Effect of the Phosphodiesterase 5 Inhibitor Sildenafil on Ischemia-Reperfusion-Induced Muscle Mitochondrial Dysfunction and Oxidative Stress

Lower-limb ischemia-reperfusion (IR) is frequent and associated with significant morbidity and mortality. Phosphodiesterase 5 inhibitors demonstrated antioxidant and beneficial effects in several organs submitted to IR, but their effects on muscle mitochondrial functions after lower-limb IR are unknown. Unilateral hindlimb IR (2 h tourniquet followed by 2 h reperfusion) without or with sildenafil (1mg/kg ip 30 minutes before ischemia) was performed in 18 mice. Maximal oxidative capacity (VMax), relative contribution of the mitochondrial respiratory chain complexes, calcium retention capacity (CRC)—a marker of apoptosis—and reactive oxygen species (ROS) production were determined using high-resolution respirometry, spectrofluorometry, and electron paramagnetic resonance in gastrocnemius muscles from both hindlimbs. IR significantly reduced mitochondrial VMax (from 11.79 ± 1.74 to 4.65 ± 1.11 pmol/s*mg wet weight (ww), p < 0.05, −50.2 ± 16.3%) and CRC (from 2.33 ± 0.41 to 0.84 ± 0.18 µmol/mg dry weight (dw), p < 0.05; −61.1 ± 6.8%). ROS tended to increase in the ischemic limb (+64.3 ± 31.9%, p = 0.08). Although tending to reduce IR-related ROS production (−42.4%), sildenafil failed to reduce muscle mitochondrial dysfunctions (−63.3 ± 9.2%, p < 0.001 and −55.2 ± 7.6% p < 0.01 for VMax, and CRC, respectively). In conclusion, lower limb IR impaired skeletal muscle mitochondrial function, but, despite tending to reduce ROS production, pharmacological preconditioning with sildenafil did not show protective effects.


Introduction
Peripheral arterial disease (PAD) is frequent and associated with significant morbidity and mortality [1,2]. PAD overall prevalence ranges from 3% to 10%, increasing to 15-20% in persons older than 70 years of age [3]. The annual incidence of critical limb ischemia in patients admitted to hospital between 2003 and 2011 was ∼150 per 100,000 people in the United States [4]. The causes

Experimental Design
All mice were subjected to 2 h ischemia through a tourniquet placed on the right hindlimb, followed by 2 h reperfusion ( Figure 1). The left non-ischemic hindlimb served as a control, since previous data demonstrated that unilateral limb ischemia did not significantly affect the contralateral limb [34]. Thirty minutes before ischemia, sham mice (n = 8) received intraperitoneal NaCl 9 (5 µL/g), and sildenafil mice (n = 10 mice) received intraperitoneally sildenafil/NaCl 9 (1 mg/kg). Mice were then placed in a hermetic anaesthetic induction cage, ventilated with a gas mixture of 4% isoflurane (AERRANE ® , BAXTER S.A.S.) and oxygen and placed on heating blankets (MINERVE ® , Esternay, France) at 37 • C. Spontaneous ventilation was allowed through an oxygen-delivering mask, with different concentrations of isoflurane depending on the surgical stage (2% during painful stimuli and 1% during latent periods). At the end of the experiment, left and right gastrocnemius muscles were dissected and immediately immersed in Krebs solution at 4 • C for the extemporaneous analyses of mitochondrial functions. Commission on Life Sciences, National Research Council. Washington: National Academy Press, 1996.

