Effects of SZV-2649, a new multiple ion channel inhibitor mexiletine analogue.

The antiarrhythmic and cardiac electrophysiological effects of SZV-2649 that contains a 2,6-diiodophenoxy moiety but lacks the benzofuran ring system present in amiodarone, were studied in mammalian cell line, rat and dog cardiac preparations. SZV-2649 exerted antiarrhythmic effects against coronary artery occlusion/reperfusion induced ventricular arrhythmias in rats and in acetylcholine- and burst stimulation induced atrial fibrillation in dogs. SZV-2649 inhibited hERG and GIRK currents in HEK cells (IC50: 342 and 529 nM, respectively). In canine ventricular myocytes, SZV-2649 (10 µM) decreased the densities of IKr, and Ito outward and INaL and ICaL inward currents. The compound (2.5-10 µM) elicited Class IB type Vmax reducing and Class III type action potential duration prolonging effects in dog right ventricular muscle preparations. In canine atrial muscle, SZV-2629 (2.5-10 µM) moderately prolonged action potential duration and this effect was greatly augmented in preparations pretreated with 1 µM carbachol. In conclusion, SZV-2649, has antiarrhythmic effects based on its multiple ion channel blocking properties. Since its chemical structure substantially differs from that of amiodarone, it is expected that SZV-2649 would exhibit fewer adverse effects than the currently used most effective multichannel inhibitor drug amiodarone and may be a promising molecule for further development.

The Properties of the Transient Outward, Inward Rectifier and Acetylcholine-Sensitive Potassium Currents in Atrial Myocytes from Dogs in Sinus Rhythm and Experimentally Induced Atrial Fibrillation Dog Models.

AIMS: Atrial fibrillation (AF) is the most common chronic/recurrent arrhythmia, which significantly impairs quality of life and increases cardiovascular morbidity and mortality. Therefore, the aim of the present study was to investigate the properties of three repolarizing potassium currents which were shown to contribute to AF-induced electrical remodeling, i.e., the transient outward (Ito), inward rectifier (IK1) and acetylcholine-sensitive (IK,ACh) potassium currents in isolated atrial myocytes obtained from dogs either with sinus rhythm (SR) or following chronic atrial tachypacing (400/min)-induced AF. METHODS: Atrial remodeling and AF were induced by chronic (4-6 weeks of) right atrial tachypacing (400/min) in dogs. Transmembrane ionic currents were measured by applying the whole-cell patch-clamp technique at 37 °C. RESULTS: The Ito current was slightly downregulated in AF cells when compared with that recorded in SR cells. This downregulation was also associated with slowed inactivation kinetics. The IK1 current was found to be larger in AF cells; however, this upregulation was not statistically significant in the voltage range corresponding with atrial action potential (-80 mV to 0 mV). IK,ACh was activated by the cholinergic agonist carbachol (CCh; 2 µM). In SR, CCh activated a large current either in inward or outward directions. The selective IK,ACh inhibitor tertiapin (10 nM) blocked the outward CCh-induced current by 61%. In atrial cardiomyocytes isolated from dogs with AF, the presence of a constitutively active IK,ACh was observed, blocked by 59% with 10 nM tertiapin. However, in "AF atrial myocytes", CCh activated an additional, significant ligand-dependent and tertiapin-sensitive IK,ACh current. CONCLUSIONS: In our dog AF model, Ito unlike in humans was downregulated only in a slight manner. Due to its slow inactivation kinetics, it seems that Ito may play a more significant role in atrial repolarization than in ventricular working muscle myocytes. The presence of the constitutively active IK,ACh in atrial myocytes from AF dogs shows that electrical remodeling truly developed in this model. The IK,ACh current (both ligand-dependent and constitutively active) seems to play a significant role in canine atrial electrical remodeling and may be a promising atrial selective drug target for suppressing AF.

Cardiovascular autonomic and peripheral sensory neuropathy in women with obesity.

