Sickle cell disease (SCD) is characterized by painful vaso-occlusive crisis. While there are several metabolic abnormalities potentially associated with muscular ischemia–reperfusion cycles that could be harmful in the context of SCD, the metabolic consequences of such events are still unknown. Ten controls (HbAA), thirteen heterozygous (HbAS), and ten homozygous (HbSS) SCD mice were submitted to a standardized protocol of rest–ischemia–reperfusion of the left leg during which adenosine triphosphate, phosphocreatine, and inorganic phosphate concentrations as well as intramuscular pH were measured using phosphorous magnetic resonance spectroscopy (MRS). Forty-eight hours later, skeletal muscles were harvested. Oxidative stress markers were then measured on the tibialis anterior. At the end of the ischemic period, HbSS mice had a lower pH value as compared with the HbAA and HbAS groups (P<0.01). During the reperfusion period, the initial rate of phosphocreatine resynthesis was lower in HbSS mice as compared with HbAA (P<0.05) and HbAS (P<0.01) animals. No significant difference among groups was observed regarding oxidative stress markers. HbSS mice displayed a higher intramuscular acidosis during the ischemic period while their mitochondrial function was impaired as compared with their HbAA and HbAS counterparts. These metabolic abnormalities could worsen the complications related to the pathology of SCD.

Introduction

Sickle cell disease (SCD) is a genetic hemoglobinopathy related to the production of abnormal hemoglobin (HbS) [1]. In its deoxygenated form, HbS tends to polymerize, leading to the sickling of red blood cells (RBC) [2]. Sickled RBC are more rigid and less deformable as compared with RBC containing normal Hb (HbA) so that they tend to remain trapped in the capillaries, thereby reducing or eventually interrupting blood supply to tissues [1]. The resulting very painful vaso-occlusive crisis (VOC) are considered as the most frequent and severe clinical manifestations of the disease [3]. VOC are particularly deleterious because the resulting ischemia can compromise organs function [4]. If this type of episode has been acknowledged in many tissues [1], skeletal muscle is not spared since painful muscular crisis can occur and could even lead to myonecrosis [5–11].

In a wide range of pathophysiological conditions, skeletal muscle ischemia–reperfusion (I/R) cycles (an event analogous to transient VOC) have been associated with several defects including microvascular dysfunction, impaired mitochondrial function, and cell death [12–15], with a strong impact on morbidity and mortality [12,15]. In addition, I/R in skeletal muscle can lead to a significant acidosis [16] and enhance oxidative stress and inflammation [17,18]. It is noteworthy that acidosis, oxidative stress, and microvascular dysfunction, among others, have been recognized as strong contributors to SCD complications, especially VOC [17–19]. Therefore, we can reasonably assume that VOC-related I/R (and the associated metabolic disorders) could, in turn, aggravate the severity of such crisis and lead to the entry into a vicious cycle previously recognized in SCD [20]. Nevertheless, the metabolic consequences of a muscular VOC have never been investigated in SCD mice. One could wonder whether repeated ischemic insults of skeletal muscle could worsen or, by adaptation, limit the metabolic changes and the oxidative stress occurring during an additional ischemic episode. In peripheral arterial disease patients, recurrent ischemic episodes have been related to a largely impaired skeletal muscle energetics more particularly resulting from a mitochondrial dysfunction [15,21]. On the other hand, ischemic preconditioning has been shown to reduce I/R-related injuries in rat skeletal muscle and more particularly diminishes the extent of oxidative stress [22]. Overall, the effects of repeated ischemic insults of skeletal muscle are controversial given that both deleterious and protective effects have been reported. On that basis, additional investigations are warranted in order to clarify this issue more particularly in the pathological context of SCD.

The aim of the present study was to evaluate the metabolic changes resulting from a standardized arterial occlusion in SCD mice with a particular attention to the processes potentially involved in the pathophysiology of the disease. We hypothesized that SCD mice would display metabolic abnormalities and a high oxidative stress status in response to ischemia–reperfusion due to the repeated VOC associated with the pathology as compared with (males) heterozygous and control counterparts.

Experimental

Animal care and feeding

We used the Townes model of humanized SCD mice [23] that represents an interesting exploratory tool given that these mice express only human hemoglobin (Hb) and display the typical clinical phenotype of patients with SCD including anemia, sickling, and organs dysfunction [23]. Five-month-old control (HbAA; n=10), heterozygous (HbAS; n=13), and homozygous (HbSS; n=10) male mice were investigated in agreement with the French guidelines for animal care and in conformity with the European convention for the protection of vertebrate animals used for experimental purposes and institutional guidelines n° 86/609/ CEE 24 November 1986. All animal experiments were approved by the Institutional Animal Care Committee of Aix-Marseille University (permit number: 01835.02). Mice were housed in an environment-controlled facility (12/12 h light–dark cycle, 22°C) and received water and standard food ad libitum.

Animal preparation

Mice were anesthetized in an induction chamber (Equipement vétérinaire Minerve, Esternay, France) using 4% isoflurane in 33% O2 and 66% N2O and weighted. Anesthetized animal was placed supine within a custom built cradle, especially designed for a strictly non-invasive investigation of posterior hindlimb muscles anatomy and energetics [24]. The belly of the posterior hindlimb muscles was located above an elliptic (8 × 12 mm) radio-frequency surface coil tuned to the phosphorus (31P) frequency. Corneas were protected from drying by applying ophthalmic cream, and the animal’s head was placed in a home-built facemask supplying continuously isoflurane throughout the experiment for anesthesia maintenance. Animal body temperature was controlled and maintained at a physiological value during anesthesia using a feedback loop as previously described [24]. Breathing rate was controlled throughout the protocol. The setup integrates a reversible ischemia inductor composed of a nylon thread loop positioned around the animal thigh and connected to a force transducer. Ischemia was induced by tightening the nylon thread around the thigh at a given force controlled by the force transducer. In preliminary experiments, we determined the suitable force allowing to induce a complete arterial occlusion so that any further pressure increase would not lead to further phosphocreatine and pH changes during a 30-min ischemia, suggesting no further reduction in oxygen supply.

Study design

Muscle energetics was assessed using 31P-magnetic resonance spectroscopy (MRS) in response to a standardized rest (5 min) – ischemia (30 min) – reperfusion (25 min) protocol. Forty-eight hours later, mice were anesthetized intraperitoneally with a Ketamine (110 ml/kg) – Xylazine (10 ml/kg) mixture and weighted. Posterior hindlimb (i.e. gastrocnemius, soleus, and plantaris) and anterior hindlimb (i.e. tibialis anterior and extensor digitorum longus) muscles of the two legs were harvested, weighed, frozen in liquid nitrogen, and then stored at −80°C. The total hindlimb muscles weight was calculated as the sum of all the muscles weight. Afterward, mice were submitted to a cervical dislocation. The tibialis anterior muscle was used for subsequent biochemical analyses because oxidative stress parameters of control mice (not submitted to I/R) have been measured on this muscle.

