title: Silencing of cardiac mitochondrial NHE1 prevents mitochondrial permeability transition pore opening.
abstract: Inhibition of Na(+)/H(+) exchanger 1 (NHE1) reduces cardiac ischemia-reperfusion (I/R) injury and also cardiac hypertrophy and failure. Although the mechanisms underlying these NHE1-mediated effects suggest delay of mitochondrial permeability transition pore (MPTP) opening, and reduction of mitochondrial-derived superoxide production, the possibility of NHE1 blockade targeting mitochondria has been incompletely explored. A short-hairpin RNA sequence mediating specific knock down of NHE1 expression was incorporated into a lentiviral vector (shRNA-NHE1) and transduced in the rat myocardium. NHE1 expression of mitochondrial lysates revealed that shRNA-NHE1 transductions reduced mitochondrial NHE1 (mNHE1) by ∼60%, supporting the expression of NHE1 in mitochondria membranes. Electron microscopy studies corroborate the presence of NHE1 in heart mitochondria. Immunostaining of rat cardiomyocytes also suggests colocalization of NHE1 with the mitochondrial marker cytochrome c oxidase. To examine the functional role of mNHE1, mitochondrial suspensions were exposed to increasing concentrations of CaCl(2) to induce MPTP opening and consequently mitochondrial swelling. shRNA-NHE1 transduction reduced CaCl(2)-induced mitochondrial swelling by 64 ± 4%. Whereas the NHE1 inhibitor HOE-642 (10 μM) decreased mitochondrial Ca(2+)-induced swelling in rats transduced with nonsilencing RNAi (37 ± 6%), no additional HOE-642 effects were detected in mitochondria from rats transduced with shRNA-NHE1. We have characterized the expression and function of NHE1 in rat heart mitochondria. Because mitochondria from rats injected with shRNA-NHE1 present a high threshold for MPTP formation, the beneficial effects of NHE1 inhibition in I/R resulting from mitochondrial targeting should be considered.
authors.affiliation: Centro de Investigaciones Cardiovasculares, CONICET Facultad de Ciencias Médicas, Universidad Nacional de La Plata, Argentina.
authors.forename: María C
authors.initials: MC
authors.lastname: Villa-Abrille
authors.forename: Eugenio
authors.initials: E
authors.lastname: Cingolani
authors.forename: Horacio E
authors.initials: HE
authors.lastname: Cingolani
authors.forename: Bernardo V
authors.initials: BV
authors.lastname: Alvarez
mesh_terms: D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000648:ultrastructure; D015870:Gene Expression / Q000187:drug effects; D020868:Gene Silencing; D006146:Guanidines / Q000494:pharmacology; D008297:Male; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000648:ultrastructure; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects*; D000083162:Mitochondrial Permeability Transition Pore; D008933:Mitochondrial Swelling / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013450:Sulfones / Q000494:pharmacology
chemical_list: D000889:Anti-Arrhythmia Agents; D006146:Guanidines; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D034741:RNA, Small Interfering; C452850:Slc9a1 protein, rat; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; C093373:cariporide; D003576:Electron Transport Complex IV; D002118:Calcium