Compromised muscle energetics and intracellular O₂ availability during dynamic contraction in the plantar flexor muscles of patients with systemic sclerosis

Abstract

The fundamental intracellular mechanisms underlying exercise intolerance and muscle weakness in patients with systemic sclerosis (SSc) are poorly understood. Thus, in 6 SSc patients (55 ± 11 yrs) and 6 age-matched healthy controls (50 ± 13 yrs), we assessed both skeletal muscle metabolism and intracellular O₂ availability in vivo during submaximal contractions. Specifically, during dynamic plantar flexion exercise at 40% of maximal aerobic power, we used a multi-modal approach combining phosphorus (³¹P) and proton (¹H) MRS of deoxymyoglobin, to examine both skeletal muscle energy pathways and intracellular partial pressure of O₂ (iPO₂). Both myoglobin O₂ levels (SSc: 70 ± 7 % and controls: 79 ± 4%; P=0.03;) and end-exercise iPO₂ (SSc: 8 ± 4 mmHg and controls: 17 ± 8 mmHg; P=0.021; d = 1.36) were lower in SSc than healthy controls. The rate of ATP synthesis from oxidative phosphorylation was also lower in SSc than controls (SSc: 5.2 ± 3.4 mM.min⁻¹; Controls: 11.8 ± 3.6 mM. min⁻¹; P=0.015) whereas PCr depletion was augmented (SSc: 55 ± 15 % from resting baseline; Controls: 77 ± 9 % from resting baseline; P=0.015). This shift toward a greater reliance on anaerobic metabolism led to an accumulation of inorganic phosphate diprotonated during exercise (SSc: 2.7 ± 1.2 mM and Controls: 1.4 ± 0.5 mM; P=0.037). Together, these findings indicate that critically low iPO₂ severely limits oxidative phosphorylation rate even during moderate-intensity dynamic plantar flexion exercise in patients with SSc. The magnitude of the disease-induced shift in metabolism and iPO₂ were so extensive that they were greater than the metabolic alterations found in patients suffering from advanced chronic cardiovascular diseases. In addition, the compensatory increase in anaerobic metabolism promoted the accumulation of inorganic phosphate diprotonated, which could amplify the susceptibility to muscle fatigue and mediate premature exercise cessation in patients with SSc.

Graphical Abstract

Introduction.

Systemic sclerosis (SSc) is a rare systemic autoimmune disorder with poor prognosis as the median survival is ~11 years post-diagnosis (1–3). This limited life expectancy is primarily mediated by greater cardiopulmonary morbidity and mortality (4), which may account for up to 60% of SSc-related deaths (5). The pathogenesis of SSc involves small vessel vasculopathy and progressive fibrosis of the skin and internal organs caused by excessive collagen production (6). Multiple organs and systems (pulmonary, cardiovascular, musculoskeletal, and gastrointestinal) are affected by the disease resulting in a broad range of debilitating symptoms (1, 7–9).

Common manifestations of SSc include fatigue and exercise intolerance, which are associated with poor quality of life (10–12). The low exercise capacity of SSc patients has traditionally been attributed to cardio-pulmonary limitations (12–14). However, a growing number of evidence suggests that peripheral dysfunction may also play an important role in the development of exercise intolerance in these patients. Muscle weakness, myalgia, and lower muscle mass have been documented in a large proportion of individuals with SSc (10, 11, 15, 16). During incremental cycling exercise, peak VO₂ of SSc patients was ~30 % lower in patients with SSc than healthy controls (17). Interestingly, in this study, the lower peak VO₂ of SSc patients was attributed to lower tissue O₂ extraction, which points tower peripheral limitations in O₂ transfer and skeletal muscle metabolic abnormalities (17).

