BackgroundhERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening.Methods and FindingsTetramethylrhodamine-5-maleimide (TMRM) fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449) in the S1–S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the V½ of activation to −27.5±2.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1–S2 linker cysteines with valines allowed unobstructed recording of S3–S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-VON, with V½ ,1 = −37.8±1.7 mV, and V½ ,2 = 43.5±7.9 mV. The first phase, V½ ,1, was ∼20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-V½ = −18.3±1.2 mV), and relatively unchanged in a non-inactivating E519C:S620T mutant (V½ = −34.4±1.5 mV), suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarization (V½ = −20.6±1.2, k = 11.4 mV) and L520C quenching during depolarization (V½ = −26.8±1.0, k = 13.3 mV) matched the respective voltage dependencies of hERG ionic tails, and deactivation time constants from −40 to −110 mV, suggesting they detected pore-S4 rearrangements related to ionic current flow during pore opening and closing.ConclusionThe data indicate: 1) that rapid environmental changes occur at the outer end of S4 in hERG channels that underlie channel activation gating, and 2) that secondary slower changes reflect channel pore opening during sustained depolarizations, and channel closing upon repolarization. 3) No direct evidence was obtained of conformational changes related to inactivation from fluorophores attached at the outer end of S4.

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Introduction: Group 2 pulmonary hypertension (PH), defined as a mean pulmonary arterial pressure ≥25 mmHg with elevated pulmonary capillary wedge pressure >15 mmHg, has no approved therapy and patients often die from right ventricular failure (RVF). Alterations in mitochondrial metabolism, notably impaired glucose oxidation, and increased mitochondrial fission, contribute to right ventricle (RV) dysfunction in PH. We hypothesized that the impairment of RV and left ventricular (LV) function in group 2 PH results in part from a proglycolytic isoform switch from pyruvate kinase muscle (PKM) isoform 1 to 2 and from increased mitochondrial fission, due either to upregulation of expression of dynamin-related protein 1 (Drp1) or its binding partners, mitochondrial dynamics protein of 49 or 51 kDa (MiD49 or 51).Methods and Results: Group 2 PH was induced by supra-coronary aortic banding (SAB) in 5-week old male Sprague Dawley rats. Four weeks post SAB, echocardiography showed marked reduction of tricuspid annular plane systolic excursion (2.9 ± 0.1 vs. 4.0 ± 0.1 mm) and pulmonary artery acceleration time (24.3 ± 0.9 vs. 35.4 ± 1.8 ms) in SAB vs. sham rats. Nine weeks post SAB, left and right heart catheterization showed significant biventricular increases in end systolic and diastolic pressure in SAB vs. sham rats (LV: 226 ± 15 vs. 103 ± 5 mmHg, 34 ± 5 vs. 7 ± 1 mmHg; RV: 40 ± 4 vs. 22 ± 1 mmHg, and 4.7 ± 1.5 vs. 0.9 ± 0.5 mmHg, respectively). Picrosirius red staining showed marked biventricular fibrosis in SAB rats. There was increased muscularization of small pulmonary arteries in SAB rats. Confocal microscopy showed biventricular mitochondrial depolarization and fragmentation in SAB vs. sham cardiomyocytes. Transmission electron microscopy confirmed a marked biventricular reduction in mitochondria size in SAB hearts. Immunoblot showed marked biventricular increase in PKM2/PKM1 and MiD51 expression. Mitofusin 2 and mitochondrial pyruvate carrier 1 were increased in SAB LVs.Conclusions: SAB caused group 2 PH. Impaired RV function and RV fibrosis were associated with increases in mitochondrial fission and expression of MiD51 and PKM2. While these changes would be expected to promote increased production of reactive oxygen species and a glycolytic shift in metabolism, further study is required to determine the functional consequences of these newly described mitochondrial abnormalities.

