The benefits of using selenium in the treatment of Chagas disease: prevention of right ventricle chamber dilatation and reversion of Trypanosoma cruzi-induced acute and chronic cardiomyopathy in mice

Cardiac damage is a frequent manifestation of Chagas disease, which is caused by the parasite Trypanosoma cruzi. Selenium (Se) is an essential micronutrient, the deficiency of which has been implicated in the development of cardiomyopathy. Our group has previously demonstrated that Se supplementation prevents myocardial damage during acute T. cruzi infection in mice. In this study, we analyzed the effect of Se treatment in cases of T. cruzi infection using prevention and reversion schemes. In the Se prevention scheme, mice were given Se supplements (2 ppm) starting two weeks prior to inoculation with T. cruzi(Brazil strain) and continuing until 120 days post-infection (dpi). In the Se reversion scheme, mice were treated with Se (4 ppm) for 100 days, starting at 160 dpi. Dilatation of the right ventricle was observed in the infected control group at both phases of T. cruzi infection, but it was not observed in the infected group that received Se treatment. Surviving infected mice that were submitted to the Se reversion scheme presented normal P wave values and reduced inflammation of the pericardium. These data indicate that Se treatment prevents right ventricular chamber increase and thus can be proposed as an adjuvant therapy for cardiac alterations already established by T. cruzi infection.     

Trypanosoma cruzi infection disrupts vinculin costameres in cardiomyocytes

Chagas' disease cardiomyopathy is an important manifestation of Trypanosoma cruzi infection, leading to cardiac dysfunction and serious arrhythmias. We have here investigated by indirect immunofluorescence assay the distribution of vinculin, a focal adhesion protein with a major role in the transmission of contraction force, during the T. cruzi-cardiomyocyte infection in vitro and in vivo. No change in vinculin distribution was observed after 24 h of infection, where control and T. cruzi-infected cardiomyocytes displayed vinculin localized at costameres and intercalated discs. On the other hand, a clear disruption of vinculin costameric distribution was noted after 72 h of infection. A significant reduction in the levels of vinculin expression was observed at all times of infection. In murine experimental Chagas' disease, alteration in the vinculin distribution was also detected in the infected myocardium, with no costameric staining in infected myocytes and irregular alignment of intercalated discs in cardiac fibers. These data suggest that the disruption of costameric vinculin distribution and the enlargement of interstitial space due to inflammatory infiltration may contribute to the reduction of transmission of cardiac contraction force, leading to alterations in the heart function in Chagas' disease.     

Nitric oxide synthase-2 modulates chemokine production by Trypanosoma cruzi-infected cardiac myocytes

An intense inflammatory process is associated with Trypanosoma cruzi infection. We investigated the mediators that trigger leukocyte activation and migration to the heart of infected mice. It is known that nitric oxide (NO) modulates the inflammatory response. During T. cruzi infection, increased concentrations of NO are produced by cardiac myocytes (CMs) in response to IFN-gamma and TNF. Here, we investigated whether NO, IFN-gamma and TNF regulate chemokine production by T. cruzi-infected CMs. In addition, we examined the effects of the NOS2 deficiency on chemokine expression both in cultured CMs and in hearts obtained from infected mice. After infection of cultured WT CMs with T. cruzi, the addition of IFN-gamma and TNF increased both mRNA and protein levels of the chemokines CXCL1, CXCL2, CCL2, CCL3, CCL4 and CCL5. Interestingly, T. cruzi-infected NOS2-deficient CMs produced significantly higher levels of CCL2, CCL4, CCL5 and CXL2 in the presence of IFN-gamma and TNF. Infection of NOS2-null mice resulted in a significant increase in the expression of both chemokine mRNA and protein levels in the heart of, compared with hearts obtained from, infected WT mice. Our data indicate that NOS2 is a potent modulator of chemokine expression which is critical to triggering the generation of the inflammatory infiltrate in the heart during T. cruzi infection.     

