Fluvastatin inhibits Rab5-mediated IKs internalization caused by chronic Ca2+-dependent PKC activation.

Statins, in addition to their cholesterol lowering effects, can prevent isoprenylation of Rab GTPase proteins, a key protein family for the regulation of protein trafficking. Rab-GTPases have been shown to be involved in the control of membrane expression level of ion channels, including one of the major cardiac repolarizing channels, IKs. Decreased IKs function has been observed in a number of disease states and associated with increased propensity for arrhythmias, but the mechanism underlying IKs decrease remains elusive. Ca2+-dependent PKC isoforms (cPKC) are chronically activated in variety of human diseases and have been suggested to acutely regulate IKs function. We hypothesize that chronic cPKC stimulation leads to Rab-mediated decrease in IKs membrane expression, and that can be prevented by statins. In this study we show that chronic cPKC stimulation caused a dramatic Rab5 GTPase-dependent decrease in plasma membrane localization of the IKs pore forming subunit KCNQ1, reducing IKs function. Our data indicates fluvastatin inhibition of Rab5 restores channel localization and function after cPKC-mediated channel internalization. Our results indicate a novel statin anti-arrhythmic effect that would be expected to inhibit pathological electrical remodeling in a number of disease states associated with high cPKC activation. Because Rab-GTPases are important regulators of membrane trafficking they may underlie other statin pleiotropic effects.

Posttranslational modifications of mitochondrial fission and fusion proteins in cardiac physiology and pathophysiology.

Mitochondrial fragmentation frequently occurs in chronic pathological conditions as seen in various human diseases. In fact, abnormal mitochondrial morphology and mitochondrial dysfunction are hallmarks of heart failure (HF) in both human patients and HF animal models. A link between mitochondrial fragmentation and cardiac pathologies has been widely proposed, but the physiological relevance of mitochondrial fission and fusion in the heart is still unclear. Recent studies have increasingly shown that posttranslational modifications (PTMs) of fission and fusion proteins are capable of directly modulating the stability, localization, and/or activity of these proteins. These PTMs include phosphorylation, acetylation, ubiquitination, conjugation of small ubiquitin-like modifier proteins, O-linked-N-acetyl-glucosamine glycosylation, and proteolysis. Thus, understanding the PTMs of fission and fusion proteins may allow us to understand the complexities that determine the balance of mitochondrial fission and fusion as well as mitochondrial function in various cell types and organs including cardiomyocytes and the heart. In this review, we summarize present knowledge regarding the function and regulation of mitochondrial fission and fusion in cardiomyocytes, specifically focusing on the PTMs of each mitochondrial fission/fusion protein. We also discuss the molecular mechanisms underlying abnormal mitochondrial morphology in HF and their contributions to the development of cardiac diseases, highlighting the crucial roles of PTMs of mitochondrial fission and fusion proteins. Finally, we discuss the future potential of manipulating PTMs of fission and fusion proteins as a therapeutic strategy for preventing and/or treating HF.

Role of mitochondrial Ca2+ homeostasis in cardiac muscles.

Recent discoveries of the molecular identity of mitochondrial Ca2+ influx/efflux mechanisms have placed mitochondrial Ca2+ transport at center stage in views of cellular regulation in various cell-types/tissues. Indeed, mitochondria in cardiac muscles also possess the molecular components for efficient uptake and extraction of Ca2+. Over the last several years, multiple groups have taken advantage of newly available molecular information about these proteins and applied genetic tools to delineate the precise mechanisms for mitochondrial Ca2+ handling in cardiomyocytes and its contribution to excitation-contraction/metabolism coupling in the heart. Though mitochondrial Ca2+ has been proposed as one of the most crucial secondary messengers in controlling a cardiomyocyte's life and death, the detailed mechanisms of how mitochondrial Ca2+ regulates physiological mitochondrial and cellular functions in cardiac muscles, and how disorders of this mechanism lead to cardiac diseases remain unclear. In this review, we summarize the current controversies and discrepancies regarding cardiac mitochondrial Ca2+ signaling that remain in the field to provide a platform for future discussions and experiments to help close this gap.

