The electrophysiological effects of cannabidiol on action potentials and transmembrane potassium currents in rabbit and dog cardiac ventricular preparations.

Cannabis use is associated with known cardiovascular side effects such as cardiac arrhythmias or even sudden cardiac death. The mechanisms behind these adverse effects are unknown. The aim of the present work was to study the cellular cardiac electrophysiological effects of cannabidiol (CBD) on action potentials and several transmembrane potassium currents, such as the rapid (IKr) and slow (IKs) delayed rectifier, the transient outward (Ito) and inward rectifier (IK1) potassium currents in rabbit and dog cardiac preparations. CBD increased action potential duration (APD) significantly in both rabbit (from 211.7 ± 11.2. to 224.6 ± 11.4 ms, n = 8) and dog (from 215.2 ± 9.0 to 231.7 ± 4.7 ms, n = 6) ventricular papillary muscle at 5 µM concentration. CBD decreased IKr, IKs and Ito (only in dog) significantly with corresponding estimated EC50 values of 4.9, 3.1 and 5 µM, respectively, without changing IK1. Although the EC50 value of CBD was found to be higher than literary Cmax values after CBD smoking and oral intake, our results raise the possibility that potassium channel inhibition by lengthening cardiac repolarization might have a role in the possible proarrhythmic side effects of cannabinoids in situations where CBD metabolism and/or the repolarization reserve is impaired.

The electrophysiological effect of cannabidiol on hERG current and in guinea-pig and rabbit cardiac preparations.

Cannabis use is associated with cardiovascular adverse effects ranging from arrhythmias to sudden cardiac death. The exact mechanism of action behind these activities is unknown. The aim of our work was to study the effect of cannabidiol (CBD), tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol on cellular cardiac electrophysiological properties including ECG parameters, action potentials, hERG and IKr ion channels in HEK cell line and in rabbit and guinea pig cardiac preparations. CBD increased action potential duration in rabbit and guinea pig right ventricular papillary muscle at lower concentrations (1 µM, 2.5 µM and 5 µM) but did not significantly change it at 10 µM. CBD at high concentration (10 µM) decreased inward late sodium and L-type calcium currents as well. CBD inhibited hERG potassium channels with an IC50 value of 2.07 µM at room temperature and delayed rectifier potassium current with 6.5 µM at 37 °C, respectively. The frequency corrected QT interval (QTc) was significantly lengthened in anaesthetized guinea pig without significantly changing other ECG parameters. Although the IC50 value of CBD was higher than literary Cmax values after CBD smoking and oral intake, our results raise the possibility that hERG and potassium channel inhibition might have a role in the possible proarrhythmic adverse effects of cannabinoids in situations where metabolism of CBD impaired and/or the repolarization reserve is weakened.

Personalised health education against health damage of COVID-19 epidemic in the elderly Hungarian population (PROACTIVE-19): protocol of an adaptive randomised controlled clinical trial.

BACKGROUND: Early reports indicate that COVID-19 may require intensive care unit (ICU) admission in 5-26% and overall mortality can rise to 11% of the recognised cases, particularly affecting the elderly. There is a lack of evidence-based targeted pharmacological therapy for its prevention and treatment. We aim to compare the effects of a World Health Organization recommendation-based education and a personalised complex preventive lifestyle intervention package (based on the same WHO recommendation) on the outcomes of the COVID-19. METHODS: PROACTIVE-19 is a pragmatic, randomised controlled clinical trial with adaptive "sample size re-estimation" design. Hungarian population over the age of 60 years without confirmed COVID-19 will be approached to participate in a telephone health assessment and lifestyle counselling voluntarily. Volunteers will be randomised into two groups: (A) general health education and (B) personalised health education. Participants will go through questioning and recommendation in 5 fields: (1) mental health, (2) smoking habits, (3) physical activity, (4) dietary habits, and (5) alcohol consumption. Both groups A and B will receive the same line of questioning to assess habits concerning these topics. Assessment will be done weekly during the first month, every second week in the second month, then monthly. The composite primary endpoint will include the rate of ICU admission, hospital admission (longer than 48 h), and mortality in COVID-19-positive cases. The estimated sample size is 3788 subjects per study arm. The planned duration of the follow-up is a minimum of 1 year. DISCUSSION: These interventions may boost the body's cardiovascular and pulmonary reserve capacities, leading to improved resistance against the damage caused by COVID-19. Consequently, lifestyle changes can reduce the incidence of life-threatening conditions and attenuate the detrimental effects of the pandemic seriously affecting the older population. TRIAL REGISTRATION: The study has been approved by the Scientific and Research Ethics Committee of the Hungarian Medical Research Council (IV/2428- 2 /2020/EKU) and has been registered at clinicaltrials.gov ( NCT04321928 ) on 25 March 2020.