Conceptually, a decrease in muscle O₂ extraction can stem from altered microcirculation (capillary density, red blood cell transit time) and/or skeletal muscle metabolism. Accordingly, recent studies indicated that reactive hyperemia, a marker of microcirculatory function, was blunted in the forearm and the calf muscles of SSc patients (18, 19). In sublingual capillaries, SSc patients also demonstrated lower microvascular density and shorter longitudinal distance for gas and nutrient exchange between red blood cells and surrounding tissues (20). Together, these findings imply that impairments in microcirculation likely account for some of the disease-related decline in muscle O₂ extraction. Interestingly, whether SSc also alters skeletal muscle metabolism during exercise is still largely unknown. One study assessed muscle energetics in the calf muscle of SSc patients during resting conditions (21), and documented greater inorganic phosphate to phosphocreatine (Pi/PCr) ratio measured in vivo by phosphorus (³¹P) magnetic resonance spectroscopy (³¹P-MRS). Metabolically, this altered ratio could either reflect an elevated skeletal muscle basal metabolism or greater metabolic disturbance for a given metabolic rate (altered metabolic control) due, perhaps, to limited O₂ availability. During muscle contractions, large adjustment in adenosine triphosphate (ATP) supply from the main energy pathways occurs to maintain ATP homeostasis, which results in the accumulation of metabolites (hydrogen ions, inorganic phosphate, adenosine diphosphate). Despite the central role of energy supply and metabolites accumulation in the development of muscle fatigue, the effect of SSc on muscle energy production are, however, yet to be determined.

Overall, the fundamental intracellular mechanisms underlying the disease-related development of exercise intolerance and muscle weakness with systemic sclerosis are poorly understood. Therefore, using a multi-modal approach combining ³¹P and proton (¹H) MRS of deoxymyoglobin (dMb), the purpose of this study was to examine in vivo both skeletal muscle energetics and intracellular O₂ availability during submaximal dynamic plantar flexion exercise, which minimized cardiopulmonary limitations, in SSc patients and age-matched healthy controls. We hypothesized that, in comparison to the healthy controls, SSc patients would 1) exhibit a lower intracellular O₂ availability that will limit the rate of oxidative phosphorylation, and 2) this will result in both a greater reliance on anaerobic metabolism for ATP supply and metabolic disturbance at the same relative exercise-intensity.

Method

Subjects

Six adults with systemic scleroderma and six healthy controls volunteered to participate in this study after signing a written informed consent (IRB# 40212). All healthy control participants were not taking any medications known to affect muscle function or blood flow. Additionally, these subjects were nonsmokers and were free of diabetes and overt cardiovascular, peripheral vascular, neuromuscular, or pulmonary disease. The study was approved by the Human Research Protection Program of the University of Utah and the Salt Lake City VAMC.

Patient and Public Involvement statement

No patients or public members were involved in the design, conduct, or dissemination of this study

Experimental Protocol

After familiarization with the equipment, individual maximum work rate (WR_max) was determined by performing incremental dynamic plantar flexion exercise until exhaustion (1 min stages). On a separate day, subjects performed two bouts of submaximal dynamic plantar flexion at ~ 40% of WRmax (frequency of 1 Hz) in the whole-body MR system to assess skeletal muscle metabolism and intracellular oxygenation, respectively. Specifically, the exercise protocol entailed 1 min of rest, 4 min of the constant-load sub-maximal plantar flexion, followed by 5 min of recovery while ³¹P-MRS was continuously recorded. After ~20 min of rest, this protocol was repeated using ¹H-MRS of deoxy-myoglobin (dMb). At the end of this second protocol, an air-cuff wrapped above the knee was inflated to 250 mmHg for 8 min to achieve complete vascular occlusion and determine the maximum dMb signal. Due to safety concerns (risk of thrombosis), the circulatory occlusion was not completed in one of the SSc patients and their data was not included in the analysis for ¹H-MRS. All experimental trials were performed in a thermoneutral environment with participants in an overnight fasted state.

