John A. Jansen

Cardiac connexins and impulse propagation

Gap junctions form the intercellular pathway for cell-to-cell transmission of the cardiac impulse from its site of origin, the sinoatrial node, along the atria, the atrioventricular conduction system to the ventricular myocardium. The component parts of gap junctions are proteins called connexins (Cx), of which three main isoforms are found in the conductive and working myocardial cells: Cx40, Cx43, and Cx45. These isoforms are regionally expressed in the heart, which suggests a specific role or function of a specific connexin in a certain part of the heart. Using genetically modified mice, the function of these connexins in the different parts of the heart have been assessed in the past years. This review will follow the cardiac impulse on its path through the heart and recapitulate the role of the different connexins in the different cardiac compartments.

Reduction of fibrosis-related arrhythmias by chronic renin-angiotensin-aldosterone system inhibitors in an aged mouse model

Myocardial fibrosis increases arrhythmia vulnerability of the diseased heart. The renin-angiotensin-aldosterone system (RAAS) governs myocardial collagen synthesis. We hypothesized that reducing cardiac fibrosis by chronic RAAS inhibition would result in reduced arrhythmia vulnerability of the senescent mouse heart. Wild-type mice (52 wk old) were treated for 36 wk: 1) untreated control (C); 2) eplerenone (E); 3) losartan (L); and 4) cotreatment with eplerenone and losartan (EL). Ventricular epicardial activation mapping was performed on Langendorff-perfused hearts. Arrhythmia inducibility was tested by one to three premature stimuli and burst pacing. Longitudinal and transverse conduction velocity and dispersion of conduction were determined during pacing at a basic cycle length of 150 ms. Sirius red staining (collagen) was performed. As a result, in the RV of mice in the E, L, and EL groups, transverse conduction velocity was significantly increased and anisotropic ratio was significantly decreased compared with those values of mice in the C group. Anisotropic reentrant arrhythmias were induced in 52% of untreated mice and significantly reduced to 22%, 26%, and 16% in the E, L, and EL groups, respectively. Interstitial fibrosis was significantly decreased in both the RV and LV of all treated groups. Scattered patches of replacement fibrosis were found in 90% of untreated hearts, which were significantly reduced in the E, L, and EL groups. A strong correlation between the abundance of patchy fibrosis and arrhythmia inducibility was found. In conclusion, chronic RAAS inhibition limited aging-related interstitial fibrosis. The lower arrhythmogeneity of treated mice was directly correlated to the reduced amount of patchy fibrosis.

Heterogeneous Connexin43 distribution in heart failure is associated with dispersed conduction and enhanced susceptibility to ventricular arrhythmias

Sudden arrhythmogenic cardiac death is a major cause of mortality in patients with congestive heart failure (CHF). To investigate determinants of the increased arrhythmogenic susceptibility, we studied cardiac remodelling and arrhythmogenicity in CHF patients and in a mouse model of chronic pressure overload.

Dominant arrhythmia vulnerability of the right ventricle in senescent mice

BACKGROUND Several cardiac disorders affect the right ventricle (RV) and left ventricle (LV) equally, but nevertheless, RV vulnerability to conduction slowing and arrhythmias exceeds that of the LV. OBJECTIVE This study sought to assess the mechanism of dominant RV arrhythmia vulnerability in senescent mice as a model of general reduced myocardial integrity. METHODS Epicardial ventricular activation mapping was performed on senescent (22 months) and adult (3 months) Langendorff perfused mouse hearts. Arrhythmia inducibility was tested by programmed stimulation. Conduction velocity longitudinal and transversal (CVT) to fiber orientation, conduction heterogeneity, and effective refractory period were determined. Subsequently, hearts were processed for immunohistochemistry, Western blotting, and Sirius red staining. RESULTS In senescent RV, but not LV, CVT was reduced and wavelength decreased, whereas anisotropic ratio and conduction heterogeneity increased. Arrhythmias, based on anisotropic reentry, were induced in 55% of senescent hearts only and predominantly in RV. In senescent mice, Connexin 43 (Cx43) and Cardiac Sodium Channel (Nav1.5) were decreased and interstitial fibrosis increased comparably in RV and LV. However, in senescent mice, heterogeneously distributed patches of replacement fibrosis were present throughout the entire RV myocardium, but only in midendocardium and subendocardium of LV. Cx43 expression in these areas was disrupted. CONCLUSION Widespread presence of replacement fibrosis in senescent RV compared with LV, combined with Cx43 and Nav1.5 disruption, potentiate shorter wavelength, conduction slowing, and conduction heterogeneity in RV, resulting in greater vulnerability of senescent RV to arrhythmias.