Transgenic LQT2, LQT5, and LQT2-5 rabbit models with decreased repolarisation reserve for prediction of drug-induced ventricular arrhythmias.

BACKGROUND AND PURPOSE: Reliable prediction of pro-arrhythmic side effects of novel drug candidates is still a major challenge. Although drug-induced pro-arrhythmia occurs primarily in patients with pre-existing repolarisation disturbances, healthy animals are employed for pro-arrhythmia testing. To improve current safety screening, transgenic long QT (LQTS) rabbit models with impaired repolarisation reserve were generated by overexpressing loss-of-function mutations of human HERG (HERG-G628S, loss of IKr ; LQT2), KCNE1 (KCNE1-G52R, decreased IKs ; LQT5), or both transgenes (LQT2-5) in the heart. EXPERIMENTAL APPROACH: Effects of K+ channel blockers on cardiac repolarisation and arrhythmia susceptibility were assessed in healthy wild-type (WT) and LQTS rabbits using in vivo ECG and ex vivo monophasic action potential and ECG recordings in Langendorff-perfused hearts. KEY RESULTS: LQTS models reflect patients with clinically "silent" (LQT5) or "manifest" (LQT2 and LQT2-5) impairment in cardiac repolarisation reserve: they were more sensitive in detecting IKr -blocking (LQT5) or IK1 /IKs -blocking (LQT2 and LQT2-5) properties of drugs compared to healthy WT animals. Impaired QT-shortening capacity at fast heart rates was observed due to disturbed IKs function in LQT5 and LQT2-5. Importantly, LQTS models exhibited higher incidence, longer duration, and more malignant types of ex vivo arrhythmias than WT. CONCLUSION AND IMPLICATIONS: LQTS models represent patients with reduced repolarisation reserve due to different pathomechanisms. As they demonstrate increased sensitivity to different specific ion channel blockers (IKr blockade in LQT5 and IK1 and IKs blockade in LQT2 and LQT2-5), their combined use could provide more reliable and more thorough prediction of (multichannel-based) pro-arrhythmic potential of novel drug candidates.

Definition of hidden drug cardiotoxicity: paradigm change in cardiac safety testing and its clinical implications.

Unexpected cardiac adverse effects are the leading causes of discontinuation of clinical trials and withdrawal of drugs from the market. Since the original observations in the mid-90s, it has been well established that cardiovascular risk factors and comorbidities (such as ageing, hyperlipidaemia, and diabetes) and their medications (e.g. nitrate tolerance, adenosine triphosphate-dependent potassium inhibitor antidiabetic drugs, statins, etc.) may interfere with cardiac ischaemic tolerance and endogenous cardioprotective signalling pathways. Indeed drugs may exert unwanted effects on the diseased and treated heart that is hidden in the healthy myocardium. Hidden cardiotoxic effects may be due to (i) drug-induced enhancement of deleterious signalling due to ischaemia/reperfusion injury and/or the presence of risk factors and/or (ii) inhibition of cardioprotective survival signalling pathways, both of which may lead to ischaemia-related cell death and/or pro-arrhythmic effects. This led to a novel concept of 'hidden cardiotoxicity', defined as cardiotoxity of a drug that manifests only in the diseased heart with e.g. ischaemia/reperfusion injury and/or in the presence of its major comorbidities. Little is known on the mechanism of hidden cardiotoxocity, moreover, hidden cardiotoxicity cannot be revealed by the routinely used non-clinical cardiac safety testing methods on healthy animals or tissues. Therefore, here, we emphasize the need for development of novel cardiac safety testing platform involving combined experimental models of cardiac diseases (especially myocardial ischaemia/reperfusion and ischaemic conditioning) in the presence and absence of major cardiovascular comorbidities and/or cotreatments.

Genomic upregulation of cardiac Cav1.2α and NCX1 by estrogen in women.

