The intracellular acidosis occurring during

The intracellular acidosis occurring during ischemia-reperfusion produces the activation of Na+/H+ exchanger isoform 1 (NHE-1) and HCO3– -dependent transports (BT) which lead to an increase of intracellular Na+ (Vaughan-Jones et al., 2016) and secondarily an increase of intracellular Ca2+ (Bernink et al., 2017). Carbonic anhydrases (CAs), associated to NHE-1 and BT, provide the substrates (H+ and HCO3−) for both transports (Li et al., 2002; Sterling et al., 2002) thus contributing to generation of Ca2+ overload. This increase of Ca2+ is a key event in the cardiomyocyte death occurring after ischemia-reperfusion (Feissner et al., 2002). Therefore, interventions that reduce Ca2+ overload are effective for minimizing the myocardial damage. In this sense, recent data from our laboratory show that CA blockade with benzolamide (BZ) limits the infarct size produced by 30 min of global ischemia and 60 min of reperfusion (Ciocci Pardo et al., 2017). Hearts treated with BZ also exhibit an improvement of post-ischemic myocardial function confirming previous results observed in a model of permanent coronary artery occlusion (Vargas et al., 2016). We also found that p38MAPK-dependent pathways are participating in the beneficial effects observed after BZ treatment (Ciocci Pardo et al., 2017).
Materials and methods
Results Forty minutes of coronary artery occlusion followed by 60 min of reperfusion in rat hearts without any treatment caused an infarct size (IS) of ~35% of the risk area. A significant PF-5274857 in IS was obtained when BZ was added to the perfusate (~5%). This beneficial effect was annulled by L-NAME treatment detecting an IS similar to untreated hearts (Fig. 2). At the end of 120 min, non-ischemic hearts exhibited a decrease of contractility of approximately 25%. After 40 min of ischemia and 60 min of reperfusion (IC group) LVDP decreased to approximately 40% of the pre-ischemic value. Fig. 3 shows the beneficial effects of BZ on left ventricular pressure (LVP) at the end of reperfusion period and its attenuation in presence of L-NAME. The mean data indicate that the addition of BZ improved post-ischemic recovery reaching. LVDP values of ~80% at the end of reperfusion. L-NAME treatment did not modify the contractility detected in ischemic control hearts but annulled the actions of BZ acquiring LVDP values up to 40% (Fig. 4 A). A similar pattern was observed when +dP/dtmax was analysed (Fig. 4 B). LVEDP, as an index of diastolic stiffness, was approximately 13 mmHg at the end of the stabilization period. In IC hearts, this parameter increased reaching a value of approximately 35 mmHg at the end of reperfusion. A significant reduction in LVEDP was obtained when BZ was added to the perfusate. At the end of reperfusion LVEDP was 13 ± 2 mmHg. This effect was lost when NO synthesis was inhibited by L-NAME showing LVEDP similar values to those observed in IC hearts (Fig. 4C). Examining -dP/dtmax an improvement of relaxation velocity after treatment with BZ was also evident (82 ± 7% vs. 46 ± 7% in IC hearts) and this effect was abolished with L-NAME (Fig. 4 D).