Mohamed Boulaksil

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.

Combined reduction of intercellular coupling and membrane excitability differentially affects transverse and longitudinal cardiac conduction

AIMS Reduced excitability and gap junction expression are commonly found in electrically remodelled diseased hearts, but their contribution to slow conduction and arrhythmias is unclear. In this study, we have investigated the effect of isolated and combined reductions in membrane excitability and intercellular coupling on impulse propagation and arrhythmogeneity in genetically modified mice. METHODS AND RESULTS Cx43 and Scn5a(1798insD/+) heterozygous (HZ) mice were crossbred to create a mixed offspring: wild-type (WT, n = 15), Cx43 HZ (n = 14), Scn5a(1798insD/+) (Scn5a) HZ (n = 17), and Cx43/Scn5a(1798insD/+) (Cx43/Scn5a) HZ (n = 15) mice. After ECG recording, epicardial activation mapping (208 recording sites) was performed on Langendorff-perfused hearts. Arrhythmia inducibility was tested by one to three premature stimuli and burst pacing. Conduction velocity longitudinal (CV(L)) and transverse (CV(T)) to fibre orientation and dispersion of conduction were determined during S1-S1 pacing (150 ms). Connexin43 (Cx43) and sodium channel Nav1.5 protein expression and myocardial tissue collagen content were determined by immunohistology. Compared with WT animals, P, QRS, and QTc intervals were prolonged in Scn5a HZ and Cx43/Scn5a HZ, but not in Cx43 HZ animals. Scn5a HZ mice showed decreased CV(L) in right ventricle (RV) but not in left ventricle compared with WT. In the RV of Cx43/Scn5a HZ, CV(T) was reduced, but CV(L) was not different from WT. Arrhythmia inducibility was low and not increased in either single- or double-mutant mice. CONCLUSION Reduction of both electrical coupling and excitability results in normal conduction velocity parallel to fibre orientation but in pronounced conduction slowing transverse to fibre orientation in RV only, although this does not affect arrhythmogeneity.

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.

Drug-induced torsade de pointes arrhythmias in the chronic AV block dog are perpetuated by focal activity

Background— The electrically remodeled canine heart after chronic AV block (CAVB) has a high susceptibility for drug-induced torsade de pointes (TdP) arrhythmias. Although focal mechanisms have been considered for initiation, there is still controversy about whether reentry is the dominant mechanism for perpetuation of TdP. In this animal model with known nonuniform prolongation of repolarization, the mechanism of perpetuation of TdP arrhythmia was explored. Methods and Results— Seventeen TdP-sensitive CAVB and 10 sinus rhythm (SR) dogs were studied. In 6 animals, 66 needle electrodes were evenly distributed transmurally to record 240 unipolar local electrograms simultaneously. Activation times and activation recovery intervals were determined before and during ibutilide-induced TdP. In 12 CAVB and 9 SR dogs, left ventricular (LV) and right ventricular (RV) epicardial electrograms were recorded with a 208-point multiterminal grid electrode allowing conduction velocity (CV) and ventricular effective refractory period (VERP) measurements. Biopsy specimens were processed for connexin43 (Cx43) expression and collagen content. Ventricular myocytes were isolated to determine sodium current (INa) density and cell dimensions. Computer simulations were used to assess the effects of changes therein. In CAVB, VERP and ARI were increased, whereas CV was unaltered in LV. Transversal but not longitudinal CV was increased in RV. INa was reduced by 37% in LV but unaltered in RV. LV and RV cell size were increased, but collagen and Cx43 content remained unchanged. Simulations showed increase in CV of RV as a consequence of increased cell size at normal INa. Ibutilide increased ARI, ERP, and maximal transmural dispersion of ERP (45±25 to 120±65 ms; P<0.05). Twenty-eight of 47 episodes of self-terminating TdP (43±72 beats) were analyzed. The majority (>90%) of beats were focal; reentry was observed only occasionally. Conclusions— Focal activity is the dominant mechanism involved in perpetuation of ibutilide-induced TdP in CAVB dogs based on detailed 3D mapping. This conclusion is in line with unaltered conduction and documented increase in VERP.

In calcineurin-induced cardiac hypertrophy expression of Nav1.5, Cx40 and Cx43 is reduced by different mechanisms

Alterations in expression levels of Na(v)1.5, Cx43 and Cx40 have been frequently reported in cardiac disease and are associated with the development of arrhythmias, but little is known about the underlying molecular mechanisms. In this study we investigated electrical conduction and expression of Na(v)1.5, Cx43 and Cx40 in hearts of transgenic mice overexpressing a constitutively active form of calcineurin (MHC-CnA). ECG recordings showed that atrial, atrioventricular and ventricular activation were significantly prolonged in MHC-CnA hearts as compared to wildtype (WT) littermates. Epicardial activation and arrhythmia susceptibility analysis revealed increased ventricular activation thresholds and arrhythmia vulnerability. Moreover, epicardial ventricular activation patterns in MHC-CnA mice were highly discontinuous with multiple areas of block. These impaired conduction properties were associated with severe reductions in Na(v)1.5, Cx43 and Cx40 protein expression in MHC-CnA hearts as visualized by immunohistochemistry and immunoblotting. Real-time RT-PCR demonstrated that the decreased protein levels for Na(v)1.5 and Cx40, but not for Cx43, were accompanied by corresponding reductions at the RNA level. Cx43 RNA isoform analysis indicated that the reduction in Cx43 protein expression is caused by a post-transcriptional mechanism rather than by RNA isoform switching. In contrast, RNA isoform analysis for Cx40 and Na(v)1.5 provided additional evidence that in calcineurin-induced hypertrophy the downregulation of these proteins originates at the transcriptional level. These results provide the molecular rationale for Na(v)1.5, Cx43 and Cx40 downregulation in this model of hypertrophy and failure and the development of the pro-arrhythmic substrate.