Introduction: A higher incidence of neural dysfunction in people with obesity has been described. We determined the prevalence of neuropathic lesions in obese women and evaluated their potential association with anthropometric and laboratory parameters. Patients and methods: In our cross-sectional study, we enrolled female patients with obesity and without diabetes before obesity treatment. Voluntary female subjects were controls with a normal body mass index (BMI). Autonomic function was assessed by Ewing's cardiovascular reflex tests, while comprehensive peripheral neuropathic assessments were conducted utilizing the Neurometer®, Tiptherm®, Monofilament®, and Rydel-Seiffer tuning fork tests. Sudomotor function was assessed by the Neuropad®-test. Body composition was examined using the InBody 770. Results: 71 patients (mean ± SD; age: 36.1 ± 8.3 years; BMI: 40.2 ± 8.5 kg/m2) and 36 controls (age: 36.4 ± 13.3 years; BMI: 21.6 ± 2.1 kg/m2) were enrolled. Patients had significantly higher systolic (patients vs. controls; 137.5 ± 16.9 vs. 114.6 ± 14.8 mmHg, p<0.001) and diastolic (83.0 ± 11.7 vs.69.8 ± 11.2 mmHg, p<0.001) blood pressure compared to controls. Among autonomic tests, only the heart rate response to Valsalva maneuver (Valsalva-ratio) revealed significant impairment in patients (1.4 ± 0.2 vs. 1.7 ± 0.4, p<0.001). Neurometer® at the median nerve revealed increased current perception threshold (CPT) values at all stimulating frequencies in patients (CPT at 2000 Hz: 204.6 ± 70.9 vs. 168.1 ± 66.9, p=0.013; 250 Hz: 84.4 ± 38.9 vs. 56.5 ± 34.8, p<0.001; CPT at 5 Hz: 58.5 ± 31.2 vs 36.9 ± 29.1, p<0.001). The Rydel-Seiffer tuning fork test has revealed a significant impairment of vibrational sensing on the lower limb in patients (right hallux: 6.8 ± 0.9 vs. 7.4 ± 0.8, p=0.030; left hallux: 6.9 ± 0.8 vs. 7.3 ± 0.9, p=0.029). The Neuropad® testing showed a significant impairment of sudomotor function in women with obesity. A negative correlation was found in patients between BMI and the 25-hydroxy-D3/D2-vitamin levels (r=-0.41, p=0.00126) and a positive correlation between the BMI and resting systolic blood pressure (r=0.26, p=0.0325). Conclusion: Peripheral sensory neuronal and sudomotor function impairments were detected in female patients with obesity compared to the controls with normal BMI. Cardiovascular autonomic dysfunction was also revealed by the Valsalva-ratio in these patients, suggesting the presence of parasympathetic dysfunction. The negative correlation between BMI and the 25-hydroxy-D3/D2-vitamin highlights the potential deficiency of vitamin D in the population affected by obesity.

Elucidating the Benefit of Perforated vs Non-Perforated Membranes in Guided Bone Regeneration: An in Vivo Histologic Evaluation and Histomorphometric Analysis.

BACKGROUND: Non-perforated Polytetrafluoroethylene (PTFE) membranes are effectively utilized in guided bone regeneration (GBR) but may hinder cell migration due to limited interaction with the periosteum. This study compared bone regeneration using occlusive or perforated membranes combined with acellular collagen sponge (ACS) and recombinant human bone morphogenic protein-2 (rhBMP-2) in a canine mandibular model. MATERIAL AND METHODS: Male beagle dogs (n=3) received two mandibular defects each to compare ACS/rhBMP-2 with experimental (perforated group) and control (non-perforated group) membranes (n=3 defects/group). Tissue healing was assessed histomorphologically, histomorphometrically and through volumetric reconstruction using microcomputed tomography. RESULTS: The perforated group showed increased bone formation and reduced soft tissue formation compared to the non-perforated group. For the primary outcome, histomorphometric analysis revealed significantly greater total regenerated bone in the perforated group (67.08 ± 6.86%) relative to the nonperforated group (25.18 ± 22.44%) (p = 0.036). Perforated membranes had less soft tissue infiltration (32.91 ± 6.86%) compared to non-perforated membranes (74.82 ± 22.44%) (p = 0.036). CONCLUSION: The increased permeability of membranes in the perforated group potentially enabled periosteal precursor cells greater accessibility to rhBMP-2. The availability may have accelerated their differentiation into mature bone-forming cells, contributing to the stimulation of new bone production, relative to the non-perforated group.

Cellular electrophysiological effects of the citrus flavonoid hesperetin in dog and rabbit cardiac ventricular preparations.