BACKGROUND: Women have a higher risk of lethal arrhythmias than men in long QT syndrome type 2 (LQTS2), but the mechanisms remain uncertain due to the limited availability of healthy control human tissue. We have previously reported that in female rabbits, estrogen increases arrhythmia risk in drug-induced LQTS2 by upregulating L-type Ca2+ (ICa,L) and sodium-calcium exchange (INCX) currents at the base of the epicardium by a genomic mechanism. This study investigates if the effects of estrogen on rabbit ICa,L and INCX apply to human hearts. METHODS: Postmortem human left ventricular tissue samples were probed with selective antibodies for regional heterogeneities of ion channel protein expression and compared to rabbit myocardium. Functionally, ICa,L and INCX were measured from female and male cardiomyocytes derived from human induced pluripotent stem cells (iPS-CMs) with the voltage-clamp technique from control and estrogen-treated iPS-CMs. RESULTS: In women (n = 12), Cav1.2α (primary subunit of the L-type calcium channel protein 1) and NCX1 (sodium-calcium exchange protein) levels were higher at the base than apex of the epicardium (40 ± 14 and 81 ± 30%, respectively, P < 0.05), but not in men (n = 6) or postmenopausal women (n = 6). Similarly, in cardiomyocytes derived from female human iPS-CMs, estrogen (1 nM, 1-2 days) increased ICa,L (31%, P < 0.05) and INCX (7.5-fold, - 90 mV, P < 0.01) and their mRNA levels (P < 0.05). Moreover, in male human iPS-CMs, estrogen failed to alter ICa,L and INCX. CONCLUSIONS: The results show that estrogen upregulates cardiac ICa,L and INCX in women through genomic mechanisms that account for sex differences in Ca2+ handling and spatial heterogeneities of repolarization due to base-apex heterogeneities of Cav1.2α and NCX1. By analogy with rabbit studies, these effects account for human sex-difference in arrhythmia risk.

Nucleoside Diphosphate Kinase-C Suppresses cAMP Formation in Human Heart Failure.

BACKGROUND: Chronic heart failure (HF) is associated with altered signal transduction via ß-adrenoceptors and G proteins and with reduced cAMP formation. Nucleoside diphosphate kinases (NDPKs) are enriched at the plasma membrane of patients with end-stage HF, but the functional consequences of this are largely unknown, particularly for NDPK-C. Here, we investigated the potential role of NDPK-C in cardiac cAMP formation and contractility. METHODS: Real-time polymerase chain reaction, (far) Western blot, immunoprecipitation, and immunocytochemistry were used to study the expression, interaction with G proteins, and localization of NDPKs. cAMP levels were determined with immunoassays or fluorescent resonance energy transfer, and contractility was determined in cardiomyocytes (cell shortening) and in vivo (fractional shortening). RESULTS: NDPK-C was essential for the formation of an NDPK-B/G protein complex. Protein and mRNA levels of NDPK-C were upregulated in end-stage human HF, in rats after long-term isoprenaline stimulation through osmotic minipumps, and after incubation of rat neonatal cardiomyocytes with isoprenaline. Isoprenaline also promoted translocation of NDPK-C to the plasma membrane. Overexpression of NDPK-C in cardiomyocytes increased cAMP levels and sensitized cardiomyocytes to isoprenaline-induced augmentation of contractility, whereas NDPK-C knockdown decreased cAMP levels. In vivo, depletion of NDPK-C in zebrafish embryos caused cardiac edema and ventricular dysfunction. NDPK-B knockout mice had unaltered NDPK-C expression but showed contractile dysfunction and exacerbated cardiac remodeling during long-term isoprenaline stimulation. In human end-stage HF, the complex formation between NDPK-C and Gαi2 was increased whereas the NDPK-C/Gαs interaction was decreased, producing a switch that may contribute to an NDPK-C-dependent cAMP reduction in HF. CONCLUSIONS: Our findings identify NDPK-C as an essential requirement for both the interaction between NDPK isoforms and between NDPK isoforms and G proteins. NDPK-C is a novel critical regulator of ß-adrenoceptor/cAMP signaling and cardiac contractility. By switching from Gαs to Gαi2 activation, NDPK-C may contribute to lower cAMP levels and the related contractile dysfunction in HF.

Effects of Chelidonium majus extracts and major alkaloids on hERG potassium channels and on dog cardiac action potential - a safety approach.