Activation of Anoctamin-1 Limits Pulmonary Endothelial Cell Proliferation via p38-Mitogen-activated Protein Kinase-Dependent Apoptosis.

Hyperproliferative endothelial cells (ECs) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Anoctamin (Ano)-1, a calcium-activated chloride channel, can regulate cell proliferation and cell cycle in multiple cell types. However, the expression and function of Ano1 in the pulmonary endothelium is unknown. We examined whether Ano1 was expressed in pulmonary ECs and if altering Ano1 activity would affect EC survival. Expression and localization of Ano1 in rat lung microvascular ECs (RLMVECs) was assessed using immunoblot, immunofluorescence, and subcellular fractionation. Cell counts, flow cytometry, and caspase-3 activity were used to assess changes in cell number and apoptosis in response to the small molecule Ano1 activator, Eact. Changes in mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine, iodide (mitochondrial membrane potential dye) and mitochondrial ROS dye, respectively. Ano1 is expressed in RLMVECs and is enriched in the mitochondria. Activation of Ano1 with Eact reduced RLMVEC counts through increased apoptosis. Ano1 knockdown blocked the effects of Eact. Ano1 activation increased mtROS, reduced mitochondrial membrane potential, increased p38 phosphorylation, and induced release of apoptosis-inducing factor. mtROS inhibition attenuated Eact-mediated p38 phosphorylation. Pulmonary artery ECs isolated from patients with idiopathic PAH (IPAH) had higher expression of Ano1 and increased cell counts compared with control subjects. Eact treatment reduced cell counts in IPAH cells, which was associated with increased apoptosis. In summary, Ano1 is expressed in lung EC mitochondria. Activation of Ano1 promotes apoptosis of pulmonary ECs and human IPAH-pulmonary artery ECs, likely via increased mtROS and p38 phosphorylation, leading to apoptosis.

Protein kinase D activation induces mitochondrial fragmentation and dysfunction in cardiomyocytes.

KEY POINTS: Abnormal mitochondrial morphology and function in cardiomyocytes are frequently observed under persistent Gq protein-coupled receptor (Gq PCR) stimulation. Cardiac signalling mechanisms for regulating mitochondrial morphology and function under pathophysiological conditions in the heart are still poorly understood. We demonstrate that a downstream kinase of Gq PCR, protein kinase D (PKD) induces mitochondrial fragmentation via phosphorylation of dynamin-like protein 1 (DLP1), a mitochondrial fission protein. The fragmented mitochondria enhance reactive oxygen species generation and permeability transition pore opening in mitochondria, which initiate apoptotic signalling activation. This study identifies a novel PKD-specific substrate in cardiac mitochondria and uncovers the role of PKD on cardiac mitochondria, with special emphasis on the molecular mechanism(s) underlying mitochondrial injury with abnormal mitochondrial morphology under persistent Gq PCR stimulation. These findings provide new insights into the molecular basis of cardiac mitochondrial physiology and pathophysiology, linking Gq PCR signalling with the regulation of mitochondrial morphology and function. ABSTRACT: Regulation of mitochondrial morphology is crucial for the maintenance of physiological functions in many cell types including cardiomyocytes. Small and fragmented mitochondria are frequently observed in pathological conditions, but it is still unclear which cardiac signalling pathway is responsible for regulating the abnormal mitochondrial morphology in cardiomyocytes. Here we demonstrate that a downstream kinase of Gq protein-coupled receptor (Gq PCR) signalling, protein kinase D (PKD), mediates pathophysiological modifications in mitochondrial morphology and function, which consequently contribute to the activation of apoptotic signalling. We show that Gq PCR stimulation induced by α1 -adrenergic stimulation mediates mitochondrial fragmentation in a fission- and PKD-dependent manner in H9c2 cardiac myoblasts and rat neonatal cardiomyocytes. Upon Gq PCR stimulation, PKD translocates from the cytoplasm to the outer mitochondrial membrane (OMM) and phosphorylates a mitochondrial fission protein, dynamin-like protein 1 (DLP1), at S637. PKD-dependent phosphorylation of DLP1 initiates DLP1 association with the OMM, which then enhances mitochondrial fragmentation, mitochondrial superoxide generation, mitochondrial permeability transition pore opening and apoptotic signalling. Finally, we demonstrate that DLP1 phosphorylation at S637 by PKD occurs in vivo using ventricular tissues from transgenic mice with cardiac-specific overexpression of constitutively active Gαq protein. In conclusion, Gq PCR-PKD signalling induces mitochondrial fragmentation and dysfunction via PKD-dependent DLP1 phosphorylation in cardiomyocytes. This study is the first to identify a novel PKD-specific substrate, DLP1 in mitochondria, as well as the functional role of PKD in cardiac mitochondria. Elucidation of these molecular mechanisms by which PKD-dependent enhanced fission mediates cardiac mitochondrial injury will provide novel insight into the relationship among mitochondrial form, function and Gq PCR signalling.

