Aging Exacerbates Ischemia-Reperfusion-Induced Mitochondrial Respiration Impairment in Skeletal Muscle

Cycles of ischemia-reperfusion (IR) that occur during peripheral arterial disease (PAD) are associated with significant morbi-mortality, and aging is an irreversible risk factor of PAD. However, the effects of advanced age on IR-induced skeletal muscle mitochondrial dysfunction are not well known. Young and aged mice were therefore submitted to hindlimb IR (2 h ischemia followed by 2 h reperfusion). Skeletal muscle mitochondrial respiration, calcium retention capacity (CRC) and reactive oxygen species (ROS) production were determined using high resolution respirometry, spectrofluorometry and electronic paramagnetic resonance. IR-induced impairment in mitochondrial respiration was enhanced in old animals (VADP; from 33.0 ± 2.4 to 18.4 ± 3.8 and 32.8 ± 1.3 to 5.9 ± 2.7 pmol/s/mg wet weight; −44.2 ± 11.4% vs. −82.0 ± 8.1%, in young and aged mice, respectively). Baseline CRC was lower in old animals and IR similarly decreased the CRC in both groups (from 11.8 ± 0.9 to 4.6 ± 0.9 and 5.5 ± 0.9 to 2.1 ± 0.3 µmol/mg dry weight; −60.9 ± 7.3 and −60.9 ± 4.6%, in young and aged mice, respectively). Further, IR-induced ROS production tended to be higher in aged mice. In conclusion, aging exacerbated the deleterious effects of IR on skeletal muscle mitochondrial respiration, potentially in relation to an increased oxidative stress.


Introduction
Ischemia-reperfusion (IR) cycles that occur during peripheral arterial disease (PAD) induce skeletal muscle injury resulting in lifestyle-limiting disability, including exercise intolerance and in the most severe cases, limb amputation. PAD is also often associated with other cardiovascular diseases including myocardial infarction, stroke and cognitive dysfunctions. With more than 200 million people diagnosed in the world, PAD is a public health issue. PAD prevalence is high in aged people and

Processing of Skeletal Muscle Tissue
The superficial gastrocnemius muscles (i.e., glycolytic), characterized by a greater susceptibility to IR-induced damage [24], were harvested and immediately placed in a Krebs-HEPES buffer (NaCl 99 mM, KCl 4.69 mM, CaCl2 2.5 mM, MgSO4 1.2 mM, NaHCO3 25 mM, KH2PO4 1.04 mM, D(+) glucose 5.6 mM, Na-HEPES 20 mM, pH 7.4 at 4 °C) allowing preservation of muscle fibers in a physiological environment at 4 °C. Samples were either gently dissected on ice under a dissecting microscope for mitochondrial respiration and CRC measurements or cut into 1-2 mm 3 slices for assessment of ROS production.

Permeabilization of Skeletal Muscle Fibers
After dissection, muscle fibers were permeabilized by incubation under stirring for 30 min at 4 °C in a buffer S (CaK2EGTA 2.77 mM, K2EGTA 7.23 mM, Na2ATP 6.04 mM, MgCl2 6.56 mM, taurine 20 mM, Na2Phosphocreatine 12.3 mM, imidazole 20 mM, dithiothreitol 0.5 mM, K-methane sulfonate 50 mM, pH 7.0 at 4 °C) with saponin (50 µg/mL). Then fibers were rinsed with agitation for 10 min at 4 °C in the buffer S. This technique allowed mitochondrial morphology to be kept and the functional cellular environment to be preserved [25,26].

Study of Mitochondrial Respiration by Oxymetry
Measuring oxygen consumption in permeabilized fibers was performed using a Clark electrode in a thermostated oxygraphic chamber at 37 °C with continuous stirring (Oxygraph-2k, Oroboros instruments, Innsbruck, Austria). Briefly, fibers (3-4 mg wet weight) were incubated twice for 5 min with agitation at 4 °C in a buffer R+ (CaK2EGTA 2.77 mM, K2EGTA 7.23 mM, MgCl2 1.38 mM, imidazole 20 mM, taurine 20 mM, dithiothreitol 0.5 mM, K-methane sulfonate 90 mM, Na-methane sulfonate 10 mM, glutamate 5 mM, malate 2 mM, K2HPO4 3 mM, and bovin serum albumin 2 mg/mL, pH 7.0 at 22.1 °C). Then fibers were placed in 2 mL of buffer R+ in the oxygraphic chamber and basal oxygen consumption (V0) was measured. Subsequently, several substrates were added in order to specifically activate different complexes of the mitochondrial electron transport chain (ETC). Addition of a saturating amount of adenosine diphosphate (ADP) (2 mM) allowed measuring complex I, III and IV activity (VADP). Then, a saturating amount of succinate (25 mM) allowed studying complex I, II, III and IV activity (VSucc). Finally, addition of N,N,N′,N′-tetramethyl-pphenylenediamine (TMPD; 0.5 mM) and ascorbate (0.5 mM) specifically activated complex IV (VTMPD/Asc). The ratio VADP/V0 was calculated to evaluate the degree of coupling between oxidation and phosphorylation. Results were expressed as pmol/s/mg wet weight.