MR data acquisition and processing

Investigations were performed using a 47/30 Biospec Avance MR system (Bruker, Karlsruhe, Germany) equipped with a Bruker 120-mm BGA12SL (200 mT/m) gradient insert. Ten consecutive non-contiguous axial scout slices (1 mm thickness, spaced 1 mm) covering the region from the knee to the ankle were selected across the lower hindlimb. RARE images of these slices (rare factor = 4, effective echo time = 22.52 ms, actual echo time = 19.597 ms, repetition time = 1000 ms, six accumulations, 42 × 42 mm field of view, 256 × 192 matrix size, acquisition time = 4 min 48 s) were recorded at rest in order to assess muscle volume. The total hindlimb muscles region was manually delineated on each slice and then muscle volume (mm3) was calculated as the sum of the four volumes included between the five consecutive largest slices. The reproducibility of this method has already been demonstrated [24].

31P spectra (8-kHz sweep width; 2048 data points) from the posterior hindlimb muscles region were acquired continuously throughout the standardized experimental protocol consisting of 5 min of rest, 30 min of ischemia, and 25 min of reperfusion. A fully relaxed spectrum (12 scans, 20 s repetition time) was acquired at rest in order to calculate absolute p-metabolite concentrations taking into account a 5 mM β-ATP concentration [25]. Then, a total of 1800 partially saturated (2 s repetition time) free induction decays (FID) were recorded and summed as block of 150 (n=1) at rest and of 90 (n=10, temporal resolution of 3 min) during the ischemia period. The remaining 750 FIDs recorded during the post ischemia–reperfusion period were summed as blocks of 30 (n=4, temporal resolution of 1 min) for the initial phase, of 60 (n=5, temporal resolution of 2 min) for the intermediate phase, then of 120 (n=1, temporal resolution of 4 min), and of 210 (n=1, temporal resolution of 7 min) for the final phase. 31P-MRS data were processed using a proprietary software developed using IDL (Interactive Data Language, Research System, Inc., Boulder, CO, U.S.A.) [26]. For each spectrum, the relative concentrations of phosphocreatine (PCr), inorganic phosphate (Pi), and adenosine triphosphate (ATP) were calculated using a time-domain fitting routine based on the AMARES-MRUI Fortran code and appropriate prior knowledge of the ATP multiplets. [PCr] and [Pi] were corrected of potential movement artifacts considering that PCr + Pi content is constant during the whole protocol as previously suggested [27]. Intracellular pH (pHi) was calculated from the chemical shift of the Pi signal relative to PCr [28]. H+ concentration (M) was calculated with the following equation:

 
formula
(1)

Time courses of ATP, H+, and PCr concentrations and pHi were used to quantitate the corresponding extent of changes during the ischemic procedure. The time course of PCr depletion during ischemia was fitted using a linear model and used to determine the PCr depletion rate (mM/min). The PCr recovery kinetic parameters were determined by fitting the [PCr] time-dependent changes during the recovery period to a mono-exponential curve described by the equation:

 
formula
(2)

where [PCr]end is the concentration of PCr measured at the end of exercise, Ap refers to the amount of PCr resynthesized during the post-exercise recovery period and τ the PCr resynthesis time-constant. The initial rate of PCr resynthesis (ViPCr) was calculated as follows:

 
formula
(3)

where [PCr]cons indicates the amount of PCr consumed at the end of exercise and k the rate constant which equals to 1/τ [29]. This parameter was considered as a marker of oxidative capacity [30].

Anatomical data analysis

Body weight (BW) of each mouse was evaluated twice, just before and 48 h after the ischemia–reperfusion protocol. While only the left hindlimb was submitted to the ischemia protocol, muscles of both hindlimbs were sampled. The comparison between the left (ischemia) and the right (no ischemia) hindlimbs allowed to assess the effects of ischemia on muscle mass.

Oxidative stress and antioxidant assessment

For oxidative stress and antioxidant assessment, the mice were compared with data already published [31] measured in mice from the same groups (HbAA, HbAS, and HbSS) that were not submitted to the ischemia–reperfusion protocol. Nevertheless, all the experiments were performed using the same protocol and exactly at the same time, that makes comparison of the different groups possible.

Tibialis anterior muscle of all the mice was homogenized (10%, w/v) in PBS 1× + EDTA 0.5 mM in ice. After centrifugation (10 min, 12000 g, 4°C), the supernatant was collected for oxidative stress and antioxidant markers measurements as previously described [32]. Homogenated aliquots were stored at −80°C. Protein concentrations were determined using the BCA protein assays Kit (Novagen, Darmstadt, Germany) in accordance to the manufacturer’s instructions.

Advanced oxidation protein products (AOPP), malondialdehyde (MDA), and ferric-reducing antioxidant power (FRAP) concentrations and glutathione peroxidase (GPx), xanthine oxidase (XO), NADPH oxidase (NOX), superoxide dismutase (SOD), and catalase activities were determined as previously described [31]. Uric acid (UA) concentration was determined using a commercially available kit (Bio-Quant, San Diego, CA). Spectrophotometric measurements were performed on TECAN Infinite 2000 plate reader (Männedorf, Switzerland). Results were standardized per mg of total protein.

Statistical analysis

Data are presented as mean ± S.D. Statistical analyses were performed with Statistica software version 9 (StatSoft, Tulsa, OK, U.S.A.). Normality was checked using a Kolmogorov–Smirnov test. One-way ANOVA was used to compare parameters, and repeated measures ANOVA were used to compare time courses of parameters. When a main effect or a significant interaction (group × time) was found, Newman–Keuls post hoc analysis was used. For oxidative stress assessments, two-way ANOVA was used to compare parameters. When a main effect or a significant interaction (group × ischemia–reperfusion protocol) was found, Newman–Keuls post hoc analysis was used. Statistical significance was accepted when P<0.05.

Results

Anatomical properties

Anatomical characteristics of the different groups are summarized in Table 1. BW was measured just before and 48 h after the I/R protocol. No group effect was observed, whereas mice were lighter after the ischemic intervention (P<0.001, Table 1). In order to assess the effects of I/R on muscle weight, we compared the muscles of both legs, keeping in mind that only the left leg had been submitted to the I/R protocol. The total hindlimb muscles weight normalized by BW was lower for the hindlimb submitted to the protocol (P<0.001, Table 1). However, considering the statistical interaction between group and ischemia (P<0.05), the lower total hindlimb muscles weight was only significant for HbSS mice (P<0.001) (Table 1). Muscle volumes calculated from magnetic resonance imaging (MRI) data and the total hindlimb muscles weight normalized by BW were lower in HbAS mice as compared with both HbAA and HbSS mice (P<0.05, Table 1).