Myofibroblasts cause heterogeneous Cx43 reduction and are unlikely to be coupled to myocytes in the healing canine infarct

Following myocardial infarction (MI) inflammatory responses transform cardiac fibroblasts to myofibroblasts, which in vitro studies show form heterocellular gap junctions with cardiac myocytes via Connexin43 (Cx43). The ability to form heterocellular junctions in the intact heart and the impact of these junctions on propagation is unclear. We used a canine model of MI and characterized the distribution and quantity of myofibroblasts in surviving epicardial cells [epicardial border zone (EBZ)]. We found a significant increase in myofibroblasts within the EBZ and no gap junction plaques between myofibroblasts and myocytes. Because myofibroblasts produce IL-1β, which downregulates Cx43, we asked whether myofibroblast proliferation causes loss of Cx43 near myofibroblast clusters. In vitro studies showed that IL-1β caused loss of Cx43 and reduced coupling. Western blot showed a significant increase of IL-1β in the EBZ, and immunohistochemistry showed a loss of Cx43 in regions of myofibroblasts in the intact heart. Additionally, dye studies in intact heart showed no coupling between myocytes and myofibroblasts. To quantify the effect of myofibroblasts on propagation we used a two-dimensional subcellular computer model of the EBZ, which showed that heterogeneities in myofibroblast density lead to conduction abnormalities. In conclusion, an increase of myofibroblasts in the infarcted heart causes heterogeneous Cx43 levels, possibly as a result of the release of IL-1β and decreased cell-cell communication, which leads to conduction abnormalities following MI.     

Enhanced Connexin-43 and α-Sarcomeric Actin Expression in Cultured Heart Myocytes Exposed to Triiodo-l-thyronine

This study examined whether triiodo-L-thyronine (T3) affects the expression of the major intercellular channel protein, connexin-43, and contractile protein alpha-sarcomeric actin. Cultured cardiomyocytes from newborn rats were treated on day three in culture with 10 or 100 nM T3 and examined 48 and 72 h thereafter. Treated and untreated cells were examined by immunofluorescence and electron microscopy. Expression levels of Cx43 and sarcomeric alpha-actin were monitored by Western blot analysis. Immunofluorescence labeling showed cell membrane location of Cx43 in punctuate gap junctions, whereby fluorescence signal area was significantly higher in cultured cardiomyocytes exposed to T3. This correlated with electron microscopical findings showing increased numbers and size of gap junction profiles, as well as with a significant dose-dependent increase of Cx43 expression detected by Western blot. Immunofluorescence of sarcomeric a-actin was enhanced and its expression increased dose- and time-dependently in T3-treated cultured heart myocytes. However, exposure to the higher dosage (100 nM) of T3 caused mild disintegration of sarcomeric a-actin in some myocytes, suggesting an over-dosage. The results indicate that T3 up-regulates Cx43 and accelerates gap junction formation in cultured neonatal cardiomyocytes. They suggest that thyroid status cannot only modulate the mechanical function of cardiomyocytes but also cell-to-cell communication essential for myocardial electrical and metabolic synchronizations.     

Behavior of atrial natriuretic factor in an experimental model of Trypanosoma cruzi infection in rats

Enhanced atrial natriuretic factor (ANF) production by the heart is related to hemodynamic overload, cardiac growth, and hypertrophy. The heart is one of the most affected organs during Trypanosoma cruzi infection. We tested the hypothesis that myocarditis produced by parasite infection alters the natriuretic peptide system by investigating the behavior of plasma ANF during the acute and chronic stages of T. cruzi infection in rats. Sprague-Dawley rats were infected with T. cruzi clone Sylvio-X10/7. Cardiac morphology showed damage to myocardial cells and lymphocyte infiltration in the acute phase; and fibrosis and cell atrophy in the chronic period. Plasma ANF levels (radioimmunoassay) were significantly higher in acute (348 +/- 40 vs. 195 +/- 36 pg/ml, P < 0.05, n = 17) and chronic T. cruzi myocarditis (545 +/- 81 vs. 229 +/- 38 pg/ml, P < 0.001, n = 11) than in their respective controls (n = 10 and 14). Rats in the chronic phase also showed higher levels than rats in the acute phase (P < 0.01). The damage of myocardial cells produced by the parasite and the subsequent inflammatory response could be responsible for the elevation of plasma ANF during the acute period of T. cruzi infection. The highest plasma ANF levels found in chronically infected rats could be derived from the progressive failure of cardiac function.     