Chelidonium majus or greater celandine is spread throughout the world, and it is a very common and frequent component of modern phytotherapy. Although C. majus contains alkaloids with remarkable physiological effect, moreover, safety pharmacology properties of this plant are not widely clarified, medications prepared from this plant are often used internally. In our study the inhibitory effects of C. majus herb extracts and alkaloids on hERG potassium current as well as on cardiac action potential were investigated. Our data show that hydroalcoholic extracts of greater celandine and its alkaloids, especially berberine, chelidonine and sanguinarine have a significant hERG potassium channel blocking effect. These extracts and alkaloids also prolong the cardiac action potential in dog ventricular muscle. Therefore these compounds may consequently delay cardiac repolarization, which may result in the prolongation of the QT interval and increase the risk of potentially fatal ventricular arrhythmias.

Characterization of a novel multifunctional resveratrol derivative for the treatment of atrial fibrillation.

BACKGROUND AND PURPOSE: Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with an increased risk for stroke, heart failure and cardiovascular-related mortality. Candidate targets for anti-AF drugs include a potassium channel K(v)1.5, and the ionic currents I(KACh) and late I(Na), along with increased oxidative stress and activation of NFAT-mediated gene transcription. As pharmacological management of AF is currently suboptimal, we have designed and characterized a multifunctional small molecule, compound 1 (C1), to target these ion channels and pathways. EXPERIMENTAL APPROACH: We made whole-cell patch-clamp recordings of recombinant ion channels, human atrial I(Kur), rat atrial I(KACh), cellular recordings of contractility and calcium transient measurements in tsA201 cells, human atrial samples and rat myocytes. We also used a model of inducible AF in dogs. KEY RESULTS: C1 inhibited human peak and late K(v)1.5 currents, frequency-dependently, with IC50 of 0.36 and 0.11 µmol·L(-1) respectively. C1 inhibited I(KACh)(IC50 of 1.9 µmol·L(-1)) and the Na(v)1.5 sodium channel current (IC50s of 3 and 1 µmol·L(-1) for peak and late components respectively). C1 (1 µmol·L(-1)) significantly delayed contractile and calcium dysfunction in rat ventricular myocytes treated with 3 nmol·L(-1) sea anemone toxin (ATX-II). C1 weakly inhibited the hERG channel and maintained antioxidant and NFAT-inhibitory properties comparable to the parent molecule, resveratrol. In a model of inducible AF in conscious dogs, C1 (1 mg·kg(-1)) reduced the average and total AF duration. CONCLUSION AND IMPLICATIONS: C1 behaved as a promising multifunctional small molecule targeting a number of key pathways involved in AF.

Involvement of large-conductance Ca(2+) -activated K(+) channels in both nitric oxide and endothelium-derived hyperpolarization-type relaxation in human penile small arteries.

Large-conductance Ca(2+) -activated K(+) channels (BKC a ), located on the vascular smooth muscle, play an important role in regulation of vascular tone. In penile corpus cavernosum tissue, opening of BKC a channels leads to relaxation of corporal smooth muscle, which is essential during erection; however, there is little information on the role of BKC a channels located in penile vascular smooth muscle. This study was designed to investigate the involvement of BKC a channels in endothelium-dependent and endothelium-independent relaxation of human intracavernous penile arteries. In human intracavernous arteries obtained in connection with transsexual operations, change in isometric force was recorded in microvascular myographs, and endothelium-dependent [nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-type] and endothelium-independent (NO-donor) relaxations were measured in contracted arteries. In penile small arteries contracted with phenylephrine, acetylcholine evoked NO- and EDH-type relaxations, which were sensitive to iberiotoxin (IbTX), a selective blocker of BKC a channels. Iberiotoxin also inhibited relaxations induced by a NO-donor, sodium nitroprusside. NS11021, a selective opener of BKC a channels, evoked pronounced relaxations that were inhibited in the presence of IbTX. NS13558, a BKC a -inactive analogue of NS11021, failed to relax human penile small arteries. Our results show that BKC a channels are involved in both NO- and EDH-type relaxation of intracavernous penile arteries obtained from healthy men. The effect of a selective opener of BKC a channels also suggests that direct activation of the channel may be an advantageous approach for treatment of impaired endothelium-dependent relaxation often associated with erectile dysfunction.

Remodeling of the cardiac sodium channel, connexin43, and plakoglobin at the intercalated disk in patients with arrhythmogenic cardiomyopathy.

