The importance of nitric oxide availability in myocardial ischemia-reperfusion injury : regulatory roles of arginase, L-arginine and tetrahydrobiopterin

Background. Maintenance of nitric oxide (NO) availability is crucial for cardiovascular homeostasis and protection against myocardial ischemia-reperfusion (IR) injury. The mechanisms underlying reduced NO availability are multifactorial and involve de ciency of the NO synthase (NOS) substrate L-arginine and co-factor tetrahydrobiopterin (BH4) as well as increased NO inactivation by reactive oxygen species. Increased activity of arginase has emerged as a key factor behind reduced NO availability by competing with NOS for L-arginine as substrate. The aims of the studies were to investigate: 1) the potential of arginase and RhoA/Rho associated kinase (ROCK) inhibition, supplementation of L-arginine with BH4 and remote ischemic perconditioning (RIPerc) to induce cardioprotection, 2) the mechanisms behind their effects and 3) the in uence of diabetes on the cardioprotective effects.

Study I. Arginase inhibition by N-omega-hydroxy-nor-L-arginine (nor-NOHA) prior to reperfusion in rats subjected to myocardial IR reduced infarct size (IS). This effect was abolished by inhibition of NOS, protein kinase C epsilon (PKCε) and blocking of the mitochondrial ATP- dependent potassium channel. The effect of nor-NOHA was associated with enhanced myocardial expression of PKCε. Study II. Combined administration of L-arginine and BH4 before and during reperfusion reduced IS in rat and pig models of myocardial IR. At the same time individual administration of L-arginine or BH4 failed to evoke cardioprotection in both species. NOS inhibition abrogated the cardioprotective effect of L-arginine and BH4. Myocardial BH4 levels were higher in pigs given BH4 with or without L-arginine. The generation of superoxide in the ischemic-reperfused myocardium was reduced only in pigs treated with the combination of L-arginine and BH4.

Studies III-IV. Pharmacological enhancement of peroxynitrite decomposition and inhibition of ROCK protected from IR injury and attenuated myocardial ROCK and arginase activity in rats subjected to IR. Rats with type 1 diabetes had increased myocardial arginase activity, arginase 2 expression and ROCK activity. In addition, ROCK and arginase inhibition protected against myocardial IR in rats with type 1 diabetes. RIPerc induced by bilateral femoral artery occlusion resulted in reduction of IS, myocardial peroxynitrite formation, ROCK and arginase activity and upregulated myocardial endothelial NOS. The cardioprotective effect of RIPerc and associated changes in arginase and ROCK activity were absent in rats with type 1 diabetes. The cardioprotective effects of RIPerc, ROCK and arginase inhibition were abolished by inhibition of NOS.

Conclusions. Inhibition of arginase before the onset of reperfusion reduced IS via a mechanism dependent on NOS activity, PKCε expression and activation of mitochondrial ATP-dependent potassium channels. Supplementation of L-arginine and BH4 during late ischemia and reperfusion reduced IS via a NOS-dependent pathway and reduced the generation of superoxide. Peroxynitrite and ROCK signaling pathways are involved in the upregulation of arginase activity during myocardial IR. Inhibition of either ROCK or arginase protected against myocardial IR injury via a NOS–dependent mechanism both in the presence and absence of type 1 diabetes. This suggests that reduction in arginase activity, as a result of reduced formation of peroxynitrite and ROCK activity, is of importance for the NOS-dependent cardioprotective effect of RIPerc. The cardioprotective effect of RIPerc and associated signaling effects on arginase, ROCK and NOS are abolished in type 1 diabetes. Arginase inhibition, supplementation of BH4 and L-arginine, inhibition of ROCK and RIPerc are potential tools in the treatment of acute myocardial infarction.

List of scientific papers

I. Yahor Tratsiakovich, Adrian T. Gonon, Anna Krook, Jiangning Yang, Alexey Shemyakin, Per-Ove Sjöquist, John Pernow. Arginase inhibition reduces infarct size via nitric oxide, proteinkinase C epsilon and mitochondrial ATP-dependent K+ channels. European Journal of Pharmacology. 2013; 712, 16–21.
https://doi.org/10.1016/j.ejphar.2013.04.044

II. Yahor Tratsiakovich, Adrian T. Gonon, Attila Kiss, Jiangning Yang, Felix Böhm, Per Tornvall, Magnus Settergren, Keith M. Channon, Per-Ove Sjöquist, John Pernow. Myocardial protection by co-administration of L-arginine and tetrahydrobiopterin during ischemia and reperfusion. International Journal of Cardiology. 2013; 169, 83–88.
https://doi.org/10.1016/j.ijcard.2013.08.075

III. Attila Kiss, Yahor Tratsiakovich, Adrian T. Gonon, Olga Fedotovskaya, Johanna T. Lanner, Daniel C. Andersson, Jiangning Yang, John Pernow.The role of arginase and Rho kinase in cardioprotection from remote ischemic perconditioning in non-diabetic and diabetic rat in vivo. PLoS ONE. 2014; 9, e104731.
https://doi.org/10.1371/journal.pone.0104731

IV. Yahor Tratsiakovich, Attila Kiss, Adrian T. Gonon, Jiangning Yang, Per-Ove Sjöquist, John Pernow. Inhibition of Rho kinase protects from ischemia-reperfusion injury via regulation of arginase activity and nitric oxide synthase in type 1 diabetes. [Manuscript]