³¹P MRS data collection and analysis

MRS was performed using a clinical 3T MRI system (Tim-Trio, Siemens Medical Solutions) operating at 49.9 MHz for ³¹P resonance. ³¹P MRS data were acquired with a dual ³¹P-¹H surface coil with linear polarization (Rapid Biomedical, Rimpar, Germany) positioned under the calf at its maximum diameter. The ³¹P single loop coil diameter was 125 mm, surrounding a 110-mm ¹H coil loop. After three-plane scout imaging, advanced localized volume shimming was performed. Before each experiment, two fully relaxed spectra were acquired at rest with three averages per spectrum and a repetition time (TR) of 30 s. ³¹P-MRS data acquisition was then performed throughout the rest–exercise–recovery protocol using an FID (free-induction-decay) pulse sequence with a 2.56 ms adiabatic-half-passage excitation RF (radio frequency) pulse and the following parameters: repetition time=2 s, receiver bandwidth=5 kHz, 1024 data points and three averages per spectrum. Saturation factors were quantified from the comparison between fully relaxed (TR = 30 s) and partially relaxed (TR = 2 s) spectra.

As described previously(22), relative concentrations of [PCr], [Pi] and [ATP] were obtained by a time-domain-fitting routine using the AMARES algorithm(23) incorporated into the CSIAPO software(24). Intracellular pH was calculated from the chemical shift difference between the P_i and PCr signals. The free cytosolic [ADP] was calculated from [PCr] and pH using the creatine kinase equilibrium constant (K_ck =1.66 x 10⁹ M⁻¹) and the assumption that PCr represents 85% of the total creatine content (25). The concentration of the diprotonated form of inorganic phosphate (H₂PO₄⁻) was calculated as H₂PO₄⁻ = [Pi] / (1+10^pH-6.75). The resting concentrations were calculated from the average peak areas of the two relaxed spectra (TR=30 s; n=3) recorded at rest and assuming an 8.2 mM [ATP] under these conditions. Free energy of ATP hydrolysis (ΔG_ATP) was calculated using the following equation and constants according to Kemp et al. (26):

$$\Delta G_{ATP}=\Delta G_{ATP}^0+RT\ln \frac{\left[ADP\right]\cdot \left[P_i\right]}{\left[ATP\right]}$$

$$\Delta G_{ATP}^0=RT\left[\ln \left(K_{ATP}^{true}\right)+\ln \left(\frac{f_{ATP}}{f_{ADP}\cdot f_{P_i}}\cdot \frac{1}{\left[H^{+}\right]}\right)\right]$$

With $K_{ATP}^{true}=0.722$ (27), R = 8.3145 K⁻¹M⁻¹ and T = 310 K.

To ensure that our findings were not confounded by the model of metabolic control utilized, the rate of oxidative ATP production at the end of exercise was estimated from the initial rate of [PCr] recovery, as previously described (28) and validated (method #1 in (29)). The initial rate of PCr resynthesis (Vi), which reflects the rate of oxidative ATP production at the end of exercise was calculated as follows:

$$\text{Vi}=\text{k}·{\left[\mathrm{P}\mathrm{C}\mathrm{r}\right]}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{s}}$$

in which [PCr]_cons represents the amount of PCr consumed at the end of exercise and the rate constant, k = 1/τ.

PCr recovery kinetics was determined by fitting PCr time-dependent changes during the recovery period to a monoexponential curve described by the following equation:

$$\left[\mathrm{P}\mathrm{C}\mathrm{r}\right]\left(\mathrm{t}\right)={\left[\mathrm{P}\mathrm{C}\mathrm{r}\right]}_{\mathrm{e}\mathrm{n}\mathrm{d}}+{\left[\mathrm{P}\mathrm{C}\mathrm{r}\right]}_{\mathrm{r}\mathrm{e}\mathrm{s}}(1-e^{-(t/\mathrm{\tau })})$$

Where [PCr]_end is the concentration of [PCr] measured at the end of exercise and [PCr]_res refers to the amount of PCr resynthesized during the recovery and $\tau$ reflects the time constant of the recovery (30, 31).

