The tight junction protein CAR regulates cardiac conduction and cell–cell communication

The Coxsackievirus-adenovirus receptor (CAR) is known for its role in virus uptake and as a protein of the tight junction. It is predominantly expressed in the developing brain and heart and reinduced upon cardiac remodeling in heart disease. So far, the physiological functions of CAR in the adult heart are largely unknown. We have generated a heart-specific inducible CAR knockout (KO) and found impaired electrical conduction between atrium and ventricle that increased with progressive loss of CAR. The underlying mechanism relates to the cross talk of tight and gap junctions with altered expression and localization of connexins that affect communication between CAR KO cardiomyocytes. Our results indicate that CAR is not only relevant for virus uptake and cardiac remodeling but also has a previously unknown function in the propagation of excitation from the atrium to the ventricle that could explain the association of arrhythmia and Coxsackievirus infection of the heart

Function of the Coxsackievirus-Adenovirus-Receptor (CAR) in the mouse heart

Der Coxsackievirus-Adenovirus-Rezeptor (CAR) wurde zunächst als ein Virusrezeptor beschrieben, welcher die Bindung und Internalisierung von CVB und einigen Adenoviren vermittelt (Bergelson et al., 1997; Tomko et al., 1997). Als Tight Junction-Protein in Epithelzellen (Coyne et al., 2005) sorgt CAR durch Homo-Dimerisierung für die Aufrechterhaltung der interzellulären Diffusionsbarriere (Honda et al., 2000; Cohen et al., 2001). In den Kardiomyozyten ist CAR Bestandteil der Glanzstreifen. Erkrankungen des Herzens, wie Herzinfarkt oder Kardiomyopathie, können zu einer verstärkten CAR-Expression führen (Fechner et al., 2003). Die physiologische Funktion von CAR im gesunden Herzen war bislang aber nur unzureichend charakterisiert. In dieser Arbeit sollte die biologische Funktion von CAR im murinen Herzen durch die Analyse von zwei konditionellen CAR-KO-Tiermodellen untersucht werden. Es standen ein herzspezifisches und ein induzierbares, herzspezifisches CAR-KO- Modell für die Analyse zur Verfügung. CAR ist für eine normale Herzentwicklung essentiell. Eine CAR-Deletion in Kardiomyozyten führte zur embryonalen Letalität am Tag E12.5 mit einem beginnenden perikardialen Ödem, aber mit unveränderter Herzmorphologie. Verstärkte Proliferation oder Apoptose konnten in CAR-defizienten Embryonen nicht beobachtet werden. Um die Funktion von CAR im adulten Herzen zu untersuchen, wurde ein induzierbares, herzspezifisches CAR-KO-Modell analysiert. Die CAR-Defizienz im adulten Myokard führte bei den Tieren zu einem progressiven AV-Block und zu einer Sinusknotendysfunktion. Untersuchungen an CAR-defizienten Embryonen konnten ebenfalls eine AV- Erregungsleitungsstörung im Herzen nachweisen. Diese Ergebnisse weisen auf eine bisher unbekannte Rolle von CAR im Reizleitungssystem des Herzens hin. Der molekulare Mechanismus könnte auf der Interaktion von CAR mit Gap Junction-Proteinen beruhen. Wird die Interaktion von CAR mit Cx45 über ZO-1 unterbrochen (Lim et al., 2008), ist eine gestörte Kommunikation zwischen den Zellen des Reizleitungssystems und damit eine gestörte Erregungsleitung zu erwarten. Die veränderte Kommunikation über Gap Junctions kann zu unterschiedlichen Effekten im Herzen führen. Im Reizleitungssystem entwickelten sich ein AV-Block und das Syndrom des kranken Sinusknotens, während ventrikuläre Arrhythmien nicht auftraten. Eine mögliche Erklärung sind die zellulären und molekularen Unterschiede zwischen Schrittmacherzellen und Kardiomyozyten des Arbeitsmyokards, welche Zellform, elektrische Erregung, Proteinexpression und CAR-Lokalisation betreffen. Bisher ist kein genetischer Defekt im CAR-Gen als krankheitsrelevant beschrieben worden. Die neuen tierexperimentellen Erkenntnisse implizieren, dass CAR in der Pathogenese von Herzrhythmusstörungen unklarer Genese eine Rolle spielt. Weiterhin könnten Arrhythmien bei Kardiomyopathie-Patienten die Folge einer Autoimmunreaktion gegen CAR sein. Damit empfiehlt sich die Berücksichtigung von CAR und Proteinen der Tight Junctions in der Entwicklung von Screening-Strategien für Arrhythmie-Patienten und als mögliche Zielproteine für therapeutische Ansätze.The Coxsackievirus-Adenovirus-Receptor (CAR) was discovered as a virus receptor for coxsackie- and adenoviruses and mediates both virus binding and internalization (Bergelson et al., 1997; Tomko et al., 1997). It is closely associated with the tight junction in epithial cells (Coyne et al., 2005) and it is important for cell-cell attachment by homo-dimerization (Honda et al., 2000; Cohen et al., 2001). In the heart CAR is a component of the intercalated disk in cardiomyocytes and in cardiac diseases, such as myocardial infarction or cardiomyopathy, CAR is up-regulated (Fechner et al., 2003), but the function in the healthy myocardium, has not been well characterized. Aim of this study was to investigate the biological function of CAR in the heart with the help of two conditional CAR KO models. One is a heart specific KO with expression of the Cre recombinase under the control of αMHC promoter. The other is a heart specific inducible CAR KO (dependent on tamoxifen-treatment). After verification of the two KO models the cardiac function was evaluated. CAR deletion in cardiomyocytes results in embryonic lethality at E12.5 with lightly signs of pericardial edema, but it was not related to altered organization of myofibrils and increased proliferation or apoptosis. The inducible CAR KO model was used to investigate the role of CAR in the adult myocardium. Functional analysis of the CAR deficient adult mice revealed progressive atrio-ventricular block and sinus node dysfunction that developed in parallel with the loss of CAR. Echocardiography of cardiac KO embryos showed a similar atrio-ventricular conduction defect. These novel findings indicated a specific function of CAR in the conduction system of the heart. The underlying mechanism could involve the interaction between CAR and gap junction proteins. The disruption of the published interaction between CAR and Cx45 via ZO-1 (Lim et al., 2008) is expected to affect communication between the cells of the conduction system and thus cause an impaired electrical conduction. An altered cell-cell communication can cause differential effects in the heart - on the one hand the development of an AV block, on the other hand an increased coupling in the ventricle. A possible explanation could be derived from the cellular and molecular differences between cells of the conduction system and the ventricular myocardium, which include differences in cell shape, action potential propagation, protein expression, and localization of CAR. So far, no genetic defect underlying human disease has been described in the literature. The in vivo data imply CAR in the pathogenesis of arrhythmia of unknown cause. Furthermore, they raise the possibility that CAR directed auto-antibodies might contribute to the development of arrhythmia in patients suffering from cardiomyopathy. These novel mechanistic insights into the interaction of CAR and gap junction proteins could help explain the association of viral disease and arrhythmia and suggest CAR as a drug target not only in viral myocarditis, but also in arrhythmia

