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

Abstract

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 (V_Max), 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 V_Max (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 V_Max, 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.

Keywords: calcic retention capacity; cyclic nucleotide phosphodiesterase; ischemia; mitochondria; muscle; oxidative stress; peripheral arterial disease; reactive oxygen species; reperfusion; sildenafil.

Figure 1

Experimental design. Top (NaCl): Ischemia-reperfusion (IR) animals underwent 2 h of unilateral hind limb tourniquet ischemia (red bar), followed by 2 h of reperfusion (open bar). 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.

Figure 2

Mitochondrial respiration in ischemic and non-ischemic gastrocnemius in mice treated (n = 10) or not (n = 8) with sildenafil; (a): V₀ corresponds to the basal O₂ consumption, with glutamate and malate as substrates. (b): V _max corresponds to the ADP-stimulated respiration, with glutamate and malate as substrates. (c): V_succ represents the activation of all complexes (I, II, III, IV, V). (d): V_asc/TMPD represents the complex IV contribution to the global mitochondrial respiratory rate. For both NaCl and Sildenafil groups: left column, non-ischemic contralateral limb. Right column: Ischemic limb. Results are expressed as means ± SEM. *: p < 0.05; **: p < 0.01 and ***: p < 0.001, as compared to the contralateral limb of the same group.

Figure 3

Calcium retention capacity (CRC) in ischemic and non-ischemic gastrocnemius in mice treated (n = 7) or not (n = 9) with sildenafil. For both NaCl and Sildenafil groups: left column, non-ischemic contralateral limb. Right column: Ischemic limb. Results are expressed as means ± SEM. **: p < 0.01.

Figure 4

Reactive Oxygen species (ROS) production in ischemic and non-ischemic gastrocnemius in mice treated (n = 8) or not (n = 8) with sildenafil. ROS level was obtained using electron paramagnetic resonance. For both NaCl and Sildenafil groups: left column, non-ischemic contralateral limb. Right column: Ischemic limb. Results are expressed as means ± SEM. p = 0.08 in the NaCl group.