Calcium Retention Capacity Measurements in Ghost Fibers
Opening of the mPTP was assessed by monitoring CRC of skeletal muscle mitochondria under energized conditions, as previously described [13]. Briefly, permeabilized fibers (5-6 mg wet weight) were incubated for 30 min under stirring at 4 °C in buffer R+ supplemented with KCl (800 mM) to

Processing of Skeletal Muscle Tissue
The superficial gastrocnemius muscles (i.e., glycolytic), characterized by a greater susceptibility to IR-induced damage [24], were harvested and immediately placed in a Krebs-HEPES buffer (NaCl 99 mM, KCl 4.69 mM, CaCl 2 2.5 mM, MgSO 4 1.2 mM, NaHCO 3 25 mM, KH 2 PO 4 1.04 mM, D(+) glucose 5.6 mM, Na-HEPES 20 mM, pH 7.4 at 4 • C) allowing preservation of muscle fibers in a physiological environment at 4 • C. Samples were either gently dissected on ice under a dissecting microscope for mitochondrial respiration and CRC measurements or cut into 1-2 mm 3 slices for assessment of ROS production.

Permeabilization of Skeletal Muscle Fibers
After dissection, muscle fibers were permeabilized by incubation under stirring for 30 min at 4 • C in a buffer S (CaK 2 EGTA 2.77 mM, K 2 EGTA 7.23 mM, Na 2 ATP 6.04 mM, MgCl 2 6.56 mM, taurine 20 mM, Na 2 Phosphocreatine 12.3 mM, imidazole 20 mM, dithiothreitol 0.5 mM, K-methane sulfonate 50 mM, pH 7.0 at 4 • C) with saponin (50 µg/mL). Then fibers were rinsed with agitation for 10 min at 4 • C in the buffer S. This technique allowed mitochondrial morphology to be kept and the functional cellular environment to be preserved [25,26].

Study of Mitochondrial Respiration by Oxymetry
Measuring oxygen consumption in permeabilized fibers was performed using a Clark electrode in a thermostated oxygraphic chamber at 37 • C with continuous stirring (Oxygraph-2k, Oroboros instruments, Innsbruck, Austria). Briefly, fibers (3-4 mg wet weight) were incubated twice for 5 min with agitation at 4 • C in a buffer R+ (CaK 2 EGTA 2.77 mM, K 2 EGTA 7.23 mM, MgCl 2 1.38 mM, imidazole 20 mM, taurine 20 mM, dithiothreitol 0.5 mM, K-methane sulfonate 90 mM, Na-methane sulfonate 10 mM, glutamate 5 mM, malate 2 mM, K 2 HPO 4 3 mM, and bovin serum albumin 2 mg/mL, pH 7.0 at 22.1 • C). Then fibers were placed in 2 mL of buffer R+ in the oxygraphic chamber and basal oxygen consumption (V 0 ) was measured. Subsequently, several substrates were added in order to specifically activate different complexes of the mitochondrial electron transport chain (ETC). Addition of a saturating amount of adenosine diphosphate (ADP) (2 mM) allowed measuring complex I, III and IV activity (V ADP ). Then, a saturating amount of succinate (25 mM) allowed studying complex I, II, III and IV activity (V Succ ). Finally, addition of N,N,N ,N -tetramethyl-p-phenylenediamine (TMPD; 0.5 mM) and ascorbate (0.5 mM) specifically activated complex IV (V TMPD/Asc ). The ratio V ADP /V 0 was calculated to evaluate the degree of coupling between oxidation and phosphorylation. Results were expressed as pmol/s/mg wet weight.

