INTRACELULLAR pH MICRODOMAINS CHARACTERIZED BY GENETICALLY ENCODED pH SENSORS

SOFÍA CIARROCCHI; DELFINA GALLO; ROMINA ALEJANDRA DI MATTÍA; ERNESTO ALEJANDRO AIELLO; ALEJANDRO ORLOWSKI

Mendoza

Congreso; 2023 Annual Meeting of ISHR Lat; 2023

International Society for Heart Research - Latinoamerican section

Introduction: Intra- and extracellular pH regulation is a key function of all cells and tissues and a prerequisite for normal physiological function. Proton microdomains are generated beat-to-beat in cardiomyocytes, presumably due to ATP hydrolysis that occurs during myofibril contraction. In addition, spatial non-uniformity of intracellular pH could be generated due to the differential subcellular distribution of the Na+/H+ exchanger (NHE1) and the Na+/HCO3- cotransporter (NBC).Aim: To study proton microdomains in male Wistar rats using a cardiotropic adenoactivated virus.Materials and Methods: We generated a proton sensor fluorescent protein (pHluorin2) fused with canonical targeting signals to the endoplasmic reticulum, mitochondrial matrix, and junctional cleft of cardiac myocytes (cleft-targeted pH sensors were generated by fusing pHluorin2 with FKBP12.6). We also developed a cardiotropic adenoassociated virus (AAV9) to express pH sensors in cardiac ventricular myocytes and induced their expression in male rats by lateral vein tail injection.Results: We initially corroborated the localization of the pH sensors by confocal microscopy of transfected HEK293T cells. The ammonia prepulse technique was used to create an intracellular acid load. pH recoveries were obtained from the cytosol, mitochondria, and endoplasmic reticulum lumen. NHE inhibition by amiloride drastically reduced pH recovery in HEPES buffer (A.U. cito: 0.144±0.004, n=24; cito+amiloride: 0.066±0.005*, n=23; ER: 0.098±0.005, n=24; ER+amiloride, 0.011±0.005* n=24; *p