Recent experimental data shows that hesperetin, a citrus flavonoid, affects potassium channels and can prolong the QTc interval in humans. Therefore, in the present study we investigated the effects of hesperetin on various transmembrane ionic currents and on ventricular action potentials. Transmembrane current measurements and action potential recordings were performed by patch-clamp and the conventional microelectrode techniques in dog and rabbit ventricular preparations. At 10 µM concentration hesperetin did not, however, at 30 µM significantly decreased the amplitude of the IK1, Ito, IKr potassium currents. Hesperetin at 3-30 µM significantly and in a concentration-dependent manner reduced the amplitude of the IKs current. The drug significantly decreased the amplitudes of the INaL and ICaL currents at 30 µM. Hesperetin (10 and 30 µM) did not change the action potential duration in normal preparations, however, in preparations where the repolarization reserve had been previously attenuated by 100 nM dofetilide and 1 µg/ml veratrine, caused a moderate but significant prolongation of repolarization. These results suggest that hesperetin at close to relevant concentrations inhibits the IKs outward potassium current and thereby reduces repolarization reserve. This effect in certain specific situations may prolong the QT interval and consequently may enhance proarrhythmic risk.

A three-month physical training program improves cardiovascular autonomic function in patients with metabolic syndrome with and without diabetes - a pilot study.

Introduction: Vascular complications and neuropathy may develop in the presence of metabolic syndrome. The aim of our study was to measure the cardiovascular autonomic function following physical training in patients with metabolic syndrome with and without diabetes. Subjects and methods: 56 patients with metabolic syndrome (32 men/24 women, 40 non-diabetic patients (NDMetS)/16 diabetic patients (DMetS) [mean ± SD]: age: 50.35 ± 8.03 vs. 56.8 ± 9.30 years, p=0.023; baseline BMI: 32.2 ± 7.03 vs. 32.8 ± 5.94 kg/m2, p=0.739) were involved in our study. All tests and measurements were carried out before and following a 3-month physical training period. Autonomic function was assessed by means of five standard cardiovascular reflex tests. ECG repolarization parameters, including short-term QT variability and stress-ECG were also measured. Results: In the whole population, Valsalva-ratio (VR) and the autonomic score (AS) improved following training (VR: 1.49 ± 0.24 vs. 1.64 ± 0.34, p=0.001; AS: 2.05 ± 1.73 vs. 1.41 ± 1.36, p=0.015) accompanied by the significant decrease of the systolic (150.3 ± 16.12 vs. 134.1 ± 16.67 mmHg, p<0.001) and diastolic (90.64 ± 12.8 vs. 82.79 ± 11.1 mmHg, p<0.001) blood pressure. An improvement in VR was detected in NDMetS patients following training (1.51 ± 0.24 vs. 1.67 ± 0.31, p= 0.002). No significant changes could be detected in autonomic tests' results in the DMetS patient group following training. The applied exercise training program did not lead to significant changes in ECG repolarization. The stress-ECG test in the whole study population yielded a significant increase in the test duration (12.9 ± 3.76 vs. 15.1 ± 2.96 min, p<0.001) and in the test load (10.5 ± 2.78 vs. 11.6 ± 2.39 MET, p<0.001). The load capability improved significantly in both subgroups: 11.1 ± 2.04 vs. 12.1 ± 1.82, (p<0.001) and 9.0 ± 3.64 vs. 10.4 ± 3.05, (p=0.033) in subpopulations of NDMetS and DMetS, respectively. The DMetS patients achieved a significantly lower MET score at baseline (p=0.039) and following training (p=0.044) in comparison to the NDMetS patients. Conclusion: The three-month exercise program improved the Valsalva-ratio and the AN score in the MetS patients, that is potentially protective against cardiovascular events. The training had some beneficial effect on blood pressure and the results of the stress-ECG tests in both groups. The absence of significant change in the reflex tests in DMetS group reflects an impaired adaptation compared to the NDMestS group.

Selective Inhibition of Cardiac Late Na+ Current Is Based on Fast Offset Kinetics of the Inhibitor.

The present study was designed to test the hypothesis that the selectivity of blocking the late Na+ current (INaL) over the peak Na+ current (INaP) is related to the fast offset kinetics of the Na+ channel inhibitor. Therefore, the effects of 1 µM GS967 (INaL inhibitor), 20 µM mexiletine (I/B antiarrhythmic) and 10 µM quinidine (I/A antiarrhythmic) on INaL and INaP were compared in canine ventricular myocardium. INaP was estimated as the maximum velocity of action potential upstroke (V+max). Equal amounts of INaL were dissected by the applied drug concentrations under APVC conditions. The inhibition of INaL by mexiletine and quinidine was comparable under a conventional voltage clamp, while both were smaller than the inhibitory effect of GS967. Under steady-state conditions, the V+max block at the physiological cycle length of 700 ms was 2.3% for GS967, 11.4% for mexiletine and 26.2% for quinidine. The respective offset time constants were 110 ± 6 ms, 456 ± 284 ms and 7.2 ± 0.9 s. These results reveal an inverse relationship between the offset time constant and the selectivity of INaL over INaP inhibition without any influence of the onset rate constant. It is concluded that the selective inhibition of INaL over INaP is related to the fast offset kinetics of the Na+ channel inhibitor.