Table 1
Anatomical properties
HbAA (n=10)HbAS (n=13)HbSS (n=10)GroupI/RGroup × I/R
IschemiaNo I/RPost I/RNo I/RPost I/RNo I/RPost I/R
BW (g) 31.6 ± 1.9 31.1 ± 1.8 30.4 ± 1.1 30.1 ± 1.5 32.3 ± 2.9 31.4 ± 2.8 NS *** NS 
Muscle W/BW (mg/g) 7.0 ± 0.7 6.8 ± 0.6 6.5 ± 0.3 6.4 ± 0.4 7.1 ± 0.2 6.6 ± 0.2$$$ *** 
Muscle volume (mm3408 ± 42 NA 371 ± 23 NA 402 ± 36 NA NA NA 
HbAA (n=10)HbAS (n=13)HbSS (n=10)GroupI/RGroup × I/R
IschemiaNo I/RPost I/RNo I/RPost I/RNo I/RPost I/R
BW (g) 31.6 ± 1.9 31.1 ± 1.8 30.4 ± 1.1 30.1 ± 1.5 32.3 ± 2.9 31.4 ± 2.8 NS *** NS 
Muscle W/BW (mg/g) 7.0 ± 0.7 6.8 ± 0.6 6.5 ± 0.3 6.4 ± 0.4 7.1 ± 0.2 6.6 ± 0.2$$$ *** 
Muscle volume (mm3408 ± 42 NA 371 ± 23 NA 402 ± 36 NA NA NA 

Data are presented as mean ± S.D. BW, body weight; I/R, ischemia-reperfusion; NA, non applicable; NS, non significant; No I/R, before the ischemia protocol (BW and muscle volume) or in the hindlimb not submitted to the protocol (muscles weight); Post I/R, 48 h after the ischemia protocol (BW and muscle weights) in the hindlimb submitted to the protocol; W, weight.

* and ***, significant effect (P<0.05 and P<0.001, respectively).

$$$, significantly different from the same group before ischemia (P<0.001).

€, significantly different from HbAA and HbSS groups (P<0.05).

Metabolic parameters at rest

At rest, both [PCr]/[ATP] and [PCr]/[Pi] were similar among groups (Table 2). In the same line, no group effect was identified concerning intramuscular pH (Table 2).

Table 2
Metabolic variables during the rest–ischemia–reperfusion protocol
ParameterHbAA (n=10)HbAS (n=13)HbSS (n=10)
Rest 
[PCr]/[ATP] 4 ± 1 4 ± 1 3 ± 1 
[PCr]/[Pi17 ± 5 16 ± 7 12 ± 6 
pHi 7.14 ± 0.01 7.15 ± 0.02 7.10 ± 0.04 
Ischemia 
[ATP] variation (mM) −0.02 ± 1.15 −0.27 ± 1.31 −0.23 ± 1.63 
PCr depletion (relative to rest) (mM) 6.5 ± 2.2 7.1 ± 1.4 6.7 ± 1.5 
PCr depletion rate (mM/min) 0.21 ± 0.07 0.25 ± 0.06 0.23 ± 0.05 
pHend 7.02 ± 0.06 7.00 ± 0.08 6.89 ± 0.09,§ 
∆ H+ (M) 2.4.10−8 ± 1.2.10−8 3.0.10−8 ± 2.1.10−8 4.9.10−8 ± 2.0.10−8 *, 
Reperfusion 
τPCr (min) 2.9 ± 1.0 2.4 ± 0.6 5.6 ± 2.6¶,║ 
ViPCr (mM/min) 2.6 ± 1.1 3.0 ± 0.9 1.7 ± 0.8*,§ 
ParameterHbAA (n=10)HbAS (n=13)HbSS (n=10)
Rest 
[PCr]/[ATP] 4 ± 1 4 ± 1 3 ± 1 
[PCr]/[Pi17 ± 5 16 ± 7 12 ± 6 
pHi 7.14 ± 0.01 7.15 ± 0.02 7.10 ± 0.04 
Ischemia 
[ATP] variation (mM) −0.02 ± 1.15 −0.27 ± 1.31 −0.23 ± 1.63 
PCr depletion (relative to rest) (mM) 6.5 ± 2.2 7.1 ± 1.4 6.7 ± 1.5 
PCr depletion rate (mM/min) 0.21 ± 0.07 0.25 ± 0.06 0.23 ± 0.05 
pHend 7.02 ± 0.06 7.00 ± 0.08 6.89 ± 0.09,§ 
∆ H+ (M) 2.4.10−8 ± 1.2.10−8 3.0.10−8 ± 2.1.10−8 4.9.10−8 ± 2.0.10−8 *, 
Reperfusion 
τPCr (min) 2.9 ± 1.0 2.4 ± 0.6 5.6 ± 2.6¶,║ 
ViPCr (mM/min) 2.6 ± 1.1 3.0 ± 0.9 1.7 ± 0.8*,§ 

Data are presented as mean ± S.D. τPCr, PCr resynthesis time-constant; ATP, adenosine triphosphate; Pi, inorganic phosphate; PCr, phosphocreatine; pHi, intramuscular pH; pHend, intramuscular pH at the end of the ischemia period; ViPCr, initial rate of PCr resynthesis.

*, and , significantly different from the HbAA group (P<0.05, P<0.01, and P<0.001, respectively).

, §, and , significantly different from the HbAS group (P<0.05, P<0.01, and P<0.001, respectively).

Metabolic parameters during the ischemic period

As illustrated in Figure 1A, neither group nor time effect was observed regarding the time course of [ATP] during the ischemic intervention, and the [ATP] changes were not different among groups (Table 2). [PCr] decreased almost linearly throughout the ischemic period, with a significant time effect (P<0.001) but neither group nor group × time interaction was identified (Figure 1B). Both PCr depletion and PCr depletion rate were similar among groups (Table 2). Time-dependent changes of [Pi] mirrored those from PCr with a significant time effect (Figure 1C). Concerning pHi, a significant drop was observed during the protocol (P<0.001), and a group effect (P<0.05, Figure 1D) was identified. The intramuscular pH measured at the end of the ischemic period (pHend) was significantly lower in HbSS mice as compared with the values recorded in HbAA and HbAS mice (P<0.01, Table 2). In the same line, H+ accumulation during the ischemic period was larger in HbSS mice compared with both HbAA and HbAS counterparts (P<0.05, Table 2).

Time courses of metabolic variables during the ischemic period

Figure 1
Time courses of metabolic variables during the ischemic period

Concentrations of ATP (A), PCr (B) and Pi (C), and intracellular pH (D) during arterial occlusion in HbAA, HbAS, and HbSS mice.

G × T, interaction between group and time; G, group effect; T, time effect. Data are presented as mean ± S.E.

Figure 1
Time courses of metabolic variables during the ischemic period

Concentrations of ATP (A), PCr (B) and Pi (C), and intracellular pH (D) during arterial occlusion in HbAA, HbAS, and HbSS mice.

G × T, interaction between group and time; G, group effect; T, time effect. Data are presented as mean ± S.E.