The Mitochondrial Ca2+ Uniporter: Structure, Function, and Pharmacology.

Mitochondrial Ca2+ uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca2+ uptake and our current understanding of mitochondrial Ca2+ homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca2+ uniporter complex.

The mitochondrial Ca2+ uniporter: regulation by auxiliary subunits and signal transduction pathways.

Mitochondrial Ca(2+) homeostasis, the Ca(2+) influx-efflux balance, is responsible for the control of numerous cellular functions, including energy metabolism, generation of reactive oxygen species, spatiotemporal dynamics of Ca(2+) signaling, and cell growth and death. Recent discovery of the molecular identity of the mitochondrial Ca(2+) uniporter (MCU) provides new possibilities for application of genetic approaches to study the mitochondrial Ca(2+) influx mechanism in various cell types and tissues. In addition, the subsequent discovery of various auxiliary subunits associated with MCU suggests that mitochondrial Ca(2+) uptake is not solely regulated by a single protein (MCU), but likely by a macromolecular protein complex, referred to as the MCU-protein complex (mtCUC). Moreover, recent reports have shown the potential role of MCU posttranslational modifications in the regulation of mitochondrial Ca(2+) uptake through mtCUC. These observations indicate that mtCUCs form a local signaling complex at the inner mitochondrial membrane that could significantly regulate mitochondrial Ca(2+) handling, as well as numerous mitochondrial and cellular functions. In this review we discuss the current literature on mitochondrial Ca(2+) uptake mechanisms, with a particular focus on the structure and function of mtCUC, as well as its regulation by signal transduction pathways, highlighting current controversies and discrepancies.

Impaired IKs channel activation by Ca(2+)-dependent PKC shows correlation with emotion/arousal-triggered events in LQT1.

BACKGROUND: The most common inherited cardiac arrhythmia, LQT1, is due to IKs potassium channel mutations and is linked to high risk of adrenergic-triggered cardiac events. We recently showed that although exercise-triggered events are very well treated by ß-blockers for these patients, acute arousal-triggered event rate were not significantly reduced after beta-blocker treatment, suggesting that the mechanisms underlying arousal-triggered arrhythmias may be different from those during exercise. IKs is strongly regulated by ß-adrenergic receptor (ß-AR) signaling, but little is known about the role of α1-AR-mediated regulation. METHODS AND RESULTS: Here we show, using a combination of cellular electrophysiology and computational modeling, that IKs phosphorylation and α1-AR regulation via activation of calcium-dependent PKC isoforms (cPKC) may be a key mechanism to control channel voltage-dependent activation and consequently action potential duration (APD) in response to adrenergic-stimulus. We show that simulated mutation-specific combined adrenergic effects (ß+α) on APD were strongly correlated to acute stress-triggered cardiac event rate for patients while ß-AR effects alone were not. CONCLUSION: We were able to show that calcium-dependent PKC signaling is key to normal QT shortening during acute arousal and when impaired, correlates with increased rate of sudden arousal-triggered cardiac events. Our study suggests that the acute α1-AR-cPKC regulation of IKs is important for QT shortening in "fight-or-flight" response and is linked to decreased risk of sudden emotion/arousal-triggered cardiac events in LQT1 patients.

Isoform-specific dynamic translocation of PKC by α1-adrenoceptor stimulation in live cells.

Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated downstream of Gq-protein coupled receptor (GqPCR) signaling and translocate to various cellular compartments including plasma membrane (PM). Recent reports suggested that different types of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is one of the GqPCRs highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, ßI, ßII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (<30 s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCßI and PKCßII, to the PM (<30 s), with sustained membrane localization. One novel PKC (nPKC), PKCε, but not another nPKC, PKCδ, was translocated by α1-AR stimulation to the PM (<30 s) and its membrane localization was also sustained. Finally, α1-AR stimulation did not cause a diacylglycerol-insensitive atypical PKC, PKCζ translocation. Our data suggest that PKCα, ß and ε activation may underlie physiological and pathophysiological responses of α1-AR signaling for the phosphorylation of membrane-associated substrates including ion-channel and transporter proteins in the cardiovascular system.

Adrenergic signaling controls RGK-dependent trafficking of cardiac voltage-gated L-type Ca2+ channels through PKD1.

RATIONALE: The Rad-Gem/Kir-related family (RGKs) consists of small GTP-binding proteins that strongly inhibit the activity of voltage-gated calcium channels. Among RGKs, Rem1 is strongly and specifically expressed in cardiac tissue. However, the physiological role and regulation of RGKs, and Rem1 in particular, are largely unknown. OBJECTIVE: To determine if Rem1 function is physiologically regulated by adrenergic signaling and thus impacts voltage-gated L-type calcium channel (VLCC) activity in the heart. METHODS AND RESULTS: We found that activation of protein kinase D1, a protein kinase downstream of α(1)-adrenergic signaling, leads to direct phosphorylation of Rem1 at Ser18. This results in an increase of the channel activity and plasma membrane expression observed by using a combination of electrophysiology, live cell confocal microscopy, and immunohistochemistry in heterologous expression system and neonatal cardiomyocytes. In addition, we show that stimulation of α(1)-adrenergic receptor-protein kinase D1-Rem1 signaling increases transverse-tubule VLCC expression that results in increased L-type Ca(2+) current density in adult ventricular myocytes. CONCLUSION: The α(1)-adrenergic stimulation releases Rem1 inhibition of VLCCs through direct phosphorylation of Rem1 at Ser18 by protein kinase D1, resulting in an increase of the channel activity and transverse-tubule expression. Our results uncover a novel molecular regulatory mechanism of VLCC trafficking and function in the heart and provide the first demonstration of physiological regulation of RGK function.

Adeno-associated virus-based approach for genetic modification of cardiac fibroblasts in adult rat hearts.

Cardiac fibroblasts (CFs) are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is the key to develop successful therapies for treating the pressure-overloaded heart. CF-specific knockout (KO) mouse lines with a Cre recombinase under the control of human TCF21 (hTCF21) promoter and/or an adeno-associated virus serotype 9 (AAV9)-hTCF21 system provide a powerful tool for understanding CF biology in vivo. Although a variety of rat disease models are vital for the research of cardiac fibrosis similar to mouse models, there are few rat models that employ cardiac cell-specific conditional gene modification, which has hindered the development and translational relevance of cardiac disease models. In addition, to date, there are no reports of gene manipulation specifically in rat CFs in vivo. Here, we report a simplified CF-specific rat transgenic model using an AAV9-hTCF21 system that achieved a CF-specific expression of transgene in adult rat hearts. Moreover, we successfully applied this approach to specifically manipulate mitochondrial morphology in quiescent CFs. In summary, this model will allow us to develop fast and simple rat CF-specific transgenic models for studying cardiovascular diseases in vivo.

Mutations in cytoplasmic loops of the KCNQ1 channel and the risk of life-threatening events: implications for mutation-specific response to ß-blocker therapy in type 1 long-QT syndrome.