Effects of cytochrome P450 inhibitors on agonist-induced Ca2+ responses and production of NO and PGI2 in vascular endothelial cells

The gap junction protein connexin-43 (Cx43) exists mainly in the phosphorylated state in the normal heart, while ischemia induces dephosphorylation. Phosphatase(s) involved in cardiac Cx43 dephosphorylation have not as yet been identified. We examined the acute effects of ischemia on the dephosphorylation of the gap junction protein connexin-43 in isolated adult cardiomyocytes and isolated perfused hearts. In addition we tested the effectiveness of protein phosphatase 1 and 2A (PP1/2A) inhibitors in preventing Cx43 dephosphorylation. In both models, significant accumulation of the 41 kDa non-phosphorylated Cx43, accompanied by decreased relative levels of the 43–46 kDa phosphorylated Cx43, was observed at 30 min of ischemia. Okadaic acid decreased ischemia-induced Cx43 dephosphorylation; it also decreased the accumulation of non-phosphorylated Cx43 at the intercalated discs of myocytes in the whole heart. Calyculin A, but not fostriecin, also decreased ischemia-induced Cx43 dephosphorylation in isolated cardiomyocytes. It is concluded that isolated adult myocytes respond to ischemia in a manner similar to whole hearts and that ischemia-induced dephosphorylation of Cx43 is mediated, at least in part, by PP1-like phosphatase(s).     

Gap junction remodeling and altered connexin43 expression in the failing human heart

Gap junctions (GJ) are important determinants of cardiac conduction and the evidence has recently emerged that altered distribution of these junctions and changes in the expression of their constituent connexins (Cx) may lead to abnormal coupling between cardiomyocytes and likely contribute to arrhythmogenesis. However, it is largely unknown whether changes in the expression and distribution of the major cardiac GJ protein, Cx43, is a general feature of diverse chronic myocardial diseases or is confined to some particular pathophysiological settings. In the present study, we therefore set out to investigate qualitatively and quantitatively the distribution and expression of Cx43 in normal human myocardium and in patients with dilated (DCM), ischemic (ICM), and inflammatory cardiomyopathies (MYO). Left ventricular tissue samples were obtained at the time of cardiac transplantation and investigated with immunoconfocal and electron microscopy. As compared with the control group, Cx43 labeling in myocytes bordering regions of healed myocardial infarction (ICM), small areas of replacement fibrosis (DCM) and myocardial inflammation (MYO) was found to be highly disrupted instead of being confined to the intercalated discs. In all groups, myocardium distant from these regions showed an apparently normal Cx43 distribution at the intercalated discs. Quantitative immunoconfocal analyis of Cx43 in the latter myocytes revealed that the Cx43 area per myocyte area or per myocyte volume is significantly decreased by respectively 30 and 55% in DCM, 23 and 48% in ICM, and by 21 and 40% in MYO as compared with normal human myocardium. In conclusion, focal disorganization of GJ distribution and down-regulation of Cx43 are typical features of myocardial remodeling that may play an important role in the development of an arrhythmogenic substrate in human cardiomyopathies.     

Characterization of the Connexin45 Carboxyl-Terminal Domain Structure and Interactions with Molecular Partners

Mechanisms underlying the initiation and persistence of lethal cardiac rhythms are of significant clinical and scientific interests. Gap junctions are principally involved in forming the electrical connections between myocytes, and changes in distribution, density, and properties are consistent characteristics in arrhythmic heart disease. Therefore, understanding the structure and function of gap junctions during normal and abnormal impulse propagation are essential in the control of arrhythmias. For example, Cx45 is predominately expressed in the specialized myocytes of the impulse generation and conduction system. In both ventricular and atrial human working myocytes, Cx45 is present in very low quantities. However, a reduction in Cx43 coupled with an increased Cx45 protein levels within the ventricles have been observed after myocardial infarction and end-stage heart failure. Cx45 may influence electrical and/or metabolic coupling as a result of pathophysiological overexpression. Our goal was to identify mechanisms that could cause cellular coupling to be different between the cardiac connexins. Based upon the conserved transmembrane and extracellular loop segments, our focus was on identifying features within the divergent cytoplasmic portions. Here, we biophysically characterize the carboxyl-terminal domain of Cx45 (Cx45CT). Purification revealed the possibility of oligomeric species, which was confirmed by analytical ultracentrifugation experiments. Sedimentation equilibrium and circular dichroism studies of different Cx45CT constructs identified one region of α-helical structure (A333-N361) that mediates CT dimerization through hydrophobic contacts. Interestingly, the binding affinity of Cx45CT dimerization is 1000-fold stronger than Cx43CT dimerization. Cx45CT resonance assignments were also used to identify the binding sites and affinities of molecular partners involved in the Cx45 regulation; although none disrupted dimerization, many of these proteins interacted within one intrinsically disordered region (P278-P285). This domain has similarities with other cardiac connexins, and we propose they constitute a master regulatory domain, which contains overlapping molecular partner binding, cis-trans proline isomerization, and phosphorylation sites.     

