Riyaz A. Kaba

Genetic variation in SCN10A influences cardiac conduction

To identify genetic factors influencing cardiac conduction, we carried out a genome-wide association study of electrocardiographic time intervals in 6,543 Indian Asians. We identified association of a nonsynonymous SNP, rs6795970, in SCN10A (P = 2.8 × 10−15) with PR interval, a marker of cardiac atrioventricular conduction. Replication testing among 6,243 Indian Asians and 5,370 Europeans confirmed that rs6795970 (G>A) is associated with prolonged cardiac conduction (longer P-wave duration, PR interval and QRS duration, P = 10−5 to 10−20). SCN10A encodes NaV1.8, a sodium channel. We show that SCN10A is expressed in mouse and human heart tissue and that PR interval is shorter in Scn10a−/− mice than in wild-type mice. We also find that rs6795970 is associated with a higher risk of heart block (P < 0.05) and a lower risk of ventricular fibrillation (P = 0.01). Our findings provide new insight into the pathogenesis of cardiac conduction, heart block and ventricular fibrillation.

Immunocytochemical analysis of connexin expression in the healthy and diseased cardiovascular system.

Gap junctions play essential roles in the normal function of the heart and arteries, mediating the spread of the electrical impulse that stimulates synchronized contraction of the cardiac chambers, and contributing to co‐ordination of activities between cells of the arterial wall. In common with other multicellular systems, cardiovascular tissues express multiple connexin isotypes that confer distinctive channel properties. This review highlights how state‐of‐the‐art immunocytochemical and cellular imaging techniques, as part of a multidisciplinary approach in gap junction research, have advanced our understanding of connexin diversity in cardiovascular cell function in health and disease. In the heart, spatially defined patterns of expression of three connexin isotypes—connexin43, connexin40, and connexin45—underlie the precisely orchestrated patterns of current flow governing the normal cardiac rhythm. Derangement of gap junction organization and/or reduced expression of connexin43 are associated with arrhythmic tendency in the diseased human ventricle, and high levels of connexin40 in the atrium are associated with increased risk of developing atrial fibrillation after coronary by‐pass surgery. In the major arteries, endothelial gap junctions may simultaneously express three connexin isotypes, connexin40, connexin37, and connexin43; underlying medial smooth muscle, by contrast, predominantly expresses connexin43, with connexin45 additionally expressed at restricted sites. In normal arterial smooth muscle, the abundance of connexin43 gap junctions varies according to vascular site, and shows an inverse relationship with desmin expression and positive correlation with the quantity of extracellular matrix. Increased connexin43 expression between smooth muscle cells is closely linked to phenotypic transformation in early human coronary atherosclerosis and in the response of the arterial wall to injury. Current evidence thus suggests that gap junctions in both their guises, as pathways for cell‐to‐cell signaling in the vessel wall and as pathways for impulse conduction in the heart, contribute to the initial pathogenesis and eventual clinical manifestation of human cardiovascular disease. Microsc. Res. Tech. 52:301–322, 2001.

Comparison of connexin expression patterns in the developing mouse heart and human foetal heart.

Heart muscle cells are electrically coupled by gap junctions, clusters of low-resistance transmembrane channels composed of connexins (Cx). The expression of the three major connexins (Cx43, Cx40 and Cx45) present in cardiac myocytes is known to be developmentally regulated but it is not clear how the patterns in the human heart compare with those found in the mouse. This issue is of importance given the wide use of transgenic mice to investigate gene function with the aim of extrapolating the results to human. In the present study we applied immunoconfocal microscopy to investigate the spatial distribution of the three connexins in the developing mouse heart and foetal human heart. Although Cx45 labelling was present at low levels throughout the developing mouse heart and human foetal (9-week) heart, it was most prominent in the conduction tissues. In the developing mouse heart, Cx40 was widely expressed at embryonic day 12.5 (E12.5) but at E17.5 expression was restricted to the conduction tissues and atria. In the 9-week human foetal heart, the Cx40 labelling pattern was similar to the E15 mouse heart, being far more abundant in conduction tissues (bundle branches to Purkinje fibres) and atria than in the ventricular muscle. Cx43 labelling became more apparent in the ventricular myocardium as development of the mouse heart progressed but was virtually undetectable in the central conduction system. In the human foetal heart Cx43 was virtually undetectable in the atria but was the predominant connexin in the ventricles. We conclude that, at least in some key aspects, the pattern of connexin expression in the developing mouse heart parallels that found in the human embryonic heart.

