Assessment of mitochondrial respiratory capacity using minimally invasive and noninvasive techniques in persons with spinal cord injury.

PURPOSE: Muscle biopsies are the gold standard to assess mitochondrial respiration; however, biopsies are not always a feasible approach in persons with spinal cord injury (SCI). Peripheral blood mononuclear cells (PBMCs) and near-infrared spectroscopy (NIRS) may alternatively be predictive of mitochondrial respiration. The purpose of the study was to evaluate whether mitochondrial respiration of PBMCs and NIRS are predictive of respiration of permeabilized muscle fibers after SCI.
METHODS: Twenty-two individuals with chronic complete and incomplete motor SCI between 18-65 years old were recruited to participate in the current trial. Using high-resolution respirometry, mitochondrial respiratory capacity was measured for PBMCs and muscle fibers of the vastus lateralis oxidizing complex I, II, and IV substrates. NIRS was used to assess mitochondrial capacity of the vastus lateralis with serial cuff occlusions and electrical stimulation.
RESULTS: Positive relationships were observed between PBMC and permeabilized muscle fibers for mitochondrial complex IV (r = 0.86, P < 0.0001). Bland-Altman displayed agreement for complex IV (MD = 0.18, LOA = -0.86 to 1.21), between PBMCs and permeabilized muscles fibers. No significant relationships were observed between NIRS mitochondrial capacity and respiration in permeabilized muscle fibers.
CONCLUSIONS: This is the first study to explore and support the agreement of less invasive clinical techniques for assessing mitochondrial respiratory capacity in individuals with SCI. The findings will assist in the application of PBMCs as a viable alternative for assessing mitochondrial health in persons with SCI in future clinical studies.

Introduction

Within most eukaryotic cells, mitochondria serve a pivotal role as a robust ATP-generating system [1, 2]. Dysregulation of mitochondria’s dynamic process of biogenesis, remodeling, and degradation, result in unfavorable physiological outcomes including diminished energy production, increased reactive oxygen species, and even cell death [3]. Mitochondrial dysfunction may contribute to obesity and insulin resistance which can lead to the development of cardiovascular disease and T2DM, chronic diseases that are commonplace within the spinal cord injury (SCI) population [4, 5]. As such, methods for evaluating mitochondrial function hold critical importance during the initial stages of diagnosis as well as determining patient prognosis. Knowledge regarding changes in mitochondrial activity following SCI is limited. SCI is accompanied by cellular, metabolic, and body composition changes [6, 7]. An increase in adipose tissue combined with inactivity and skeletal muscle atrophy decreases daily energy expenditure and contributes to the obesity commonly observed in the SCI population [6, 8]. Furthermore, previous work suggests that mitochondrial mass and activity are decreased in older individuals with SCI when compared to those who are younger [9]. A previous study demonstrated that during an acute bout of electrical stimulation, persons with SCI heavily rely on carbohydrate utilization compared to fat utilization as demonstrated by the respiratory exchange ratio, which may indirectly suggest diminished mitochondrial activity [10]. However, it is still unclear whether mitochondrial dysfunction contributes to this observed pattern of substrate utilization after SCI. Multiple studies have shown that electrical stimulation training of muscle may yield increased muscle endurance, function increases in oxidative enzyme activity and gene expression [11-14]. These results suggest that improved muscle function may be mediated by mitochondrial capacity and highlights the need for clinical evaluation tools to assist in monitoring skeletal muscle function in this population [15]. An improved understanding of the cellular response of skeletal muscles in chronic SCI may help clinicians better formulate therapeutic and rehabilitative regimens for improving the long-term health and quality of life for persons living with SCI [13, 16].