Metabolic parameters during the post-occlusion reperfusion period

[ATP] was rather stable throughout the 25 min of reperfusion, with no significant effect of time or group (Figure 2A). As soon as the occlusion was released, [PCr] increased rapidly until resting values and then plateaued (P<0.001, Figure 2B). Although no group effect was observed, a tendency to an interaction between group and time was noticed (P<0.06, Figure 2B). PCr resynthesis time-constant (τPCr) was indeed longer in HbSS mice as compared with HbAA and HbAS groups (P<0.001, Table 2). Similarly, the initial rate of PCr resynthesis (ViPCr) was slower in HbSS mice as compared with their HbAA (P<0.05) and HbAS (P<0.01) counterparts (Table 2). Time-dependent changes of Pi mirrored those from PCr with significant time effect (P<0.001) and group × time interaction (P<0.05) suggesting a slower Pi disappearance in HbSS mice (Figure 2C). pHi increased throughout the reperfusion period until resting values with significant time (P<0.001) and group (P<0.05) effects (Figure 2D).

Time courses of metabolic variables during the reperfusion period

Figure 2
Time courses of metabolic variables during the reperfusion period

Concentrations of ATP (A), PCr (B) and Pi (C), and intracellular pH (D) during the post ischemic–reperfusion period in HbAA, HbAS, and HbSS mice.

G × T, interaction between group and time; G, group effect; T, time effect. Data are presented as mean ± S.E.

Figure 2
Time courses of metabolic variables during the reperfusion period

Concentrations of ATP (A), PCr (B) and Pi (C), and intracellular pH (D) during the post ischemic–reperfusion period in HbAA, HbAS, and HbSS mice.

G × T, interaction between group and time; G, group effect; T, time effect. Data are presented as mean ± S.E.

Oxidative stress and antioxidant assessment

While neither group nor protocol effect has been observed concerning AOPP (Figure 3A), MDA concentrations were significantly lower in mice submitted to the I/R protocol (P<0.05, Figure 3B). XO activity tended to be lower after the I/R protocol whatever the group (P<0.1, Figure 3C). Concerning NOX activity, no difference among groups was observed (Figure 3D). No difference was measured for the antioxidant enzymes activities, i.e. CAT (Figure 3E), GPx (Figure 3F), and SOD (Figure 3G). FRAP (Figure 3H) and UA (Figure 3I) concentrations were significantly higher in the mice submitted to the I/R protocol (P<0.01 and P<0.001, respectively).

Oxidative stress and antioxidant markers (mean ± S.D.)

Figure 3
Oxidative stress and antioxidant markers (mean ± S.D.)

AOPP (advanced oxidation protein products, A) and MDA (malondialdehyde, B) concentrations, XO (xanthine oxidase, C), NOX (NADPH oxidase, D), CAT (catalase, E), GPx (glutathione peroxidase, F) and SOD (superoxide dismutase, G) activities, and FRAP (ferric-reducing antioxidant power, H) and UA (uric acid, I) concentrations measured in the tibialis anterior.

G × I/R, group × ischemia-reperfusion interaction; G, group effect; I/R, ischemia-reperfusion effect.

Figure 3
Oxidative stress and antioxidant markers (mean ± S.D.)

AOPP (advanced oxidation protein products, A) and MDA (malondialdehyde, B) concentrations, XO (xanthine oxidase, C), NOX (NADPH oxidase, D), CAT (catalase, E), GPx (glutathione peroxidase, F) and SOD (superoxide dismutase, G) activities, and FRAP (ferric-reducing antioxidant power, H) and UA (uric acid, I) concentrations measured in the tibialis anterior.

G × I/R, group × ischemia-reperfusion interaction; G, group effect; I/R, ischemia-reperfusion effect.

Discussion

The present study aimed at determining the metabolic changes resulting from a standardized ischemia–reperfusion paradigm in SCD mice as compared with heterozygous and controls counterparts. The main findings were that HbSS mice displayed a larger intramuscular acidosis during the ischemic period and that the ischemia–reperfusion cycle was associated with a significant mitochondrial dysfunction as compared with what measured in HbAA and HbAS counterparts.

Exacerbated ischemia-induced acidosis in SCD mice

During the ischemic period, we recorded a progressive acidosis in all groups, as previously described during a similar protocol [16]. A major result of the present study is that HbSS mice displayed a larger acidosis after 30 min of ischemia as compared with HbAA and HbAS mice. Intracellular acidosis is a multifactorial phenomenon involving production, buffering, and transport of protons [33]. It is noteworthy that PCr depletion is associated with H+ consumption by buffer systems [34] and should consequently influence acidosis magnitude. Considering that the PCr breakdown kinetics was not different among groups, one can reasonably rule out the associated proton buffering as an accounting factor of the deeper acidosis in SCD mice. In a previous study based on protein content assessments, we observed that the muscle content of some proteins involved in intramuscular proton buffering and extrusion were similar in HbSS animals as compared with HbAA and HbAS mice [35]. On that basis, proton buffering and transport can also be ruled out as a factor in the more severe acidosis displayed by the HbSS mice. Consequently, one can hypothesize that a larger proton production is involved in the more marked acidosis in SCD mice under I/R. If the underlying mechanisms accounting for muscle H+ generation are still debated, it has been largely acknowledged that H+ appearance is closely linked to muscle glycolytic activity coupled to ATP production and hydrolysis [36–38]. Accordingly, our results suggest that the glycolytic flux would be higher in HbSS mice than in both HbAA and HbAS groups during the ischemic period. This result is supported by the higher activity of enolase, a glycolytic enzyme, and the greater exercise-induced acidosis we previously observed in HbSS mice during a standardized stimulation protocol [35].

The higher glycolytic flux observed in SCD mice and illustrated by the exacerbated intramuscular acidosis would be supportive of an enhanced ATP turnover inasmuch as [ATP] was fairly stable throughout the protocol and that PCr kinetic parameters were similar among groups. Given that ATP demand was only related to the basal metabolism during our protocol, our results suggest that the resting energy expenditure rate would be higher in SCD mice. This result and its inference are reminiscent with previous studies using indirect calorimetry to demonstrate higher resting metabolic rate in human SCD [39–46]. The adequacy between the results that we obtained in mice and those reported in patients would further confirm that the Townes model of SCD is a valuable tool for investigating energy defects in SCD.

