Oscar Casis

Loss-of-Function Mutations in the Cardiac Calcium Channel Underlie a New Clinical Entity Characterized by ST-Segment Elevation, Short QT Intervals, and Sudden Cardiac Death

Background— Cardiac ion channelopathies are responsible for an ever-increasing number and diversity of familial cardiac arrhythmia syndromes. We describe a new clinical entity that consists of an ST-segment elevation in the right precordial ECG leads, a shorter-than-normal QT interval, and a history of sudden cardiac death. Methods and Results— Eighty-two consecutive probands with Brugada syndrome were screened for ion channel gene mutations with direct sequencing. Site-directed mutagenesis was performed, and CHO-K1 cells were cotransfected with cDNAs encoding wild-type or mutant CACNB2b (Cav&bgr;2b), CACNA2D1 (Cav&agr;2&dgr;1), and CACNA1C tagged with enhanced yellow fluorescent protein (Cav1.2). Whole-cell patch-clamp studies were performed after 48 to 72 hours. Three probands displaying ST-segment elevation and corrected QT intervals ≤360 ms had mutations in genes encoding the cardiac L-type calcium channel. Corrected QT ranged from 330 to 370 ms among probands and clinically affected family members. Rate adaptation of QT interval was reduced. Quinidine normalized the QT interval and prevented stimulation-induced ventricular tachycardia. Genetic and heterologous expression studies revealed loss-of-function missense mutations in CACNA1C (A39V and G490R) and CACNB2 (S481L) encoding the &agr;1- and &bgr;2b-subunits of the L-type calcium channel. Confocal microscopy revealed a defect in trafficking of A39V Cav1.2 channels but normal trafficking of channels containing G490R Cav1.2 or S481L Cav&bgr;2b-subunits. Conclusions— This is the first report of loss-of-function mutations in genes encoding the cardiac L-type calcium channel to be associated with a familial sudden cardiac death syndrome in which a Brugada syndrome phenotype is combined with shorter-than-normal QT intervals.

A novel rare variant in SCN1Bb linked to Brugada syndrome and SIDS by combined modulation of Nav1.5 and Kv4.3 channel currents

BACKGROUND Cardiac sodium channel β-subunit mutations have been associated with several inherited cardiac arrhythmia syndromes. OBJECTIVE To identify and characterize variations in SCN1Bb associated with Brugada syndrome (BrS) and sudden infant death syndrome (SIDS). METHODS All known exons and intron borders of the BrS-susceptibility genes were amplified and sequenced in both directions. Wild type (WT) and mutant genes were expressed in TSA201 cells and studied using co-immunoprecipitation and whole-cell patch-clamp techniques. RESULTS Patient 1 was a 44-year-old man with an ajmaline-induced type 1 ST-segment elevation in V1 and V2 supporting the diagnosis of BrS. Patient 2 was a 62-year-old woman displaying a coved-type BrS electrocardiogram who developed cardiac arrest during fever. Patient 3 was a 4-month-old female SIDS case. A R214Q variant was detected in exon 3A of SCN1Bb (Na(v)1B) in all three probands, but not in any other gene previously associated with BrS or SIDS. R214Q was identified in 4 of 807 ethnically-matched healthy controls (0.50%). Co-expression of SCN5A/WT + SCN1Bb/R214Q resulted in peak sodium channel current (I(Na)) 56.5% smaller compared to SCN5A/WT + SCN1Bb/WT (n = 11-12, P<0.05). Co-expression of KCND3/WT + SCN1Bb/R214Q induced a Kv4.3 current (transient outward potassium current, I(to)) 70.6% greater compared with KCND3/WT + SCN1Bb/WT (n = 10-11, P<0.01). Co-immunoprecipitation indicated structural association between Na(v)β1B and Na(v)1.5 and K(v)4.3. CONCLUSION Our results suggest that R214Q variation in SCN1Bb is a functional polymorphism that may serve as a modifier of the substrate responsible for BrS or SIDS phenotypes via a combined loss of function of sodium channel current and gain of function of transient outward potassium current.

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.

Mechanism of Block of Cardiac Transient Outward K+ Current (Ito) by Antidepressant Drugs

Imipramine, amitriptyline, mianserine, maprotiline, and trazodone are five widely used antidepressant drugs with different chemical structures. Imipramine and amitriptyline are tricyclics, mianserine and maprotiline are tetracyclics, and trazodone is a triazolopyridine derivative. We studied the effects of these drugs on the transient outward K+ current (I(to)) and the interaction mechanisms within the drug molecules and the channel-binding site. The transient outward K+ current is mainly responsible for action-potential repolarization in the rat ventricle, and all of the five drugs studied block I(to), but in different manners. Cyclic drugs block I(to) in the open state of the channel with very little block in the rested or inactivated states or both. Trazodone blocks the channel in a state-independent manner. From these results, we suggest that a relation exists between drug structure and preference for the different channel conformations.

Kv7 channels can function without constitutive calmodulin tethering.

M-channels are voltage-gated potassium channels composed of Kv7.2-7.5 subunits that serve as important regulators of neuronal excitability. Calmodulin binding is required for Kv7 channel function and mutations in Kv7.2 that disrupt calmodulin binding cause Benign Familial Neonatal Convulsions (BFNC), a dominantly inherited human epilepsy. On the basis that Kv7.2 mutants deficient in calmodulin binding are not functional, calmodulin has been defined as an auxiliary subunit of Kv7 channels. However, we have identified a presumably phosphomimetic mutation S511D that permits calmodulin-independent function. Thus, our data reveal that constitutive tethering of calmodulin is not required for Kv7 channel function.

Macrophage-dependent IL-1β production induces cardiac arrhythmias in diabetic mice

Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1β in DM mice. IL-1β causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1β-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1β axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1β as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.

Effects of fluoxetine administration on mu-opoid receptor immunostaining in the rat forebrain

Fluoxetine is a selective serotonin reuptake inhibitor. Analysis of mu-opioid receptor immunostaining after chronic fluoxetine administration in rats revealed an increase in the density of cells expressing mu-opioid receptors in the caudatus-putamen, the dentate gyrus, the lateral septum and the frontal, parietal and piriform cortices. These data suggest that mu-opioid receptor expression in the rat forebrain is altered by in vivo chronic fluoxetine treatment.

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.