Sources of mitochondrial dysfunction identified through respirometry include impaired mitochondrial membrane transport, substrate utilization issues, and disrupted fatty acid metabolism [17]. Prior studies have established protocols measuring mitochondrial activity from as little as 2–4 mg of skeletal muscle tissue [18-20]; however, these methods have not been extensively utilized in studies involving skeletal muscle biopsies from individuals with SCI. This invasive procedure requires a skilled team and technical expertise not available in most clinical settings. The use of muscle biopsies, although very helpful in studying mitochondria, is not a feasible strategy in clinical trials with large sample sizes as individuals may not consent to this procedure. Furthermore, many individuals may be concerned about the discomfort or complications associated with this invasive procedure. Less or noninvasive methods can also be used to quantify mitochondrial activity such as the measurement of blood cell mitochondrial function and skeletal muscle oxygenation [19, 21]. However, it remains unknown whether these procedures are representative of skeletal muscle mitochondrial function after SCI.

Recent research suggests that mitochondrial activity in blood cells such as peripheral blood mononuclear cells (PBMCs) may be representative of overall mitochondrial health [19, 22–26]. PBMCs are a subpopulation of white blood cells and include mostly lymphocytes and monocytes. Because of their exposure to inflammatory signals released from skeletal muscle in times of metabolic stress, these cells may be predictive of metabolic health [27-29]. Aging individuals, as well as those with T2DM or chronic kidney disease have dysfunctional white blood cell mitochondria similar to that seen in other tissues [22, 23]. Additionally, PBMC mitochondrial activity has been shown to be related to skeletal muscle mitochondrial activity in older adults [24]. A recent in vivo study performed using non-human primates demonstrated that blood based cellular respirometry is significantly correlated with bioenergetic measurements generated by skeletal and cardiac muscle [19]. Despite the relative promise of these earlier studies, it is still unknown if PBMC mitochondrial function is associated with oxygen consumption from skeletal muscle biopsies in humans with SCI.

A noninvasive tool helpful in measuring mitochondrial function is near-infrared spectroscopy (NIRS) [30]. NIRS measures tissue oxygenation through the oxygen-dependent absorption of near-infrared light by hemoglobin and myoglobin [30]. This technology was combined with methodologies originally adapted for use with magnetic resonance spectroscopy to assess the oxidative capacity of skeletal muscle following brief exercise [31-35]. Compared to magnetic resonance spectroscopy, the NIRS technique is more convenient and relatively inexpensive [30]. This NIRS serial arterial occlusion technique has subsequently been validated in comparison with magnetic resonance spectroscopy [31-35] as well as invasive skeletal muscle biopsy techniques [20]. Within SCI [12, 36] and other clinical populations [37, 38], this approach has been successfully implemented; however, these examples are limited and have not been validated for specific mitochondrial complexes following SCI.

The present study seeks to evaluate whether mitochondrial respiration of PBMCs and skeletal muscle oxygenation measured noninvasively by NIRS are predictive of permeabilized muscle fiber respiration in individuals with chronic SCI. We hypothesized that mitochondrial respiratory capacity measured by both high-resolution respirometry of PBMCs and NIRS would be correlated with those observed in high-resolution respirometry of permeabilized skeletal muscle fibers. The focus is on using these less-invasive techniques to measure cellular activity with the hope of decreasing the need for invasive techniques such as muscle biopsies.

Material and methods

Ethical approval

All aspects of this study were approved by the McGuire VA Medical Center institutional review board and conducted in the McGuire VA Medical Center. All procedures were conducted in accordance with the ethical standards of the Helsinki Declaration of 1964 and its later amendments. Participants provided written informed consent as part of an ongoing clinical trial, registered at clinicaltrials.gov (NCT# NCT02660073). Data presented in this study are cross-sectional prior to conducting any intervention.

Participants

Individuals with chronic SCI (≥ 1-year post injury), aged between 18–65 years, characterized by low levels of physical activity, and with a body mass index (BMI) ≤ 30 kg/m2 were recruited into the study. BMI was measured as each subject’s weight divided by their height squared [39]. All were asked to read and sign a consent form approved by local ethics committees. Participants were classified using the American Spinal Injury Association impairment scale (AIS) as either motor complete (A) or incomplete (B or C) as determined through a comprehensive physical examination by a board-certified SCI physiatrist. Subjects were excluded if they presented with a current or prior history of any of the following: cardiovascular disease (e.g., myocardial infarction, heart failure, etc.), type 1 diabetes, non-optimally treated T2DM (HbA1c > 5%), uncontrolled hypertension (BP > 130/80 mmHg), and/or insulin dependence.