BACKGROUND: Arrhythmogenic cardiomyopathy (AC) is closely associated with desmosomal mutations in a majority of patients. Arrhythmogenesis in patients with AC is likely related to remodeling of cardiac gap junctions and increased levels of fibrosis. Recently, using experimental models, we also identified sodium channel dysfunction secondary to desmosomal dysfunction. OBJECTIVE: To assess the immunoreactive signal levels of the sodium channel protein NaV1.5, as well as connexin43 (Cx43) and plakoglobin (PKG), in myocardial specimens obtained from patients with AC. METHODS: Left and right ventricular free wall postmortem material was obtained from 5 patients with AC and 5 controls matched for age and sex. Right ventricular septal biopsies were taken from another 15 patients with AC. All patients fulfilled the 2010 revised Task Force Criteria for the diagnosis of AC. Immunohistochemical analyses were performed using antibodies against Cx43, PKG, NaV1.5, plakophilin-2, and N-cadherin. RESULTS: N-cadherin and desmoplakin immunoreactive signals and distribution were normal in patients with AC compared to controls. Plakophilin-2 signals were unaffected unless a plakophilin-2 mutation predicting haploinsufficiency was present. Distribution was unchanged compared to that in controls. Immunoreactive signal levels of PKG, Cx43, and NaV1.5 were disturbed in 74%, 70%, and 65% of the patients, respectively. CONCLUSIONS: A reduced immunoreactive signal of PKG, Cx43, and NaV1.5 at the intercalated disks can be observed in a large majority of the patients. Decreased levels of Nav1.5 might contribute to arrhythmia vulnerability and, in the future, potentially could serve as a new clinically relevant tool for risk assessment strategies.

Prolonged antispasmodic effect in isolated radial artery graft and pronounced platelet inhibition induced by the inodilator drug, levosimendan.

Radial artery frequently develops spasm and requires vasodilator therapy during coronary artery bypass graft surgery (CABG). Levosimendan was recently shown to oppose 5-hydroxytryptamine-induced contraction of radial artery (RA) grafts. The aim of the present study was to explore whether levosimendan retains its vasodilatory capacity following in vitro pre-incubation of RA segments with the inodilator. A possible cumulative effect of the drug in human platelets was also studied. Human isolated RA segments were pre-incubated in 0.16 µmol/L levosimendan containing solution or in 0.9% NaCl, Bretschneider, 5% albumin and a 5% human serum protein solution (Biseko) as controls for 45 min. Contractions were induced by three consecutive administrations of 5-hydroxytryptamine (0.31 µM) 45, 90 and 120 min. after exchanging the pre-incubation solutions with Krebs-Henseleit solution, uniformly. Receptor-independent contractions (KCl, 80 mmol/L), endothelium-dependent (acetylcholine, 1 µmol/L) and independent relaxations (papaverine, 100 µmol/L) were also investigated. Washed human platelets were pre-incubated with levosimendan (0.06 µmol/L) for 2 or 15 min. and aggregated with thrombin (0.1 IU/mL). Contractions of RA grafts induced by 5-hydroxytryptamine were significantly smaller 45 min. and 90 min. after the replacement of levosimendan with Krebs-Henseleit solution. Biseko solution also decreased the contraction of the graft at 45 min. Contractions did not change in time following the pre-incubations of radial arteries with 0.9% NaCl, Bretschneider and 5% albumin solutions. The grafts remained intact as assessed by their maximum contractions and endothelium-dependent and endothelium-independent relaxations at the end of the investigations. Platelets revealed larger anti-aggregatory effect to levosimendan following the enhancement of the incubation time. Results indicate that the antispasmodic and anti-aggregatory effects of levosimendan cumulate in the vascular tissue and in platelets. The storage of RA with the inodilator before implantation may help to prevent the intraoperative spasm of the graft and also thrombotic occlusion during CABG surgery.

Reorganized PKA-AKAP associations in the failing human heart.