Reduced Cx43 expression triggers increased fibrosis due to enhanced fibroblast activity.

Background— Arrhythmogenic ventricular remodeling is hallmarked by both reduced gap junction expression and increased collagen deposition. We hypothesized that reduced connexin43 (Cx43) expression is responsible for enhanced fibrosis in the remodeled heart, resulting in an arrhythmogenic substrate. Therefore, we investigated the effect of normal or reduced Cx43 expression on the formation of fibrosis in a physiological (aging) and pathophysiological (transverse aortic constriction [TAC]) mouse model. Methods and Results— The Cx43fl/fl and Cx43CreER(T)/fl mice were aged 18 to 21 months or, at the age of 3 months, either TAC or sham operated and euthanized after 16 weeks. Epicardial activation mapping of the right and left ventricles was performed on Langendorff perfused hearts. Sustained ventricular arrhythmias were induced in 0 of 11 aged Cx43fl/fl and 10 of 15 Cx43Cre-ER(T)/fl mice (P<0.01). Cx43 expression was reduced by half in aged Cx43CreER(T)/fl compared with aged Cx43fl/fl mice, whereas collagen deposition was significantly increased from 1.1±0.2% to 7.4±1.3%. Aged Cx43CreER(T)/fl mice with arrhythmias had significantly higher levels of fibrosis and conduction heterogeneity than aged Cx43CreER(T)/fl mice without arrhythmias. The TAC operation significantly increased fibrosis in control compared with sham (4.0±1.2% versus 0.4±0.06%), but this increase was significantly higher in Cx43CreER(T)/fl mice (10.8±1.4%). Discoidin domain receptor 2 expression was unchanged, but procollagen peptide I and III expression and collagen type 1&agr;2 mRNA levels were higher in TAC–operated Cx43HZ mice. Conclusions— Reduced cellular coupling results in more excessive collagen deposition during aging or pressure overload in mice due to enhanced fibroblast activity, leading to increased conduction in homogeneity and proarrhythmia.

Lysosome mediated Kir2.1 breakdown directly influences inward rectifier current density

The inward rectifier current generated by Kir2.1 ion channel proteins is primarily responsible for the stable resting membrane potential in various excitable cell types, like neurons and myocytes. Tight regulation of Kir2.1 functioning prevents premature action potential formation and ensures optimal repolarization times. While Kir2.1 forward trafficking has been addressed in a number of studies, its degradation pathways are thus far unknown. Using three different lysosomal inhibitors, NH(4)Cl, chloroquine and leupeptin, we now demonstrate involvement of the lysosomal degradation pathway in Kir2.1 breakdown. Upon application of the inhibitors, increased steady state protein levels are detectable within few hours coinciding with intracellular granular Kir2.1 accumulation. Treatment for 24h with either chloroquine or leupeptin results in increased plasmamembrane originating inward rectifier current densities, while current-voltage characteristics remain unaltered. We conclude that the lysosomal degradation pathway contributes to Kir2.1 mediated inward rectifier current regulation.

Inhibition of Cardiomyocyte Automaticity by Electrotonic Application of Inward Rectifier Current from Kir2.1 Expressing Cells

A biological pacemaker might be created by generation of a cellular construct consisting of cardiac cells that display spontaneous membrane depolarization, and that are electrotonically coupled to surrounding myocardial cells by means of gap junctions. Depending on the frequency of the spontaneously beating cells, frequency regulation might be required. We hypothesized that application of Kir2.1 expressing non-cardiac cells, which provide IK1 to spontaneously active neonatal cardiomyocytes (NCMs) by electrotonic coupling in such a cellular construct, would generate an opportunity for pacemaker frequency control. Non-cardiac Kir2.1 expressing cells were co-cultured with spontaneously active rat NCMs. Electrotonic coupling between the two cell types resulted in hyperpolarization of the cardiomyocyte membrane potential and silencing of spontaneous activity. Either blocking of gap-junctional communication by halothane or inhibition of IK1 by BaCl2 restored the original membrane potential and spontaneous activity of the NCMs. Our results demonstrate the power of electrotonic coupling for the application of specific ion currents into an engineered cellular construct such as a biological pacemaker.