Metabolic impairments in SCD mice during the post-ischemic–reperfusion period

Similar to what we observed during the ischemic period, [ATP] level remained stable during the reperfusion phase. The onset of PCr resynthesis occurred concomitantly to the blood flow restoration and the corresponding kinetics followed an exponential time-dependent evolution as previously described [16,47]. Interestingly, both the PCr resynthesis time-constant (τPCr) and the initial rate of PCr resynthesis (ViPCr), commonly used as biomarkers of muscle oxidative capacity [48,49], were altered in HbSS mice. According to a previous study, the larger intramuscular acidosis observed in response to ischemia in SCD mice could account for this inhibition of oxidative phosphorylation [50]. However, while τPCr has been reported to vary with respect to both end of exercise acidosis and PCr consumption [48,49], ViPCr has been considered as independent of the end-of-exercise metabolic status [48,49]. On that basis, the slower ViPCr measured in SCD mice would illustrate an impaired oxidative capacity even when acidosis is taken into consideration. It has been well described that I/R injuries can initiate a muscle mitochondrial dysfunction leading to a reduced oxidative capacity [51]. The present results suggest that the I/R paradigm was much more deleterious for skeletal muscle in SCD mice as compared with their control counterparts. A first impression might be that mitochondrial function of SCD skeletal muscle is impaired as a result of the multiple I/R episodes occurring as part of the SCD phenotype [1]. However, this idea can be ruled out given that oxidative function, illustrated by ViPCr measurements, performed subsequently to a post-stimulation protocol was unchanged in SCD mice [35]. Therefore, the present results are more likely the results of acute deleterious effects of I/R on mitochondrial function in SCD mice. In addition to intramuscular acidosis, other elements could be involved in I/R-related mitochondrial dysfunction, such as oxidative stress.

Oxidative stress and antioxidant mechanisms are increased after I/R

Given that it has been suggested that oxidative stress could be strongly involved in I/R-related muscle injuries [51,52], we have assessed some mechanisms illustrating the oxidative stress status. Among the end-products of oxidative stress and pro- or antioxidant enzymes or compounds studied in the present study, some did not change (AOPP, NOx, CAT, GPX, and SOD), while several others were altered in response to I/R. Interestingly, the UA concentration was higher in mice submitted to I/R. As a end-product of purine metabolism during reoxygenation, the UA content reflects the prior XO pathway activation known to increase superoxide production [18]. The higher [UA] suggested that the I/R paradigm was responsible for an increased oxidative stress, as previously described in several conditions [12]. The higher FRAP concentration observed in mice submitted to I/R could be related to the [UA] increase as previously suggested [53]. At first sight, the reduced MDA (end-product of poly-insatured fatty acids) and pro-oxidant XO activity [18,54] in the mice groups subjected to I/R are therefore quite surprising. However, since muscle sampling was done 48 h after I/R protocol, it could also suggest that skeletal muscles have developed adaptive mechanisms during this period. This hypothesis is further supported by previous reports suggesting that ischemic preconditioning generally protects skeletal muscle against future I/R, especially improving ROS buffering capacity and reducing ROS production and mitochondrial dysfunctions [22,51]. Of interest, no significant difference among genotypes has been observed concerning all these parameters. Consequently, it seems that oxidative stress would not be responsible for the exacerbated mitochondrial dysfunction observed in SCD mice, contrary to what we expected based on other physiological and pathological conditions [51,52].

Importantly, oxidative stress markers have been measured on the tibialis anterior, a muscle mostly composed of fast twitch fibers. Because oxidative skeletal muscles are more protected against I/R-related oxidative stress [52], the impacts would be lower on a muscle mostly composed of slow oxidative fibers, such as the soleus.

Effects of ischemia–reperfusion on anatomical properties

BW was measured just before and 48 h after the standardized rest–ischemia–reperfusion protocol. Interestingly, BW was significantly lower after the mice were submitted to the protocol, whatever the group. The comparative analysis of muscles weight with respect to side indicated that the left hindlimb submitted to ischemia was lighter as compared with the right one. This is in agreement with the lower BW reported in a rat model of intermittent femoral ischemia, which has been proposed to be related to an increased protein turnover related to a metabolic defect [55]. Interestingly, the reduction in muscle weights was much more pronounced in HbSS mice than in the HbAS and HbAA counterparts. Collectively, these results illustrate that a 30-min arterial occlusion period is associated with a significant alteration in body composition, and that SCD mice muscles suffer particularly of I/R. The exacerbated body composition alterations related to I/R observed in SCD mice could be explained by the higher metabolic changes observed in these mice. Further studies are warranted to confirm this hypothesis.

Clinical implications

The aim of the present study was to better describe the metabolic repercussions of a muscular VOC with a particular interest on the potentially associated clinical implications. A major result of our study was that SCD mice displayed an exacerbated intramuscular H+ accumulation in response to a 30-min ischemic period. Considering that a large part of H+ is expected to be extruded from muscle to blood [33] and that blood acidosis is a well-described risk factor of HbS polymerization and RBC sickling [19], this metabolic consequence of I/R in SCD could reinforce the VOC. In this line, it appears that reducing the VOC-related metabolic acidosis could be of interest. In accordance, it has been shown that a single administration of an alkalizing solution to SCD patients during a spontaneous VOC was associated with a decrease in the proportion of sickled RBC and a pain reduction [56,57]. In addition, we have observed a higher UA concentration as a result of the I/R paradigm, likely associated with an increased ROS production. Given that oxidative stress status is strongly involved in SCD complications [17,18], VOC-related ROS production could aggravate the pathology.

We have also observed that I/R was associated with mitochondrial dysfunction in SCD mice relative to their control and heterozygous counterparts. As it is the case in some pathological conditions, such as peripheral artery disease, this impaired mitochondrial function could be associated with high morbidity and mortality rates [12,15]. Therefore, further therapeutic strategies should target VOC-related mitochondrial defects in SCD as proposed in other pathologies [12].

In our protocol, a 30-min ischemic period was sufficient to induce muscle atrophy. Interestingly, this atrophy was much more pronounced in SCD mice. In the long term, muscle mass deficit would be associated with increased cardiovascular risks and is a strong predictor of disability and mortality in several conditions [58–61]. These elements also need to be taken into account for the well-being, the quality of life, and the life expectancy of SCD patients. As previously proposed, the issue of body composition abnormalities in SCD should be targeted as part of a therapeutic approach [62].

Overall, the metabolic abnormalities observed in the present study in response to I/R (and so to VOC) could in turn trigger further complications, according to the idea of the vicious cycle of SCD [20].

We definitely provided results showing the larger sensitivity of SCD muscle to an I/R episode as compared with HbAS and HbAA muscles. This major result could bring interesting clinical perspectives for the care of SCD patients. Further studies, including in vitro analyses, should be conducted to better describe the underlying mechanisms and the potential relationships with the tissue damages previously described in SCD patients after a VOC [6,8,10,11].

Conclusion

The main finding of the present study was the exacerbated metabolic impairments displayed by SCD mice in response to a muscular ischemia–reperfusion intervention. The metabolic acidosis, mitochondrial dysfunction, ROS production, and muscle mass deficit related to I/R could in turn induce further complications and favor VOC maintenance and aggravation. Further studies aiming at evaluating the underlying cellular and molecular mechanisms and at dampening the metabolic consequences of VOC are warranted.

Clinical perspectives

  • Sickle cell disease patients suffer from severe vaso-occlusive crisis.