Experimental Design
All mice were subjected to 2 hour ischemia through a tourniquet placed on the right hindlimb, followed by 2 hour reperfusion ( Figure 1). The left non-ischemic hindlimb served as a control, since previous data demonstrated that unilateral limb ischemia did not significantly affect the contralateral limb [34]. Thirty minutes before ischemia, sham mice (n = 8) received intraperitoneal NaCl 9‰ (5µl/g), and sildenafil mice (n = 10 mice) received intraperitoneally sildenafil/NaCl 9‰ (1 mg/kg). Mice were then placed in a hermetic anaesthetic induction cage, ventilated with a gas mixture of 4% isoflurane (AERRANE ® , BAXTER S.A.S.) and oxygen and placed on heating blankets (MINERVE ® , Esternay, France) at 37 °C. Spontaneous ventilation was allowed through an oxygen-delivering mask, with different concentrations of isoflurane depending on the surgical stage (2% during painful stimuli and 1% during latent periods). At the end of the experiment, left and right gastrocnemius muscles were dissected and immediately immersed in Krebs solution at 4 °C for the extemporaneous analyses of mitochondrial functions. The right, non-ischemic hind limb served as a control, and Nacl was administered ip 30 min before ischemia. Bottom (Sildenafil + IR): the same protocol was performed, but 1mg/kg sildenafil/NaCl ip was administered.
Permeabilized fibers were placed in chambers to record the basal oxygen consumption (V0) with glutamate (10 mM) and malate (2.5 mM). Then, maximal respiration rate (VMax) was measured under continuous stirring in the presence of saturating amount of ADP (2 mM) as a phosphate acceptor. Succinate injection (25 mM, Vsucc) allowed the activation of all complexes (I, II, III, IV, V). Finally, ascorbate (0.5 mM) and N, N, N ', N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD, 0.5 mM) were injected, Vasc/TMPD representing the complex IV contribution. Results were expressed as pmol/sec/mg wet weight. . The right, non-ischemic hind limb served as a control, and Nacl was administered ip 30 min before ischemia. Bottom (Sildenafil + IR): the same protocol was performed, but 1mg/kg sildenafil/NaCl ip was administered.
Permeabilized fibers were placed in chambers to record the basal oxygen consumption (V 0 ) with glutamate (10 mM) and malate (2.5 mM). Then, maximal respiration rate (V Max ) was measured under continuous stirring in the presence of saturating amount of ADP (2 mM) as a phosphate acceptor. Succinate injection (25 mM, V succ ) allowed the activation of all complexes (I, II, III, IV, V). Finally, ascorbate (0.5 mM) and N, N, N ', N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD, 0.5 mM) were injected, V asc/TMPD representing the complex IV contribution. Results were expressed as pmol/sec/mg wet weight.

Calcium Retention Capacity (CRC) Measurements in Gastrocnemius Ghost Fibers
The time to the opening of the mitochondrial permeability transition pore (mPTP) following Ca 2+ challenge (5 µL of Ca 2+ , 1 mM, pulses performed every 5 min) was determined in permeabilized "ghost" muscle fibers [23]. The amount of Ca 2+ needed to trigger a massive Ca 2+ release by the mitochondria due to mPTP opening was calculated from a standard curve relating [Ca 2+ ] to the fluorescence of calcium green and expressed as µmol/mg dry weight.

Statistical Analysis
All results were expressed as means ± SEM. Data were analysed using Prism software (GraphPad Prism 5, Graph Pad Software, Inc., San Diego, CA, USA), and differences between groups were assessed using two-way ANOVA test, followed by the Bonferroni post-test.
To determine the precise p value when considering ROS, paired t-test was performed. A p value < 0.05 was considered significant.
The time to the opening of the mitochondrial permeability transition pore (mPTP) following Ca 2+ challenge (5 µ l of Ca 2+ , 1 mM, pulses performed every 5 min) was determined in permeabilized "ghost" muscle fibers [23]. The amount of Ca 2+ needed to trigger a massive Ca 2+ release by the mitochondria due to mPTP opening was calculated from a standard curve relating [Ca 2+ ] to the fluorescence of calcium green and expressed as µmol/mg dry weight.

Statistical Analysis
All results were expressed as means ± SEM. Data were analysed using Prism software (GraphPad Prism 5, Graph Pad Software, Inc., San Diego, CA, USA), and differences between groups were assessed using two-way ANOVA test, followed by the Bonferroni post-test.
To determine the precise p value when considering ROS, paired t-test was performed. A p value < 0.05 was considered significant.

4.2.
Sildenafil did not protect skeletal muscle mitochondrial respiration and calcium retention capacity, albeit tending to reduce ROS production.
The sildenafil-related reduction in ROS production (−42.4%, as compared to the NaCl group) failed to reach statistical significance.

Discussion
The main findings of this study are that lower limb IR impaired mitochondrial oxidative capacity and decreased calcium retention capacity in skeletal muscle. Further, albeit reducing ROS production, acute preconditioning with the phosphodiesterase 5 inhibitor sildenafil did not reduce IR-induced muscle injuries.