Alterations in Cardiac Connexin Expression in Cardiomyopathies

Gap junctions, assembled from connexins, form the cell-to-cell pathways for propagation of the precisely orchestrated patterns of current flow that govern the synchronized rhythm of the healthy heart. As in most tissues and organs, multiple connexin types are co-expressed in the heart; the connexins Cx43, Cx40 and Cx45 are found in distinctive combinations and relative quantities in different, functionally specialized subsets of cardiomyocytes. Alterations in connexin expression and gap junction organization, now a well-documented feature of human cardiomyopathies, potentially contribute to the pro-arrhythmic substrate. In the diseased ventricle, the most consistently reported quantitative alteration involves heterogeneous reduction in Cx43 expression and disruption of the normal ordered pattern of Cx43 gap junction distribution. Additional studies suggest that upregulation of Cx40 and Cx45 may also feature in the failing ventricle, the former restricted to ischemic cardiomyopathy and localized to the subendocardial region. By correlating data from studies on the human patient with those from animal and cell models, alterations in connexin expression and gap junction organization have emerged as important factors to be considered in understanding the pro-arrhythmic substrate found in human cardiomyopathies.

Differential Expression of Connexin43 and Desmin Defines Two Subpopulations of Medial Smooth Muscle Cells in the Human Internal Mammary Artery

Upregulation of connexin43-gap junctions is associated with transition of contractile vascular smooth muscle cells (SMCs) to the synthetic state. To determine whether phenotypically distinct subpopulations of medial SMCs differentially express connexin43, we investigated the human distal internal mammary artery, a structurally heterogeneous vessel with features ranging from elastic to elastomuscular to muscular. Immunoconfocal microscopy combined with quantitative analysis and complemented by in situ hybridization showed that SMCs in the elastic medial regions expressed high levels of connexin43 but low levels of desmin, whereas those of muscular medial regions expressed low levels of connexin43 but high levels of desmin. Ultrastructurally, SMCs of both regions were of the contractile phenotype, but the former cells were irregular in shape with relatively prominent synthetic organelles whereas the latter were spindle shaped with fewer synthetic organelles. Vimentin, smooth muscle alpha-actin, calponin, h-caldesmon, and myosin heavy chains (SM1 and SM2) were equally highly expressed by most cells in both subpopulations. The connexin43/desmin expression pattern of SMCs in regions of intimal thickening resembled those of elastic medial regions. These findings refine the view suggested from previous studies that high levels of connexin43 expression are associated with SMCs of a less contractile/more synthetic phenotype. In the internal mammary artery, the 2 subpopulations of SMCs with markedly different connexin43 expression levels both represent a differentiated contractile phenotype, but the subpopulation showing high levels of connexin43-gap junctions is characterized by low levels of desmin and structural features that reflect a more synthetic tendency.

Detection of the diastolic pathway, circuit morphology, and inducibility of human postinfarction ventricular tachycardia from mapping in sinus rhythm.

OBJECTIVE The purpose of this study was to determine whether sinus rhythm activation maps could be used to detect the origin and characteristics of reentrant ventricular tachycardia in postinfarction patients. METHODS In each of 11 post-myocardial infarction patients, unipolar electrograms were acquired at 256 virtual endocardial sites using noncontact mapping. Electrograms were marked for activation time and mapped on a three-dimensional grid. Spatial differences in sinus rhythm activation time were correlated to isthmus characteristics and to activation through the diastolic pathway during tachycardia on the basis of the presence of contiguous lines of slow conduction and block. RESULTS Twenty tachycardia morphologies were analyzed. Fourteen sustained reentrant circuit morphologies occurred in nine patients, with dual morphologies having a shared isthmus occurring in five of nine patients. Dual morphologies were caused by changes in entrance-exit point location about a common isthmus. One transient circuit morphology of <10 beats occurred in three of nine patients also having sustained reentry. The estimated isthmus determined from sinus rhythm activation overlapped the diastolic pathway determined from tachycardia maps with 83.8% sensitivity and 89.2% specificity. The mean difference in sinus rhythm activation time across the isthmus border was larger in transient compared with sustained morphologies (32.8 +/- 9.5 ms vs. 22.8 +/- 1.8 ms), with smaller isthmus size (4.8 +/- 1.1 cm(2) vs. 10.0 +/- 1.1 cm(2); P < .05), narrower entrance-exit points (7.0 +/- 1.5 mm vs. 9.3 +/- 0.8 mm; P < .05), and greater activation time difference across them (16.3 +/- 3.5 ms vs. 10.1 +/- 1.0 ms; P < .05). CONCLUSION In post-myocardial infarction patients, the reentry isthmus can be localized in the endocardial border zone from sinus rhythm activation maps. Nonsustained reentry occurs when isthmus size is small and entrance-exit points are narrow and more electrically discontinuous.