Sex and age differences in AMPK phosphorylation, mitochondrial homeostasis, and inflammation in hearts from inflammatory cardiomyopathy patients.

Linked to exacerbated inflammation, myocarditis is a cardiovascular disease, which may lead to dilated cardiomyopathy. Although sex and age differences in the development of chronic myocarditis have been postulated, underlying cellular mechanisms remain poorly understood. In the current study, we aimed to investigate sex and age differences in mitochondrial homeostasis, inflammation, and cellular senescence. Cardiac tissue samples from younger and older patients with inflammatory dilated cardiomyopathy (DCMI) were used. The expression of Sirt1, phosphorylated AMPK, PGC-1α, Sirt3, acetylated SOD2, catalase, and several mitochondrial genes was analyzed to assess mitochondrial homeostasis. The expression of NF-κB, TLR4, and interleukins was used to examine the inflammatory state in the heart. Finally, several senescence markers and telomere length were investigated. Cardiac AMPK expression and phosphorylation were significantly elevated in male DCMI patients, whereas Sirt1 expression remained unchanged in all groups investigated. AMPK upregulation was accompanied by a preserved expression of all mitochondrial proteins/genes investigated in older male DCMI patients, whereas the expression of TOM40, TIM23, and the mitochondrial oxidative phosphorylation genes was significantly reduced in older female patients. Mitochondrial homeostasis in older male patients was further supported by the reduced acetylation of mitochondrial proteins as indicated by acetylated SOD2. The inflammatory markers NF-κB and TLR4 were downregulated in older male DCMI patients, whereas the expression of IL-18 was increased in older female patients. This was accompanied by progressed senescence in older DCMI hearts. In conclusion, older women experience more dramatic immunometabolic disorders on the cellular level than older men.

Cardiac electrophysiological remodeling associated with enhanced arrhythmia susceptibility in a canine model of elite exercise.

The health benefits of regular physical exercise are well known. Even so, there is increasing evidence that the exercise regimes of elite athletes can evoke cardiac arrhythmias including ventricular fibrillation and even sudden cardiac death (SCD). The mechanism of exercise-induced arrhythmia and SCD is poorly understood. Here, we show that chronic training in a canine model (12 sedentary and 12 trained dogs) that mimics the regime of elite athletes induces electrophysiological remodeling (measured by ECG, patch-clamp, and immunocytochemical techniques) resulting in increases of both the trigger and the substrate for ventricular arrhythmias. Thus, 4 months sustained training lengthened ventricular repolarization (QTc: 237.1±3.4 ms vs. 213.6±2.8 ms, n=12; APD90: 472.8±29.6 ms vs. 370.1±32.7 ms, n=29 vs. 25), decreased transient outward potassium current (6.4±0.5 pA/pF vs. 8.8±0.9 pA/pF at 50 mV, n=54 vs. 42), and increased the short-term variability of repolarization (29.5±3.8 ms vs. 17.5±4.0 ms, n=27 vs. 18). Left ventricular fibrosis and HCN4 protein expression were also enhanced. These changes were associated with enhanced ectopic activity (number of escape beats from 0/hr to 29.7±20.3/hr) in vivo and arrhythmia susceptibility (elicited ventricular fibrillation: 3 of 10 sedentary dogs vs. 6 of 10 trained dogs). Our findings provide in vivo, cellular electrophysiological and molecular biological evidence for the enhanced susceptibility to ventricular arrhythmia in an experimental large animal model of endurance training.

Phosphodiesterase 8 governs cAMP/PKA-dependent reduction of L-type calcium current in human atrial fibrillation: a novel arrhythmogenic mechanism.

