Mónica Gallego

Effects of diabetic cardiomyopathy on regional electrophysiologic characteristics of rat ventricle

Aims/hypothesis. To identify the possible causes of the lengthening of the action potential duration described in patients affected by diabetes mellitus.¶Methods. We studied the effects of streptozotocin-induced diabetes on the current density of the repolarising potassium currents Ito, IK, Iss and IK1 in enzymatically isolated myocytes from three different regions of rat heart: total right ventricle, subepicardium at the apex of the left ventricle and subendocardium at the base of the left ventricle.¶Results. No changes in IK1 were found due to diabetes, but there was a uniform decrease in Ito (50 %) and Iss (40 %) current densities in the three regions. In contrast, IK diminished unevenly, with the greatest decrease in the subendocardium at the base of the left ventricle (48 %), followed by the subepicardium at the apex of the left ventricle (32 %) and right ventricle (10 %).¶Conclusion/interpretation. These findings suggest the existence of regional differences in ion channel expression associated with diabetes. The decrease of these repolarising currents could account for the lengthening of action potential and the consequent change in the Q-T interval of the ECG observed in diabetic rats. [Diabetologia (2000) 43: 101–109]

DIFFERENCES IN REGIONAL DISTRIBUTION OF K+ CURRENT DENSITIES IN RAT VENTRICLE

The objective of the present work is to study the ionic mechanisms for the regional differences in action potential duration in rat ventricle. This regional diversity has been related to differences in the regional distribution of some potassium currents in several species. Single cells were obtained by enzymatic dispersion of tissue segments from rat ventricular muscle. Whole cell voltage-clamp methods were used to identify the K+ currents involved in action potential repolarisation in the different regions. 4-Aminopiridine, TEA and voltage protocols were used to isolate the following potassium currents: transient outward, Ito, delayed rectifier, Ik, and sustained current, Iss. In the present work, we have studied the distribution of these three repolarising currents, and that of the inward rectifier, Ikl, in the free wall of the right ventricle, the subepicardium of the apex of the left ventricle and in the subendocardium of the base of the left ventricle. Action potential duration was longer in the left than in the right ventricle, and in the former it was longer in the subendocardium of the base than in the subepicardium of the apex. The main difference was in the phase 1, suggesting the implication of Ito. This was confirmed with voltage-clamp experiments. In conclusion, this work shows that Ito current density is higher in the regions with the shorter action potential, whereas there are no differences in the regional distribution of Ik, Iss or Ikl.

Restoration of cardiac transient outward potassium current by norepinephrine in diabetic rats.

Abstract. In cardiac ventricle, the density of the transient outward potassium current, Ito, is clearly related to sympathetic nervous system integrity. This sympathetic regulation of Ito expression may be greatly significant to the genesis of cardiac complications of several diseases such us diabetes mellitus. Autonomic neuropathy, including cardiac neuropathy, is a complication of chronic diabetes. The objective of the present study was to identify the possible role of cardiac sympathetic neuropathy in the reduction of Ito current density in diabetic ventricular myocardium. Thus, we employed the patch-clamp technique to test whether Ito can be restored in diabetic myocytes incubated with norepinephrine. We also measured, using HPLC, the catecholamine content of the stellate ganglion, which is responsible for cardiac sympathetic innervation, in normal and diabetic animals. The main result of the present study was to show that a 24-h incubation of diabetic cells with norepinephrine restores Ito density to control values. The restoration of Ito current density by norepinephrine suggests that the diabetes-induced reduction of Ito is at least partially attributable to a reduced trophic effect of norepinephrine on the expression of Ito.

Regulation of cardiac transient outward potassium current by norepinephrine in normal and diabetic rats

α‐Adrenergic stimulation regulates cardiac contractility by reducing repolarising K+ currents. Despite this, no published work exists on the effects of norepinephrine on isolated cardiac transient outward current, responsible for action potential duration in the rat and human. Besides, diabetes alters cardiac inotropic responses to sympathetic innervation, and this can result from altered responsiveness of the transient outward current to norepinephrine.

Ionic channels underlying the ventricular action potential in zebrafish embryo

Over the last years zebrafish has become a popular model in the study of cardiac physiology, pathology and pharmacology. Recently, the application of the 3Rs regulation and the characteristics of the embryo have reduced the use of adult zebrafish use in many studies. However, the zebrafish embryo cardiac physiology is poorly characterized since most works have used indirect techniques and direct recordings of cardiac action potential and ionic currents are scarce. In order to optimize the zebrafish embryo model, we used electrophysiological, pharmacological and immunofluorescence tools to identify the characteristics and the ionic channels involved in the ventricular action potentials of zebrafish embryos. The application of Na(+) or T-type Ca(+2) channel blockers eliminated the cardiac electrical activity, indicating that the action potential upstroke depends on Na(+) and T-type Ca(+2) currents. The plateau phase depends on L-type Ca(+2) channels since it is abolished by specific blockade. The direct channel blockade indicates that the action potential repolarization and diastolic potential depends on ERG K(+) channels. The presence in the embryonic heart of the Nav1.5, Cav1.2, Cav3.2 and ERG channels was also confirmed by immunofluorescence, while the absence of effect of specific blockers and immunostaining indicate that two K(+) repolarizing currents present in human heart, Ito and IKs, are absent in the embryonic zebrafish heart. Our results describe the ionic channels present and its role in the zebrafish embryo heart and support the use of zebrafish embryos to study human diseases and their use for drug testing.

