Here we show that propagation of the electrical impulse is significantly slowed in the right ventricle of ZDF rats compared to ZDL rats. To our knowledge, this is the first study to report decreased cardiac CV in an animal model of type 2 diabetes.
Our finding is in accordance with studies performed in STZ induced type 1 diabetic Wistar rats, which show decreased CV at unstressed conditions  and a decreased conduction reserve [17–19, 35]. In addition, QRS prolongation, which may occur due to decreased CV, is found in both STZ treated rats  and ZDF rats , as well as in diabetic humans . This indicates that decreased CV may be an independent risk factor for development of ventricular arrhythmias in both type 1 and 2 diabetic patients.
CV slowing can occur by either reduced excitability, increased internal electrical resistance or by obstacles to conduction such as fibrosis . CV is normally very stable presumably due to the existence of a conduction reserve and therefore large alterations are needed to slow CV (for review see ). The internal electrical resistance is determined by both the electrical resistance of the cell interior and by the gap junction mediated coupling between the cells. Previous studies on rats with STZ induced type 1 diabetes indicate changes in expression, localization, and phosphorylation of the major ventricular gap junction protein C×43, but the reports are contradicting. Some studies report unchanged levels of C×43 [19, 20], whereas other report reduced [17, 18] or increased levels [20, 21]. The discrepancies between studies may be design dependent; Lin et al. showed that C×43 levels were increased 5 weeks after STZ treatment, but C×43 levels were back to normal after 10 weeks . In this study, we found no difference in the level of C×43 between ZDF and ZDL rats, but we did see a statistically significant, increase in C×43 lateralization, measured as the fraction of C×43 not localized in close proximity of N-cadherin, in ZDF rat hearts. Lateralization of C×43 is frequently reported in cardiac diseases, and it is often assumed that lateralized C×43 is nonfunctional. When analyzing C×43 localization based on the shape of the cardiomyocytes, it should, however, be noted that gap junctions, which are located at the “long side” as opposed to the “end” of the cell (where the endplates are located), may none the less still be located in intercalated discs, and thus be functional. In this regard, it is of interest to note that one study has found that lateralization of C×43 improves lateral coupling , which indicates that lateralized gap junctions may not be nonfunctional. Furthermore, it was previously shown that interaction between connexins and the N-cadherin-catenin complex found in intercalated discs, is required to maintain gap junction functionality . Therefore, we chose to evaluate gap junction lateralization as the fraction of C×43 that did not appear in close proximity of N-cadherin. The observed increase in lateralization of C×43 in ZDF rats may account for part of the observed decrease in CV. Studies in heterozygous C×43 knockout mice have shown contradicting effects on CV following a 50% reduction in C×43 expression; some studies report a decreased CV [41–43], whereas other studies did not detect significant decreases in CV [44, 45]. Based on the above, we find it unlikely that the 17% decrease (from 70 to 58%) in C×43 found in the intercalated discs solely accounts for the observed decrease in CV. In contrast to C×43 lateralization, it is well documented that C×43 phosphorylation plays an important role in gap junction coupling [46, 47] and dephosphorylation of C×43 has been related to electrical uncoupling in ischemia [48, 49]. Judged by the electrophoretic mobility of C×43, we did not observe any differences in overall C×43 phosphorylation between ZDF and ZDL rat hearts. This is in contrast to studies on STZ rats where mobility shifts indicates hyperphosphorylation [17, 20, 21]. However, since more than twenty phosphorylation sites are present in C×43, we cannot exclude that some changes in C×43 phosphorylation may have occurred in the ZDF rats and a complete phosphorylation analysis may be an interesting subject for future work. Nonetheless, our data indicate that changes in C×43 expression, localization or phosphorylation cannot by itself explain the observed decrease in CV in ZDF rats. This interpretation is further supported by the fact that AAP10 did not affect CV in the ZDF rats. Previous studies have shown that the AAP analogue, rotigaptide, prevents electrical uncoupling  and CV slowing during ischemic conditions with no effect under unstressed conditions  and that it also effectively reverts established CV slowing . AAP10 and its analogs are believed to affect gap junction coupling through changes in C×43 phosphorylation , which is a rapid process occurring within minutes. Therefore, the lack of an AAP10 effect in the ZDF hearts implies that the mechanism of CV slowing in diabetes is different from that seen in ischemia, where gap junction uncoupling by C×43 dephosphorylation is a major contributor.
In addition to gap junction coupling, cell size is also important for CV because the cell dimensions determine both how many high resistance barriers (cell-cell junctions) the impulse has to cross per unit length and the cross sectional area of the cell available for conducting current. Cell size is reduced in STZ induced diabetes due to removal of the hypertrophic effect of insulin . Type 2 diabetes on the other hand is characterized by initial hyperinsulinemia that together with the increase in blood pressure in ZDF rats (Table 1) may lead to hypertrophy (for review see ). In the present study, we found no change in cell dimensions, ruling out that atrophy or hypertrophy causes the observed changes in the propagation of the electrical impulse.
A mild increase in cardiac fibrosis has previously been reported for ZDF rats at both 7, 14 and 21 weeks of age . Fibrosis may compose an obstacle to electrical conduction and thereby contribute to a decrease in conduction velocity. Our histological analysis, however, showed absolutely no histomorphological differences between ZDF and ZDL rats.
Since the CV slowing we observe in ZDF rats does not seem to be fully explained by changes in either gap junction remodeling, changes in cell size or other morphological changes, we hypothesize that other factors are involved in the development of conduction disturbances in type 2 diabetes. Lipotoxicity due to ectopic lipid accumulation in the heart is a well known phenomenon in diabetes. Lipotoxicity has mainly been connected to impairment of cardiac energy metabolism and lipoapoptosis (for recent review see ), but a number of studies suggests that altered energy metabolism may compromise the function of cardiac ion channels (for review see ). Our lipid staining revealed significant intramyocardial lipid accumulation in ZDF compared to ZDL rats, which has also previously been shown by chloroform-methanol extraction [25, 26]. Therefore, we hypothesize that intramyocardial lipid accumulation and/or altered lipid metabolism may be involved in development of conduction disturbances in ZDF rats. Previous studies have shown that the fatty acid metabolite palmitoylcarnitine causes a concentration dependent decrease in the transient outward (Ito) K+ current , as well as the Na+ current  in isolated cardiomyocytes. In addition, mice with cardiac-specific over expression of the peroxisome proliferator-activated receptor α (PPARα) (a key player in the regulation of cardiac lipid metabolism) also displays decreased Ito density . Changes in the Ito current are not directly related to conduction disturbances, but decreased Na+ current density reduces cardiomyocytes excitability and may thereby slow conduction. Diabetes also correlates to reduced function of the sarcolemmal Na+/K+ pump, due to changes in ATP levels . Altered Na+/K+ pump function may lead to intracellular Na+ accumulation and K+ depletion, which may depolarize the resting membrane potential and increase the fraction of inactivated Na+ channels. A combination of reduced Na+ channel density and increased fraction of inactivated channels could contribute to the decreased CV observed in ZDF rats. It does, however, remain an important question for future research whether the metabolic alterations are sufficient to cause altered ion channel function and/or disturbances in the trans-membrane ion gradients, which is of a magnitude sufficient to explain the observed decrease in CV.