BACKGROUND: ß-Adrenergic stimulation is the main trigger for cardiac events in type 1 long-QT syndrome (LQT1). We evaluated a possible association between ion channel response to ß-adrenergic stimulation and clinical response to ß-blocker therapy according to mutation location. METHODS AND RESULTS: The study sample comprised 860 patients with genetically confirmed mutations in the KCNQ1 channel. Patients were categorized into carriers of missense mutations located in the cytoplasmic loops (C loops), membrane-spanning domain, C/N terminus, and nonmissense mutations. There were 27 aborted cardiac arrest and 78 sudden cardiac death events from birth through 40 years of age. After multivariable adjustment for clinical factors, the presence of C-loop mutations was associated with the highest risk for aborted cardiac arrest or sudden cardiac death (hazard ratio versus nonmissense mutations=2.75; 95% confidence interval, 1.29-5.86; P=0.009). ß-Blocker therapy was associated with a significantly greater reduction in the risk of aborted cardiac arrest or sudden cardiac death among patients with C-loop mutations than among all other patients (hazard ratio=0.12; 95% confidence interval, 0.02-0.73; P=0.02; and hazard ratio=0.82; 95% confidence interval, 0.31-2.13; P=0.68, respectively; P for interaction=0.04). Cellular expression studies showed that membrane spanning and C-loop mutations produced a similar decrease in current, but only C-loop mutations showed a pronounced reduction in channel activation in response to ß-adrenergic stimulation. CONCLUSIONS: Patients with C-loop missense mutations in the KCNQ1 channel exhibit a high risk for life-threatening events and derive a pronounced benefit from treatment with ß-blockers. Reduced channel activation after sympathetic activation can explain the increased clinical risk and response to therapy in patients with C-loop mutations.

Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca2+-induced ATP production in cardiac H9c2 myoblasts.

Ca(+) influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca(2+) influx is mainly mediated by the mitochondrial Ca(2+) uniporter (MCU). Growing evidence also indicates that mitochondrial Ca(2+) influx mechanisms are regulated not solely by MCU but also by multiple channels/transporters. We have previously reported that skeletal muscle-type ryanodine receptor (RyR) type 1 (RyR1), which expressed at the mitochondrial inner membrane, serves as an additional Ca(2+) uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca(2+) influx mechanism is the dominant regulator of mitochondrial morphology/dynamics and energetics in cardiomyocytes. To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology/function in cardiac cells, RyR1 was transiently or stably overexpressed in cardiac H9c2 myoblasts. We found that overexpressed RyR1 was partially localized in mitochondria as observed using both immunoblots of mitochondrial fractionation and confocal microscopy, whereas RyR2, the main RyR isoform in the cardiac sarcoplasmic reticulum, did not show any expression at mitochondria. Interestingly, overexpression of RyR1 but not MCU or RyR2 resulted in mitochondrial fragmentation. These fragmented mitochondria showed bigger and sustained mitochondrial Ca(2+) transients compared with basal tubular mitochondria. In addition, RyR1-overexpressing cells had a higher mitochondrial ATP concentration under basal conditions and showed more ATP production in response to cytosolic Ca(2+) elevation compared with nontransfected cells as observed by a matrix-targeted ATP biosensor. These results indicate that RyR1 possesses a mitochondrial targeting/retention signal and modulates mitochondrial morphology and Ca(2+)-induced ATP production in cardiac H9c2 myoblasts.

Alpha1-adrenenoceptor stimulation inhibits cardiac excitation-contraction coupling through tyrosine phosphorylation of beta1-adrenoceptor.

Adrenoceptor stimulation is a key determinant of cardiac excitation-contraction coupling mainly through the activation of serine/threonine kinases. However, little is known about the role of protein tyrosine kinases (PTKs) activated by adrenergic signaling on cardiac excitation-contraction coupling. A cytoplasmic tyrosine residue in ß1-adrenoceptor is estimated to regulate Gs-protein binding affinity from crystal structure studies, but the signaling pathway leading to the phosphorylation of these residues is unknown. Here we show α1-adrenergic signaling inhibits ß-adrenergically activated Ca(2+) current, Ca(2+) transients and contractile force through phosphorylation of tyrosine residues in ß1-adrenoceptor by PTK. Our results indicate that inhibition of ß-adrenoceptor-mediated Ca(2+) elevation by α1-adrenoceptor-PTK signaling serves as an important regulatory feedback mechanism when the catecholamine level increases to protect cardiomyocytes from cytosolic Ca(2+) overload.

Intermittent Fasting Activates AMP-Kinase to Restructure Right Ventricular Lipid Metabolism and Microtubules.

