Cardiovascular Effects of Necrostatin-1 (Nec-1)

Cardiovascular Effects of Necrostatin-1 (Nec-1) Abstract Necrostatins have been identified as cardioprotective agents to reduce reperfusion injury after ischemia, preventing necroptosis due to their RIPK1 inhibitory effect. In this study basal cardiovascular effects of Nec-1 and its inactive analog Nec-1i was investigated in healthy rats under anesthesia. Relatively low doses of Nec-1 and Nec-1i were administered (0.8mg/kg and 0.846 mg/kg, respectively) in line with the in vivo dose response model described by Takahashi et al (2012). Basal heart functions were recorded namely, systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), P interval, PR interval and QTc interval. The results of this study indicated that administration of Nec-1 but not Nec-1i raised systolic and diastolic blood pressure, heart rate, while PR interval was depressed. No statistically significant effect on P interval, and QTc was observed by administration of both necrostatins. The effectiveness of Nec-1 reveals a dual cardiovascular effects, exerting both vasodilator and vasoconstrictor actions as well as a positive inotropic effect on cardiomyocytes. Since no conditions of ischemia or any other oxidative stress are present, which means that no type of programmed cell death is triggered (apoptosis, necrosis, or necroptosis), it can be assumed that Nec-1 acts in a RIPK1-independent manner. Thus, this action of Nec-1 under normal heart conditions remains to be clarified at a cellular level investigating its involvement in signaling pathways (e.g. NO pathway, ÃŽ ²-AR pathway) of all cell types involved cardiovascular function (endothelial cells and smooth muscle cells of blood vessels), and cardiomyocytes as well. In general, after the elucidation of the exact mechanisms of action of Nec-1 at a molecular basis, Nec-1 could be applied as a positive inotrope that enhances basal cardiac function in pathological conditions. Discussion Degterev et al (2005) initially introduced necrostatins as therapeutic agents for ischemic brain injury through chemical inhibition of non apoptotic cell death. Three years later the same research group identified RIPK1 as a specific cellular target of necrostatins (Degterev et al, 2008). In parallel necrostatins were also reported as potential cardioprotective agents by Smith et al (2007) as they reduced ischemia reperfusion injury in their experimental trials. Although, current research has used necrostatins as potential inhibitors of necroptosis in ischemic heart experimental designs in vitro and in vivo (Smith et al 2007, Takahashi et al 2012), basal effects of these substances on heart function and haemodynamics have not yet been investigated. In this study administration of necrostatins (Nec-1 and its inactive analog Nec-1i) in healthy rats under anesthesia was performed. The results of this study indicated that administration of Nec-1 but not Nec-1i raised systolic and diastol ic blood pressure, heart rate, while PR interval was depressed. No statistically significant effect on P interval, and QTc was observed by administration of both necrostatins. Interpreting the results of this study is a complicated issue as the availability of comparable data is restricted and sometimes contradictory. The effectiveness of Nec-1 on increasing mean systolic and diastolic blood pressure and heart rate, reveals a dual cardiovascular effects, exerting both vasodilator and vasoconstrictor actions. When evaluating the effectiveness of Nec-1 using in vivo murine disease models, Nec-1 was suggested to reduce ischemia reperfusion injury as an RIPK1 inhibitor in the TNF signalling pathway, thus preventing necroptosis (Smith et al 2007). But when no conditions of ischemia or any other oxidative stress are present, which means that no type of programmed cell death is triggered (apoptosis, necrosis, or necroptosis), it can be assumed that Nec-1 acts in a RIPK1-independent manner. Thus, this action of Nec-1 under normal heart conditions remains to be clarified at a cellular level investigating its involvement in signaling pathways of all cell types involved cardiovascular function (endothelial cells and smooth muscle cells of blood vessels), and cardiomyocytes as well. Interestingly, Eefting et al (2004) reviewed the role of apoptosis in reperfusion injury discussing both pharmacological as well as genetic interventions in animal models. Nitric oxide (NO) appeared to increase myocardial contractility, myocardial function and endothelial function in many reports of this review. NO is a well known regulator of excitation-contraction coupling in myocardial function (Ziolo et al, 2001a) and ÃŽ ²-adrenergic receptor (ÃŽ ²-AR) signalling (Ziolo et al, 2001b). In line with this notion, a recent study in rats demonstrated that renal vasodilatation was induced through the NO pathway with a pharmacological agent (Garcia-Pedraza et al, 2015). Thus, further research could aim in identifying potential