Effects of Ischemia-Reperfusion
Using different substrates, we observed that the functional activities of the main mitochondrial respiratory chain complexes were impaired by IR. The gastrocnemius muscle was chosen in view of its susceptibility to IR injuries [35,36], allowing it to be a key target when investigating new therapeutic approaches. Thus, main mitochondrial complexes showed a significant decrease in their oxidative capacity, and ROS production tended to be increased. These data are in accordance with results previously obtained with lower limb IR models, secondary to aortic ligation or unilateral leg IR, which demonstrated reduced skeletal muscle oxidative capacities together with increased ROS production [13][14][15][16][17]19,37,38]. Accordingly, oxidative damage in the gastrocnemius has been observed in patients with peripheral artery disease [39]. Interestingly, IR also induced a reduction in mitochondrial calcium retention capacity. This corresponded to a rapid opening of the mitochondrial permeability transition pore, leading thereby to cell apoptosis.

Effects of Sildenafil
Several studies previously reported a protective effect of sildenafil in IR settings. Thus, sildenafil protection was reported during acute cardiac IR in mouse [40], in the human heart [25], as well in the kidney [26], lung [27], liver [28], and brain [29]. Further, another phosphodiesterase 5 inhibitor, vardenafil, improved vascular graft function [41]. Taken together and considering the fact that PDE5 expression has been observed in skeletal muscles [21,30,42], these data suggest that PDE5 might also be implicated in skeletal muscle IR.
Accordingly, chronic administration of sildenafil improved ischemia-induced neovascularization in hypercholesterolemic apolipoprotein E-deficient mice, reduced TNF alpha staining in the femoral artery, and reduced inflammation and oxidative stress in skeletal muscle [31][32][33].
Nevertheless, our study shows that, although tending to decrease ROS production in line with literature data obtained both in skeletal and in cardiac muscles [31,43], sildenafil did not overcome the deleterious effects of acute IR on skeletal muscle. This was unexpected, since we previously observed that BNP, also known to increase the second messenger cGMP, protected skeletal muscle in a similar setting [19].
One explanation might be that chronic treatment might be mandatory. Indeed, beneficial effects of sildenafil were observed after daily, weekly, or even longer administration [31][32][33]. However, acute sildenafil administration also demonstrated some degrees of protection [44][45][46]. In these cases, the PDE5 inhibitor was administered either prior to ischemia or 30 min before reperfusion and after release of the clamps [44][45][46]. Similarly, we administrated sildenafil 30 minutes before ischemia to allow a large diffusion in muscles. Indeed, maximal concentration occurred 1 h after a single intravenous administration, and the half-life was around 0.4-1.3 h in rodents [47]. These studies, nevertheless, investigated mainly environmental changes in muscles, focusing on the inflammatory status. Thus, muscular changes consisted principally in reduced inflammatory cells and TNF alpha staining [44,45]. Necrosis was not reduced when using sildenafil alone [45]. Particularly, no previous data have been reported concerning the specific effect of sildenafil on IR-induced skeletal muscle mitochondrial dysfunctions, but, interestingly, sildenafil reduced caspase 3, which is considered a marker of apoptosis, only at a late stage (24 h) of reperfusion. No significant change was observed at 4 h of reperfusion [46].
In line with our results, Nio et al. [48] reported only a slight mRNA expression of PDE5 in gastrocnemius and, contrary to data obtained in the lung and the heart, oral sildenafil at a single dose of 30 mg/kg did not increase cGMP in such muscle [48]. Thus, a low physiological level of intracellular cGMP and/or a low amount of PDE type 5 in the gastrocnemius muscle might suggest a longer or greater exposure to sildenafil in order to protect skeletal muscle.
Thus, higher sildenafil doses might deserve further discussion. Higher dosages (40 and 10 mg/Kg) of oral sildenafil have been previously used [31,33]. However, 1 mg/Kg sildenafil also showed beneficial effects using different administration modalities (per os, ip, iv) [32,[44][45][46]. In our setting, administering 1 mg/Kg sildenafil for two times and combining pre-and post-conditioning might deserve further investigations.

Conclusions
Lower limb IR significantly impairs skeletal muscle mitochondrial functions. However, although beneficial in other IR settings and despite its tendency to decrease ROS production, acute pharmacologic preconditioning with the phosphodiesterase 5 inhibitor sildenafil did not reduce IR-induced impairments in mitochondrial oxidative and calcium retention capacities in skeletal muscle. Further studies will be useful to further determine the functional roles of PDE and cGMP and to investigate mitochondria-targeted antioxidants in normal and ischemic skeletal muscles [49].