At the onset of exercise, ATP synthesis from oxidative phosphorylation is negligible for such that anaerobic ATP production accounts for the entire ATP synthesis (32). Accordingly, the rate of ATP synthesis at the onset of exercise was derived from the initial changes in PCr concentration over the first two time points. The corresponding amount scaled to power output provides a reliable estimate of the ATP cost of contraction (29, 33)

Model variables were determined with an iterative process by minimizing the sum of squared residuals between the fitted function and the observed values. The goodness of fit was assessed by visual inspection of the residual plot, and the frequency plot distribution of the residuals, χ² values and the coefficient of determination (r²) were calculated using the following equation [33]:

$$r^2=1-({SS}_{reg}/{SS}_{tot})$$

with SS_reg is the sum of squares of the residuals from the fit and SS_tot is the sum of squares of the residuals from the mean.

¹H-MRS of deoxy-myoglobin data collection and analysis

¹H-MRS data were acquired with a 4-Channel Flex coil (Siemens Biomedical, Germany) positioned around the calf at its maximum diameter. After three-plane scout imaging, advanced localized volume shimming was performed. The n-delta proton of the proximal histidine of F8 of myoglobin in the deoxygenated state was selectively excited using a Gaussian pulse (500 μs duration, 10 kHz carrier frequency offset) (34–37). ¹H-MRS spectra were acquired using the following parameters (TR = 17 ms, receiver bandwidth = 10 kHz, acquisition duration = 12 ms, nominal flip angle = 70°, 128 data points, 600 averages per spectrum, effective time resolution = 10s).

The quantification of dMb signal was performed on CSIAPO by manual integration of the peak after apodization. The fraction of dMb was determined by normalizing signal areas to the average signal acquired during the 7^th and 8^th min of ischemia at the pressure of 250 mmHg as previously described (38).

Conversion of myoglobin oxygenation into intracellular PO₂ values was calculated from the myoglobin oxygen-binding curve as follows:

$${\text{PO}}_2=\left[\frac{\text{1}-f}{f}\right].P_{\text{50}}$$

Where 1 – f is the fraction of myoglobin that is oxygenated, f is the fraction of myoglobin that is not oxygenated, and P₅₀ is the O₂ pressure where half of the myoglobin binding sites are bound to O₂. The temperature-dependent myoglobin P50 of 3.2 mmHg was used, based on an approximate muscle temperature of 39°C (39).

Statistical Analysis

After testing for homoscedasticity, the assessment of differences between patients with systemic scleroderma and healthy controls was performed with either independent Student’s t-tests or nonparametric Mann-Whitney tests, where appropriate (GraphPad Prism, Version 9.4.1; San Diego, CA). Statistical significance was accepted at P < 0.05. Effect sizes were determined by calculating the Cohen’s d. Results are means ± SD in tables and figures (with individual data).

Results

Participant characteristics

Participants with scleroderma and controls were matched for age, sex, and body stature (P>0.05, Table 1). Five SSc patients were on Ca²⁺ Channel Blockers and three were on immunomodulator medications. SSc patients’ diagnosis was based ACR/EULAR Classification Criteria (6). All patients were ANA positive with a SSc-specific antibody with a disease duration of less than 7 years disease duration from first non-Raynaud’s phenomenon. No patients were classified as a myositis overlap.

Table 1.

Participants characteristics.

	Healthy Controls		Systemic Sclerosis	P-value
n (Female/Male)	6 (1/5)		6 (2/4)
Age (y)	50 ± 13		55 ± 11	0.49
Anthropometric characteristics
Height (cm)	164 ± 7		170 ± 7	0.23
Weight (kg)	68 ± 14		80 ± 12	0.12
BMI	25 ± 4		28 ± 5	0.28
Medications
Ca2+ Channel Blockers			5
Immunomodulator			3

Values expressed as mean ± SD. Body mass index, BMI;

Mechanical output during exercise testing:

Plantar flexor power output during the constant load exercise at 40% of WRmax was significantly lower in SSc than Controls (SSc: 2.2 ± 1.0 watts and Controls: 3.2 ± 1.6 watts; P = 0.04, d = 1.35).