Study design

The current study is based on data gathered at baseline prior to intervention from an ongoing clinical trial. Following an overnight fast (10–12 hours), participants came into the lab, voided their bladder, then underwent a muscle biopsy procedure in the early afternoon. Participants returned to the lab 3–4 days later following another overnight fast and to have a small sample of blood collected for the PBMCs isolation. Shortly that same morning, subjects began NIRS testing. In addition to the overnight fast, participants were asked to abstain from any level of physical activity for 2–3 day prior to coming on site.

Skeletal muscle biopsy & tissue preparation

Muscle biopsy samples were obtained from all subjects in the morning following an overnight fast. Briefly, muscle biopsy specimens were collected from the right vastus lateralis (VL) using a 14-gauge Tru-Cut needle (Merit Medical Systems, South Jordan, UT, USA) under local anesthesia (2% lidocaine). Immediately following the biopsy procedure, ~20 mg was placed in ice cold biopsy preserving solution (BioPS media, 2.77 mM CaK2EGTA, 7.23 mM K2EGTA, 5.77 mM Na2ATP, 6.56 mM MgCl2 6 H2O, 20 mM taurine, 15 mM Na2Phosphocreatine, 20 mM imidazole, 0.5 mM dithiothreitol, 50 mM MES hydrate, pH 7.1) for high-resolution respirometry. Muscle fibers were separated along the longitudinal axis using needle-tipped forceps under magnification yielding between 1–10 mg (average 4.9 ± 0.5 mg) of viable tissue. Due the presence of fat infiltration that is associated in individuals with SCI, an excess amount of muscle was utilized [7]. The plasma membrane of muscle fibers was permeabilized by gentle agitation for 20 min at 4°C in BioPS containing 50 μg/ml saponin [40] followed by two 4-min washes in mitochondrial respiration buffer (miR05, 0.5 mM EGTA, 3 mM MgCl2 6H2O, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM D–sucrose, 1g/L bovine serum albumin, pH 7.1) [13].

High resolution respirometry measurement of permeabilized muscle fibers

High-resolution O2 consumption measurements of separated muscle fibers suspended in 2 ml of miR05 buffer were measured at 37°C using the OROBOROS Oxygraph-2k (Oroboros, Innsbruck, Austria). Oxygen concentration and flux were recorded with DatLab software (Oroboros, Innsbruck, Austria). Respiration was measured using the following protocol: 10 mM glutamate + 5 mM malate (complex I substrates), followed by sequential additions of 0.1 and 1 mM ADP, 10 μM cytochrome c (to test for membrane integrity), 1 μM rotenone (complex I inhibitor), 10 mM succinate (complex II substrate), 40 μM 2-thenoyltrifluoroacetone (TTFA) (complex II inhibitor), 0.5 mM tetramethyl-p-phenylenediamine (TMPD)– 10 mM sodium ascorbate (complex IV substrates), and 10 mM sodium azide (complex IV inhibitor); Oxygen flux was expressed as pmol·sec -1 normalized to mg weight of the fiber bundle [13]. Mitochondrial respiration rates were determined after correcting for non-mitochondrial oxygen consumption following the addition of the corresponding inhibitor for complex I, II, and IV. Respiration rates were expressed as inhibitor-sensitive rates to eliminate the contribution of oxygen consumption not related to oxidation of the specific substrate [41]. A representative bioenergetic profile for skeletal muscle is shown in Fig 1.

Fig 1

Representative bioenergetic profile for peripheral blood mononuclear cells and permeabilized skeletal muscle fibers from one individual.