  • A single ischemia–reperfusion cycle would lead to exacerbated intramuscular acidosis, reactive oxygen species production, and mitochondrial dysfunction in sickle cell mice.

  • Vaso-occlusive crisis could have harmful metabolic effects in sickle cell disease since acidosis, oxidative stress, and mitochondrial dysfunction could worsen the severity of the disease. Overall, the metabolic abnormalities observed in the present study in response to a simulated vaso-occlusive crisis could in turn trigger further complications.

Funding

The present study was supported by Centre National de la Recherche Scientifique (CNRS UMR 7339) and a grant of Société Française de Myologie and Genzyme. This work was performed by a laboratory member of France Life Imaging network [grant number ANR-11-INBS-0006]. The funders had no role in study design, data collection,and analysis, decision to publish, or preparation of the manuscript.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Author contribution

B.C., D.B., L.M., V.P., and M.B. designed the study. B.C., C.V., and V.P. performed the experiments. B.C., D.B., L.M., V.P., and M.B. wrote the manuscript. All authors critically revised the manuscript.

Abbreviations

     
  • τPCr

    PCr resynthesis time-constant

  •  
  • AOPP

    advanced oxidation protein products

  •  
  • BW

    body weight

  •  
  • FID

    free induction decays

  •  
  • FRAP

    ferric-reducing antioxidant power

  •  
  • GPx

    glutathione peroxidase

  •  
  • Hb

    hemoglobin

  •  
  • HbA

    normal Hb

  •  
  • HbS

    abnormal Hb

  •  
  • HbAA

    controls

  •  
  • HbAS

    heterozygous form of sickle cell disease

  •  
  • HbSS

    homozygous form of sickle cell disease

  •  
  • I/R

    ischemia–reperfusion

  •  
  • MDA

    malondialdehyde

  •  
  • MRS

    magnetic resonance spectroscopy

  •  
  • NOX

    NADPH oxidase

  •  
  • PCr

    phosphocreatine

  •  
  • Pi

    inorganic phosphate

  •  
  • pHi

    intracellular pH

  •  
  • RBC

    red blood cells

  •  
  • SCD

    sickle cell disease

  •  
  • SOD

    superoxide dismutase

  •  
  • UA

    uric acid

  •  
  • VOC

    vaso-occlusive crisis

  •  
  • XO

    xanthine oxidase

References

References
1
Rees
D.C.
,
Williams
T.N.
and
Gladwin
M.T.
(
2010
)
Sickle-cell disease
.
Lancet
376
,
2018
2031
,
[PubMed]
2
Vekilov
P.G.
(
2007
)
Sickle-cell haemoglobin polymerization: is it the primary pathogenic event of sickle-cell anaemia?
Br. J. Haematol.
139
,
173
184
[PubMed]
3
Alexy
T.
,
Sangkatumvong
S.
,
Connes
P.
,
Pais
E.
,
Tripette
J.
,
Barthelemy
J.C.
et al
(
2010
)
Sickle cell disease: selected aspects of pathophysiology
.
Clin. Hemorheol. Microcirc.
44
,
155
166
,
[PubMed]
4
Piel
F.B.
,
Steinberg
M.H.
and
Rees
D.C.
(
2017
)
Sickle cell disease
.
N. Engl. J. Med.
376
,
1561
1573
[PubMed]
5
Schumacher
H.R.
Jr.
,
Murray
W.M.
and
Dalinka
M.K.
(
1990
)
Acute muscle injury complicating sickle cell crisis
.
Semin. Arthritis Rheum.
19
,
243
247
[PubMed]
6
Malekgoudarzi
B.
and
Feffer
S.
(
1999
)
Myonecrosis in sickle cell anemia
.
N. Engl. J. Med.
340
,
483
[PubMed]
7
Vicari
P.
,
Achkar
R.
,
Oliveira
K.R.
,
Miszpupten
M.L.
,
Fernandes
A.R.
,
Figueiredo
M.S.
et al
(
2004
)
Myonecrosis in sickle cell anemia: case report and review of the literature
.
South. Med. J.
97
,
894
896
[PubMed]
8
Turaga
L.P.
,
Boddu
P.
,
Kipferl
S.
,
Basu
A.
and
Yorath
M.
(
2017
)
Myonecrosis in sickle cell anemia: case study
.
Am. J. Case Rep.
18
,
100
103
9
Chatel
B.
,
Messonnier
L.A.
and
Bendahan
D.
(
2017
)
Exacerbated in vivo metabolic changes suggestive of a spontaneous muscular vaso-occlusive crisis in exercising muscle of a sickle cell mouse
.
Blood Cells Mol. Dis.
65
,
56
59
[PubMed]
10
Tageja
N.
,
Racovan
M.
,
Valent
J.
and
Zonder
J.
(
2010
)
Myonecrosis in sickle cell anemia-overlooked and underdiagnosed
.
Case Rep. Med.
2010
,
659031
11
Mani
S.
and
Duffy
T.P.
(
1993
)
Sickle myonecrosis revisited
.
Am. J. Med.
95
,
525
530
[PubMed]
12
Eltzschig
H.K.
and
Eckle
T.
(
2011
)
Ischemia and reperfusion – from mechanism to translation
.
Nat. Med.
17
,
1391
1401
,
[PubMed]
13
Jerome
S.N.
,
Kong
L.
and
Korthuis
R.J.
(
1994
)
Microvascular dysfunction in postischemic skeletal muscle
.
J. Invest. Surg.
7
,
3
16
14
Pipinos
I.I.
,
Swanson
S.A.
,
Zhu
Z.
,
Nella
A.A.
,
Weiss
D.J.
,
Gutti
T.L.
et al
(
2008
)
Chronically ischemic mouse skeletal muscle exhibits myopathy in association with mitochondrial dysfunction and oxidative damage
.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
295
,
R290
R296
[PubMed]
15
Ryan
T.E.
,
Schmidt
C.A.
,
Green
T.D.
,
Brown
D.A.
,
Neufer
P.D.
and
McClung
J.M.
(
2015
)
Mitochondrial regulation of the muscle microenvironment in critical limb ischemia
.
Front. Physiol.
6
,
336
[PubMed]
16
Pathare
N.
,
Vandenborne
K.
,
Liu
M.
,
Stevens
J.E.
,
Li
Y.
,
Frimel
T.N.
et al
(
2008
)
Alterations in inorganic phosphate in mouse hindlimb muscles during limb disuse
.