AIMS: Atrial fibrillation (AF) is associated with altered cAMP/PKA signaling and an AF-promoting reduction of L-type Ca2+-current (ICa,L), the mechanisms of which are poorly understood. Cyclic-nucleotide phosphodiesterases (PDEs) degrade cAMP and regulate PKA-dependent phosphorylation of key calcium-handling proteins, including the ICa,L-carrying Cav1.2α1C subunit. The aim was to assess whether altered function of PDE type-8 (PDE8) isoforms contributes to the reduction of ICa,L in persistent (chronic) AF (cAF) patients. METHODS AND RESULTS: mRNA, protein levels, and localization of PDE8A and PDE8B isoforms were measured by RT-qPCR, western blot, co-immunoprecipitation and immunofluorescence. PDE8 function was assessed by FRET, patch-clamp and sharp-electrode recordings. PDE8A gene and protein levels were higher in paroxysmal AF (pAF) vs. sinus rhythm (SR) patients, whereas PDE8B was upregulated in cAF only. Cytosolic abundance of PDE8A was higher in atrial pAF myocytes, whereas PDE8B tended to be more abundant at the plasmalemma in cAF myocytes. In co-immunoprecipitation, only PDE8B2 showed binding to Cav1.2α1C subunit which was strongly increased in cAF. Accordingly, Cav1.2α1C showed a lower phosphorylation at Ser1928 in association with decreased ICa,L in cAF. Selective PDE8 inhibition increased Ser1928 phosphorylation of Cav1.2α1C, enhanced cAMP at the subsarcolemma and rescued the lower ICa,L in cAF, which was accompanied by a prolongation of action potential duration at 50% of repolarization. CONCLUSION: Both PDE8A and PDE8B are expressed in human heart. Upregulation of PDE8B isoforms in cAF reduces ICa,L via direct interaction of PDE8B2 with the Cav1.2α1C subunit. Thus, upregulated PDE8B2 might serve as a novel molecular mechanism of the proarrhythmic reduction of ICa,L in cAF.

Disease- and sex-specific differences in patients with heart valve disease: a proteome study.

Pressure overload in patients with aortic valve stenosis and volume overload in mitral valve regurgitation trigger specific forms of cardiac remodeling; however, little is known about similarities and differences in myocardial proteome regulation. We performed proteome profiling of 75 human left ventricular myocardial biopsies (aortic stenosis = 41, mitral regurgitation = 17, and controls = 17) using high-resolution tandem mass spectrometry next to clinical and hemodynamic parameter acquisition. In patients of both disease groups, proteins related to ECM and cytoskeleton were more abundant, whereas those related to energy metabolism and proteostasis were less abundant compared with controls. In addition, disease group-specific and sex-specific differences have been observed. Male patients with aortic stenosis showed more proteins related to fibrosis and less to energy metabolism, whereas female patients showed strong reduction in proteostasis-related proteins. Clinical imaging was in line with proteomic findings, showing elevation of fibrosis in both patient groups and sex differences. Disease- and sex-specific proteomic profiles provide insight into cardiac remodeling in patients with heart valve disease and might help improve the understanding of molecular mechanisms and the development of individualized treatment strategies.

A Possible Explanation for the Low Penetrance of Pathogenic KCNE1 Variants in Long QT Syndrome Type 5.

Long QT syndrome (LQTS) is an inherited cardiac rhythm disorder associated with increased incidence of cardiac arrhythmias and sudden death. LQTS type 5 (LQT5) is caused by dominant mutant variants of KCNE1, a regulatory subunit of the voltage-gated ion channels generating the cardiac potassium current IKs. While mutant LQT5 KCNE1 variants are known to inhibit IKs amplitudes in heterologous expression systems, cardiomyocytes from a transgenic rabbit LQT5 model displayed unchanged IKs amplitudes, pointing towards the critical role of additional factors in the development of the LQT5 phenotype in vivo. In this study, we demonstrate that KCNE3, a candidate regulatory subunit of IKs channels minimizes the inhibitory effects of LQT5 KCNE1 variants on IKs amplitudes, while current deactivation is accelerated. Such changes recapitulate IKs properties observed in LQT5 transgenic rabbits. We show that KCNE3 accomplishes this by displacing the KCNE1 subunit within the IKs ion channel complex, as evidenced by a dedicated biophysical assay. These findings depict KCNE3 as an integral part of the IKs channel complex that regulates IKs function in cardiomyocytes and modifies the development of the LQT5 phenotype.

Ca2+/calmodulin-dependent protein kinase II and protein kinase G oxidation contributes to impaired sarcomeric proteins in hypertrophy model.