Exercise-induced changes in myoglobin oxygenation and intracellular partial pressure of O₂

Figure 1 displays myoglobin oxygen saturation (MbO₂) kinetics during constant-load exercise and the corresponding mean MbO₂ along with end-exercise iPO₂ levels. In both groups, MbO₂ levels dropped rapidly reaching a nadir of 55 ± 8 % in SSc and 68 ± 7% in controls (P = 0.02; d = 1.81), for which the corresponding iPO₂ are displayed in Figure 1C. Both end-exercise MbO₂ (SSc: 70 ± 7 % and controls: 79 ± 4%; P = 0.03; d = 1.81) and end-exercise iPO₂ (d = 1.36, Figure 1D) were lower in SSc than healthy controls.

Figure 1.

Time-dependent changes in myoglobin oxygen saturation during dynamic plantar flexion exercise (A) and the corresponding mean saturation levels (B), intracellular partial pressure of O₂ nadir at the onset of exercise (iPO₂, C), and iPO₂ at the end of the exercise (D) in healthy controls and systemic sclerosis patients. Values are presented as mean ± SD with individual data (n = 6 in healthy control; n = 5 in SSc).

Exercise-induced changes in high-energy phosphate compounds and intracellular pH

Figure 2 illustrates phosphocreatine kinetics during the constant-load exercise and the corresponding end-exercise concentrations. PCr levels during the last minute of the exercise were significantly different between SSc and healthy control (d = 1.79). The intracellular metabolite concentrations and pH in the plantar flexor muscles at the end of the exercise are illustrated in Figure 3. Intracellular pH, [Pi], and [ADP] were not significantly different between groups. However, the exercise-induced changes in H₂PO₄⁻ were higher in SSc than controls (d = 1.39, Figure 3C). ΔG_ATP was not significantly different between SSc and healthy controls (P = 0.13, d = 0.82, Figure 3E).

Figure 2.

Time-dependent changes in phosphocreatine concentration during dynamic plantar flexion exercise (left panel)) and the corresponding individual phosphocreatine consumption at the end of the exercise (right panel) in healthy controls and systemic sclerosis (SSc) patients. Values are presented as mean ± SD with individual data (n = 6 in both groups).

Figure 3.

Intracellular pH (A), inorganic phosphate (B), changes in diprotonated inorganic phosphate (C), adenosine diphosphate (D), free energy of ATP hydrolysis (E), and the rate of ATP synthesis by oxidative phosphorylation (F) at the end of the constant-load plantar flexion exercise in healthy controls and systemic sclerosis (SSc) patients. Values are presented as mean ± SD with individual data (n = 6 in both groups).

Oxidative phosphorylation was lower in SSc than healthy controls when expressed as absolute rates (d = 1.88, Figure 1F) or relative to total ATP synthesis (SSc: 39 ± 21 %; Controls: 63 ± 17 %; P = 0.059, d = 1.24). The overall ATP cost of contraction was, however, not significantly different between groups (SSc: 13 ± 16 mM.min⁻¹.W⁻¹; Controls: 10 ± 9 mM. min⁻¹.W⁻¹; P = 0.99, d = 0.23).

Discussion

The fundamental intracellular mechanisms underlying exercise intolerance and muscle weakness in patients with SSc are still elusive. Therefore, using a non-invasive multinuclear approach combining ³¹P/¹H MRS, this study examined both skeletal muscle energetics and intracellular O₂ availability in vivo during dynamic plantar flexion exercise in SSc patients and age-matched healthy controls. The principal novel findings of this study were that 1) SSc patients demonstrated a greater Mb desaturation resulting in lower intracellular PO₂ than controls in the exercising muscles, 2) the rate of ATP synthesis from oxidative phosphorylation was lower in SSc patients than controls whereas PCr breakdown was augmented, and 3) the ATP cost of contraction was similar between groups. Overall, these results indicate a significant shift in the main ATP synthesis pathways toward a greater dependence on the creatine kinase reaction to buffer the ATP demand from skeletal muscle contractions. This increased reliance on anaerobic metabolism in SSc patients was a compensatory response to the attenuated oxidative phosphorylation rate due to limited intracellular O₂ availability rather than a change in the energy cost of muscle contraction. These metabolic alterations during exercise were also associated with the accumulation of a metabolic by-product (H₂PO₄⁻) linked to muscle fatigue, which may thus play a role in the lower exercise capacity of SSc patients.