interplay of Nec-1 through other signaling pathways starting from the NO pathway. Another approach could be the potential relation of Nec-1 with vascular ATP levels, as the vasodilatory action of intravascular ATP in the coronary circulation was reported to be attributed to the dual and e qual activities of adenosine and ADP acting at purinergic P1 and P2Y1 receptors, respectively (Korchazhkina et al, 1999). Nevertheless, the results of this study indicate that Nec-1 exerts a positive effect on basal cardiac function, by raising heart rate, blood pressure and by depressing PR interval. Since, stimulation of ÃŽ ²-adrenergic receptor (ÃŽ ²-AR) pathway has been reported as the most important regulator of cardiac contractility (Bers and Ziolo, 2001), it could be assumed that Nec-1 affects electrical signal transduction in some way of this pathway. In general, activation of ÃŽ ²-AR activates the cAMP-dependent protein kinase A (PKA) leading to the phosphorylation of several target proteins within the cardiomyocyte, such as Ica (L-type membrane Ca+2 channels), RyR (ryanodine receptors of the sarcoplasmic reticulum), TnI (troponin I), and PLB (phospholamban). Interstingly, it has been suggested that PLB phosphorylation at Ser 16 by PKA is one the major factors affecting positively cardiomyocyte contraction after ÃŽ ²-AR stimulation (Kohr et al, 2012). In line with these observations, Nec-1 coul d somehow affect the phosphorylation status of proteins responsible for cardiac function. The observed depression of PR interval, in this study, after administration of Nec-1, which represents not only atrial depolarization but also the beginning of ventricular depolarization, reflects that the signal conductance through AV node/His bundle was increased possibly through increased phosphorylation of intracellular proteins involved in cardiac contractility. Noteworthy, Nec-1s has been shown to stimulate directly cardiac contractility through myosine binding protein C (MYBP-C) phosphorylation (Szobi et al. 2015, unpublished data) in animal experiments. Regulation of cardiac contractility by MYBP-C through phosphorylation has been reviewed by Saul Winegrad (1999), along with its role in the formation of the sarcomeric myofibril as a result of binding to myosin and titin. Although, not statistically significant in this study, QTc interval tended to be shortened under the effect of Nec-1. Preliminary data from animal experiments with Nec-1s after ischemia reperfusion indicated decreased QTc intervals, suggesting that Nec-1s might be protective on ventricular arrhythmias (Szobi et al. 2015, unpublished data). The comparison of Nec-1 and its inactive demethylated derivative Nec-1i as factors effecting basal haemodynamics and heart function appears confusing, as previous experimental approaches were applied on disease models using in vivo and/or in vitro ischemic conditions (Degterev et al 2005, Degterev et al 2008, Smith et al 2007, Takahashi et al 2012), and not healthy animals under anesthesia. Additionally, the effectiveness of these substances in previous reports varied not only in a time and dose dependent manner, but also regarding species specificity (Takahashi et al 2012). Nonetheless, in this study Nec-1 but not Nec-1i influenced positively systolic and diastolic blood pressure and heart rate, while PR interval was depressed. These results could be compared with the ones obtained from the in vivo murine model of ischemia-reperfusion injury reported by Smith et al (2007), where Nec-1 (1.65 mg/kg) reduced infarct size whilst Nec-1i (1.74 mg/kg) was ineffective. On the other hand, in vivo Nec-1i was as protective as Nec-1 against lethality associated with TNF-induced necroptosis in high doses (6mg/kg) (Takahashi et al 2012). As both necrostatins were proven to inhibit human IDO as predicted by molecular modeling by Takahashi et al (2012), it can be assumed that Nec-1i cannot be used us a reliable ineffective control for Nec-1. Alternatively, Nec-1s which is a more specific RIPK1 inhibitor lacking the IDO-targeting effect, was suggested by Takahashi et al (2012) as a control substance in experiments investigating the potent effect of necrosatins (Vandenabeele et al 2013). Interestingly, in vitro Nec-1i exhibited paradoxically higher cardioprotection that Nec-1 at high doses (100ÃŽ ¼ÃŽÅ") (Smith et al 2007). In the present in vivo study, relatively low doses of Nec-1 and Nec-1i were used (0.8mg/kg and 0.846 mg/kg, respectively) in line with the in vivo dose response model described by Takahashi et al (2012). Although, low doses (0.6mg/kg) of both Nec-1 and Nec-1i had a toxic effect increasing lethality during TNF-induced necroptosis, suggesting that RIPK1/RIPK3- dependent pathway drives TNF-induced mortality (Takahashi et al 2012), this toxicity effect was not observed in the present study. Of course, in their experiments necrostatins were administered during TNF-induced mortality, demonstrating that low doses were toxic in terms that they