Transient outward potassium channel regulation in healthy and diabetic hearts.

Diabetic patients have a higher incidence of cardiac arrhythmias, including ventricular fibrillation and sudden death, and show important alterations in the electrocardiogram, most of these related to the repolarization. In myocytes isolated from diabetic hearts, the transient outward K+ current (Ito) is the repolarizing current that is mainly affected. Type 1 diabetes alters Ito at 3 levels: the recovery of inactivation, the responsiveness to physiologic regulators, and the functional expression of the channel. Diabetes slows down Ito recovery of inactivation because it triggers the switching from fast-recovering Kv4.x channels to the slow-recovering Kv1.4. Diabetic animals also have decreased responsiveness of Ito towards the sympathetic nervous system; thus, the diabetic heart develops a resistance to its physiologic regulator. Finally, diabetes impairs support of various trophic factors required for the functional expression of the channel and reduces Ito amplitude by decreasing the amount of Kv4.2 and Kv4.3 proteins.

Toll-like receptor 4 activation promotes cardiac arrhythmias by decreasing the transient outward potassium current (Ito) through an IRF3-dependent and MyD88-independent pathway

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca(2+) transients, as well as the L-type Ca(2+) current (ICaL) and the transient outward K(+) current (Ito), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, Ito amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The Ito decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca(2+) transients and ICaL were not affected by LPS; however, Na(+)/Ca(2+) exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased Ito, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca(2+) efflux via NCX, could explain the presence of arrhythmias in the LPS group.

α1-Adrenoreceptors regulate only the caveolae-located subpopulation of cardiac KV4 channels

In ventricular myocytes, α1-AR stimulates Gas proteins and reduces the transient outward K+ current (Ito) via a cAMP/PKA-mediated pathway and thus regulates cardiac contraction and excitability. This Ito reduction is compartmentalized and limited to discrete membrane regions since PKA-dependent phosphorylation of the Ito channels after α1-AR stimulation requires the integrity of both the sarcoplasmic membrane and the cytoskeleton. The aim of this work was to investigate the mechanisms involved in the compartmentalization of the PKA-dependent modulation of Ito in response to α1-AR activation. Ito current recordings were performed by the Patch-Clamp technique. Membrane rafts from isolated ventricular myocytes were extracted by centrifugation in a sucrose density gradient. The different proteins were visualized by western blot and protein-protein interactions determined by coimmunoprecipitation experiments. Localization of Ito channel in caveolae, particular subtypes of membrane rafts, was achieved by electron microscopy. Patch-Clamp recordings show that a functional supramolecular complex, kept together by the A kinase anchoring protein AKAP100, exist in caveolae in living myocytes. Density gradients and immunoprecipitation experiments show that the components of the a1-AR/Ito pathway localize in caveolae, forming two different groups of proteins. The KV4.2/KV4.3 channel forms a supramolecular complex with PKA through AKAP100 and is attached to caveolae by interacting with caveolin-3. On the other hand, α1-AR, Gas and adenylate cyclase gather in a second group also connected to caveolin-3. Therefore, both groups of preassembled proteins are maintained in close proximity by caveolin-3. A different Ito channel population localizes in non-caveolar membrane rafts and is not sensitive to a1-adrenergic regulation.

Improvement of the metabolic status recovers cardiac potassium channel synthesis in experimental diabetes

The fast transient outward current, Ito,fast, is the most extensively studied cardiac K+ current in diabetic animals. Two hypotheses have been proposed to explain how type‐1 diabetes reduces this current in cardiac muscle. The first one is a deficiency in channel expression due to a defect in the trophic effect of insulin. The second one proposes flawed glucose metabolism as the cause of the reduced Ito,fast. Moreover, little information exists about the effects and possible mechanisms of diabetes on the other repolarizing currents of the human heart: Ito,slow, IKr, IKs, IKur, IKslow and IK1.

Effects of amphetamine on calcium and potassium currents in rat heart

We used the patch-clamp technique to study the effects of amphetamine on the membrane currents responsible for rat cardiac action-potential duration. Amphetamine has no effect on the slow inward Ca2+ current (I(Ca)-L), the inwardly rectifying K+ current (I(K1) and the outward K+ delayed rectifier (I(K)) and sustained (I(SS)) currents. Amphetamine blocks the transient outward K+ current (I(to)) both in the open and in the rested state. The transient outward K+ current is largely responsible for action-potential repolarization and for the regional differences in action-potential duration in rat ventricle. Therefore, the reduction of the transient outward K+ current (I(to)) caused by amphetamine may facilitate the appearance of ventricular tachycardia and fibrillation, a reported cause of death in amphetamine users.