Here we reveal that the characterization of large-scale re-arrangements of signaling scaffolds induced by heart failure can serve as a novel concept to identify more specific therapeutic targets. In the mammalian heart, the cAMP pathway, with the cAMP-dependent protein kinase (PKA) in a central role, acts directly downstream of adrenergic receptors to mediate cardiac contractility and rhythm. Heart failure, characterized by severe alterations in adrenergic stimulation is, amongst other interventions, often treated with ß-blockers. Contrasting results, however, have shown both beneficial and detrimental effects of decreased cAMP levels in failing hearts. We hypothesize that the origin of this behavior lies in the complex spatiotemporal organization of the regulatory subunit of PKA (PKA-R), which associates tightly with various A-kinase anchoring proteins (AKAPs) to specifically localize PKA's activity. Using chemical proteomics directly applied to human patient and control heart tissue we demonstrate that the association profile of PKA-R with several AKAPs is severely altered in the failing heart, for instance effecting the interaction between PKA and the novel AKAP SPHKAP was 6-fold upregulated upon failing heart conditions. Also a significant increase in captured cGMP-dependent protein kinase (PKG) and phosphodiesterase 2 (PDE2) was observed. The observed altered profiles can already explain many aspects of the aberrant cAMP-response in the failing human heart, validating that this dataset may provide a resource for several novel, more specific, treatment options. This article is part of a Special Issue entitled "Local Signaling in Myocytes".

Left-to-right atrial inward rectifier potassium current gradients in patients with paroxysmal versus chronic atrial fibrillation.

BACKGROUND: Recent evidence suggests that atrial fibrillation (AF) is maintained by high-frequency reentrant sources with a left-to-right-dominant frequency gradient, particularly in patients with paroxysmal AF (pAF). Unequal left-to-right distribution of inward rectifier K(+) currents has been suggested to underlie this dominant frequency gradient, but this hypothesis has never been tested in humans. METHODS AND RESULTS: Currents were measured with whole-cell voltage-clamp in cardiomyocytes from right atrial (RA) and left (LA) atrial appendages of patients in sinus rhythm (SR) and patients with AF undergoing cardiac surgery. Western blot was used to quantify protein expression of I(K1) (Kir2.1 and Kir2.3) and I(K,ACh) (Kir3.1 and Kir3.4) subunits. Basal current was ≈2-fold larger in chronic AF (cAF) versus SR patients, without RA-LA differences. In pAF, basal current was ≈2-fold larger in LA versus RA, indicating a left-to-right atrial gradient. In both atria, Kir2.1 expression was ≈2-fold greater in cAF but comparable in pAF versus SR. Kir2.3 levels were unchanged in cAF and RA-pAF but showed a 51% decrease in LA-pAF. In SR, carbachol-activated (2 µmol/L) I(K,ACh) was 70% larger in RA versus LA. This right-to-left atrial gradient was decreased in pAF and cAF caused by reduced I(K,ACh) in RA only. Similarly, in SR, Kir3.1 and Kir3.4 proteins were greater in RA versus LA and decreased in RA of pAF and cAF. Kir3.1 and Kir3.4 expression was unchanged in LA of pAF and cAF. CONCLUSION: Our results support the hypothesis that a left-to-right gradient in inward rectifier background current contributes to high-frequency sources in LA that maintain pAF. These findings have potentially important implications for development of atrial-selective therapeutic approaches.

Gender-related differences in ion-channel and transporter subunit expression in non-diseased human hearts.

Gender-related differences in ventricular electrophysiology are known to be important determinants of human arrhythmic risk, but the underlying molecular basis is poorly understood. The present work aims to provide the first detailed analysis of gender-related cardiac ion-channel gene-distribution, based on samples from non-diseased human hearts. By using a high-throughput quantitative approach, we investigated at a genome-scale the expression of 79 genes encoding ion-channel and transporter subunits in epicardial and endocardial tissue samples from non-diseased transplant donors (10 males, 10 females). Gender-related expression differences involved key genes implicated in conduction and repolarization. Female hearts showed reduced expression for a variety of K(+)-channel subunits with potentially important roles in cardiac repolarization, including HERG, minK, Kir2.3, Kv1.4, KChIP2, SUR2 and Kir6.2, as well as lower expression of connexin43 and phospholamban. In addition, they demonstrated an isoform switch in Na(+)/K(+)-ATPase, expressing more of the alpha1 and less of the alpha3 subunit than male hearts, along with increased expression of calmodulin-3. Iroquois transcription factors (IRX3, IRX5) were more strongly expressed in female than male epicardium, but the transmural gradient remained. Protein-expression paralleled transcript patterns for all subunits examined: HERG, minK, Kv1.4, KChIP2, IRX5, Nav1.5 and connexin43. Our results indicate that male and female human hearts have significant differences in ion-channel subunit composition, with female hearts showing decreased expression for a number of repolarizing ion-channels. These findings are important for understanding sex-related differences in the susceptibility to ventricular arrhythmias, particularly for conditions associated with repolarization abnormalities like Brugada and Long QT syndrome.