were not sufficient enough to protect form induced mortality. In contrast, in this in vivo study, evaluating the comparative effect of low doses of both necrostatins, it could be assumed that these substances maintain their profile of active (Nec-1) and inactive (Nec-1i) factors when administered under normal-non stressful condition. But which signaling pathway is triggered, under the effect of Nec-1 but not Nec-1i, leading to this elevated systolic and diastolic blood pressure, heart rate) remains to be clarified. In general, as demonstrated in this study, enhancement of basal cardiac performance by Nec-1, evidenced by increased systolic and diastolic blood pressure heart rate, and depressed PR interval could be a double-edged sword. Although Nec-1 has been introduced as an agent reducing injury after reperfusion in brain and heart (Degeterev et al 2005, Smith et al 2007, Takahashi et al 2012), the effect of this substance under normal conditions must be further investigated before its administration in other pathological conditions. Thus, examining the response of all cell types (cardiomyocytes, smooth muscle and endothelial cells of blood vessels) under the effect of Nec-1 could be a first step under this point of view. Each cell type, and eventually the overall response to any pharmacological administration, will depend on several distinct or sometimes overlapping factors, such as changes in metabolic conditions (pH, calcium levels, ATP levels), or even active (under phosphorylation or not) signaling molecules and transcription factors. Additionally, in order to clarify the molecular mechanism of action of Nec-1 on basal cardiac function, the comparative effect of other necrostatins apart from Nec-1i, e.g. Nec-1s which lacks the IDO inhibitor effect, would provide meaningful insights. Conclusions Although Nec-1 was introduced to prevent necroptosis as a RIPK1 inhibitor reducing ischemia reperfusion injury, the enhancement of basal cardiac activity by Nec-1 in healthy anesthetized rats, as demonstrated by this study, indicates that this molecule may also act in an RIPK1-independent manner. Thus, further research is needed in order to clarify the molecular mechanism underlying this effect. For example, future directions could aim at identifying the potential interplay of Nec-1 in signaling pathways, such as the NO pathway and the ÃŽ ²-AR pathway, of all cell types involved cardiovascular function. The biochemistry of this involvement could be comparatively analyzed by administration of other necorstatinsm, such as Nec-1s. Finally, Nec-1 after evaluating all these parameters, Nec-1 could be used a positive inotropic agent in cases of cardiomyopathy, congestive heart failure, heart attack or cardiogenic shock. References Bers DM, Ziolo MT. (2001). When is cAMP not cAMP? Effects of compartmentalization. Circ.Res. 89, 373–375. Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 2005; 1: 112–119. Degterev A, Hitomi J, Germscheid M, Ch’en IL, Korkina O, Teng X et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol 2008; 4: 313–321. Eefting F, Rensing B, Wigman J, Pannekoek WJ , Liu WM, Cramer MJ, Lips DJ, Doevendans PA. Role of apoptosis in reperfusion injury. Cardiovascular Research 61 (2004) 414– 426. Garcia-Pedraza JA, Garcia M, Martin ML, Moran A, Pharmacological evidence that 5-HT1D activation induces renal vasodilation by NO pathway in rats. Clin Exp Pharmacol Physiol. 2015 doi: 10.1111/1440-1681.12397. Korchazhkina O, Wright G, Exley C. Intravascular ATP and coronary vasodilation in the isolated working rat heart. British Journal of Pharmacology (1999) 127, 701  ± 708 Kohr MJ, Roof SR, Zweier JL, Ziolo MT. Modulation of myocardial contraction by peroxynitrite. Frontiers in Physiology (2012);3:468(1-10). Smith CCT, Davidson SM, Lim SY, Simpkin JC, . Hothersall JS, Yellon DM. Necrostatin: A Potentially Novel Cardioprotective Agent? Cardiovasc Drugs Ther (2007) 21:227–233. Takahashi N, Duprez L, Grootjans S, Cauwels A, Nerinckx W, DuHadaway JB, Goossens V, Roelandt R, Van HauwermeirenF, Libert C, Declercq W, Callewaert N, Prendergast GC, Degterev A, Yuan J and Vandenabeele P. Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models. Cell Death and Disease (2012) 3, e437; doi:10.1038/cddis.2012.176. Vandenabeele P, Grootjans S, Callewaert N , Takahashi N. Necrostatin-1 blocks both RIPK1 and IDO: consequences for the study of cell death in experimental disease models. Cell Death and Differentiation (2013) 20, 185–187. Winegrad S. Cardiac Myosin Binding Protein C Circ Res. 1999;84:1117-1126.) Ziolo MT, Katoh H, and Bers DM. (2001a). Expression of inducible nitric oxide synthase depresses beta-adrenergic-stimulated calcium release from the sarcoplasmic reticulum in intact ventricular myocytes. Circulation 104, 2961–2966. Ziolo MT, Katoh H, and Bers DM. (2001b). Positive and negative effects of nitric oxide on Ca(2+) sparks: influence of beta-adrenergic stimulation. Am.J. Physiol.HeartCirc.Physiol. 281, H2295–H2303.