Characterization of the Connexin45 Carboxyl-Terminal Domain Structure and Interactions with Molecular Partners

Mechanisms underlying the initiation and persistence of lethal cardiac rhythms are of significant clinical and scientific interests. Gap junctions are principally involved in forming the electrical connections between myocytes, and changes in distribution, density, and properties are consistent characteristics in arrhythmic heart disease. Therefore, understanding the structure and function of gap junctions during normal and abnormal impulse propagation are essential in the control of arrhythmias. For example, Cx45 is predominately expressed in the specialized myocytes of the impulse generation and conduction system. In both ventricular and atrial human working myocytes, Cx45 is present in very low quantities. However, a reduction in Cx43 coupled with an increased Cx45 protein levels within the ventricles have been observed after myocardial infarction and end-stage heart failure. Cx45 may influence electrical and/or metabolic coupling as a result of pathophysiological overexpression. Our goal was to identify mechanisms that could cause cellular coupling to be different between the cardiac connexins. Based upon the conserved transmembrane and extracellular loop segments, our focus was on identifying features within the divergent cytoplasmic portions. Here, we biophysically characterize the carboxyl-terminal domain of Cx45 (Cx45CT). Purification revealed the possibility of oligomeric species, which was confirmed by analytical ultracentrifugation experiments. Sedimentation equilibrium and circular dichroism studies of different Cx45CT constructs identified one region of α-helical structure (A333-N361) that mediates CT dimerization through hydrophobic contacts. Interestingly, the binding affinity of Cx45CT dimerization is 1000-fold stronger than Cx43CT dimerization. Cx45CT resonance assignments were also used to identify the binding sites and affinities of molecular partners involved in the Cx45 regulation; although none disrupted dimerization, many of these proteins interacted within one intrinsically disordered region (P278-P285). This domain has similarities with other cardiac connexins, and we propose they constitute a master regulatory domain, which contains overlapping molecular partner binding, cis-trans proline isomerization, and phosphorylation sites.     

The Expression, Phosphorylation, and Localization of Connexin 43 and Gap-Junctional Intercellular Communication during the Establishment of a Synchronized Contraction of Cultured Neonatal Rat Cardiac Myocytes

We analyzed the expression, phosphorylation, and localization of the major cardiac gap-junction protein connexin 43 (Cx43) during the establishment of a synchronized contraction in confluent monolayers of primary cultured neonatal rat cardiac myocytes, combined with a functional assay of gap junctions by the microinjection-dye transfer method. Monitoring of the beating rate and synchronization by Fotonic Sensor showed that at Day 1 of culture cardiac myocytes contracted spontaneously but irregularly, that the contractile rate increased with culture time, and that a synchronized contraction was gradually formed. At Day 7, the confluent cells exhibited synchronous contraction with a relatively constant rate (125 ± 20 beats/min). Cardiac myocytes expressed a large amount of Cx43 mRNA even at Day 1 and maintained the expression until at least Day 7. Immunofluorescence of Cx43 showed that the localization of Cx43-positive spots was mostly restricted to cell-cell contacts between myocytes and that few Cx43-positive spots were present between myocytes and fibroblasts or between fibroblasts. The amount of Cx43 protein, the proportion of phosphorylated forms to the nonphosphorylated one, and the number and total area of Cx43-positive spots increased with culture time. Gap-junctional intercellular communication measured by dye transfer assay was also increased with culture time and correlated well with the number and total area of Cx43-positive spots. Our systematic study suggests that a concerted action of the expression, phosphorylation, and localization of Cx43 and gap-junctional intercellular communication plays a major role in the reestablishment of synchronous beating of cultured neonatal rat cardiac myocytes.     