Longitudinal arrhythmogenic remodelling in a mouse model of longstanding pressure overload

Introduction. Sudden arrhythmogenic cardiac death is a major cause of mortality in patients with congestive heart failure due to adverse electrical remodelling. To establish whether abnormal conduction is responsible for arrhythmogenic remodelling in progressed stages of heart failure, we have monitored functional, structural and electrical remodelling in a murine model of heart failure, induced by longstanding pressure overload.Methods. Mice were subjected to transverse aortic constriction (TAC; n=18) or sham operated (n=19) and monitored biweekly by echocardiography and electrocardiography. At the 16-week endpoint, electrical mapping was performed to measure epicardial conduction velocity and susceptibility to arrhythmias. Finally, tissue sections were stained for Cx43 and fibrosis.Results. In TAC mice, fractional shortening decreased gradually and was significantly lower compared with sham at 16 weeks. Left ventricular hypertrophy was significant after six weeks. TAC mice developed PQ prolongation after 12 weeks, QT prolongation after 16 weeks and QRS prolongation after two weeks. Right ventricular conduction velocity was slowed parallel to fibre orientation. In 8/18 TAC hearts, polymorphic ventricular tachyarrhythmias were provoked and none in sham hearts. TAC mice had more interstitial fibrosis than sham. Immunohistology showed that Cx43 levels were similar but highly heterogeneous in TAC mice. All parameters were comparable in TAC mice with and without arrhythmias, except for Cx43 heterogeneity, which was significantly higher in arrhythmogenic TAC mice.Conclusion. Chronic pressure overload resulted in rapid structural and electrical remodelling. Arrhythmias were related to heterogeneous expression of Cx43. This may lead to functional block and unstable reentry, giving rise to polymorphic ventricular tachyarrhythmias. (Neth Heart J 2010;18:509-15.)

Passive ventricular remodeling in cardiac disease: focus on heterogeneity

Passive ventricular remodeling is defined by the process of molecular ventricular adaptation to different forms of cardiac pathophysiology. It includes changes in tissue architecture, such as hypertrophy, fiber disarray, alterations in cell size and fibrosis. Besides that, it also includes molecular remodeling of gap junctions, especially those composed by Connexin43 proteins (Cx43) in the ventricles that affect cell-to-cell propagation of the electrical impulse, and changes in the sodium channels that modify excitability. All those alterations appear mainly in a heterogeneous manner, creating irregular and inhomogeneous electrical and mechanical coupling throughout the heart. This can predispose to reentry arrhythmias and adds to a further deterioration into heart failure. In this review, passive ventricular remodeling is described in Hypertrophic Cardiomyopathy (HCM), Dilated Cardiomyopathy (DCM), Ischemic Cardiomyopathy (ICM), and Arrhythmogenic Cardiomyopathy (ACM), with a main focus on the heterogeneity of those alterations mentioned above.

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.

Spatial Heterogeneity of Cx43 is an Arrhythmogenic Substrate of Polymorphic Ventricular Tachycardias during Compensated Cardiac Hypertrophy in Rats

Background Ventricular remodeling increases the propensity of ventricular tachyarrhythmias and sudden death in patients. We studied the mechanism underlying these fatal arrhythmias, electrical and structural cardiac remodeling, as well as arrhythmogeneity during early, compensated hypertrophy in a rat model of chronic pressure overload. Methods Twenty-six Wistar rats were subjected to transverse aortic constriction (TAC) (n = 13) or sham operation (n = 13). Four weeks postoperative, echo- and electrocardiography was performed. Epicardial (208 or 455 sites) and transmural (30 sites) ventricular activation mapping was performed on Langendorff perfused hearts. Subsequently, hearts were processed for (immuno)histological and molecular analyses. Results TAC rats showed significant hypertrophy with preserved left ventricular (LV) function. Epicardial conduction velocity (CV) was similar, but more dispersed in TAC. Transmural CV was slowed in TAC (37.6 ± 2.9 cm s−1) compared to sham (58.5 ± 3.9 cm s−1; P < 0.01). Sustained polymorphic ventricular tachycardias were induced from LV in 8/13 TAC and in 0/13 sham rats. During VT, electrical activation patterns showed variable sites of earliest epicardial activation and altering sites of functional conduction block. Wandering epicardial reentrant activation was sporadically observed. Collagen deposition was significantly higher in TAC compared to sham, but not different between arrhythmogenic and non-arrhythmogenic TAC animals. Connexin43 (Cx43) expression was heterogeneous with a higher prevalence of non-phosphorylated Cx43 in arrhythmogenic TAC animals. Conclusion In TAC rats with compensated cardiac hypertrophy, dispersion of conduction correlated to arrhythmogenesis, an increased heterogeneity of Cx43, and a partial substitution with non-phosphorylated Cx43. These alterations may result in the increased vulnerability to polymorphic VTs.