remodeling. Ca2+ (ICaL) and K+ currents were not altered by AT1R overexpression (AT1R at 50 days). However, ICaL density and CaV1.2 messenger RNA expression were reduced by structural remodeling (AT1R at 6 months). Conversely, Na+ current (INa) was reduced (-65%) by AT1R overexpression (AT1R at 50 days) and the presence of structural remodeling (AT1R at 6 months) yields no further effect. The reduced INa density was not explained by lower NaV1.5 expression but was rather associated with an increase in sarcolemmal protein kinase C alpha expression in the atria, suggesting that chronic AT1R activation reduced INa through protein kinase C alpha activation. Furthermore, connexin 40 expression was reduced in AT1R mice at 50 days and 6 months. These changes were associated with delayed atrial conduction time, as evidenced by prolonged P-wave duration. Conclusions Chronic AT1R activation leads to slower atrial conduction caused by reduced INa density and connexin 40 expression.

Pregnancy-induced increased heart rate is independent of thyroid hormones.

BACKGROUND: The heart rate increases by 10-20 beats per minute (bpm) throughout pregnancy in women, reaching maximum heart rate in the third trimester. During pregnancy, important changes in thyroid hormones also occur, with increases of up to 50% in the levels of triiodothyronine (T3), the biological active thyroid hormone. In addition, T3 has been shown to regulate cardiac electrophysiology. OBJECTIVE: Thus, in the present study the potential contribution of T3 in pregnancy-induced increased heart rate was explored. METHODS: We compared the heart rate between nonpregnant and pregnant mice under control conditions and after altering thyroid hormone levels with T3 and propylthiouracil (PTU, an antithyroid drug) treatments. RESULTS: Consistent with the clinical data, we found a 58% rise in T3 levels during pregnancy in mice. Although pregnant mice had a higher baseline heart rate (607 ± 8 bpm, P = .004) and higher T3 levels (1.9 ± 0.4 nM, P = .0005) than nonpregnant mice (heart rate: 546 ± 16 bpm; T3 levels: 1.2 ± 0.1 nM), their heart rate responded similarly to T3 treatment as nonpregnant mice (nonpregnant: Δ130 ± 22 bpm; pregnant: Δ126 ± 17 bpm, P = .858). Additionally, the heart rate remained significantly elevated (607 ± 11 bpm, P = .038) and comparable to untreated pregnant mice, after the use of the antithyroid drug PTU, although T3 levels (1.3 ± 0.2 nM, P = .559) returned to nonpregnant values. CONCLUSION: Based on these results, it is unlikely that T3 contributes significantly to the pregnancy-induced increased heart rate.

MK2-Deficient Mice Are Bradycardic and Display Delayed Hypertrophic Remodeling in Response to a Chronic Increase in Afterload.

Background Mitogen-activated protein kinase-activated protein kinase-2 (MK2) is a protein serine/threonine kinase activated by p38α/ß. Herein, we examine the cardiac phenotype of pan MK2-null (MK2-/-) mice. Methods and Results Survival curves for male MK2+/+ and MK2-/- mice did not differ (Mantel-Cox test, P=0.580). At 12 weeks of age, MK2-/- mice exhibited normal systolic function along with signs of possible early diastolic dysfunction; however, aging was not associated with an abnormal reduction in diastolic function. Both R-R interval and P-R segment durations were prolonged in MK2-deficient mice. However, heart rates normalized when isolated hearts were perfused ex vivo in working mode. Ca2+ transients evoked by field stimulation or caffeine were similar in ventricular myocytes from MK2+/+ and MK2-/- mice. MK2-/- mice had lower body temperature and an age-dependent reduction in body weight. mRNA levels of key metabolic genes, including Ppargc1a, Acadm, Lipe, and Ucp3, were increased in hearts from MK2-/- mice. For equivalent respiration rates, mitochondria from MK2-/- hearts showed a significant decrease in Ca2+ sensitivity to mitochondrial permeability transition pore opening. Eight weeks of pressure overload increased left ventricular mass in MK2+/+ and MK2-/- mice; however, after 2 weeks the increase was significant in MK2+/+ but not MK2-/- mice. Finally, the pressure overload-induced decrease in systolic function was attenuated in MK2-/- mice 2 weeks, but not 8 weeks, after constriction of the transverse aorta. Conclusions Collectively, these results implicate MK2 in (1) autonomic regulation of heart rate, (2) cardiac mitochondrial function, and (3) the early stages of myocardial remodeling in response to chronic pressure overload.

GATA6 is a regulator of sinus node development and heart rhythm.

The sinus node (SAN) is the primary pacemaker of the human heart, and abnormalities in its structure or function cause sick sinus syndrome, the most common reason for electronic pacemaker implantation. Here we report that transcription factor GATA6, whose mutations in humans are linked to arrhythmia, is highly expressed in the SAN and its haploinsufficiency in mice results in hypoplastic SANs and rhythm abnormalities. Cell-specific deletion reveals a requirement for GATA6 in various SAN lineages. Mechanistically, GATA6 directly activates key regulators of the SAN genetic program in conduction and nonconduction cells, such as TBX3 and EDN1, respectively. The data identify GATA6 as an important regulator of the SAN and provide a molecular basis for understanding the conduction abnormalities associated with GATA6 mutations in humans. They also suggest that GATA6 may be a potential modifier of the cardiac pacemaker.