AIMS: Volume overload (VO) induced hypertrophy is one of the hallmarks to the development of heart diseases. Understanding the compensatory mechanisms involved in this process might help preventing the disease progression. METHODS AND RESULTS: Therefore, the present study used 2 months old Wistar rats, which underwent an aortocaval fistula to develop VO-induced hypertrophy. The animals were subdivided into four different groups, two sham operated animals served as age-matched controls and two groups with aortocaval fistula. Echocardiography was performed prior termination after 4- and 8-month. Functional and molecular changes of several sarcomeric proteins and their signalling pathways involved in the regulation and modulation of cardiomyocyte function were investigated. RESULTS: The model was characterized with preserved ejection fraction in all groups and with elevated heart/body weight ratio, left/right ventricular and atrial weight at 4- and 8-month, which indicates VO-induced hypertrophy. In addition, 8-months groups showed increased left ventricular internal diameter during diastole, RV internal diameter, stroke volume and velocity-time index compared with their age-matched controls. These changes were accompanied by increased Ca2+ sensitivity and titin-based cardiomyocyte stiffness in 8-month VO rats compared with other groups. The altered cardiomyocyte mechanics was associated with phosphorylation deficit of sarcomeric proteins cardiac troponin I, myosin binding protein C and titin, also accompanied with impaired signalling pathways involved in phosphorylation of these sarcomeric proteins in 8-month VO rats compared with age-matched control group. Impaired protein phosphorylation status and dysregulated signalling pathways were associated with significant alterations in the oxidative status of both kinases CaMKII and PKG explaining by this the elevated Ca2+ sensitivity and titin-based cardiomyocyte stiffness and perhaps the development of hypertrophy. CONCLUSIONS: Our findings showed VO-induced cardiomyocyte dysfunction via deranged phosphorylation of myofilament proteins and signalling pathways due to increased oxidative state of CaMKII and PKG and this might contribute to the development of hypertrophy.

Species-dependent differences in the inhibition of various potassium currents and in their effects on repolarization in cardiac ventricular muscle.

Even though rodents are accessible model animals, their electrophysiological properties are deeply different from those of humans, making the translation of rat studies to humans rather difficult. We compared the mechanisms of ventricular repolarization in various animal models to those of humans by measuring cardiac ventricular action potentials from ventricular papillary muscle preparations using conventional microelectrodes and applying selective inhibitors of various potassium transmembrane ion currents. Inhibition of the IK1 current (10 µmol/L barium chloride) significantly prolonged rat ventricular repolarization, but only slightly prolonged it in dogs, and did not affect it in humans. On the contrary, IKr inhibition (50 nmol/L dofetilide) significantly prolonged repolarization in humans, rabbits, and dogs, but not in rats. Inhibition of the IKur current (1 µmol/L XEN-D0101) only prolonged rat ventricular repolarization and had no effect in humans or dogs. Inhibition of the IKs (500 nmol/L HMR-1556) and Ito currents (100 µmol/L chromanol-293B) elicited similar effects in all investigated species. We conclude that dog ventricular preparations have the strongest translational value and rat ventricular preparations have the weakest translational value in cardiac electrophysiological experiments.

In vivo and cellular antiarrhythmic and cardiac electrophysiological effects of desethylamiodarone in dog cardiac preparations.

BACKGROUND AND PURPOSE: The aim of the present study was to study the antiarrhythmic effects and cellular mechanisms of desethylamiodarone (DEA), the main metabolite of amiodarone (AMIO), following acute and chronic 4-week oral treatments (25-50 mg·kg-1 ·day-1 ). EXPERIMENTAL APPROACH: The antiarrhythmic effects of acute iv. (10 mg·kg-1 ) and chronic oral (4 weeks, 25 mg·kg-1 ·day-1 ) administration of DEA were assessed in carbachol and tachypacing-induced dog atrial fibrillation models. Action potentials were recorded from atrial and right ventricular tissue following acute (10 µM) and chronic (p.o. 4 weeks, 50 mg·kg-1 ·day-1 ) DEA application using the conventional microelectrode technique. Ionic currents were measured by the whole cell configuration of the patch clamp technique in isolated left ventricular myocytes. Pharmacokinetic studies were performed following a single intravenous dose (25 mg·kg-1 ) of AMIO and DEA intravenously and orally. In chronic (91-day) toxicological investigations, DEA and AMIO were administered in the oral dose of 25 mg·kg-1 ·day-1 ). KEY RESULTS: DEA exerted marked antiarrhythmic effects in both canine atrial fibrillation models. Both acute and chronic DEA administration prolonged action potential duration in atrial and ventricular muscle without any changes detected in Purkinje fibres. DEA decreased the amplitude of several outward potassium currents such as IKr , IKs , IK1 , Ito , and IKACh , while the ICaL and late INa inward currents were also significantly depressed. Better drug bioavailability and higher volume of distribution for DEA were observed compared to AMIO. No neutropenia and less severe pulmonary fibrosis was found following DEA compared to that of AMIO administration. CONCLUSION AND IMPLICATIONS: Chronic DEA treatment in animal experiments has marked antiarrhythmic and electrophysiological effects with better pharmacokinetics and lower toxicity than its parent compound. These results suggest that the active metabolite, DEA, should be considered for clinical trials as a possible new, more favourable option for the treatment of cardiac arrhythmias including atrial fibrillation.

