Peripheral arterial disease (PAD) is frequent and associated with significant morbidity and mortality [1
]. 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 of PAD are multiple, related to general (wound, contusion, compression) or local arterial injuries (atherosclerosis, embolic events, thrombosis, dissections of the arterial wall) and result finally in vessel obstruction leading to limb ischemia [5
]. Lower limb ischemia requires surgical revascularization, possibly associated with thrombolysis, but revascularization might be impossible in some cases, highlighting the need for new therapeutic approaches [2
Recent improvement in the knowledge of PAD pathophysiology might be useful to open new therapeutic approaches. Thus, skeletal muscle mitochondrial dysfunctions, together with increased reactive oxygen species (ROS) production are key factors. Mitochondria play a central role in cell homeostasis, since they are the primary sites of energy production through ATP synthesis. Besides cellular energy metabolism adaptation, such organelles also modulate the production of ROS and consequently cell apoptosis, which is increased when mitochondrial calcic retention capacity (CRC) is impaired [7
]. Interestingly, if ischemia per se is clearly deleterious and urges to perform revascularization procedures, the reperfusion period is also responsible for deleterious effects relying importantly on the amount of ROS released. Thus, after aortic cross-clamping, increased oxidative stress precedes mitochondrial dysfunction [11
Ischemic preconditioning generally demonstrated to be protective in lower limb ischemia-reperfusion [12
]. Ischemic post-conditioning can also reduce oxidative stress and preserve antioxidant defense in an experimental model of aortic clamping [14
] but it has also been shown to be deleterious [15
]. These data pave the way to develop pharmacological approaches. Cyclosporine A demonstrated promising results by protecting muscle in young rats but was disappointing when investigating old animals submitted to lower limb ischemia-reperfusion (IR) with tourniquet use [16
]. Interestingly, confirming data previously obtained in the setting of myocardial infarction [18
], we observed that the cardiac hormone brain natriuretic peptide (BNP) reduced skeletal muscle mitochondrial dysfunction and oxidative stress after acute lower limb IR [19
]. Since BNP main actions are mediated by an increase in the second intracellular messenger cyclic guanylyl monophosphate (cGMP), another approach aiming to reduce cGMP degradation might be interesting.
In this view, phosphodiesterase inhibition deserves to be investigated. Cyclic nucleotide phosphodiesterase families (PDEs) control cyclic nucleotide levels and play a major role in the control of normal and pathological cellular signaling [20
]. Particularly, the PDE5 family hydrolyzes specifically cGMP, and its inhibition increases cGMP levels. Sildenafil, a well-known PDE5 inhibitor, is widely used in humans mainly for treating pulmonary hypertension and erectile dysfunctions [23
]. It also induced protective effects during ischemia-reperfusion on several organs [24
], supporting its potential usefulness in IR settings. PDE5 has been also shown to be expressed in skeletal muscle [21
], and, accordingly, sildenafil reduced muscle oxidative stress and/or increased muscle angiogenesis and reduced inflammation 7 or 30 days after either femoral artery removal or unilateral hindlimb IR [31
The aim of this study was, therefore, to investigate whether pharmacological pre-conditioning with the PDE5 inhibitor might, through its anti-oxidant effect, protect skeletal muscle mitochondrial oxidative capacity and calcium retention capacity, using the model of tourniquet-induced lower limb IR.
2. Material and Methods
Eighteen male Swiss mice (12–16 week old), provided by JANVIER Labs (Saint Berthevin, France), were housed in a thermo-neutral environment at 22 ± 2 °C on a 12 h day/night cycle and were provided food and water ad libitum. The protocol was approved by the Regional Committee of Ethics in Animal Experimentation of Strasbourg (C.R.E.M.E.A.S) and the Ministry of Higher Education and Research (CREMEAS n°2018022716192465v3), and the animal care complied with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. Washington: National Academy Press, 1996.
2.2. 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.
2.3. Mitochondrial Respiratory Chain Complex Activities
Muscle oxygen consumption was determined using a high-resolution oxygraph (Oxygraph 2K, Oroboros instruments, Innsbruck, Austria), in 2 ml of Miro5 + Cr thermostated at 37 °C, containing EGTA (0.5 mM), MgCl2 (3 mM), K lactobionate (60 mM), taurine (20 mM), KH2PO4 (10 mM), HEPES (20 mM), sucrose (110 mM), creatine (20 mM), BSA (1 g/L).
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.
2.4. Calcium Retention Capacity (CRC) Measurements in Gastrocnemius Ghost Fibers
The time to the opening of the mitochondrial permeability transition pore (mPTP) following Ca2+
challenge (5 µL of Ca2+
, 1 mM, pulses performed every 5 min) was determined in permeabilized “ghost” muscle fibers [23
]. The amount of Ca2+
needed to trigger a massive Ca2+
release by the mitochondria due to mPTP opening was calculated from a standard curve relating [Ca2+
] to the fluorescence of calcium green and expressed as μmol/mg dry weight.
2.5. Production of Reactive Oxygen Species Using Electron Paramagnetic Resonance
Immediately after harvesting, gastrocnemius muscles were placed in Krebs solution containing NaCl 99 mM, KCl 4.69 mM, CaCl2 2.5 mM, MgSO4 1.2 mM, NaHCO3 25 mM, KH2PO4 1.03 mM, D(+) glucose 5.6 mM, Na-Hepes 20 mM, deferoxamine 25 µM, and DETC 5 µM. Tissues were cut into 1 to 2 mm3 slices and incubated for 30 min with 1-hydroxy-3-methoxycarbonyl-2, 2, 5, 5-tetramethylpyrrolidine HCl (CMH) in a thermo-regulated incubator (37 °C) under controlled pressure (20 mmHg) and gas mix (N2: 97.8%, O2: 2.8%) (Noxygen®, Germany). EPR spectroscopy (Bruker Win-EPR®, Bruker Analytik, GmbH) was used to determine ROS production, expressed in µmol/min/mg dry weight.
3. 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.