The rate of oxidative phosphorylation for PBMCs (A) and permeabilized skeletal muscle fiber (B) was measured in a high-resolution respirometer OROBOROS Oxygraph-2k. The blue line represents oxygen concentration in the oxygen chamber, and the red line represents the rate of oxygen consumption. 5.2 mg of skeletal muscle and 5.8 x 106 cells are represented in (A) and (B), respectively. Boxed blue lines indicate recorded data points. Glutamate + malate (GM) + 1mM ADP (ADP1), succinate, and tetramethyl-p-phenylenediamine + ascorbate (TMPD) were substrates used for complex I (a), II (b), and IV (c) substrates, respectively. Rotenone (ROT), thenoyltrifluoroacetone (TTFA), azide (AZ) were used to inhibit complex I (a), II (b), and IV (c), respectively. Respiration rates were expressed as inhibitor-sensitive rates (e.g. a—ai, b—bi, c—ci). 0.1 mM ADP (ADP0.1) and 1mM ADP (ADP1) were used to stimulate oxygen consumption. The final rate of oxidative phosphorylation pmol·sec-1 was normalized per wet weight of skeletal muscle (mg) or 1 x 106 cells.

Blood collection and mononuclear cell isolation

Vacutainers (BD Biosciences, San Jose, CA, USA) containing ethylenediaminetetraacetic acid (EDTA) were used to collect 20 ml of blood from patients following an overnight fast ~5 days apart from their muscle biopsy procedure. Blood and muscle were collected on different days due to the inherent time-intensive efforts involved with analysis for each sample. Since the study was cross-sectional, the effects of the time gap in between sample collections should be negligible. Blood was immediately diluted with an equal amount of phosphate-buffered saline (PBS), then layered over a Ficoll-Paque (Millipore-Sigma, Burlington, MA, USA) density gradient, and centrifuged at 750 x g for 30 min in room temperature. Mononuclear cells were isolated from the interface between the plasma and the Ficoll-Paque. Collection of mononuclear cells was performed via centrifugation at 350 x g for 10 min at 4°C, and washed twice with PBS.

High resolution respirometry measurement of mononuclear cells

High-resolution O2 consumption measurements of mononuclear cells were conducted using the OROBOROS Oxygraph-2k, and recorded by DatLab 4 software (OROBOROS Instruments, Innsbruck, Austria). Mononuclear cell pellets were resuspended in miR05 respiration buffer, then counted using the Countess II automated cell counter (Invitrogen, Carlsbad, CA, USA). 2 ml of suspension containing intact mononuclear cells in miR05 buffer (approximately 1 x 106–9 x 106 cells/ml) was added to the instrument’s chamber. A substrate-uncoupling inhibitor protocol was used as follows: 10 mM glutamate + 5 mM malate, followed by sequential additions of 6μg digitonin (detergent) per 1 x 106 cells (final concentration: ~10–50 μg/ml), 0.1–1 mM ADP, 10 μM cytochrome c, 1 μM rotenone, 10 mM succinate, 40 μM 2-thenoyltrifluoroacetone (TTFA), 0.5 mM tetramethyl-p-phenylenediamine (TMPD)– 10 mM and sodium ascorbate, 10 mM sodium azide; Oxygen flux was expressed as pmol·sec-1 normalized to 1 x 106 cells [40]. Respiration rates were corrected for non-mitochondrial respiration by using the oxygen consumption rates determined following the addition of the corresponding inhibitor for complex I, II, and IV. Similarly to skeletal muscle analysis, respiration rates for PBMCs were expressed as inhibitor-sensitive rates to eliminate the contribution of oxygen consumption not related to oxidation of the specific substrate [41]. A representative bioenergetic profile for PBMCs is shown in Fig 1.

Near-infrared spectroscopy measurement of mitochondrial capacity

The recovery rate of deoxygenated hemoglobin (HHb), or mitochondrial capacity, was obtained with continuous wave NIRS during a serial arterial occlusion method similar to another study which demonstrated its safety for individuals with SCI [36]. A portable NIRS device with three fixed optode distances (PortaMon, Artinis Medical Systems, Elst, Netherlands) was originally employed for testing (n = 12), while a single channel device with flexible optode distances (OxyMon, Artinis Medical Systems, Elst, Netherlands) was later used for the remaining participants (n = 6). Each tool was placed longitudinally over the right VL (~10 cm above the patella) to reduce inter-limb variability with the muscle biopsy technique. Two 8 x 10 cm2 surface electrodes (Uni-Patch, Wabasha, MI, USA) were also laterally seated superior to the right patella (~2–3 cm and ~30 cm respectively). Additionally, a rapid-inflating vascular cuff (Hokanson SC– 10D, Hokanson, Inc. Bellevue, WA, USA) was wrapped as proximally as possible on the right thigh. This cuff was controlled via a rapid-inflation system (Hokanson E20, Hokanson, Inc. Bellevue, WA, USA) to produce arterial occlusions using 250 mmHg of pressure.