Docosahexaenoic acid normalizes QT interval in long QT type 2 transgenic rabbit models in a genotype-specific fashion.

AIM: Long QT syndrome (LQTS) is a cardiac channelopathy predisposing to ventricular arrhythmias and sudden cardiac death. Since current therapies often fail to prevent arrhythmic events in certain LQTS subtypes, new therapeutic strategies are needed. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, which enhances the repolarizing IKs current. METHODS AND RESULTS: We investigated the effects of DHA in wild type (WT) and transgenic long QT Type 1 (LQT1; loss of IKs), LQT2 (loss of IKr), LQT5 (reduction of IKs), and LQT2-5 (loss of IKr and reduction of IKs) rabbits. In vivo ECGs were recorded at baseline and after 10 µM/kg DHA to assess changes in heart-rate corrected QT (QTc) and short-term variability of QT (STVQT). Ex vivo monophasic action potentials were recorded in Langendorff-perfused rabbit hearts, and action potential duration (APD75) and triangulation were assessed. Docosahexaenoic acid significantly shortened QTc in vivo only in WT and LQT2 rabbits, in which both α- and ß-subunits of IKs-conducting channels are functionally intact. In LQT2, this led to a normalization of QTc and of its short-term variability. Docosahexaenoic acid had no effect on QTc in LQT1, LQT5, and LQT2-5. Similarly, ex vivo, DHA shortened APD75 in WT and normalized it in LQT2, and additionally decreased AP triangulation in LQT2. CONCLUSIONS: Docosahexaenoic acid exerts a genotype-specific beneficial shortening/normalizing effect on QTc and APD75 and reduces pro-arrhythmia markers STVQT and AP triangulation through activation of IKs in LQT2 rabbits but has no effects if either α- or ß-subunits to IKs are functionally impaired. Docosahexaenoic acid could represent a new genotype-specific therapy in LQT2.

Transgenic rabbit models for cardiac disease research.

To study the pathophysiology of human cardiac diseases and to develop novel treatment strategies, complex interactions of cardiac cells on cellular, tissue and on level of the whole heart need to be considered. As in vitro cell-based models do not depict the complexity of the human heart, animal models are used to obtain insights that can be translated to human diseases. Mice are the most commonly used animals in cardiac research. However, differences in electrophysiological and mechanical cardiac function and a different composition of electrical and contractile proteins limit the transferability of the knowledge gained. Moreover, the small heart size and fast heart rate are major disadvantages. In contrast to rodents, electrophysiological, mechanical and structural cardiac characteristics of rabbits resemble the human heart more closely, making them particularly suitable as an animal model for cardiac disease research. In this review, various methodological approaches for the generation of transgenic rabbits for cardiac disease research, such as pronuclear microinjection, the sleeping beauty transposon system and novel genome-editing methods (ZFN and CRISPR/Cas9)will be discussed. In the second section, we will introduce the different currently available transgenic rabbit models for monogenic cardiac diseases (such as long QT syndrome, short-QT syndrome and hypertrophic cardiomyopathy) in detail, especially in regard to their utility to increase the understanding of pathophysiological disease mechanisms and novel treatment options. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc.

Arrhythmogenic Remodeling in the Failing Heart.

Chronic heart failure is a clinical syndrome with multiple etiologies, associated with significant morbidity and mortality. Cardiac arrhythmias, including ventricular tachyarrhythmias and atrial fibrillation, are common in heart failure. A number of cardiac diseases including heart failure alter the expression and regulation of ion channels and transporters leading to arrhythmogenic electrical remodeling. Myocardial hypertrophy, fibrosis and scar formation are key elements of arrhythmogenic structural remodeling in heart failure. In this article, the mechanisms responsible for increased arrhythmia susceptibility as well as the underlying changes in ion channel, transporter expression and function as well as alterations in calcium handling in heart failure are discussed. Understanding the mechanisms of arrhythmogenic remodeling is key to improving arrhythmia management and the prevention of sudden cardiac death in patients with heart failure.

Peripheral and Autonomic Neuropathy Status of Young Patients With Type 1 Diabetes Mellitus at the Time of Transition From Pediatric Care to Adult-Oriented Diabetes Care.

Introduction: The prevalence of neuropathic lesions in young patients with type 1 diabetes mellitus (T1DM) at the time of transition from pediatric care to adult-oriented diabetes care is poorly studied. A comparative study with healthy volunteers to assess the possible neuropathic condition of this special population and to identify the potential early screening needs has not been performed yet. The results may provide important feedback to pediatric diabetes care and a remarkable baseline reference point for further follow up in adult diabetes care. Patients and Methods: Twenty-nine young patients with T1DM [age: 22.4 ± 2.9 years; HbA1c: 8.5 ± 2.1%, diabetes duration: 12.2 ± 5.8 years; (mean ± SD)] and 30 healthy volunteers (age: 21.5 ± 1.6 years; HbA1c: 5.3 ± 0.3%) were involved in the study. Autonomic function was assessed by standard cardiovascular reflex tests. Complex peripheral neuropathic testing was performed by Neurometer®, Neuropad®-test, Tiptherm®, Monofilament®, and Rydel-Seiffer tuning fork tests. Results: T1DM patients had significantly higher diastolic blood pressure than controls (80 ± 9 vs. 74 ± 8 mmHg, p < 0.01), but there was no significant difference in systolic blood pressure (127 ± 26 vs. 121 ± 13 mmHg). Cardiovascular reflex tests had not revealed any significant differences between the T1DM patients and controls. No significant differences with Neurometer®, Neuropad®-test, and Monofilament® were detected between the two groups. The vibrational sensing on the radius on both sides was significantly impaired in the T1DM group compared to the controls with Rydel-Seiffer tuning fork test (right: 7.5 ± 1.0 vs. 7.9 ± 0.3; left: 7.5 ± 0.9 vs. 7.9 ± 0.3, p < 0.05). The Tiptherm®-test also identified a significant impairment in T1DM patients (11 sensing failures vs. 1, p < 0.001). In addition, the neuropathic complaints were significantly more frequently present in the T1DM patient group than in the controls (9 vs. 0, p < 0.01). Conclusion: In this young T1DM population, cardiovascular autonomic neuropathy and cardiac morphological alterations could not be found. However, Rydel-Seiffer tuning fork and Tiptherm®-tests revealed peripheral sensory neurological impairments in young T1DM patients at the time of their transition to adult diabetes care.

Canine Myocytes Represent a Good Model for Human Ventricular Cells Regarding Their Electrophysiological Properties.

Due to the limited availability of healthy human ventricular tissues, the most suitable animal model has to be applied for electrophysiological and pharmacological studies. This can be best identified by studying the properties of ion currents shaping the action potential in the frequently used laboratory animals, such as dogs, rabbits, guinea pigs, or rats, and comparing them to those of human cardiomyocytes. The authors of this article with the experience of three decades of electrophysiological studies, performed in mammalian and human ventricular tissues and isolated cardiomyocytes, summarize their results obtained regarding the major canine and human cardiac ion currents. Accordingly, L-type Ca2+ current (ICa), late Na+ current (INa-late), rapid and slow components of the delayed rectifier K+ current (IKr and IKs, respectively), inward rectifier K+ current (IK1), transient outward K+ current (Ito1), and Na+/Ca2+ exchange current (INCX) were characterized and compared. Importantly, many of these measurements were performed using the action potential voltage clamp technique allowing for visualization of the actual current profiles flowing during the ventricular action potential. Densities and shapes of these ion currents, as well as the action potential configuration, were similar in human and canine ventricular cells, except for the density of IK1 and the recovery kinetics of Ito. IK1 displayed a largely four-fold larger density in canine than human myocytes, and Ito recovery from inactivation displayed a somewhat different time course in the two species. On the basis of these results, it is concluded that canine ventricular cells represent a reasonably good model for human myocytes for electrophysiological studies, however, it must be borne in mind that due to their stronger IK1, the repolarization reserve is more pronounced in canine cells, and moderate differences in the frequency-dependent repolarization patterns can also be anticipated.