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

RATIONALE Pulmonary arterial hypertension (PAH) is a severe lethal cardio-pulmonary disease. Loss of function mutations in KCNK3 gene, which encodes an outward rectifier K+ channel, have been identified in PAH patients. OBJECTIVE We have demonstrated that KCNK3 dysfunction is common to heritable and non-heritable PAH 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 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, AKT, Src, and overexpression of HIF1-α, survivin and Von Willebrand factor. Linked with plasma membrane depolarization, reduced eNOS 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 PAH pathogenesis. This model presents new opportunities for understanding the initiating mechanisms of PH and testing biologically relevant therapeutic molecules in the context of PH.

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