Prior to testing, participants were transferred from their wheelchair to a mat via a ceiling lift. Participants then lay in a supine position for at least 10 min while the testing site was prepared by removing excess hair and cleaning the skin with alcohol pads. Each participant was also provided with a pillow for head and neck support, and if necessary, an additional popliteal bolster was provided to stabilize the knee joint. Each participant was also offered an optional pair of noise dampening headphones to wear during testing. Finally, the lower extremity was stabilized during testing using manual support.

Testing began with an initial 30-s cuff occlusion which was used to assess signal quality. Following a 3–5-min refractory period, an ischemic calibration was completed to establish maximum and minimum HHb values using a 10-s period of surface neuromuscular electrical stimulation (NMES; Theratouch 4.7; Richmar, Inola, OK, USA; biphasic waveform, 5 Hz, 175 mA, 450 μs) during an arterial occlusion lasting 3–5 min. After another 3–5-min refractory period, a 15-s bout of NMES paired with a cuff occlusion of the same length of time was executed. This was immediately followed by a series of 20 transient arterial occlusions (occlusions 1–5: 5-s on / 5-s off; occlusions 6–10: 5-s on / 10-s off; occlusions 11–20: 10-s on / 20-s off). In cases where spasm occurred during testing, this serial occlusion procedure was repeated and the trial with the least number of artifacts was chosen for data processing.

The NIRS data were collected at 10 Hz using OxySoft (3.0.103.3, Artinis Medical Systems, Elst, Netherlands) and a manufacturer recommended differential pathlength factor of 4. Data were then transferred for initial processed in Visual 3D (6.01.22, C-motion, Germantown, MD, USA). Raw HHb signals were first corrected for expected changes in blood volume as previously described [42]. The signal was then scaled to observed values during the ischemic calibration. To identify when cuff occlusions occurred during serial occlusion tests, a thresholding technique was applied to the rectified second derivative of the corrected HHb signal. These timepoints were visually confirmed. Slopes of the linear portions of the corrected HHb signal during each of the 20 arterial occlusions were calculated using linear regression. The magnitude of these slopes, along with the time for each occlusion, were exported to JMP Pro (14.3.0; SAS Institute Inc., Cary, NC, USA) and fit to a monoexponential curve. In many cases, early data points were excluded from the final curve fitting due to clearly de-trended values identified following visual inspection. Additional outliers were identified using studentized residuals (≥ 2.5) and removed from final curve fitting. In the event that > 50% of the data points were excluded or removed, then that participant’s data wereexcluded from analysis. The recovery rate constant (k), s-1, of the final monoexponential curve was indicative of HHb recovery and mitochondrial capacity [35]. These adapted methods have been reported in detail [43].

Statistical analysis

Statistical analysis was performed using SPSS 24 (Chicago, IL, USA). Outliers and normality were identified and assessed for each variable using box plots and Q-Q plots, respectively. Independent t-tests were utilized for the study participant demographic comparisons. Pearson’s correlation coefficients were used to identify associations between PBMC mitochondrial activity and skeletal muscle biopsy mitochondrial activity, as well as between mitochondrial capacity as measured by NIRS and skeletal muscle biopsy mitochondrial activity. Partial correlations were conducted to adjust for age, weight, BMI, and time since injury (TSI) between PBMCs and permeabilized muscle fibers [9, 44]. These statistical tools however, are not sensitive enough to establish agreement between different measurement techniques; thus, agreement between the compared methods was assessed using Bland-Altman analysis [45-47]. Due to the presence of different units between PBMCs and skeletal muscle respirometry, standardized Z-scores were computed for each measure prior to Bland-Altman analysis [20]. Confidence intervals ≤ 2.5 were used to determine Bland-Altman agreement between the compared methods. Bland-Altman plots graphically display the mean differences between two measurements along with their 95% confidence intervals, thereby allowing for a visual assessment of agreement [45-47]. Paired t-tests were also performed to evaluate potential mean biases between the compared methods [46]. The combination of these complementary statistical tools provides a more complete assessment of agreement between the PBMC measurement technique compared to the muscle biopsy gold standard [45-47]. Paired t-tests were performed comparing individual oxygen consumption rates following the addition of 1mM ADP and 10 uM cytochrome c to validate mitochondrial membrane integrity in PMBCs and skeletal muscles. Statistical significance levels were set to an α < 0.05.

