Lukas Nalos

Comparison of the IKr blockers moxifloxacin, dofetilide and E‐4031 in five screening models of pro‐arrhythmia reveals lack of specificity of isolated cardiomyocytes

Drug development requires the testing of new chemical entities for adverse effects. For cardiac safety screening, improved assays are urgently needed. Isolated adult cardiomyocytes (CM) and human embryonic stem cell‐derived cardiomyocytes (hESC‐CM) could be used to identify pro‐arrhythmic compounds. In the present study, five assays were employed to investigate their sensitivity and specificity for evaluating the pro‐arrhythmic properties of IKr blockers, using moxifloxacin (safe compound) and dofetilide or E‐4031 (unsafe compounds).

Efficient and specific cardiac IK1 inhibition by a new pentamidine analogue

AIMSnIn excitable cells, KIR2.x ion-channel-carried inward rectifier current (IK₁) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal KIR2.x protein levels. No specific IK1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of IK1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits IK₁ by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific IK₁ inhibitors based on the P structure.nnnMETHODS AND RESULTSnWe analysed seven pentamidine analogues (PA-1 to PA-7) for IK₁ blocking potency at 200 nM using inside-out patches from KIR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked KIR2.x currents of human and mouse with low IC₅₀ values (12-15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (ΔG -44.1 kJ/Mol) than for P (ΔG -31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect INav1.5, ICa-L, IKv4.3, IKv11.1, and IKv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of IK₁ in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 (n = 9), 26 (n = 5), and 34% (n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on KIR2.1 channel expression at concentrations from 0.1 to 3 μM. However, PA-6 at 10 μM increased KIR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 μM.nnnCONCLUSIONnPA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete IK₁ block.

Exploring Chemical Substructures Essential for hERG K+ Channel Blockade by Synthesis and Biological Evaluation of Dofetilide Analogues

In this study we followed a new approach to analyze molecular substructures required for hERG channel blockade. We designed and synthesized 40 analogues of dofetilide (1), a potent hERG potassium channel blocker, and established structure–activity relationships (SAR) for their interaction with this important cardiotoxicity‐related off‐target. Structural modifications to dofetilide were made by diversifying the substituents on the phenyl rings and the protonated nitrogen and by varying the carbon chain length. The analogues were evaluated in a radioligand binding assay and SAR data were derived with the aim to specify structural features that give rise to hERG toxicity.

Inhibition of lysosomal degradation rescues pentamidine-mediated decreases of KIR2.1 ion channel expression but not that of Kv11.1

The antiprotozoal drug pentamidine inhibits two types of cardiac rectifier potassium currents, which can precipitate life-threatening arrhythmias. Here, we use pentamidine as a tool to investigate whether a single drug affects trafficking of two structurally different potassium channels by identical or different mechanisms, and whether the adverse drug effect can be suppressed in a channel specific fashion. Whole cell patch clamp, Western blot, real time PCR, and confocal laser scanning microscopy were used to determine potassium current density, ion channel protein levels, mRNA expression levels, and subcellular localization, respectively. We demonstrate that pentamidine inhibits delayed (I(Kr)) and inward (I(K1)) rectifier currents in cultured adult canine cardiomyocytes. In HEK293 cells, pentamidine inhibits functional K(v)11.1 channels, responsible for I(Kr), by interfering at the level of full glycosylation, yielding less mature form of K(v)11.1 at the plasma membrane. In contrast, total K(IR)2.1 expression levels, underlying I(K1), are strongly decreased, which cannot be explained from mRNA expression levels. No changes in molecular size of K(IR)2.1 protein were observed, excluding interference in overt glycosylation. Remaining K(IR)2.1 protein is mainly expressed at the plasma membrane. Inhibition of lysosomal protein degradation is able to partially rescue K(IR)2.1 levels, but not those of K(v)11.1. We conclude that 1) a single drug can interfere in cardiac potassium channel trafficking in a subtype specific mode and 2) adverse drug effects can be corrected in a channel specific manner.

DIFFERENTIAL EFFECTS OF HEMOFILTRATION AND OF COUPLED PLASMA FILTRATION ADSORPTION ON CARDIAC REPOLARIZATION IN PIGS WITH HYPERDYNAMIC SEPTIC SHOCK

The aim was to investigate effects of continuous hemofiltration (CHF) and of coupled plasma filtration adsorption (CPFA) on electrophysiological properties of the septic heart. Sepsis was induced in anesthetized pigs by fecal peritonitis and continued for 22 h either without intervention (control sepsis) or with intervention (CHF or CPFA) applied for the last 10 h of this period. Electrocardiograms were recorded at baseline, before induction of peritonitis, and 22 h later, at the end of in vivo experiment. In vitro, action potentials were recorded in right ventricular trabeculae. RR, QT, and QTc (QT corrected for heart rate) intervals were shortened by sepsis. Action potential durations (APDs) were shortened by CHF, but not by CPFA, compared with control sepsis. Continuous hemofiltration prolonged APD. Coupled plasma filtration adsorption filtrate did not exert any effect on APD. Plasma separated during CPFA prolonged APD. Continuous hemofiltration shortened cardiac repolarization, and this effect was reversed by the hemofiltrate. In contrast, neither CPFA nor the CPFA filtrate influenced APD. The data indicate that some inflammatory mediators able to delay cardiac repolarization were removed from plasma to hemofiltrate by CHF but not by CPFA.ABBREVIATIONS - CHF - continuous hemofiltration; CPFA - coupled plasma filtration adsorption; APD - action potential duration

Heart Rate Variability in Porcine Progressive Peritonitis-Induced Sepsis

Accumulating evidence suggests that heart rate variability (HRV) alterations could serve as an indicator of sepsis progression and outcome, however, the relationships of HRV and major pathophysiological processes of sepsis remain unclear. Therefore, in this experimental study HRV was investigated in a clinically relevant long-term porcine model of severe sepsis/septic shock. HRV was analyzed by several methods and the parameters were correlated with pathophysiological processes of sepsis. In 16 anesthetized, mechanically ventilated, and instrumented domestic pigs of either gender, sepsis was induced by fecal peritonitis. Experimental subjects were screened up to the refractory shock development or death. ECG was continuously recorded throughout the experiment, afterwards RR intervals were detected and HRV parameters computed automatically using custom made measurement and analysis MATLAB routines. In all septic animals, progressive hyperdynamic septic shock developed. The statistical measures of HRV, geometrical measures of HRV and Poincaré plot analysis revealed a pronounced reduction of HRV that developed quickly upon the onset of sepsis and was maintained throughout the experiment. The frequency domain analysis demonstrated a decrease in the high frequency component and increase in the low frequency component together with an increase of the low/high frequency component ratio. The reduction of HRV parameters preceded sepsis-associated hemodynamic changes including heart rate increase or shock progression. In a clinically relevant porcine model of peritonitis-induced progressive septic shock, reduction of HRV parameters heralded sepsis development. HRV reduction was associated with a pronounced parasympathetic inhibition and a shift of sympathovagal balance. Early reduction of HRV may serve as a non-invasive and sensitive marker of systemic inflammatory syndrome, thereby widening the therapeutic window for early interventions.

Parasympathetic regulation of heart rate in rats after 5/6 nephrectomy is impaired despite functionally intact cardiac vagal innervation

BACKGROUNDnChronic renal failure is frequently associated with a high risk of sudden cardiac death due to dysfunction of the autonomic nervous system. The pathogenic mechanisms underlying the parasympathetic cardiac dysautonomia are not fully elucidated yet.nnnMETHODSnChronic renal failure was induced in rats by 5/6 nephrectomy. Blood pressure, resting heart rate and plasma levels of creatinine, urea and asymmetric dimethylarginine (ADMA) were measured. To characterize the parasympathetic innervation of the heart, chronotropic responses to atropine, metipranolol and to vagal stimulation in the absence or presence of ADMA were investigated in vivo. In vitro, chronotropic and inotropic effects of carbachol and ADMA and mRNA expression of muscarinic M2 receptors, high affinity choline transporter (CHT1), vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) were assessed in the isolated cardiac tissues.nnnRESULTSnIn 5/6 nephrectomy rats, the resting heart rate was significantly higher and the parasympathetic tone, measured as the effect of atropine after administration of metipranolol was significantly lower than in control animals. Plasma ADMA levels were significantly elevated in the uraemic rats and significantly inversely correlated with the effect of atropine on the heart rate. No differences were revealed in the plasma norepinephrine concentrations, negative chronotropic responses to stimulation of the vagus nerves, chronotropic and inotropic responses to carbachol and the relative expression of M2 receptors, CHT1, VAChT and ChAT.nnnCONCLUSIONnThe data suggest that cardioacceleration in chronic renal failure is caused by a diminished cardiac parasympathetic tone in the presence of a functionally intact intrinsic cardiac cholinergic signalling system.