Calmodulin/CaMKII inhibition improves intercellular communication and impulse propagation in the heart and is antiarrhythmic under conditions when fibrosis is absent

AIM In healthy hearts, ventricular gap junctions are mainly composed by connexin43 (Cx43) and localize in the intercalated disc, enabling appropriate electrical coupling. In diseased hearts, Cx43 is heterogeneously down-regulated, whereas activity of calmodulin/calcium-calmodulin protein kinase II (CaM/CaMKII) signalling increases. It is unclear if CaM/CaMKII affects Cx43 expression/localization or impulse propagation. We analysed different models to assess this. METHODS AND RESULTS AC3-I mice with CaMKII genetically inhibited were subjected to pressure overload (16 weeks, TAC vs. sham). Optical and epicardial mapping was performed on Langendorff-perfused rabbit and AC3-I hearts, respectively. Cx43 subcellular distribution from rabbit/mouse ventricles was evaluated by immunoblot after Triton X-100-based fractionation. In mice with constitutively reduced CaMKII activity (AC3-I), conduction velocity (CV) was augmented (n = 11, P < 0.01 vs. WT); in AC3-I, CV was preserved after TAC, in contrast to a reduction seen in TAC-WT mice (-20%). Cx43 expression was preserved after TAC in AC3-I mice, though arrhythmias and fibrosis were still present. In rabbits, W7 (CaM inhibitor, 10 µM) increased CV (6-13%, n= 6, P< 0.05), while susceptibility to arrhythmias decreased. Immunoconfocal microscopy revealed enlarged Cx43 cluster sizes at intercalated discs of those hearts. Total Cx43 did not change by W7 (n= 4), whereas Triton X-100 insoluble Cx43 increased (+21%, n= 4, P< 0.01). Similar findings were obtained in AC3-I mouse hearts when compared with control, and in cultured dog cardiomyocytes. Functional implication was shown through increased intercellular coupling in cultured neonatal rat cardiomyocytes. CONCLUSION Both acute and chronic CaM/CaMKII inhibition improves conduction characteristics and enhances localization of Cx43 in the intercalated disc. In the absence of fibrosis, this reduced the susceptibility for arrhythmias.

A common co-morbidity modulates disease expression and treatment efficacy in inherited cardiac sodium channelopathy

Aims Management of patients with inherited cardiac ion channelopathy is hindered by variability in disease severity and sudden cardiac death (SCD) risk. Here, we investigated the modulatory role of hypertrophy on arrhythmia and SCD risk in sodium channelopathy. Methods and results Follow-up data was collected from 164 individuals positive for the SCN5A-1795insD founder mutation and 247 mutation-negative relatives. A total of 38 (obligate) mutation-positive patients died suddenly or suffered life-threatening ventricular arrhythmia. Of these, 18 were aged >40 years, a high proportion of which had a clinical diagnosis of hypertension and/or cardiac hypertrophy. While pacemaker implantation was highly protective in preventing bradycardia-related SCD in young mutation-positive patients, seven of them aged >40 experienced life-threatening arrhythmic events despite pacemaker treatment. Of these, six had a diagnosis of hypertension/hypertrophy, pointing to a modulatory role of this co-morbidity. Induction of hypertrophy in adult mice carrying the homologous mutation (Scn5a1798insD/+) caused SCD and excessive conduction disturbances, confirming a modulatory effect of hypertrophy in the setting of the mutation. The deleterious effects of the interaction between hypertrophy and the mutation were prevented by genetically impairing the pro-hypertrophic response and by pharmacological inhibition of the enhanced late sodium current associated with the mutation. Conclusion This study provides the first evidence for a modulatory effect of co-existing cardiac hypertrophy on arrhythmia risk and treatment efficacy in inherited sodium channelopathy. Our findings emphasize the need for continued assessment and rigorous treatment of this co-morbidity in SCN5A mutation-positive individuals.