Intermittent fasting (IF) extends life span via pleotropic mechanisms, but one important molecular mediator is adenosine monophosphate-activated protein kinase (AMPK). AMPK enhances lipid metabolism and modulates microtubule dynamics. Dysregulation of these molecular pathways causes right ventricular (RV) failure in patients with pulmonary arterial hypertension. In rodent pulmonary arterial hypertension, IF activates RV AMPK, which restores mitochondrial and peroxisomal morphology and restructures mitochondrial and peroxisomal lipid metabolism protein regulation. In addition, IF increases electron transport chain protein abundance and activity in the right ventricle. Echocardiographic and hemodynamic measures of RV function are positively associated with fatty acid oxidation and electron transport chain protein levels. IF also combats heightened microtubule density, which normalizes transverse tubule structure.

Mitochondrial Ca 2+ uniporter (MCU) variants form plasma-membrane channels.

MCU is widely recognized as a responsible gene for encoding a pore-forming subunit of highly mitochondrial-specific and Ca 2+ -selective channel, mitochondrial Ca 2+ uniporter complex (mtCUC). Here, we report a novel short variant derived from the MCU gene (termed MCU-S) which lacks mitochondria-targeted sequence and forms a Ca 2+ - permeable channel outside of mitochondria. MCU-S was ubiquitously expressed in all cell-types/tissues, with particularly high expression in human platelets. MCU-S formed Ca 2+ channels at the plasma membrane, which exhibited similar channel properties to those observed in mtCUC. MCU-S channels at the plasma membrane served as an additional Ca 2+ influx pathway for platelet activation. Our finding is completely distinct from the originally reported MCU gene function and provides novel insights into the molecular basis of MCU variant-dependent cellular Ca 2+ handling.

Ion channel mechanisms related to sudden cardiac death in phenotype-negative long-QT syndrome genotype-phenotype correlations of the KCNQ1(S349W) mutation.

UNLABELLED: BACKGROUND: Data regarding possible ion channel mechanisms that predispose to ventricular tachyarrhythmias in patients with phenotype-negative long-QT syndrome (LQTS) are limited. METHODS AND RESULTS: We carried out cellular expression studies for the S349W mutation in the KCNQ1 channel, which was identified in 15 patients from the International LQTS Registry who experienced a high rate of cardiac events despite lack of significant QTc prolongation. The clinical outcome of S349W mutation carriers was compared with that of QTc-matched carriers of haploinsufficient missense (n = 30) and nonsense (n = 45) KCNQ1 mutations. The channels containing the mutant S349W subunit showed a mild reduction in current (<50%), in the haploinsuficient range, with an increase in maximal conductance compared with wild-type channels. In contrast, expression of the S349W mutant subunit produced a pronounced effect on both the voltage dependence of activation and the time constant of activation, while haploinsuficient channels showed no effect on either parameter. The cumulative probability of cardiac events from birth through age 20 years was significantly higher among S349W mutation carriers (58%) as compared with carriers of QTc-matched haploinsufficent missense (21%, P = 0.004) and nonsense (25%, P = 0.01) mutations. CONCLUSIONS: The S349W mutation in the KCNQ1 potassium channel exerts a relatively mild effect on the ion channel current, whereas an increase in conductance compensates for impaired voltage activation of the channel. The changes observed in voltage activation of the channel may underlie the mechanisms predisposing to arrhythmic risk among LQTS patients with a normal-range QTc.

Molecular basis of decreased Kir4.1 function in SeSAME/EAST syndrome.

SeSAME/EAST syndrome is a channelopathy consisting of a hypokalemic, hypomagnesemic, metabolic alkalosis associated with seizures, sensorineural deafness, ataxia, and developmental abnormalities. This disease links to autosomal recessive mutations in KCNJ10, which encodes the Kir4.1 potassium channel, but the functional consequences of these mutations are not well understood. In Xenopus oocytes, all of the disease-associated mutant channels (R65P, R65P/R199X, G77R, C140R, T164I, and A167V/R297C) had decreased K(+) current (0 to 23% of wild-type levels). Immunofluorescence demonstrated decreased surface expression of G77R, C140R, and A167V expressed in HEK293 cells. When we coexpressed mutant and wild-type subunits to mimic the heterozygous state, R199X, C140R, and G77R currents decreased to 55, 40, and 20% of wild-type levels, respectively, suggesting that carriers of these mutations may present with an abnormal phenotype. Because Kir4.1 subunits can form heteromeric channels with Kir5.1, we coexpressed the aforementioned mutants with Kir5.1 and found that currents were reduced at least as much as observed when we expressed mutants alone. Reduction of pH(i) from approximately 7.4 to 6.8 significantly decreased currents of all mutants except R199X but did not affect wild-type channels. In conclusion, perturbed pH gating may underlie the loss of channel function for the disease-associated mutant Kir4.1 channels and may have important physiologic consequences.

Life's Simple 7 cardiovascular health score and premature atrial contractions: The atherosclerosis risk in communities (ARIC) study.

BACKGROUND: Premature atrial contractions (PACs) are associated with increased risk of atrial fibrillation (AF) and ischemic stroke. Although lifestyle and risk factor modification reduces AF incidence, their relationship to PACs frequency is unclear. We assessed the association of Life's Simple 7 (LS7) and individual LS7 factors in midlife with PACs frequency in late life in the Atherosclerosis Risk in Communities (ARIC) Study. METHODS: We followed 1924 participants from ARIC clinic Visit 3 (1993--95) to Visit 6 (2016-17) when a 2-week continuous heart rhythm monitor (Zio®XT Patch) was applied. LS7 factors were assessed at Visit 3 and a composite score was calculated. PACs frequency was categorized as minimal (<0.1%), occasional (≥0.1%-5%) and frequent (>5%). Logistic regression was used to evaluate the association of LS7 score and individual factors with PACs frequency. RESULTS: Each 1-point LS7 score increase was associated with lower odds of frequent PACs vs. no PACs (OR [95% CI]: 0.87 [0.78, 0.98]) and frequent PACs vs. occasional PACs (OR [95% CI]: 0.88 [0.79, 0.98]). Of the individual LS7 factors, compared with ideal physical activity, poor physical activity was associated with 81% higher odds of frequent PACs vs. no PACs. Compared with ideal BMI, poor BMI was associated with 41% higher odds of occasional PACs vs. no PACs. CONCLUSION: Lifestyle risk factors, particularly physical activity and BMI, are associated with higher odds of PACs frequency. More research is needed to determine whether modifying these risk factors in midlife would prevent frequent PACs, and thereby prevent AF and stroke in older age.

α7 Nicotinic acetylcholine receptor mediates right ventricular fibrosis and diastolic dysfunction in pulmonary hypertension.

Right ventricular (RV) fibrosis is a key feature of maladaptive RV hypertrophy and dysfunction and is associated with poor outcomes in pulmonary hypertension (PH). However, mechanisms and therapeutic strategies to mitigate RV fibrosis remain unrealized. Previously, we identified that cardiac fibroblast α7 nicotinic acetylcholine receptor (α7 nAChR) drives smoking-induced RV fibrosis. Here, we sought to define the role of α7 nAChR in RV dysfunction and fibrosis in the settings of RV pressure overload as seen in PH. We show that RV tissue from PH patients has increased collagen content and ACh expression. Using an experimental rat model of PH, we demonstrate that RV fibrosis and dysfunction are associated with increases in ACh and α7 nAChR expression in the RV but not in the left ventricle (LV). In vitro studies show that α7 nAChR activation leads to an increase in adult ventricular fibroblast proliferation and collagen content mediated by a Ca2+/epidermal growth factor receptor (EGFR) signaling mechanism. Pharmacological antagonism of nAChR decreases RV collagen content and improves RV function in the PH model. Furthermore, mice lacking α7 nAChR exhibit improved RV diastolic function and have lower RV collagen content in response to persistently increased RV afterload, compared with WT controls. These finding indicate that enhanced α7 nAChR signaling is an important mechanism underlying RV fibrosis and dysfunction, and targeted inhibition of α7 nAChR is a potentially novel therapeutic strategy in the setting of increased RV afterload.