Heterogeneous Connexin43 distribution in heart failure is associated with dispersed conduction and enhanced susceptibility to ventricular arrhythmias.

AIMS: Sudden arrhythmogenic cardiac death is a major cause of mortality in patients with congestive heart failure (CHF). To investigate determinants of the increased arrhythmogenic susceptibility, we studied cardiac remodelling and arrhythmogenicity in CHF patients and in a mouse model of chronic pressure overload. METHODS AND RESULTS: Clinical and (immuno)histological data of myocardial biopsies from CHF patients with (VT+) and without (VT-) documented ventricular arrhythmia were compared with controls. In CHF patients, ejection fraction was decreased and QRS duration was increased. Cell size and interstitial fibrosis were increased, but Connexin43 (Cx43) levels, the most abundant gap junction in ventricular myocardium, were unchanged. No differences were found between VT+ and VT- patients, except for the distribution pattern of Cx43, which was significantly more heterogeneous in VT+. Mice were subjected to transverse aortic constriction (TAC) or sham operated. At 16 weeks, cardiac function was determined by echocardiography and epicardial ventricular activation mapping was performed. Transverse aortic constriction mice had decreased fractional shortening and prolonged QRS duration. Right ventricular conduction velocity was reduced, and polymorphic VTs were induced in 44% TAC and 0% sham mice. Interstitial fibrosis was increased and Cx43 quantity was unchanged in TAC mice with and without arrhythmias. Similar to CHF patients, heterogeneous Cx43 distribution was significantly associated with arrhythmias in TAC mice and with spatial heterogeneity of impulse conduction. CONCLUSION: Heterogeneous Cx43 expression during CHF is associated with dispersed impulse conduction and may underlie enhanced susceptibility to ventricular tachyarrhythmias.

Mechanisms of ventricular rate adaptation as a predictor of arrhythmic risk.

Protracted QT interval (QTI) adaptation to abrupt heart rate (HR) changes has been identified as a clinical arrhythmic risk marker. This study investigates the ionic mechanisms of QTI rate adaptation and its relationship to arrhythmic risk. Computer simulations and experimental recordings in human and canine ventricular tissue were used to investigate the ionic basis of QTI and action potential duration (APD) to abrupt changes in HR with a protocol commonly used in clinical studies. The time for 90% QTI adaptation is 3.5 min in simulations, in agreement with experimental and clinical data in humans. APD adaptation follows similar dynamics, being faster in mid-myocardial cells (2.5 min) than in endocardial and epicardial cells (3.5 min). Both QTI and APD adapt in two phases following an abrupt HR change: a fast initial phase with time constant < 30 s, mainly related to L-type calcium and slow-delayed rectifier potassium current, and a second slow phase of >2 min driven by intracellular sodium concentration ([Na(+)](i)) dynamics. Alterations in [Na(+)](i) dynamics due to Na(+)/K(+) pump current inhibition result in protracted rate adaptation and are associated with increased proarrhythmic risk, as indicated by action potential triangulation and faster L-type calcium current recovery from inactivation, leading to the formation of early afterdepolarizations. In conclusion, this study suggests that protracted QTI adaptation could be an indicator of altered [Na(+)](i) dynamics following Na(+)/K(+) pump inhibition as it occurs in patients with ischemia or heart failure. An increased risk of cardiac arrhythmias in patients with protracted rate adaptation may be due to an increased risk of early after-depolarization formation.

Biseko colloidal solution diminishes the vasoreactivity of human isolated radial arteries.