Results

Participant demographics

Twenty-two individuals were recruited for this study. Two participants withdrew prior to any testing for unknown personal reasons. Demographic and injury information for the remaining 20 participants are summarized in Table 1. No significant differences for age, height, weight, BMI, or TSI were observed between paraplegics (level of injury [LOI]: T1 –S5) and tetraplegics (LOI: C5 –T1), as well as between Caucasians and African Americans.

Table 1

Participant demographics.

	Tetraplegic	Paraplegic	Total
N	7	13	20
AIS, n	A = 4; B = 2; C = 1	A = 8; B = 1; C = 4	–
Age, year	39.6 ± 11.4	36.6 ± 12.2	38 ± 13.0
Weight, kg	61 ± 11.7	72.4 ± 17.0	68 ± 16
Height, cm	178.5 ± 7.8	173.1 ± 9.1	175 ± 9.0
LOI	C5 –C7	T1 –T11	–
BMI, kg/m2	19.4 ± 5.0	24.2 ± 5.5	23.0 ± 5.0
TSI, year	13.7 ± 13.9	4.8 ± 3.1	8.0 ± 11.0
Caucasian, n	3	6	9
African-American, n	4	7	11
Male	6	10	16
Female	1	3	4

n, number of participants; AIS, American spinal injury association impairment scale; LOI, level of injury; TSI, times since injury; mean ± SD.

Relationships between PBMCs and permeabilized skeletal muscle fibers

While PBMC respirometry analysis was performed on all 20 participants, due to insufficient muscle tissue in one sample (i.e. wet muscle weight less than 1 mg), permeabilized skeletal muscle respirometry analysis was conducted for 19 participants. Subsequent testing error and equipment malfunction similar to failure of the oxygen sensors in the Oxygraph-2k chamber resulted in the loss of all permeabilized skeletal muscle data for an additional 2 participants and the loss of 1 data point for PBMC complex I and IV. Initial analysis also revealed 3 data points among PBMC complex II measures, 2 data points among PBMC complex IV, and 1 data point among permeabilized skeletal muscle complex II and complex IV to be outliers using Q-Q plots. Normality was found among each of the variables. Table 2 provides mean values of mitochondrial respiration measured from PBMCs and permeabilized skeletal muscle fibers. Comparisons of 16 participants were made for complex I, while comparisons among 14 participants were made for both complex II and IV. Pearson’s correlations were used to compare bioenergetics between PBMCs and skeletal muscle fibers (Fig 2). A significant positive relationship was observed between maximal oxidative phosphorylation rates of PBMCs and permeabilized skeletal muscle for complex IV (r = 0.86, P < 0.0001). Conversely, no significant relationships were observed for complex I (r = 0.36, P = 0.18) and complex II (r = -0.23, P = 0.43). Paired t-tests performed to validate mitochondria membrane integrity yielded no significant difference between individual oxygen consumption rates after adding 1mM ADP and 10 μM cytochrome c in skeletal muscle (P = 0.72) and PMBCs (P = 0.08).

Table 2

Bioenergetic profiles of SCI study subjects.