Effect of systemic sclerosis on intracellular O₂ availability during submaximal muscle contractions

The direct in vivo quantification of intracellular O₂ availability in the exercising muscle afforded by ¹H-MRS of deoxymyoglobin is a major stength of the present study. Using this technique, a key finding was that iPO₂ in the plantar flexor muscle was consistently lower in SSc patients than controls at the same relative intensity (Figure 1). Specifically, iPO₂ of the plantar flexor muscles of SSc rapidly dropped at the onset of the exercise to a nadir of ~4 mmHg before rising to a steady state of ~8 mmHg by the end of the exercise. In contrast, the MbO₂ response of the control group closely followed a mono-exponential decay, which is consistent with the response previously observed in the calf muscle of healthy controls during dynamic moderate- and heavy-intensity plantar flexions (40). The occurrence of a MbO₂ “undershoot” reveals that the increase in blood flow (O₂ delivery) and O₂ diffusional conductance at the onset of exercise in the SSc were sluggish relative to the rise in the rate of oxidative phosphorylation. Interestingly, after ~1 min, MbO₂ in the calf muscle of SSc slowly rose and reached a steady state by the end of the exercise thus implying a delayed increase in limb blood flow and late vascular adjustments to fine tune O₂ supply to metabolic demand.

Despite the use of a moderate-intensity exercise (~40% of WRmax) known to elicit modest metabolic changes in the calf muscle (41–44), the iPO₂ markedly dropped to critically low levels ranging between 4 to 8 mmHg in SSc patients, i.e. about half the levels of the controls (~17 mmHg). Although the literature is scarce on this topic due to the technical challenges with MRS measurements of dMb, most studies in untrained adults have only documented iPO₂ <10 mmHg during exercise performed at or above the heavy-intensity domain (40, 45–47). For instance, Richardson et al reported values of ~6 mmHg in the calf muscle of healthy adults when exercise intensity reached 80% of WRmax whereas lower intensity (60% of WRmax) resulted in iPO₂ of ~11 mmHg (40). During an incremental knee-extension exercise, iPO₂ values of ~5-7mmHg were measured at exhaustion in the quadriceps muscle of sedentary adults (45, 46, 48). Together, these findings suggest that severe derangements in peripheral O₂ supply with SSc compromised intracellular O₂ availability even at low work rates. In support of this interpretation, our groups recently documented that exercised-induced hyperemia measured in conduit arteries of the forearm was blunted by as much as ~35% in SSc patients compared with healthy controls, which is consistent with impaired convective O₂ delivery (49, 50). In addition, some evidence in other organs (sublingual capillaries) lend support to the hypothesis that O₂ diffusional conductance may also be impaired with SSc as perfused microvascular density was lower in these patients and was associated with a reduction in the surface of exchange between red blood cells and the surrounding tissues (20). This study also revealed alterations of the endothelial glycocalyx barrier, a gel-like structure that projects from the endothelial cell wall toward the vessel lumen, which could disrupt the exquisite regulation of microcirculatory blood flow and O₂ supply (20).

Effect of systemic sclerosis on ATP producing energy pathways submaximal muscle contractions

Another original finding from this study was that the rate of oxidative ATP synthesis in the plantar flexor muscles was noticeably lower in SSc, i.e. ~half of the control (Figure 3D). Specifically, the relative aerobic contribution to total ATP synthesis during this moderate-intensity exercise bouts was only ~39% in SSc whereas it accounted for as much as ~63% (d = 1.24) in the controls. As the ATP cost of contraction was similar between groups, the metabolic demand was met by a greater consumption of phosphocreatine (Figure 2). These data therefore provide strong evidence that one of the metabolic manifestations of SSc is a shift from aerobic metabolism toward anaerobic ATP production in the skeletal muscle. As O₂ is a key reactant in the process of oxidative phosphorylation(51–53), the drastically lower iPO₂ observed in the skeletal muscle of SSc patients may have limited the rate of oxidative ATP synthesis even during this submaximal exercise, which was compensated by a greater reliance on the creatine kinase reaction.