Calcium Retention Capacity Measurements in Ghost Fibers
Opening of the mPTP was assessed by monitoring CRC of skeletal muscle mitochondria under energized conditions, as previously described [13]. Briefly, permeabilized fibers (5-6 mg wet weight) were incubated for 30 min under stirring at 4 • C in buffer R+ supplemented with KCl (800 mM) to extract myosin, block the calcium uptake by the sarcoplasmic reticulum, and thus allow calcium uptake only by mitochondria. Then fibers were washed 3 times 10 min in CRC buffer (Tris-Base 20 mM, saccharose 150 mM, KCl 50 mM, KH 2 PO 4 2 mM, succinate 5 mM, pH 7.4 at 23 • C) containing ethylene glycol-bis(β-aminoethyl ether)-N,N,N ,N -tetraacetic acid (EGTA) (5 µM) and bovine serum albumin (2 mg/mL).
Permeabilized ghost fibers were incubated at 24 • C in a quartz tank with continuous stirring in 1 mL of CRC buffer containing calcium green-5N fluorescent probe (5 µM; excitation 500 nm; emission 530 nm). The reaction was started by the addition of a calcium pulse (20 mM), following by calcium pulses every 5 min until it was necessary. After each pulse, a peak of extramitochondrial calcium was recorded and a rapid uptake by the mitochondria was observed, resulting in a decrease in extramitochondrial calcium concentration to near basal level. When mitochondria reached the maximal calcium loading threshold, opening of mPTP happens and mitochondrial calcium is released, resulting in an abrupt increase in extramitochondrial calcium concentration. The amount of calcium necessary to trigger mPTP opening was calculated from a standard curve relating calcium concentrations to the fluorescence of calcium green-5N. At the end of the experiment, muscle fibers were gathered, dehydrated at 150 • C for 15 min, and weighted. Results were expressed as µmol/mg dry weight.

Measurement of Intramuscular Reactive Oxygen Species Production by Electron Paramagnetic Resonance Spectroscopy
The electron paramagnetic resonance spectroscopy is one of the best techniques used to detect the "instantaneous" presence of free radical species in the samples, by oxidation from superoxide anion (O 2 · ) and other ROS of a spin probe 1-hydroxy-3-methoxycarbonyl-2, 2, 5, 5-tetramethyl-pyrrolidine The signal amplitude is calculated and the concentration of CM · is determined from standard calibration curve of CM. At the end of the experiment, muscle fragments were gathered, dehydrated at 150 • C for 15 min, and weighted. Results were expressed in µmol/min/mg dry weight.

Statistical Analysis
Values are represented by mean ± SEM. The statistical analysis was performed using a two-way ANOVA test, followed by the Tukey post-test. A p-value less than 0.05 was considered significant.
The respiratory control capacity (i.e., VADP/V0 ratio), representing the degree of coupling between oxidation and phosphorylation, remained unchanged in all groups ( Figure 2E    Considering the mitochondrial respiration through complex II-linked succinate substrate, V Succ decreased from 43.7 ± 3.0 to 35.0 ± 2.8 and from 40.1 ± 1.5 to 30.2 ± 5.2 pmol/s/mg wet weight, in sham and IR young and aged mice, respectively. A main effect of IR (p = 0.0198) was observed, without a specific effect of aging (p = 0.2683) ( Figure 2C).

Discussion
The main findings of this study demonstrate that aging exacerbates lower limb IR-induced mitochondrial respiration impairment in skeletal muscle. Such enhanced alteration was observed while ROS production tended to increase in the aged muscle. Baseline CRC was reduced in aged mice, but the further IR-induced impairment in CRC was similar in both groups.

Experimental Design and Baseline Characteristics of Young and Aged Muscle
As few data investigating the effects of age on IR-related deleterious effects on skeletal muscle oxidative capacity were reported, we used a well-known model of acute tourniquet-induced IR inducing mitochondrial respiratory chain alterations [13]. We specifically harvested the gastrocnemius muscle since this glycolytic phenotype has been described to be more sensitive to IR [24,[27][28][29], and to present a greater atrophy with aging [30][31][32].
We also selected in vivo markers of mitochondrial functions relevant to sarcopenia associated with aging and to IR injury. Specifically, we examined mitochondrial respiratory chain complex activities, mPTP opening through the mitochondrial sensitivity to calcium overload, and ROS

Discussion
The main findings of this study demonstrate that aging exacerbates lower limb IR-induced mitochondrial respiration impairment in skeletal muscle. Such enhanced alteration was observed while ROS production tended to increase in the aged muscle. Baseline CRC was reduced in aged mice, but the further IR-induced impairment in CRC was similar in both groups.