OBJECTIVE: Radial arteries are increasingly used as grafts in coronary artery bypass surgery. The surgical preparation and intraoperative management of this conduit artery may affect its early and long-term patencies. We investigated the effects of the colloidal Biseko and 5% albumin solutions as well as the crystalloid physiological saline (0.9% NaCl) and Bretschneider solutions on the contractile and relaxing capacities of isolated human radial artery grafts. METHOD: Radial artery segments were harvested using the technique with an ultrasonic scalpel, and 2.5-3mm rings were obtained from the proximal part of the artery. Arterial rings were stored in Biseko or 5% albumin solutions and in 0.9% NaCl or Bretschneider solutions for 45 min. Isometric tensions of radial arteries obtained from 26 patients were measured in isolated organ baths. Contractions were induced by 0.31 micromolL(-1) 5-hydroxytryptamine and 10 micromol L(-1) noradrenaline. Endothelium-dependent relaxations were induced by 10 micromol L(-1) acetylcholine and 1 micromol L(-1) bradykinin as well as the endothelium-independent relaxations by 10 micromol L(-1) glyceryl trinitrate and 100 micromol/l papaverine. RESULTS: Contractions of radial arteries induced by 5-hydroxytryptamine were significantly lower following storage in Biseko solution (12.6+/-4.4 mN) than in 5% albumin (37.9+/-13.0 mN, p=0.03) or in 0.9% NaCl solution (35.9+/-11.9 mN, p=0.04). Noradrenaline-induced contractions of the arteries were also diminished in Biseko solution compared to those stored in 5% albumin (32.9+/-6.2 mN vs 49.2+/-6.4 mN, p=0.01). No significant differences in relaxations were obtained between the two crystalloid and the two colloidal solutions using endothelium-dependent and independent vasorelaxants. CONCLUSION: Our results suggest that storage of radial artery in Biseko colloidal solution before coronary artery bypass grafting decreases the sensitivity of the graft to vasoconstriction, thereby decreasing the risk of intra/perioperative graft failure.

5,7,8-Trimethyl-benzopyran and 5,7,8-trimethyl-1,4-benzoxazine aminoamide derivatives as novel antiarrhythmics against ischemia-reperfusion injury.

6-Hydroxy-5,7,8-trimethyl-benzopyran derivatives and 5,7,8-trimethyl-1,4-benzoxazine analogues substituted by the lidocaine pharmacophore aminoamide functionality at C4 or N4, respectively, were synthesized and evaluated against arrhythmias associated with ischemia-reperfusion injury. The antiarrhythmic effect of substitutents at positions C2 and C6 was examined. Six out of the 11 new derivatives, exhibited arrhythmia scores 1.0-1.3 versus the control (4.5 +/- 1.2), which was also reflected to the % premature beats (0.5-3.9), control (13.7 +/- 3.6). Selected compounds were further studied by a conventional microelectrode method. 2-Diethylamino-1-(5,7,8-trimethyl-2-phenyl-2,3-dihydro-benzo[1,4]oxazin-4-yl)-ethanone (50) and the trolox-inspired 4-(2-diethylamino-acetyl)-2,5,7,8-tetramethyl-3,4-dihydro-2H-benzo[1,4]oxazine-2-carboxylic acid ethyl ester (62) suppress reperfusion arrhythmias and reduce malondialdehyde (MDA) content, leading to a fast recovery of the heart after ischemia-reperfusion. They exhibit combined class IB and class III antiarrhythmic properties, which constitutes them as promising compounds for further studies because, due to their multichannel "amiodarone like" effect, less proarrhythmic complications can be expected.

Transcriptional profiling of ion channel genes in Brugada syndrome and other right ventricular arrhythmogenic diseases.

AIMS: Brugada syndrome is an inherited sudden-death arrhythmia syndrome. Na(+)-current dysfunction is central, but mutations in the SCN5A gene (encoding the cardiac Na(+)-channel Nav1.5) are present in only 20% of probands. This study addressed the possibility that Brugada patients display specific expression patterns for ion-channels regulating cardiac conduction, excitability, and repolarization. METHODS AND RESULTS: Transcriptional profiling was performed on right-ventricular endomyocardial biopsies from 10 unrelated Brugada probands, 11 non-diseased organ-donors, seven heart-transplant recipients, 10 with arrhythmogenic right-ventricular cardiomyopathy, and nine with idiopathic right-ventricular outflow-tract tachycardia. Brugada patients showed distinct clustering differences vs. the two control and two other ventricular-tachyarrhythmia groups, including 14 of 77 genes encoding important ion-channel/ion-transporter subunits. Nav1.5 and K(+)-channels Kv4.3 and Kir3.4 were more weakly expressed, whereas the Na(+)-channel Nav2.1 and the K(+)-channel TWIK1 were more strongly expressed, in Brugada syndrome. Differences were also seen in Ca(2+)-homeostasis transcripts, including stronger expression of RYR2 and NCX1. The molecular profile of Brugada patients with SCN5A mutations did not differ from Brugada patients without SCN5A mutations. CONCLUSION: Brugada patients exhibit a common ion-channel molecular expression signature, irrespective of the culprit gene. This finding has potentially important implications for our understanding of the pathophysiology of Brugada syndrome, with possible therapeutic and diagnostic consequences.