Characterization of Kcnk3 -Mutated Rat, a Novel Model of Pulmonary Hypertension

Abstract

Rationale: Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations in KCNK3 (potassium channel subfamily K member 3) gene, which encodes an outward rectifier K + channel, have been identified in pulmonary arterial hypertension patients. Objective: We have demonstrated that KCNK3 dysfunction is common to heritable and nonheritable pulmonary arterial hypertension and to experimental pulmonary hypertension (PH). Finally, KCNK3 is not functional in mouse pulmonary vasculature. Methods and Results: Using CRISPR/Cas9 technology, we generated a 94 bp out of frame deletion in exon 1 of Kcnk3 gene and characterized these rats at the electrophysiological, echocardiographic, hemodynamic, morphological, cellular, and molecular levels to decipher the cellular mechanisms associated with loss of KCNK3. Using patch-clamp technique, we validated our transgenic strategy by demonstrating the absence of KCNK3 current in freshly isolated pulmonary arterial smooth muscle cells from Kcnk3 -mutated rats. At 4 months of age, echocardiographic parameters revealed shortening of the pulmonary artery acceleration time associated with elevation of the right ventricular systolic pressure. Kcnk3 -mutated rats developed more severe PH than wild-type rats after monocrotaline exposure or chronic hypoxia exposure. Kcnk3 -mutation induced a lung distal neomuscularization and perivascular extracellular matrix activation. Lungs of Kcnk3 -mutated rats were characterized by overactivation of ERK1/2 (extracellular signal–regulated kinase1-/2), AKT (protein kinase B), SRC, and overexpression of HIF1-α (hypoxia-inducible factor-1 α), survivin, and VWF (Von Willebrand factor). Linked with plasma membrane depolarization, reduced endothelial-NOS expression and desensitization of endothelial-derived hyperpolarizing factor, Kcnk3 -mutated rats presented predisposition to vasoconstriction of pulmonary arteries and a severe loss of sildenafil-induced pulmonary arteries relaxation. Moreover, we showed strong alteration of right ventricular cardiomyocyte excitability. Finally, Kcnk3 -mutated rats developed age-dependent PH associated with low serum-albumin concentration. Conclusions: We established the first Kcnk3 -mutated rat model of PH. Our results confirm that KCNK3 loss of function is a key event in pulmonary arterial hypertension pathogenesis. This model presents new opportunities for understanding the initiating mechanisms of PH and testing biologically relevant therapeutic molecules in the context of PH.

Total, Bioavailable, and Free Vitamin D Levels and Their Prognostic Value in Pulmonary Arterial Hypertension

“…This is consistent with the concept that vitD itself or its metabolites do not regulate DBP production [19,38]. Previous studies also reported lower serum albumin concentrations in PAH patients [39,40] and in PAH animal models [41], suggesting that reduced albumin may be a risk factor for PAH. Following the formulas given above, calculated free 25(OH)vitD is inversely related to DBP and albumin concentrations.…”

Proteomic Analysis of KCNK3 Loss of Expression Identified Dysregulated Pathways in Pulmonary Vascular Cells

“…We recently found that KCNK3 knockdown is associated with ERK-1/2 activation, HIF1α stabilization, and the over-phosphorylation of AKT, promoting the proliferation of control hPASMCs [ 8 ]. Here, with our analysis we found that Kcnk3 deficiency is in favor of cell spreading, cell movement, and cell invasion, as well as some canonical pathways linked to cell proliferation (mTOR, PI3K/AKT) and DNA damage.…”

“…Otherwise, we recently found that Kcnk3 deficiency in rats (CRISPR/Cas9 technology) leads to higher susceptibility to monocrotaline- or chronic hypoxia-induced PH, confirming that KCNK3 loss of function predisposes one to the development of PAH. We also found that KCNK3 knockdown enhances proliferation in control human PASMCs (hPASMCs) [ 8 ].…”

Tie-2 Cre-Mediated Deficiency of Extracellular Signal-Regulated Kinase 2 Potentiates Experimental Bronchopulmonary Dysplasia-Associated Pulmonary Hypertension in Neonatal Mice

“…Because increased PA pressures cause the pulmonary valve to close prematurely and decrease the PAT and PAT/ET ratio, our findings suggest that hyperoxia-induces a PH phenotype, and this phenotype is augmented by endothelial ERK2 deficiency in neonatal mice. Interventions used to rescue the PH phenotype in adult rodents decrease ERK1/2 activation [63][64][65][66], suggesting that activation of these kinases promotes the development of PH. Similarly, Young et al [67] demonstrated that inhibition of c-kit signaling mitigates hypoxia-induced PH in neonatal mice by downregulating activation of ERK1/2.…”