Human connexin31.9, unlike its orthologous protein connexin30.2 in the mouse, is not detectable in the human cardiac conduction system

In the human heart connexin(Cx)40, Cx43 and Cx45-containing gap junctional channels electrically couple cardiomyocytes, forming a functional syncytium. In the mouse heart, additionally, Cx30.2-containing gap junctions have been detected in the atrioventricular node where they are implicated, together with Cx45, in impulse delay. However, whether the human ortholog of Cx30.2, Cx31.9, is expressed in the human heart has not previously been investigated. We therefore generated Cx31.9 specific antibodies to test for the expression of Cx31.9 in the human heart. These antibodies recognized the Cx31.9 protein in HeLaCx31.9 transfectants by immunofluorescence and immunoblot analyses. However, we did not find punctate Cx31.9 specific immunofluorescence signals in the working myocardium or in the impulse generation and conduction system of adult or fetal human heart. Complementary immunoblot analyses did not reveal Cx31.9 protein in the adult atrial or ventricular myocardium. We conclude that the Cx31.9 protein, unlike its counterpart in the mouse, is not expressed in detectable quantities and is thus unlikely to contribute to the impulse generation and conduction system or the working myocardium of the human heart.

Comparison of Connexin 43, 40 and 45 Expression Patterns in the Developing Human and Mouse Hearts

The mouse is currently widely used as a model organism in the analysis of gene function but how developmentally regulated patterns of connexin gene expression in the mouse compare with those in the human is unclear. Here we compare the patterns of connexin expression in the heart during the development of the mouse (from embryonic day 12.5 to 6 weeks postpartum) and the human (at 9 weeks gestation and adult stage). The extent of connexin43 expression in the ventricles progressively increased during development of the mouse heart. The developmental pattern of expression for connexins 40 and 45 in the mouse heart was similar, but not identical, and in the ventricles showed a progressive and preferential expression in the conduction system. In general, these dynamic changes of connexins 43, 40 and 45 during mouse cardiac development appear to be mirrored in the human.

Original-clinicalDetection of the diastolic pathway, circuit morphology, and inducibility of human postinfarction ventricular tachycardia from mapping in sinus rhythm

OBJECTIVE The purpose of this study was to determine whether sinus rhythm activation maps could be used to detect the origin and characteristics of reentrant ventricular tachycardia in postinfarction patients. METHODS In each of 11 post-myocardial infarction patients, unipolar electrograms were acquired at 256 virtual endocardial sites using noncontact mapping. Electrograms were marked for activation time and mapped on a three-dimensional grid. Spatial differences in sinus rhythm activation time were correlated to isthmus characteristics and to activation through the diastolic pathway during tachycardia on the basis of the presence of contiguous lines of slow conduction and block. RESULTS Twenty tachycardia morphologies were analyzed. Fourteen sustained reentrant circuit morphologies occurred in nine patients, with dual morphologies having a shared isthmus occurring in five of nine patients. Dual morphologies were caused by changes in entrance-exit point location about a common isthmus. One transient circuit morphology of <10 beats occurred in three of nine patients also having sustained reentry. The estimated isthmus determined from sinus rhythm activation overlapped the diastolic pathway determined from tachycardia maps with 83.8% sensitivity and 89.2% specificity. The mean difference in sinus rhythm activation time across the isthmus border was larger in transient compared with sustained morphologies (32.8 +/- 9.5 ms vs. 22.8 +/- 1.8 ms), with smaller isthmus size (4.8 +/- 1.1 cm(2) vs. 10.0 +/- 1.1 cm(2); P < .05), narrower entrance-exit points (7.0 +/- 1.5 mm vs. 9.3 +/- 0.8 mm; P < .05), and greater activation time difference across them (16.3 +/- 3.5 ms vs. 10.1 +/- 1.0 ms; P < .05). CONCLUSION In post-myocardial infarction patients, the reentry isthmus can be localized in the endocardial border zone from sinus rhythm activation maps. Nonsustained reentry occurs when isthmus size is small and entrance-exit points are narrow and more electrically discontinuous.

Short-term pacing in the mouse alters cardiac expression of connexin43.

BackgroundCardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time.ResultsThe subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10–15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias.ConclusionOur data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease.