Background and purpose: Pentamidine is a drug used in treatment of protozoal infections. Pentamidine treatment may cause sudden cardiac death by provoking cardiac arrhythmias associated with QTc prolongation and U‐wave alterations. This proarrhythmic effect was linked to inhibition of hERG trafficking, but not to acute block of ion channels contributing to the action potential. Because the U‐wave has been linked to the cardiac inward rectifier current (I_K1), we examined the action and mechanism of pentamidine‐mediatedI_K1block.Experimental approach: Patch clamp measurements ofI_K1were made on cultured adult canine ventricular cardiomyocytes, K_IR2.1‐HEK293 cells and K_IR2.x inside‐out patches. Pentamidine binding to cytoplasmic amino acid residues of K_IR2.1 channels was studied by molecular modelling.Key results: Pentamidine application (24 h) decreasedI_K1in cultured canine cardiomyocytes and K_IR2.1‐HEK293 cells under whole cell clamp conditions. Pentamidine inhibitedI_K1in K_IR2.1‐HEK293 cells 10 min after application. When applied to the cytoplasmic side under inside‐out patch clamp conditions, pentamidine block ofI_K1was acute (IC₅₀= 0.17 µM). Molecular modelling predicted pentamidine‐channel interactions in the cytoplasmic pore region of K_IR2.1 at amino acids E224, D259 and E299. Mutation of these conserved residues to alanine reduced pentamidine block ofI_K1. Block was independent of the presence of spermine. K_IR2.2, and K_IR2.3 basedI_K1was also sensitive to pentamidine blockade.Conclusions and implications: Pentamidine inhibits cardiacI_K1by interacting with three negatively charged amino acids in the cytoplasmic pore region. Our findings may provide new insights for development of specificI_K1blocking compounds.

In excitable cells, K_IR2.x ion-channel-carried inward rectifier current (I_K1) 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 K_IR2.x protein levels. No specific I_K1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of I_K1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits I_K1 by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific I_K1 inhibitors based on the P structure. We analysed seven pentamidine analogues (PA-1 to PA-7) for I_K1 blocking potency at 200 nM using inside-out patches from K_IR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked K_IR2.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 I_Nav1.5, I_Ca-L, I_Kv4.3, I_Kv11.1, and I_Kv7.1/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 I_K1 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 K_IR2.1 channel expression at concentrations from 0.1 to 3 μM. However, PA-6 at 10 μM increased K_IR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 μM. PA-6 has higher efficiency and specificity to K_IR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete I_K1 block.

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.