LVDP values of at the end of reperfusion L
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).
At the end of the reperfusion period, the expression of phosphorylated forms of Akt (Fig. 5 A) and p38MAPK (Fig. 5 B) decreased in IC hearts and increased in hearts treated with BZ and BZ + L-NAME in comparison to NIC. The expression of eNOS decreased in IC hearts and increased in hearts treated with BZ. This increase was abolished when NOS was inhibited with L-NAME (Fig. 5C). Opposite results were detected in iNOS expression. The level of this enzyme increased in IC hearts and decreased in presence of BZ, in L-NAME and BZ + L-NAME groups (Fig. 5D).
Given that ROS generation and the consequent tissue damage may be responsible for myocardial reperfusion injury, we next determined the impact of BZ treatment on myocardial level of reduced glutathione (GSH) and thiobarbituric LY 2157299 reactive substances (TBARS) concentration. In IC hearts, GSH decreased and TBARS increased (Fig. 6 A and B). These changes were significantly attenuated by BZ treatment and reversed by NOS inhibition with L-NAME or BZ + L-NAME. Moreover, in hearts treated with BZ, the level of 3-nitrotyrosine significantly decreased (84 ± 5% vs 127 ± 4% in IC group) and increased in presence of L-NAME and BZ + L-NAME acquiring similar values to those observed in untreated hearts (Fig. 6 C and D).
Discussion Of the various pathological events that lead to ischemia reperfusion injury the increased cytosolic Ca2+, the abrupt ROS production, and the cross talk between both events play key roles (Braunersreuther and Jaquet, 2012; Peng and Jou, 2012). As mentioned earlier in this study, the pH normalization via activation of NHE-1 and BT associated to CAs contribute to an increase of intracellular Na+ and Ca2+ concentration (Vaughan-Jones et al., 2016; Li et al., 2002). In relation to CAs, recent data from our laboratory show that CA blockade with BZ attenuates the pH recovery of papillary muscles submitted to an acid load by reduction of the activity of both transporters (Ciocci Pardo et al., 2017). These results indicate that BZ is contributing to generate a more acidic environment which could lead to a decrease of intracellular Ca2+ concentration. It is also recognized that an increase of ROS principally generated by mitochondria and taking place initially at reperfusion produce myocardial damage (Bagheri et al., 2016; Zweier and Talukder, 2006). ROS are transformed in inactive by antioxidant systems being GSH one of the most important. In this study, ischemic control hearts showed a diminution of GSH content and an increase of lipid peroxidation (through TBARS) indicating the existence of oxidative stress. Contrarily, hearts treated with BZ showed a preservation of GSH level and a diminution of lipid peroxidation. Therefore, a diminution of Ca2+ overload and attenuation of oxidative stress appear associated to the decrease of infarct size and the increase of post-ischemic contractility detected in hearts treated with BZ. Different pathways activated by kinases are involved in the cardioprotective mechanisms afforded by ischemic and pharmacological interventions (Schulz et al., 2004; Heusch et al., 2008). Furthermore, it was also demonstrated that phosphorylation of eNOS by Akt with a subsequent increase in NO production is an important downstream effector in survival signalling in myocardial ischemia and reperfusion (Gao et al., 2002; Wang et al., 2005). Analysing the mechanisms of BZ-mediated cardioprotection we recently demonstrated the participation of p38MAPK-dependent pathways (Ciocci Pardo et al., 2017). Here, the question was: Is eNOS/NO system playing any role in the BZ-mediated protection? The eNOS is expressed constitutively and is generally regulated by Ca2+/calmodulin and by phosphorylation on several residues. Specifically, the phosphorylation of Ser1177 enhance electron flux through the oxygenase domain of eNOS and increases NO production. On the other hand, the activity of iNOS is usually determined by its expression level; that is increased iNOS expression is associated with elevated NOS activity. It has been reported that during ischemia-reperfusion the up-regulation of iNOS occurs and results in a marked increase in NO and myocardial injury (Mungrue et al., 2002; Liu et al., 2005). Contrarily, the low doses of NO derived from eNOS appear to be beneficial in ischemia-reperfusion (Jugdutt, 2002).At this point, it is necessary to consider that the uncoupling of all NOS isoforms (for example by the lack of essential co-factor BH4) generates superoxide and reduces the NO production. The interaction of superoxide with NO produces peroxynitrite a reactive specie that is capable of triggering an array of cytotoxic processes, including lipid peroxidation, protein oxidation and nitration of tyrosine residues (Katori et al., 2006). Therefore, an increase of ROS at the onset of reperfusion is closely linked to low NO bioavailability and an increase of protein nitration. In this study, ischemic control hearts exhibited an increase of 20% in iNOS expression, a decrease of approximately 40% in P-Ser1177eNOS expression and an increase of 3-nitrotyrosine level. Hence, in these conditions, the balance of NO could be negative decreasing approximately 20%. When the hearts were treated with BZ we detected an increase of eNOS (60%), a decrease of iNOS (20%) and a decrease of the 3-nitrotyrosine concentration, giving a positive NO balance (approximately 40%). Although we do not measure NO concentration these data suggest that NO bioavailability could be increased following BZ treatment. Now the question was: Is there relationship between p38MAPK and eNOS/NO pathways? It has been established that p38MAPK plays a critical role in the activation of nuclear factor-κB (NF-κB) which regulates the expression of proinflammatory genes, including iNOS (Song et al., 2013). In our experimental conditions, ischemic control hearts exhibited a decrease of P-p38MAPK and an increase of iNOS expression. Opposite changes were obtained in BZ treated hearts. These results confirm that p38MAPK is also involved in the beneficial effects of BZ in a regional ischemia model while iNOS appears responsible of the detrimental effects produced by ischemia-reperfusion.