Cardiac remodeling in rats with renal failure shows interventricular differences

Chronic renal failure (CRF) is associated with an increased incidence of cardiovascular diseases. Intensive research revealed a number of alterations in the heart during CRF; however, possible interventricular differences in CRF-induced cardiac remodeling have so far not been addressed. CRF was induced by two-stage surgical 5/6 nephrectomy (NX) in male Wistar rats. Cellular hypertrophy was quantified using immunohistological morphometric analysis. Contraction force and membrane potential were recorded in left and right ventricle papillary muscles with an isometric force transducer and high-resistance glass microelectrodes. Hypertrophy was present in the left ventricle (LV) of NX animals, but not in the right ventricle (RV) of NX animals, as documented by both ventricle/body weight ratios and cellular morphometric analysis of the cross-sectional area of myocytes. The contraction force was reduced in the LV of NX animals but increased in the RV of NX animals compared with sham-operated rats. Rest potentiation of contraction force was relatively more pronounced in the LV of NX rats. Fifty percent substitution of extracellular sodium with lithium significantly increased the contraction force only in the LV of NX animals. Action potential durations were shortened in both ventricles of CRF animals. Cardiac structural and contractile remodeling in CRF shows significant interventricular differences. CRF induces hypertrophy of the LV but not of the RV. LV hypertrophy was associated with a reduction of contraction force, whereas in the RV, the contraction force was enhanced. Partial recovery of contractile function of the LV by rest potentiation or lithium substitution indicates a role of the Na+/Ca2+ exchanger in this phenomenon.

Plasma and Tissue Levels of Neuropeptide Y in Experimental Septic Shock: Relation to Hemodynamics, Inflammation, Oxidative Stress, and Hemofiltration

Neuropeptide Y (NPY), a potent vasoconstrictor released from the sympathetic nerves, has been suggested to counterbalance sepsis-induced vasodilation. Thus, the changes in plasma and tissue NPY concentrations in relation to hemodynamic variables and inflammatory markers in a porcine model of moderate septic shock were investigated. Susceptibility of NPY to be removed by continuous hemofiltration in two settings has been also studied. Thirty-four domestic pigs were divided into five groups: (i) control group; (ii) control group with conventional hemofiltration; (iii) septic group; (iv) septic group with conventional hemofiltration; and (v) septic group with high-volume hemofiltration. Sepsis induced by fecal peritonitis continued for 22 h. Hemofiltration was applied for the last 10 h. Hemodynamic and inflammatory parameters (heart rate, mean arterial pressure, cardiac output, systemic vascular resistance, plasma concentrations of tumor necrosis factor-α, interleukin-6, and NPY) were measured before and at 12 and 22 h of peritonitis. NPY tissue levels were determined in the left ventricle and mesenteric and coronary arteries. Sepsis induced long-lasting increases in the systemic NPY levels without affecting its tissue concentrations. Continuous hemofiltration at any dose did not reduce sepsis-induced elevations in NPY plasma concentrations, nor did it affect the peptide tissue levels. The increases in NPY systemic levels were significantly correlated with changes in the systemic vascular resistance. The results support the hypothesis of NPY implication in the regulation of the vascular resistance under septic conditions and indicate that NPY clearance rate during hemofiltration does not exceed the capacity of perivascular nerves to release it.

Continuous hemofiltration in pigs with hyperdynamic septic shock affects cardiac repolarization

Objective:Sepsis has been defined as the systemic host response to infection with an overwhelming systemic production of both proinflammatory and anti-inflammatory mediators. Continuous hemofiltration has been suggested as possible therapeutic option that may remove the inflammatory mediators. However, hemodialysis and hemofiltration were reported to influence cardiac electrophysiologic parameters and to increase the arrhythmogenic risk. We hypothesize that sepsis affects electrophysiologic properties of the pig heart and that the effects of sepsis are modified by hemofiltration. Design:Laboratory animal experiments. Setting:Animal research laboratory at university medical school. Subjects:Forty domestic pigs of either gender. Interventions:In anesthetized, mechanically ventilated, and instrumented pigs sepsis was induced by fecal peritonitis and continued for 22 hours. Conventional or high-volume hemofiltration was applied for the last 10 hours of this period. Measurements and Main Results:Electrocardiogram was recorded before and 22 hours after induction of peritonitis. RR, QT, and QTc intervals were significantly shortened by sepsis. The plasma levels of interleukin-6 and tumor necrosis factor-&agr; were increased in sepsis. High-volume hemofiltration blunted the sepsis-induced increase in tumor necrosis factor-&agr;. Action potentials were recorded in isolated ventricular tissues obtained at the end of in vivo experiments. Action potential durations were significantly shortened in septic preparations at all stimulation cycle lengths tested. Both conventional and high-volume hemofiltrations lead to further shortening of action potential durations measured afterward in vitro. This action potential duration shortening was reversed by septic hemofiltrates obtained previously by conventional or high-volume hemofiltration. Tumor necrosis factor-&agr; (500 ng/L) had no effect on action potential durations in vitro. Conclusions:In a clinically relevant porcine model of hyperdynamic septic shock, both sepsis and continuous hemofiltration shortened duration of cardiac repolarization. The continuous hemofiltration was not associated with an increased prevalence of ventricular arrhythmias. Tumor necrosis factor-&agr; or interleukin-6 did not contribute to the observed changes in action potential durations.