Investigation of hypoxia induced bicarbonate transport regulation in cardiomyocytes

Intracellular pH (pHi) is a major modulator of cell function. pHi is controlled strictly by sarcolemmal acid-base transporters between tight limits (7.1-7.3) in mammalian heart. Na+/ H+ exchanger (NHE1) and Na+/HCO3- co-transporter (NBC) are the main acid extruding membrane proteins in cardiomyocytes. There are electrogenic (NBCe1 /NBCe2) and electroneutral (NBCn1) isoforms in heart tissue. The effect of hypoxia on NBC flux in mammalian cardiomyocytes is currently unknown. In this thesis, the effect of hypoxia (1% O2, 48 hours) and acute anoxia (1 mM DT, 10 minutes) on NBC flux was investigated for the first time in mouse atrial derived HL-1 cells. Intracellular acid load was achieved with ammonium prepulse method and NBC flux was measured using pH-sensitive SNARF dye. NBCe1 and NBCn1 isoforms were detected but not NBCe2 in HL-1 cells. When extracellular pH (pHo) was kept at 7.4, pHi-sensitivity of NBC flux was increased in hypoxia. This increased sensitivity was not due to buffering capacity changes under hypoxic conditions. Moreover, hypoxia did not affect NBCe1 and NBCn1 protein levels but interestingly NBCe1 mRNA levels were increased. These results were also repeated with a hypoxia mimetic agent, dimetyloxalilglycine (DMOG), that accumulates hypoxia inducible factor (Hif) in cytoplasm under normoxic conditions. According to these findings the increased sensitivity of NBC flux is Hif-mediated. In contrast, NBC flux was inhibited under acute anoxia conditions. As a result, acute anoxia and long term hypoxia regulate NBC flux by different mechanisms. Contribution of electrogenic (NBCe1) and electroneutral (NBCn1) isoforms was dissected out by the response of the acidic recovery to hyperkalemic solutions under hypoxia. Our results showed that NBCn1 isoform is responsible from the hypoxia induced Hif-dependent increase in NBC flux.