In agreement with this interpretation, experimental manipulation of tissue O₂ availability by lowering the inspired fraction of O₂ has been documented to augment PCr depletion during submaximal plantar flexion exercise in young adults (54–56). Similarly, in a canine gastrocnemius preparation in situ, decreased O₂ delivery from lowering arterial PO₂ (hypoxemia) or through circulatory occlusion resulted in greater PCr depletion and lower muscle O₂ consumption during electrically stimulated contractions (51, 57, 58). Furthermore, clinical populations with compromised O₂ delivery to the skeletal muscle due to severe pulmonary limitations (Chronic Obstructive Pulmonary Disease, COPD) or obstructed limb blood flow (peripheral artery disease) demonstrated a similar metabolic shift toward increased phosphocreatine consumption and intracellular acidification as a consequence of impaired oxidative ATP synthesis in the calf muscles (59, 60). Overall, the present data and prior studies support the interpretation that the metabolic changes in the energy producing ATP pathways of SSc are compensatory mechanisms caused by low iPO₂ rather than intrinisc metabolic anomalies of the skeletal muscle.

Effect of systemic sclerosis on metabolite accumulations during muscle contraction: potential implications for fatigue development

It is noteworthy that for the same relative intensity, the exercise-induced changes in H₂PO₄⁻ were doubled in SSc compared with controls (d = 1.39). Although the exercise intensity for this study was restricted to the moderate intensity-domain, this excessive accumulation of H₂PO₄⁻ has some potential implication for understanding the mechanisms underlying exercise intolerance and susceptibility to exercise-induced fatigue in patients with SSc (10–12). There is indeed a large body of evidence linking the accumulation of H₂PO₄⁻ to the development of peripheral fatigue (61, 62) (63). Accordingly, one can speculate that the greater reliance on anaerobic metabolism during exercise in SSc leads to excessive accumulation of H₂PO₄⁻ and be responsible for the premature development of muscle fatigue and exercise limitation in these patients. Future studies, using invasive (metabolomics from tissue biopsies (64)) or non-invasive (MRS) techniques, are therefore warranted to better determine the relative contribution of muscle metabolism and H₂PO₄⁻ accumulation to the diminished exercise tolerance of SSc patients.

Conclusion

In summary, this study provides novel mechanistic insights into the underlying cellular mechanisms of exercise intolerance and muscle weakness in patients with SSc. Using a non-invasive multinuclear approach combining ³¹P/¹H MRS during dynamic moderate-intensity plantar flexion exercise, this study revealed critically low intracellular PO₂ in the exercising muscles of SSc patients than healthy controls. This lower intracellular O₂ availability with SSc limited the rate of ATP synthesis from oxidative phosphorylation and increased the reliance on the creatine kinase reaction to buffer the ATP demand from skeletal muscle contractions. Furthermore, the shift toward anaerobic metabolism observed in SSc patients accentuated the accumulation of metabolites, which may represent an additional peripheral mechanism contributing to the greater susceptibility of SSc patients to exercise-induced fatigue.

Clinical Perspectives.

• The fundamental intracellular mechanisms underlying exercise intolerance and muscle weakness in patients with systemic sclerosis (SSc) are poorly understood.

• Critically low intracellular O₂ levels in the skeletal muscle severely limits oxidative phosphorylation rate, which leads to greater metabolic disturbance during dynamic plantar flexion exercise and augment the susceptibility to muscle fatigue in patients with SSc.

• Identifications of impairments in skeletal muscle metabolism and oxygenation is a steppingstone toward developing new interventions for the management of scleroderma and improving patient’s quality of life.

Data availability statement:

The data that support the findings of this study are available from the corresponding author upon reasonable request.