Role of S-nitrosothiol transport in the cardioprotective effects of S-nitrosocysteine in rat hearts

The objective of this study was to determine if prior exposure of rat hearts to S-nitrosocysteine (CysNO) was able to provide protection against reperfusion injury. We probed NO release using the extracellular NO scavenger oxyhemoglobin (oxyHb), and we examined the involvement of the amino acid transport system L (L-AT), a known transporter of CysNO, using the L-AT competitor, l-leucine (L-Leu). Isolated (9- to 12-week-old Wistar male) rat hearts (six to eight per group) were perfused with CysNO (10 μM) for 30 min with or without the L-AT competitor L-Leu (1 mM) before 30 min of ischemia. Cardiac function was assessed before, during, and after treatment and during 120 min of reperfusion after ischemia. Functional recovery (rate–pressure product) was significantly improved in the CysNO group compared to hearts in the CysNO + L-Leu group and the control group (p < 0.05). Necrosis, measured by triphenyltetrazolium chloride staining, was significantly reduced in CysNO hearts (p < 0.05) and this improvement was reversed by L-Leu. The NO scavenger oxyHb (20 μM) was perfused either concomitant with CysNO or just before ischemia. In neither case did oxyHb affect the cardioprotection afforded by CysNO. OxyHb alone, given in either time window, did not alter the course of ischemia–reperfusion injury. When nitrite was used in place of CysNO, no protective effects were observed. Perfusion with CysNO increased tissue S-nitrosothiol (RSNO) levels from an unmeasurable background to a value of about 15.7 ± 4.1 pmol RSNO/mg protein, as measured by triiodide-based chemiluminescence in the presence and absence of mercury(II) chloride. In the presence of l-Leu, this value dropped to 0.4 ± 0.3 pmol RSNO/mg protein. This study demonstrates that exposure to CysNO before ischemia increases tissue S-nitrosothiol levels, improves postischemic contractile dysfunction, and attenuates necrosis. The mechanism of cardioprotection requires the uptake of CysNO via the L-AT and does not seem to involve NO release either during CysNO exposure or during ischemia. This suggests that the protective effects of CysNO are mediated through the posttranslational modification of cellular proteins through an NO-independent transnitrosation mechanism.