NMR Biomed.
21
,
101
110
[PubMed]
17
Nur
E.
,
Biemond
B.J.
,
Otten
H.M.
,
Brandjes
D.P.
and
Schnog
J.J.
(
2011
)
Oxidative stress in sickle cell disease; pathophysiology and potential implications for disease management
.
Am. J. Hematol.
86
,
484
489
[PubMed]
18
Chirico
E.N.
and
Pialoux
V.
(
2011
)
Role of oxidative stress in the pathogenesis of sickle cell disease
.
IUBMB Life
64
,
72
80
,
[PubMed]
19
Greenberg
M.S.
,
Kass
E.H.
and
Castle
W.B.
(
1957
)
Studies on the destruction of red blood cells. XII. Factors influencing the role of S hemoglobin in the pathologic physiology of sickle cell anemia and related disorders
.
J. Clin. Invest.
36
,
833
843
[PubMed]
20
Verduzco
L.A.
and
Nathan
D.G.
(
2009
)
Sickle cell disease and stroke
.
Blood
114
,
5117
5125
[PubMed]
21
Brass
E.P.
,
Hiatt
W.R.
and
Green
S.
(
2004
)
Skeletal muscle metabolic changes in peripheral arterial disease contribute to exercise intolerance: a point-counterpoint discussion
.
Vasc. Med.
9
,
293
301
[PubMed]
22
Kocman
E.A.
,
Ozatik
O.
,
Sahin
A.
,
Guney
T.
,
Kose
A.A.
,
Dag
I.
et al
(
2015
)
Effects of ischemic preconditioning protocols on skeletal muscle ischemia-reperfusion injury
.
J. Surg. Res.
193
,
942
952
[PubMed]
23
Wu
L.C.
,
Sun
C.W.
,
Ryan
T.M.
,
Pawlik
K.M.
,
Ren
J.
and
Townes
T.M.
(
2006
)
Correction of sickle cell disease by homologous recombination in embryonic stem cells
.
Blood
108
,
1183
1188
,
[PubMed]
24
Giannesini
B.
,
Vilmen
C.
,
Le Fur
Y.
,
Dalmasso
C.
,
Cozzone
P.J.
and
Bendahan
D.
(
2010
)
A strictly noninvasive MR setup dedicated to longitudinal studies of mechanical performance, bioenergetics, anatomy, and muscle recruitment in contracting mouse skeletal muscle
.
Magn. Reson. Med.
64
,
262
270
[PubMed]
25
Giannesini
B.
,
Vilmen
C.
,
Amthor
H.
,
Bernard
M.
and
Bendahan
D.
(
2013
)
Lack of myostatin impairs mechanical performance and ATP cost of contraction in exercising mouse gastrocnemius muscle in vivo
.
Am. J. Physiol. Endocrinol. Metab.
305
,
E33
E40
[PubMed]
26
Le Fur
Y.
,
Nicoli
F.
,
Guye
M.
,
Confort-Gouny
S.
,
Cozzone
P.J.
and
Kober
F.
(
2010
)
Grid-free interactive and automated data processing for MR chemical shift imaging data
.
MAGMA
23
,
23
30
,
[PubMed]
27
Chance
B.
,
Eleff
S.
,
Leigh
J.S.
Jr
,
Sokolow
D.
and
Sapega
A.
(
1981
)
Mitochondrial regulation of phosphocreatine/inorganic phosphate ratios in exercising human muscle: a gated 31P NMR study
.
Proc. Natl. Acad. Sci. U.S.A.
78
,
6714
6718
28
Moon
R.B.
and
Richards
J.H.
(
1973
)
Determination of intracellular pH by 31P magnetic resonance
.
J. Biol. Chem.
248
,
7276
7278
,
[PubMed]
29
Layec
G.
,
Malucelli
E.
,
Le Fur
Y.
,
Manners
D.
,
Yashiro
K.
,
Testa
C.
et al
(
2013
)
Effects of exercise-induced intracellular acidosis on the phosphocreatine recovery kinetics: a 31P MRS study in three muscle groups in humans
.
NMR Biomed.
26
,
1403
1411
[PubMed]
30
Arnold
D.L.
,
Matthews
P.M.
and
Radda
G.K.
(
1984
)
Metabolic recovery after exercise and the assessment of mitochondrial function in vivo in human skeletal muscle by means of 31P NMR
.
Magn. Reson. Med.
1
,
307
315
,
[PubMed]
31
Chatel
B.
,
Messonnier
L.A.
,
Barge
Q.
,
Vilmen
C.
,
Noirez
P.
,
Bernard
M.
et al
(
2018
)
Endurance training reduces exercise-induced acidosis and improves muscle function in a mouse model of sickle cell disease
.
Mol. Genet. Metab.
123
,
400
410
[PubMed]
32
Charrin
E.
,
Ofori-Acquah
S.F.
,
Nader
E.
,
Skinner
S.
,
Connes
P.
,
Pialoux
V.
et al
(
2016
)
Inflammatory and oxidative stress phenotypes in transgenic sickle cell mice
.
Blood Cells Mol. Dis.
62
,
13
21
[PubMed]
33
Juel
C.
(
2008
)
Regulation of pH in human skeletal muscle: adaptations to physical activity
.
Acta Physiol. (Oxf.)
193
,
17
24
,
[PubMed]
34
Guthrie
B.M.
,
Frostick
S.P.
,
Goodman
J.
,
Mikulis
D.J.
,
Plyley
M.J.
and
Marshall
K.W.
(
1996
)
Endurance-trained and untrained skeletal muscle bioenergetics observed with magnetic resonance spectroscopy
.
Can. J. Appl. Physiol.
21
,
251
263
,
[PubMed]
35
Chatel
B.
,
Messonnier
L.A.
,
Hourde
C.
,
Vilmen
C.
,
Bernard
M.
and
Bendahan
D.
(
2017
)
Moderate and intense muscular exercises induce marked intramyocellular metabolic acidosis in sickle cell disease mice
.
J. Appl. Physiol.
122
,
1362
1369
[PubMed]
36
Robergs
R.A.
,
Ghiasvand
F.
and
Parker
D.
(
2004
)
Biochemistry of exercise-induced metabolic acidosis
.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
287
,
R502
R516
,
[PubMed]
37
Marcinek
D.J.
,
Kushmerick
M.J.
and
Conley
K.E.
(
2010
)
Lactic acidosis in vivo: testing the link between lactate generation and H+ accumulation in ischemic mouse muscle
.
J. Appl. Physiol.
108
,
1479
1486
,
[PubMed]
38
Lindinger
M.I.
,
Kowalchuk
J.M.
and
Heigenhauser
G.J.
(
2005
)
Applying physicochemical principles to skeletal muscle acid-base status
.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
289
,
R891
R894
, ,
[PubMed]
39
Singhal
A.
,
Parker
S.
,
Linsell
L.
and
Serjeant
G.
(
2002
)
Energy intake and resting metabolic rate in preschool Jamaican children with homozygous sickle cell disease