Increased co-localization of connexin43 and ZO-1 in dissociated adult myocytes

In the heart, the intercellular geometry of myocyte coupling by Connexin43-gap junctions (Cx43-gjs) is a determinant of normal and abnormal patterns of propagation of electrical excitation. ZO-1 has been suggested to play a role in determining the pattern of intercellular coupling between myocytes. We therefore investigated the co-distribution of Cx43 with ZO-1 in ventricular myocytes of the adult rat using quantitative immunoconfocal microscopy. Our data indicates that low-moderate levels of co-immunolocalization occur between Cx43 and ZO-1 in normal ventricular myocardium. However, rapid and significant increases in relative co-localization occur between Cx43 and ZO-1 following dissociation of myocytes from ventricular myocardium--a treatment inducing internalization of Cx43-gjs. This increased relative co-localization may represent an increase in Cx43-ZO-1 interaction, suggesting a role for ZO-1 in the remodeling of myocardial Cx43-gjs. A more comprehensive study, including immunoprecipitation and immunoelectron microscopy analyses has been carried out (Barker et al. Circ. Res., in press, 2002 and as presented to the 2001 International GJ Conference). This study further assesses the biological relevance of the increased association between ZO-1 and Cx43 accompanying internalization of Cx43-gjs.     

Increased Co-localization of Connexin43 and ZO-1 in Dissociated Adult Myocytes

In the heart, the intercellular geometry of myocyte coupling by Connexin43-gap junctions (Cx43-gjs) is a determinant of normal and abnormal patterns of propagation of electrical excitation. ZO-1 has been suggested to play a role in determining the pattern of intercellular coupling between myocytes. We therefore investigated the co-distribution of Cx43 with ZO-1 in ventricular myocytes of the adult rat using quantitative immunoconfocal microscopy. Our data indicates that low-moderate levels of co-immunolocalization occur between Cx43 and ZO-1 in normal ventricular myocardium. However, rapid and significant increases in relative co-localization occur between Cx43 and ZO-1 following dissociation of myocytes from ventricular myocardium-a treatment inducing internalization of Cx43-gjs. This increased relative co-localization may represent an increase in Cx43-ZO-1 interaction, suggesting a role for ZO-1 in the remodeling of myocardial Cx43-gjs. A more comprehensive study, including immuno-precipitation and immunoelectron microscopy analyses has been carried out (Barker et al. Circ. Res., in press, 2002 and as presented to the 2001 International GJ Conference). This study further assesses the biological relevance of the increased association between ZO-1 and Cx43 accompanying internalization of Cx43-gjs.     

Increased Co-localization of Connexin43 and ZO-1 in Dissociated Adult Myocytes

In the heart, the intercellular geometry of myocyte coupling by Connexin43-gap junctions (Cx43-gjs) is a determinant of normal and abnormal patterns of propagation of electrical excitation. ZO-1 has been suggested to play a role in determining the pattern of intercellular coupling between myocytes. We therefore investigated the co-distribution of Cx43 with ZO-1 in ventricular myocytes of the adult rat using quantitative immunoconfocal microscopy. Our data indicates that low-moderate levels of co-immunolocalization occur between Cx43 and ZO-1 in normal ventricular myocardium. However, rapid and significant increases in relative co-localization occur between Cx43 and ZO-1 following dissociation of myocytes from ventricular myocardium-a treatment inducing internalization of Cx43-gjs. This increased relative co-localization may represent an increase in Cx43-ZO-1 interaction, suggesting a role for ZO-1 in the remodeling of myocardial Cx43-gjs. A more comprehensive study, including immuno-precipitation and immunoelectron microscopy analyses has been carried out (Barker et al. Circ. Res., in press, 2002 and as presented to the 2001 International GJ Conference). This study further assesses the biological relevance of the increased association between ZO-1 and Cx43 accompanying internalization of Cx43-gjs.     

Connexin43 expression levels influence intercellular coupling and cell proliferation of native murine cardiac fibroblasts

Little is known about connexin expression and function in murine cardiac fibroblasts. The authors isolated native ventricular fibroblasts from adult mice and determined that although they expressed both connexin43 (Cx43) and connexin45 (Cx45), the relative abundance of Cx45 was greater than that of Cx43 in fibroblasts compared to myocytes, and the electrophoretic mobility of both Cx43 and Cx45 differed in fibroblasts and in myocytes. Increasing Cx43 expression by adenoviral infection increased intercellular coupling, whereas decreasing Cx43 expression by genetic ablation decreased coupling. Interestingly, increasing Cx43 expression reduced fibroblast proliferation, whereas decreasing Cx43 expression increased proliferation. These data demonstrate that native fibroblasts isolated from the mouse heart exhibit intercellular coupling via gap junctions containing both Cx43 and Cx45. Fibroblast proliferation is inversely related to the expression level of Cx43. Thus, connexin expression and remodeling is likely to alter fibroblast function, maintenance of the extracellular matrix, and ventricular remodeling in both normal and diseased hearts.     