Hypochlorhydria reduces mortality in heart failure caused by Kcne2 gene deletion

Heart failure (HF) is an increasing global health crisis, affecting 40 million people and causing 50% mortality within 5 years of diagnosis. A fuller understanding of the genetic and environmental factors underlying HF, and novel therapeutic approaches to address it, are urgently warranted. Here, we discovered that cardiac‐specific germline deletion in mice of potassium channel β subunit‐ encoding Kcne2 (Kcne2 CS(−/−)) causes dilated cardiomyopathy and terminal HF (median longevity, 28 weeks). Mice with global Kcne2 deletion (Kcne2 Glo(−/−)) exhibit multiple HF risk factors, yet, paradoxically survived over twice as long as Kcne2 CS(−/−) mice. Global Kcne2 deletion, which inhibits gastric acid secretion, reduced the relative abundance of species within Bacteroidales, a bacterial order that positively correlates with increased lifetime risk of human cardiovascular disease. Strikingly, the proton‐pump inhibitor omeprazole similarly altered the microbiome and delayed terminal HF in Kcne2 CS(−/−) mice, increasing survival 10‐fold at 44 weeks. Thus, genetic or pharmacologic induction of hypochlorhydria and decreased gut Bacteroidales species are associated with lifespan extension in a novel HF model

Kcne2 deletion causes early-onset nonalcoholic fatty liver disease via iron deficiency anemia.

Nonalcoholic fatty liver disease (NAFLD) is an increasing health problem worldwide, with genetic, epigenetic, and environmental components. Here, we describe the first example of NAFLD caused by genetic disruption of a mammalian potassium channel subunit. Mice with germline deletion of the KCNE2 potassium channel β subunit exhibited NAFLD as early as postnatal day 7. Using mouse genetics, histology, liver damage assays and transcriptomics we discovered that iron deficiency arising from KCNE2-dependent achlorhydria is a major factor in early-onset NAFLD in Kcne2(─/─) mice, while two other KCNE2-dependent defects did not initiate NAFLD. The findings uncover a novel genetic basis for NAFLD and an unexpected potential factor in human KCNE2-associated cardiovascular pathologies, including atherosclerosis

Kcne2 deletion causes early-onset nonalcoholic fatty liver disease via iron deficiency anemia.

Nonalcoholic fatty liver disease (NAFLD) is an increasing health problem worldwide, with genetic, epigenetic, and environmental components. Here, we describe the first example of NAFLD caused by genetic disruption of a mammalian potassium channel subunit. Mice with germline deletion of the KCNE2 potassium channel β subunit exhibited NAFLD as early as postnatal day 7. Using mouse genetics, histology, liver damage assays and transcriptomics we discovered that iron deficiency arising from KCNE2-dependent achlorhydria is a major factor in early-onset NAFLD in Kcne2(─/─) mice, while two other KCNE2-dependent defects did not initiate NAFLD. The findings uncover a novel genetic basis for NAFLD and an unexpected potential factor in human KCNE2-associated cardiovascular pathologies, including atherosclerosis

Deletion in mice of X-linked, Brugada syndrome- and atrial fibrillation-associated Kcne5 augments ventricular KV currents and predisposes to ventricular arrhythmia

KCNE5 is an X-linked gene encoding KCNE5, an ancillary subunit to voltage-gated potassium (K(V)) channels. Human KCNE5 mutations are associated with atrial fibrillation (AF)- and Brugada syndrome (BrS)-induced cardiac arrhythmias that can arise from increased potassium current in cardiomyocytes. Seeking to establish underlying molecular mechanisms, we created and studied Kcne5 knockout (Kcne5(-/0)) mice. Intracardiac ECG revealed that Kcne5 deletion caused ventricular premature beats, increased susceptibility to induction of polymorphic ventricular tachycardia (60 vs. 24% in Kcne5(+/0) mice), and 10% shorter ventricular refractory period. Kcne5 deletion increased mean ventricular myocyte K(V) current density in the apex and also in the subpopulation of septal myocytes that lack fast transient outward current (I(to,f)). The current increases arose from an apex-specific increase in slow transient outward current-1 (I(Kslow,1)) (conducted by K(V)1.5) and I(to,f) (conducted by K(V)4) and an increase in I(Kslow,2) (conducted by K(V)2.1) in both apex and septum. Kcne5 protein localized to the intercalated discs in ventricular myocytes, where K(V)2.1 was also detected in both Kcne5(-/0) and Kcne5(+/0) mice. In HL-1 cardiac cells and human embryonic kidney cells, KCNE5 and K(V)2.1 colocalized at the cell surface, but predominantly in intracellular vesicles, suggesting that Kcne5 deletion increases I(K,slow2) by reducing K(V)2.1 intracellular sequestration. The human AF-associated mutation KCNE5-L65F negative shifted the voltage dependence of K(V)2.1-KCNE5 channels, increasing their maximum current density >2-fold, whereas BrS-associated KCNE5 mutations produced more subtle negative shifts in K(V)2.1 voltage dependence. The findings represent the first reported native role for Kcne5 and the first demonstrated Kcne regulation of K(V)2.1 in mouse heart. Increased K(V) current is a manifestation of KCNE5 disruption that is most likely common to both mouse and human hearts, providing a plausible mechanistic basis for human KCNE5-linked AF and BrS

Deletion in mice of X-linked, Brugada syndrome- and atrial fibrillation-associated Kcne5 augments ventricular K-V currents and predisposes to ventricular arrhythmia

KCNE5 is an X-linked gene encoding KCNE5, an ancillary subunit to voltage-gated potassium (K(V)) channels. Human KCNE5 mutations are associated with atrial fibrillation (AF)- and Brugada syndrome (BrS)-induced cardiac arrhythmias that can arise from increased potassium current in cardiomyocytes. Seeking to establish underlying molecular mechanisms, we created and studied Kcne5 knockout (Kcne5(-/0)) mice. Intracardiac ECG revealed that Kcne5 deletion caused ventricular premature beats, increased susceptibility to induction of polymorphic ventricular tachycardia (60 vs. 24% in Kcne5(+/0) mice), and 10% shorter ventricular refractory period. Kcne5 deletion increased mean ventricular myocyte K(V) current density in the apex and also in the subpopulation of septal myocytes that lack fast transient outward current (I(to,f)). The current increases arose from an apex-specific increase in slow transient outward current-1 (I(Kslow,1)) (conducted by K(V)1.5) and I(to,f) (conducted by K(V)4) and an increase in I(Kslow,2) (conducted by K(V)2.1) in both apex and septum. Kcne5 protein localized to the intercalated discs in ventricular myocytes, where K(V)2.1 was also detected in both Kcne5(-/0) and Kcne5(+/0) mice. In HL-1 cardiac cells and human embryonic kidney cells, KCNE5 and K(V)2.1 colocalized at the cell surface, but predominantly in intracellular vesicles, suggesting that Kcne5 deletion increases I(K,slow2) by reducing K(V)2.1 intracellular sequestration. The human AF-associated mutation KCNE5-L65F negative shifted the voltage dependence of K(V)2.1-KCNE5 channels, increasing their maximum current density >2-fold, whereas BrS-associated KCNE5 mutations produced more subtle negative shifts in K(V)2.1 voltage dependence. The findings represent the first reported native role for Kcne5 and the first demonstrated Kcne regulation of K(V)2.1 in mouse heart. Increased K(V) current is a manifestation of KCNE5 disruption that is most likely common to both mouse and human hearts, providing a plausible mechanistic basis for human KCNE5-linked AF and BrS