Experimental Design and Baseline Characteristics of Young and Aged Muscle
As few data investigating the effects of age on IR-related deleterious effects on skeletal muscle oxidative capacity were reported, we used a well-known model of acute tourniquet-induced IR inducing mitochondrial respiratory chain alterations [13]. We specifically harvested the gastrocnemius muscle since this glycolytic phenotype has been described to be more sensitive to IR [24,[27][28][29], and to present a greater atrophy with aging [30][31][32].
We also selected in vivo markers of mitochondrial functions relevant to sarcopenia associated with aging and to IR injury. Specifically, we examined mitochondrial respiratory chain complex activities, mPTP opening through the mitochondrial sensitivity to calcium overload, and ROS

Discussion
The main findings of this study demonstrate that aging exacerbates lower limb IR-induced mitochondrial respiration impairment in skeletal muscle. Such enhanced alteration was observed while ROS production tended to increase in the aged muscle. Baseline CRC was reduced in aged mice, but the further IR-induced impairment in CRC was similar in both groups.

Experimental Design and Baseline Characteristics of Young and Aged Muscle
As few data investigating the effects of age on IR-related deleterious effects on skeletal muscle oxidative capacity were reported, we used a well-known model of acute tourniquet-induced IR inducing mitochondrial respiratory chain alterations [13]. We specifically harvested the gastrocnemius muscle since this glycolytic phenotype has been described to be more sensitive to IR [24,[27][28][29], and to present a greater atrophy with aging [30][31][32].
In the current study, we did not find baseline (i.e., before IR) difference in mitochondrial respiration and ROS production between young (6 months old) and aged (21 months old) sham mice. These data were similar to others studying mitochondrial respiration [47,48] and oxidative stress production [49]. However, and interestingly, we observed a decrease in mitochondrial CRC with aging. This is consistent with a previous work in glycolytic permeabilized fibers showing a reduction in mitochondrial CRC and time of mPTP opening with aging [31]. Since intracellular calcium concentration increases with aging in skeletal muscle [50], this might jeopardize the aged muscle.

Aging Exacerbated IR-Induced Skeletal Muscle Injuries
In young mice, as expected, although ROS production was not significantly increased, as has been observed previously in a similar setting reporting no ROS production at all [13,21], we observed deleterious effects of IR on both mitochondrial respiration and CRC. While impaired mitochondrial respiration is well-known, there are only several reports demonstrating an impaired mitochondrial CRC in skeletal muscle after lower limb IR [45,[51][52][53]. Such increased susceptibility of mPTP opening is thought to favor cell death, either by apoptosis [10,45], or by necrosis [54,55].
Considering aging, few studies specifically investigated a possible enhanced sensitivity of old muscle as compared to young muscles in response to lower limb IR and therefore analyzed concomitantly of both young and aged muscles in such a setting [19][20][21][22]. A recent review confirms the need of research on the effect of aging on IR-induced ROS production [56]. Here, combining the effects of aging and IR on skeletal muscle mitochondrial functions, we demonstrated that aging exacerbated IR-induced mitochondrial ETC dysfunction, mainly affecting complex I activity. Similarly, Miura et al. demonstrated that aging decreased blood flow recovery and angiogenesis after IR, through mechanisms implying damages of mitochondrial DNA, ROS production and muscle mitochondrial dysfunction [22]. Accordingly, previous data reported that IR further altered muscle morphology and force in older rats [19,20].
Interestingly, relatively little is known on the effect of aging on mPTP opening susceptibility after IR in skeletal muscle. We observed that unlike mitochondrial respiration, the IR-induced CRC decrease was not enhanced in old mice. IR-related CRC alterations were nevertheless significant but, they were similar in young and old mice. Whether this might be related to the already decreased baseline CRC value in old muscle or if it is species specific, deserves further studies. Interestingly, even if heart and skeletal muscles differ on some aspects, according to our data, isolated cardiac mitochondria of aged rats showed increased mPTP opening susceptibility after IR [57]. This might be related to a calcium surcharge in the cytoplasm of aged cardiomyocyte [58].

Limitations of the Study
Several limitations deserve to be acknowledged. We did not investigate gender-associated differences that might have modulated both aging and IR-effects on skeletal muscles. This deserves to be studied. Further, although such a preclinical model is well-characterized and exhibits pathophysiological features similar to those observed in patients, this is an experimental study performed in healthy mice without key cardiovascular risk factors such as diabetes, hypertension etc. Thus, although the message of an enhanced susceptibility to IR-damage in aging might be even stronger when taking into account other risk factors, caution should be applied when extrapolating these data to humans.

Conclusions
The present study provides evidence that aging exacerbates the deleterious effects of IR in skeletal muscle, further impairing mitochondrial respiration. Although not affecting the mitochondrial CRC, already impaired in old mice, such enhanced susceptibility to IR of aged muscles should likely be taken into account when treating older patients suffering from PAD.