Diabetes mellitus is a growing epidemic with severe cardiovascular complications. Although much is known about mechanical and electrical cardiac dysfunction in diabetes, few studies have investigated propagation of the electrical signal in the diabetic heart and the associated changes in intercellular gap junctions. This study was designed to investigate these issues, using hearts from control and diabetic rats. Diabetic conditions were induced by streptozotocin (STZ), given I.V. 7-14 days before experiments. Optical mapping with the voltage-sensitive dye di-4-ANEPPS, using hearts perfused on a Langendorff apparatus, showed little change in baseline conduction velocity in diabetic hearts, reflecting the large reserve of function. However, both the gap junction uncoupler heptanol (0.5-1 mM) and elevated potassium (9 mM, to reduce cell excitability) produced a significantly greater slowing of impulse propagation in diabetic hearts than in controls. The maximal action potential upstroke velocity (an index of the sodium current) and resting potential was similar in single ventricular myocytes from control and diabetic rats, suggesting similar electrical excitability. Immunoblotting of connexin 43 (Cx43), a major gap junction component, showed no change in total expression. However, immunofluorescence labelling of Cx43 showed a significant redistribution, apparent as enhanced Cx43 lateralization. This was quantified and found to be significantly larger than in control myocytes. Labelling of two other gap junction proteins, N-cadherin and β-catenin, showed a (partial) loss of co-localization with Cx43, indicating that enhancement of lateralized Cx43 is associated with non-functional gap junctions. In conclusion, conduction reserve is smaller in the diabetic heart, priming it for impaired conduction upon further challenges. This can desynchronize contraction and contribute to arrhythmogenesis.

Background and purpose: Aldosterone plays a major role in cardiac pathology. This study was designed to investigate the role of cardiac aldosterone in modulating K þ currents and oxidative stress in the streptozotocin-induced diabetic rat heart. Experimental approach: Transient and sustained K þ currents were measured in ventricular myocytes by voltage clamp. Plasma and cellular aldosterone were measured by ELISA. Fluorescent dihydroethidium (DHE) was used to assess superoxide ions as markers of oxidative stress. Key results: The mineralocorticoid antagonist spironolactone (1 mM, 5-9 h) significantly augmented both K þ currents in diabetic males, with a concomitant shortening of the action potential but had no effect in myocytes from control males or from diabetic females. Effects of spironolactone were restored in ovariectomized diabetic females and abolished in orchidectomized diabetic males. The aldosterone synthase inhibitor FAD286 (1 mM, 5-9 h) significantly augmented K þ currents in cells from diabetic males, but not females. Spironolactone and FAD286 significantly reduced oxidative stress in cells from diabetic males. Plasma aldosterone content was elevated in diabetic males (relative to control), but not in females. Cellular aldosterone was also elevated, but not significantly. The elevation in aldosterone was only partly dependent on a concomitant increase in cellular angiotensin II. Conclusions and implications: A gender-related, sex-hormone-dependent elevation in plasma and cardiac cell aldosterone contributed to oxidative stress and to attenuation of K þ currents in diabetic male rats. Aldosterone may thus contribute to diabetes-associated cardiac arrhythmias. Aldosterone elevation was partly related to levels of angiotensin II, but residual, angiotensin II-independent, aldosterone maintains functional relevance.

The incidence of diabetes mellitus is increasing. Cardiac dysfunction often develops, resulting in diverse arrhythmias. These arise from ion channel remodeling or from altered speed and pattern of impulse propagation. Few studies have investigated impulse propagation in the diabetic heart. We previously showed a reduced conduction reserve in the diabetic heart, with associated changes in intercellular gap junctions. The present study investigated whether these effects are sex specific. Hearts from control and streptozotocin-diabetic male and female rats were used. Optical mapping was performed with the voltage-sensitive dye di-4-ANEPPS, using Langendorff-perfused hearts. Isolated ventricular cells and tissue sections were used for immunofluorescent labeling of the gap junction protein connexin43 (Cx43). The gap junction uncoupler heptanol (0.75 mM) or elevated K+ (9 mM, to reduce cell excitability) produced significantly greater slowing of propagation in diabetic males than females. In ovariectomized diabetic females, 9 mM K+ slowed conduction significantly more than in nonovariectomized females. The subcellular redistribution (lateralization) of the gap junction protein Cx43 was smaller in diabetic females. Pretreatment of diabetic males with the angiotensin-converting enzyme inhibitor quinapril reduced Cx43 lateralization and the effects of 9 mM K+ on propagation. In conclusion, the slowing of cardiac impulse propagation in type 1 diabetes is smaller in female rats, partly due to the presence of female sex hormones. This difference is (partly) mediated by sex differences in activation of the cardiac renin-angiotensin system.