.
Am. J. Clin. Nutr.
75
,
1093
1097
[PubMed]
40
Gray
N.T.
,
Bartlett
J.M.
,
Kolasa
K.M.
,
Marcuard
S.P.
,
Holbrook
C.T.
and
Horner
R.D.
(
1992
)
Nutritional status and dietary intake of children with sickle cell anemia
.
Am. J. Pediatr. Hematol. Oncol.
14
,
57
61
41
Singhal
A.
,
Davies
P.
,
Sahota
A.
,
Thomas
P.W.
and
Serjeant
G.R.
(
1993
)
Resting metabolic rate in homozygous sickle cell disease
.
Am. J. Clin. Nutr.
57
,
32
34
[PubMed]
42
Salman
E.K.
,
Haymond
M.W.
,
Bayne
E.
,
Sager
B.K.
,
Wiisanen
A.
,
Pitel
P.
et al
(
1996
)
Protein and energy metabolism in prepubertal children with sickle cell anemia
.
Pediatr. Res.
40
,
34
40
[PubMed]
43
Singhal
A.
,
Davies
P.
,
Wierenga
K.J.
,
Thomas
P.
and
Serjeant
G.
(
1997
)
Is there an energy deficiency in homozygous sickle cell disease?
Am. J. Clin. Nutr.
66
,
386
390
[PubMed]
44
Barden
E.M.
,
Zemel
B.S.
,
Kawchak
D.A.
,
Goran
M.I.
,
Ohene-Frempong
K.
and
Stallings
V.A.
(
2000
)
Total and resting energy expenditure in children with sickle cell disease
.
J. Pediatr.
136
,
73
79
[PubMed]
45
Kopp-Hoolihan
L.E.
,
van Loan
M.D.
,
Mentzer
W.C.
and
Heyman
M.B.
(
1999
)
Elevated resting energy expenditure in adolescents with sickle cell anemia
.
J. Am. Diet. Assoc.
99
,
195
199
[PubMed]
46
Badaloo
A.
,
Jackson
A.A.
and
Jahoor
F.
(
1989
)
Whole body protein turnover and resting metabolic rate in homozygous sickle cell disease
.
Clin. Sci.
77
,
93
97
[PubMed]
47
Liu
M.
,
Walter
G.A.
,
Pathare
N.C.
,
Forster
R.E.
and
Vandenborne
K.
(
2007
)
A quantitative study of bioenergetics in skeletal muscle lacking carbonic anhydrase III using 31P magnetic resonance spectroscopy
.
Proc. Natl. Acad. Sci. U.S.A.
104
,
371
376
,
48
Roussel
M.
,
Bendahan
D.
,
Mattei
J.P.
,
Le Fur
Y.
and
Cozzone
P.J.
(
2000
)
31P magnetic resonance spectroscopy study of phosphocreatine recovery kinetics in skeletal muscle: the issue of intersubject variability
.
Biochim. Biophys. Acta
1457
,
18
26
[PubMed]
49
Layec
G.
,
Haseler
L.J.
,
Hoff
J.
,
Hart
C.R.
,
Liu
X.
,
Le Fur
Y.
et al
(
2013
)
Short-term training alters the control of mitochondrial respiration rate before maximal oxidative ATP synthesis
.
Acta Physiol. (Oxf.)
208
,
376
386
,
[PubMed]
50
Jubrias
S.A.
,
Crowther
G.J.
,
Shankland
E.G.
,
Gronka
R.K.
and
Conley
K.E.
(
2003
)
Acidosis inhibits oxidative phosphorylation in contracting human skeletal muscle in vivo
.
J. Physiol.
553
,
589
599
,
[PubMed]
51
Lejay
A.
,
Meyer
A.
,
Schlagowski
A.I.
,
Charles
A.L.
,
Singh
F.
,
Bouitbir
J.
et al
(
2014
)
Mitochondria: mitochondrial participation in ischemia-reperfusion injury in skeletal muscle
.
Int. J. Biochem. Cell Biol.
50
,
101
105
[PubMed]
52
Charles
A.L.
,
Guilbert
A.S.
,
Guillot
M.
,
Talha
S.
,
Lejay
A.
,
Meyer
A.
et al
(
2017
)
Muscles susceptibility to ischemia-reperfusion injuries depends on fiber type specific antioxidant level
.
Front. Physiol.
8
,
52
[PubMed]
53
Benzie
I.F.
and
Strain
J.J.
(
1996
)
The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay
.
Anal. Biochem.
239
,
70
76
[PubMed]
54
Paradis
S.
,
Charles
A.L.
,
Meyer
A.
,
Lejay
A.
,
Scholey
J.W.
,
Chakfe
N.
et al
(
2016
)
Chronology of mitochondrial and cellular events during skeletal muscle ischemia-reperfusion
.
Am. J. Physiol. Cell Physiol.
310
,
C968
C982
[PubMed]
55
Dodd
S.L.
,
Vrabas
I.S.
and
Stetson
D.S.
(
1998
)
Effects of intermittent ischemia on contractile properties and myosin isoforms of skeletal muscle
.
Med. Sci. Sports Exerc.
30
,
850
855
[PubMed]
56
Greenberg
M.S.
and
Kass
E.H.
(
1958
)
Studies on the destruction of red blood cells. XIII. Observations on the role of pH in the pathogenesis and treatment of painful crisis in sickle-cell disease
.
AMA Arch. Intern. Med.
101
,
355
363
[PubMed]
57
Barreras
L.
and
Diggs
L.W.
(
1971
)
Sodium citrate orally for painful sickle cell crises
.
JAMA
215
,
762
768
[PubMed]
58
Im
I.J.
,
Choi
H.J.
,
Jeong
S.M.
,
Kim
H.J.
,
Son
J.S.
and
Oh
H.J.
(
2017
)
The association between muscle mass deficits and arterial stiffness in middle-aged men
.
Nutr. Metab. Cardiovasc. Dis.
27
,
1130
1135
,
59
Kim
J.K.
,
Kim
S.G.
,
Oh
J.E.
,
Lee
Y.K.
,
Noh
J.W.
,
Kim
H.J.
et al
(
2017
)
Impact of sarcopenia on long-term mortality and cardiovascular events in patients undergoing hemodialysis
.
Korean J. Intern. Med.
60
Harimoto
N.
,
Yoshizumi
T.
,
Izumi
T.
,
Motomura
T.
,
Harada
N.
,
Itoh
S.
et al
(
2017
)
Clinical outcomes of living liver transplantation according to the presence of sarcopenia as defined by skeletal muscle mass, hand grip, and gait speed
.
Transplant. Proc.
49
,
2144
2152
,
[PubMed]
61
Springer
J.
,
Springer
J.I.
and
Anker
S.D.
(
2017
)
Muscle wasting and sarcopenia in heart failure and beyond: update 2017
.
ESC Heart Fail.
4
,
492
498
,
[PubMed]
62
Capers
P.L.
,
Hyacinth
H.I.
,
Cue
S.
,
Chappa
P.
,
Vikulina
T.
,
Roser-Page
S.
et al
(
2015
)
Body composition and grip strength are improved in transgenic sickle mice fed a high-protein diet
.
J. Nutr. Sci.
4
,
e6
[PubMed]