Contribution of estrogen to the pregnancy-induced increase in cardiac automaticity.

BACKGROUND: The heart rate progressively increases throughout pregnancy, reaching a maximum in the third trimester. This elevated heart rate is also present in pregnant mice and is associated with accelerated automaticity, higher density of the pacemaker current If and changes in Ca2+ homeostasis in sinoatrial node (SAN) cells. Strong evidence has also been provided showing that 17ß-estradiol (E2) and estrogen receptor α (ERα) regulate heart rate. Accordingly, we sought to determine whether E2 levels found in late pregnancy cause the increased cardiac automaticity associated with pregnancy. METHODS AND RESULTS: Voltage- and current-clamp experiments were carried out on SAN cells isolated from female mice lacking estrogen receptor alpha (ERKOα) or beta (ERKOß) receiving chronic E2 treatment mimicking late pregnancy concentrations. E2 treatment significantly increased the action potential rate (284 ± 24 bpm, +E2 354 ± 23 bpm, p = 0.040) and the density of If (+52%) in SAN cells from ERKOß mice. However, If density remains unchanged in SAN cells from E2-treated ERKOα mice. Additionally, E2 also increased If density (+67%) in nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM), recapitulating in a human SAN cell model the effect produced in mice. However, the L-type calcium current (ICaL) and Ca2+ transients, examined using N-hiPSC-CM and SAN cells respectively, were not affected by E2, indicating that other mechanisms contribute to changes observed in these parameters during pregnancy. CONCLUSION: The accelerated SAN automaticity observed in E2-treated ERKOß mice is explained by an increased If density mediated by ERα, demonstrating that E2 plays a major role in regulating SAN function during pregnancy.

Mechanisms of Arrhythmia and Sudden Cardiac Death in Patients With HIV Infection.

Long-term survival of HIV-infected patients has significantly improved with the use of antiretroviral therapy (ART). As a consequence, cardiovascular diseases are now emerging as an important clinical problem in this population. Sudden cardiac death is the third leading cause of mortality in HIV patients. Twenty percent of patients with HIV who died of sudden cardiac death had previous cardiac arrhythmias including ventricular tachycardia, atrial fibrillation, and other unspecified rhythm disorders. This review presents a summary of HIV-related arrhythmias, associated risk factors specific to the HIV population, and underlying mechanisms. Compared with the general population, patients with HIV have several cardiac conditions and electrophysiological abnormalities. As a result, they have an increased risk of developing severe arrhythmias, that can lead to sudden cardiac death. Possible explanations may be related to non-ART polypharmacy, electrolyte imbalances, and use of substances observed in HIV-infected patients; many of these conditions are associated with alterations in cardiac electrical activity, increasing the risk of arrhythmia and sudden cardiac death. However, clinical and experimental evidence has also revealed that cardiac arrhythmias occur in HIV-infected patients, even in the absence of drugs. This indicates that HIV itself can change the electrophysiological properties of the heart profoundly and cause cardiac arrhythmias and related sudden cardiac death. The current knowledge of the underlying mechanisms, as well as the emerging role of inflammation in these arrhythmias, are discussed here.

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.

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 Ca(2+) 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 Ca(2+) channels.

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.

Nestin is a marker of lung remodeling secondary to myocardial infarction and type I diabetes in the rat.

Upregulation of the intermediate filament protein nestin was identified in a subpopulation of fibroblasts during reactive and reparative fibrosis and directly contributed to the enhanced proliferative phenotype. The present study tested the hypothesis that nestin was expressed in lung fibroblasts and the pattern of expression represented a distinct marker of pulmonary remodeling secondary to myocardial infarction and type I diabetes. Nestin((+)) fibroblasts were detected in rat lungs and a subpopulation exhibited a myofibroblast phenotype delineated by the co-expression of smooth muscle α-actin. In the lungs of myocardial infarcted rats, interstitial collagen content and nestin mRNA/protein levels were significantly increased despite the absence of secondary pulmonary hypertension, whereas smooth muscle α-actin protein expression was unchanged. Exposure of rat pulmonary fibroblasts to pro-fibrotic stimuli angiotensin II and transforming growth factor-ß significantly increased nestin protein levels. In the lungs of type I diabetic rats, the absence of a reactive fibrotic response was associated with a significant downregulation of nestin mRNA/protein expression. Nestin was reported a target of miR-125b, albeit miR-125b levels were unchanged in pulmonary fibroblasts treated with pro-fibrotic stimuli. Nestin((+)) cells lacking smooth muscle α-actin/collagen staining were also identified in rodent lungs and a transgenic approach revealed that expression of the intermediate filament protein was driven by intron 2 of the nestin gene. The disparate regulation of nestin characterized a distinct pattern of pulmonary remodeling secondary to myocardial infarction and type I diabetes and upregulation of the intermediate filament protein in lung fibroblasts may have facilitated in part the reactive fibrotic response.

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.

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.