. Differential autocrine modulation of atrial and ventricular potassium currents and of oxidative stress in diabetic rats. Am J Physiol Heart Circ Physiol 290: H1879 -H1888, 2006. First published December 9, 2005 doi:10.1152/ajpheart.01045.2005The autocrine modulation of cardiac K ϩ currents was compared in ventricular and atrial cells (V and A cells, respectively) from Type 1 diabetic rats. K ϩ currents were measured by using whole cell voltage clamp. ANG II was measured by ELISA and immunofluorescent labeling. Oxidative stress was assessed by immunofluorescent labeling with dihydroethidium, a measure of superoxide ions. In V cells, K ϩ currents are attenuated after activation of the renin-angiotensin system (RAS) and the resulting ANG II-mediated oxidative stress. In striking contrast, these currents are not attenuated in A cells. Inhibition of the angiotensin-converting enzyme (ACE) also has no effect, in contrast to current augmentation in V cells. ANG II levels are enhanced in V, but not in A, cells. However, the high basal ANG II levels in A cells suggest that in these cells, ANG II-mediated pathways are suppressed, rather than ANG II formation. Concordantly, superoxide ion levels are lower in diabetic A than in V cells. Several findings indicate that high atrial natriuretic peptide (ANP) levels in A cells inhibit RAS activation. In male diabetic V cells, in vitro ANP (300 nM-1 M, Ͼ5 h) decreases oxidative stress and augments K ϩ currents, but not when excess ANG II is present. ANP has no effect on ventricular K ϩ currents when the RAS is not activated, as in control males, in diabetic males treated with ACE inhibitor and in diabetic females. In conclusion, the modulation of K ϩ currents and oxidative stress is significantly different in A and V cells in diabetic rat hearts. The evidence suggests that this is largely due to inhibition of RAS activation and/or action by ANP in A cells. These results may underlie chamber-specific arrhythmogenic mechanisms. diabetes; cardiac potassium currents DIABETES MELLITUS is a growing epidemic (59). Despite improved treatment, cardiovascular complications develop, becoming the leading cause of diabetes-related death (24, 59). Diabetic cardiomyopathy impairs mechanical function, (3,38) and electrical abnormalities increase the propensity for some cardiac arrhythmias (1). Cardiac action potentials are prolonged in animal models of diabetes due to attenuated transient and sustained repolarizing K ϩ currents (39,56). A reduction in current magnitude reflects downregulation of K ϩ channel expression, as determined in earlier work by us and others (33,42,43). Diminished outward currents presumably underlie the prolongation of the QT interval in the human electrocardiogram. Prolongation and dispersion of the QT interval, established predictors of arrhythmias and mortality (23), are common in diabetes (50).

Phospholamban (PLN) is a 52 amino acid integral membrane protein of the sarcoplasmic reticulum (SR) that exists in both monomeric and pentameric forms. In its unphosphorylated state, PLN inhibits the SR Ca(2+) ATPase (SERCA). This inhibition is relieved when PLN is phosphorylated as a result of β-adrenergic stimulation of the heart. Consistent with some predictions from molecular models and from functional studies of PLN incorporated into planar lipid bilayers, it has also been postulated that pentameric PLN can also form ion-selective channels. Other molecular models contradict this hypothesis, however. In the work reported here, we used the Ca(2+)-sensitive fluorescent dye Fura-2, to examine the passive Ca(2+) permeability of the SR membrane in vesicles derived from cardiac ventricle. We have found that phosphorylation of PLN by protein kinase A (PKA) leads to an increase in the rate of Ca(2+) leak from Ca(2+)-loaded SR vesicles. This enhanced rate of Ca(2+) leak from the SR is also observed when SR vesicles are incubated with a PLN specific antibody (A1) that mimics phosphorylation of PLN. The ryanodine receptor blocker ruthenium red does not affect the increased rate of Ca(2+) leak from the SR after PLN phosphorylation with PKA or after exposure to A1 antibody, arguing against a possible role of ryanodine receptors in mediating the enhanced leak. Our results are consistent with the hypothesis that phosphorylated PLN forms or regulates a Ca(2+) leak pathway in cardiac SR membranes in situ.

We determined the effects of epigallocatechin-3-gallate (EGCG) and epicatechin (EC), on pump turnover and Ca2+ transport by the cardiac form of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). Fluorescence spectroscopy was used to directly measure SERCA ATPase activity and to measure Ca2+ uptake into cardiac sarcoplasmic reticulum (SR) vesicles and microsomes derived from human embryonic kidney (HEK) cells expressing human cardiac SERCA2a. We found that EGCG reduces the maximum velocity of Ca2+ uptake into cardiac SR vesicles and increases the Ca2+-sensitivity of uptake in a concentration-dependent manner. EC is less potent than EGCG in increasing the Ca2+-sensitivity of uptake and does not affect maximum uptake velocity. The EGCG-dependent reduction in Ca2+ uptake velocity is well correlated with direct inhibition of SERCA. The effect of EGCG on the Ca2+-sensitivity of Ca2+ uptake into cardiac SR vesicles is affected by the phosphorylation status of phospholamban (PLB). When cardiac SERCA2a is expressed in HEK cells without PLB, EGCG reduces the maximum velocity of Ca2+ uptake but does not affect the Ca2+-sensitivity of uptake into microsomes derived from these cells indicating that the effect of EGCG on Ca2+-sensitivity requires the presence of PLB. Our results show that EGCG has dual effects on SERCA function in cardiac SR vesicles: it directly affects SERCA by reducing maximum uptake velocity; it increases the Ca2+-sensitivity of Ca2+ uptake in a manner that appears to depend on the interaction between SERCA and PLB.

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