Lymphoid organ alterations enhanced by sub-lethal doses of coronaviruses in experimentally induced Trypanosoma cruzi infection in mice

The effect of sub-lethal doses of coronaviruses on the course of disease in CBA mice experimentally infected with a mildly pathogenic strain of Trypanosoma cruzi was investigated. Mice were inoculated with either T. cruzi, 0.1 median lethal dose (LD50) of coronavirus (mouse hepatitis virus [MHV-3] or virus X), or both pathogens. Levels of parasitemia, mortality, and the extent of pathologic alterations in lymphoid organs were determined. Mice inoculated with T. cruzi had mild alterations in their lymphoid organs and survived infection. In contrast, mice inoculated with both pathogens died, and had significantly higher levels of parasitemia and profound alterations in lymphoid organs. These results indicate that the pathologic profile of T. cruzi infection can be profoundly altered by subclinical infection with coronaviruses.     

Influence of Trypanosoma cruzi strain on the pathogenesis of chronic myocardiopathy in mice

The murine model of chronic Chaga's myocardiopathy was developed in 201 inbred and outbred mice. The experimental groups consisted of 1st: 73 inbred AKR and A/J mice inoculated with one of the following Trypanosoma cruzi strains: Peruvian (Type I), 12 SF (Type II) or Colombian (Type III); 2nd: 128 outbred Swiss mice, chronically infected either with Type II or Type III strains isolated from human patients from different geographical areas. All T. cruzi strains were previously characterized by their morphobiological behaviour in mice and by isoenzymatic patterns. For the 1st group the inoculum was 5 x 10(4) for the Peruvian strain and 1 x 10(5) for the 12 SF and Colombian strains. In the 2nd group-Swiss mice the inoculum size varied from 2 x 10(4) to 2 x 10(5). The inbred animals were killed at a 3 time-point scale (90, 180 and 240 days) post-infection. The Swiss mice were killed from 180 to 660 days after infection. The evaluation of parasitemia and serology (xenodiagnosis and indirect immunofluorescent test) was performed. The incidence of macroscopic alterations of the heart and cardiac index were evaluated. Histopathological lesions of the myocardium were graded. The influence of T. cruzi strain on the intensity of cardiac lesions was evaluated by the Chi-square test; the incidence of inflammatory lesions and its relationship to the parasite strain was evaluated by the Fisher test. The influence of the duration of infection was evaluated by using the Gamma Coefficient of Kruskal and Goodman and its measure of significance. Slight to severe microscopic alterations occurred in 85% of the chronically infected mice. There were a clear predominance on the incidence and intensity of inflammatory and fibrotic alterations for the mice infected with Type III strains. Statistical analysis has shown significant differences among the infected groups, in the inflammatory and fibrotic lesions. Macroscopic alterations (right cavities dilatation and apex aneurism of left ventricle), differed in incidence according to mice strains; in Swiss and AKR mice, significant differences were seen in mice infected with different T. cruzi strains, but the A/J mice failed to show significant differences correlated with different parasite strains. The duration of infection, from 90 to 240 days, could not be correlated with the degree of lesions in the several groups.     

Effects of streptozotocin-induced diabetes on connexin43 mRNA and protein expression in ventricular muscle

Abnormal QT prolongation with the associated arrhythmias is a significant predictor of mortality in diabetic patients. Gap junctional intercellular communication allows electrical coupling between heart muscle cells. The effects of streptozotocin (STZ)-induced diabetes mellitus on the expression and distribution of connexin 43 (Cx43) in ventricular muscle have been investigated. Cx43 mRNA expression was measured in ventricular muscle by quantitative PCR. The distribution of total Cx43, phosphorylated Cx43 (at serine 368) and non-phosphorylated Cx43 was measured in ventricular myocytes and ventricular muscle by immunocytochemistry and confocal microscopy. There was no significant difference in Cx43 mRNA between diabetic rat ventricle and controls. Total and phosphorylated Cx43 were significantly increased in ventricular myocytes and ventricular muscle and dephosphorylated Cx43 was not significantly altered in ventricular muscle from diabetic rat hearts compared to controls. Disturbances in gap junctional intercellular communication, which in turn may be attributed to alterations in balance between total, phosphorylated and dephosporylated Cx43, might partly underlie prolongation of QRS and QT intervals in diabetic heart.