The mammalian homeostatic oxygen sensing system (HOSS) initiates changes in vascular tone, respiration, and neurosecretion that optimize oxygen uptake and tissue oxygen delivery within seconds of detecting altered environmental or arterial PO2. The HOSS includes carotid body type 1 cells, adrenomedullary cells, neuroepithelial bodies, and smooth muscle cells (SMC) in pulmonary arteries (PA), ductus arteriosus (DA) and fetoplacental arteries. Hypoxic pulmonary vasoconstriction (HPV) optimises ventilation-perfusion matching. In utero, HPV diverts placentally-oxygenated blood from the non-ventilated lung through the DA. At birth, increased alveolar and arterial oxygen tension dilate the pulmonary vasculature and, constrict the DA, respectively, thereby transitioning the newborn to an air-breathing organism. Though modulated by endothelial-derived relaxing and constricting factors, O2-sensing is intrinsic to PA- and DASMCs. Within the SMC’s dynamic mitochondrial network, changes in PO2 alter the reduction-oxidation state of redox couples (NAD+/NADH, NADP+/NADPH) and the production of reactive oxygen species, ROS (e.g. H2O2) by Complexes I and III of the electron transport chain (ETC). ROS and redox couples regulate ion channels, transporters, and enzymes, changing intracellular calcium [Ca2+]i and calcium sensitivity and eliciting homeostatic responses to hypoxia. In PASMC, hypoxia inhibits ROS production and reduces redox couples, thereby inhibiting O2-sensitive voltage-gated potassium (Kv) channels, depolarizing the plasma membrane, activating voltage-gated calcium channels (CaL), increasing [Ca2+]i and causing vasoconstriction. In DASMC, elevated PO2 causes mitochondrial fission, increasing ETC Complex I activity and ROS production. The DASMC’s downstream response to elevated PO2 (Kv channel inhibition, CaL activation, increased [Ca2+]i and rho kinase activation) is similar to the PASMC’s hypoxic response. Impaired O2-sensing contributes to human diseases, including pulmonary arterial hypertension and patent DA.

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Aim: Pulmonary arterial hypertension (PAH) results in right ventricular (RV) dysfunction due, in part, to RV ischemia. The relative contribution of RV microvascular rarefaction versus reduced right coronary artery perfusion pressure (RCA-PP) to RV ischemia is unknown. We hypothesize that increasing RCA-PP improves RV function in PAH by increasing RV systolic perfusion.Methods: Supra-coronary aortic banding (SAB) or sham surgery was performed on male Sprague-Dawley rats. 7-10 days later, rats received either monocrotaline (MCT; 60mg/kg) or saline. After one month, echocardiography, cardiac catheterization, 99m Tc-sestamibi single-photon emission computed tomography (SPECT) and microsphere infusion studies were performed. The RV was harvested for measurement of hypertrophy (RVH), fibrosis and immunoblotting, and the lung was harvested for pulmonary artery (PA) histology.Results: SAB increased systolic pressures in proximal aorta and systolic RCA-PP in SAB+MCT vs. MCT rats (114±12 vs. 5±9mmHg), without altering diastolic RCA-PP. SAB+MCT rats had improved RV function versus MCT rats, evident from their significantly increased cardiac output (CO), RV free wall (RVFW) thickening, tricuspid annular plane systolic excursion (TAPSE), and RV-PA coupling indices. RV-PA coupling indices and CO correlated directly with systolic RCA-PP. RV perfusion was increased in SAB+MCT vs. MCT rats and correlated well with CO; whereas microvascular rarefaction was unaltered. SAB+MCT rats had less RVH and fibrosis and lower PA pressures versus MCT rats. SAB+MCT rats had significantly lower RV pyruvate kinase muscle isoform 2/1 ratios than MCT rats, consistent with restoration of oxidative metabolism. Conclusion:A SAB-induced increase in systolic RCA-PP improves RV perfusion and function in MCT rats. Maintaining systolic RCA perfusion can preserve RV function in PAH.

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