Tissue	Complex	Mean	SD	Range
Permeabilized muscle fiber	I& (n = 17)	11.22	6.10	3.06–22.29
	II (n = 16)	8.09	2.51	3.97–12.65
	II# (n = 17)	8.66	3.39	3.97–17.79
	IV (n = 16)	54.06	30.67	22.71–118.53
	IV# (n = 17)	61.23	41.92	22.71–176.03
PBMC	I (n = 19)	8.08	3.57	1.74–14.11
	I# (n = 20)	9.72	8.09	1.74–40.74
	II (n = 17)	4.08	2.61	0.09–8.68
	II# (n = 20)	6.67	7.13	0.09–29.28
	IV (n = 17)	39.18	18.60	17.30–74.32
	IV# (n = 19)	47.88	33.90	17.30–160.01
NIRS (n = 12)	–	0.011	0.004	0.004–0.018

Respiration is measured as pmol·sec1·1 x 106 cells-1 [PBMCs], pmol·sec-1·mg wet weight-1 [permeabilized fibers], and s-1 [NIRS]. 1 nmol·min-1 = 1000 pmol·60sec-1. PBMC, peripheral blood mononuclear cells; NIRS, near-infrared spectroscopy; SD, standard deviation

&, data present with outliers excluded

#, data with outliers included.

Fig 2

Comparison of PBMCs and permeabilized skeletal muscle fiber measures of mitochondrial capacity.

Relationships of maximal rate of oxidative phosphorylation between PBMCs and permeabilized muscle fibers for mitochondrial complexes (A) I (n = 17), (B) II (n = 14), and (C) IV (n = 14).

Partial correlations adjusting for participants’ physical characteristics (age, weight, BMI, and TSI) did not yield additional significant relationships (Table 3). Statistically significant positive relationships between PBMCs and permeabilized muscle fibers were maintained when controlling for all physical characteristics. Bland-Altman plots comparing standardized Z-scores of PBMCs and permeabilized fibers (Fig 3) show good agreement for complex I (95% CI: 2.26) and IV (95% CI: 1.03) with no significant biases (P > 0.05). The confidence interval for complex II Z-score comparisons fell outside the a priori limits of agreement (95% CI: 3.23). Additional details regarding Bland-Altman agreement between PBMCs and permeabilized muscle fibers are provided in Table 4.

Table 3

Partial correlations between permeabilized muscle fiber and monocyte or NIRS bioenergetics after independently controlling for age, weight, BMI, or TSI.

		PBMCs			NIRS
Confounding variables	Complex	n	Pearson’s r	P-value	n	Pearson’s r	P-value
Age	I	17	0.275	0.302	9	0.504	0.202
	II	14	-0.274	0.366	8	-0.471	0.286
	IV	14	0.895	<0.0001*	9	-0.585	0.127
Weight	I	17	0.307	0.248	9	0.453	0.260
	II	14	-0.227	0.455	8	-0.270	0.558
	IV	14	0.900	<0.0001*	9	-0.477	0.232
BMI	I	17	0.296	0.265	9	0.335	0.418
	II	14	-0.228	0.454	8	-0.232	0.616
	IV	14	0.875	<0.0001*	9	-0.467	0.243
TSI	I	17	0.299	0.260	9	0.436	0.280
	II	14	-0.095	0.757	8	-0.583	0.169
	IV	14	0.851	<0.0001*	9	-0.514	0.192

“*” P-value ≤ 0.05.

Fig 3

Agreement between PBMCs and permeabilized skeletal muscle fiber measures of mitochondrial capacity.

Bland-Altman plots of normalized high resolution repirometry of PBMC and normalized permeabilized muscle fiber indices of mitochondrial respiratory capacity in individuals with SCI for mitochondrial complexes (A) I (n = 16), (B) II (n = 14), and (C) IV (n = 14). Solid black line represents the mean difference; the greyed region between the dashed black lines represents the 95% limits of agreement.

Table 4

Bland-Altman limits of agreement analysis between permeabilized muscle fibers and monocytes Z-scores.

		95% limits of agreement
	Mean difference (SD)	Lower limit (95% CI)	Upper limit (95% CI)
PBMCs
Complex I (n = 16)	-0.102 (1.154)	-2.364 (-3.414 to -1.313)	2.159 (1.108 to 3.210)
Complex II (n = 14)	-0.121 (1.648)	-3.350 (-4.976 to -1.724)	3.108 (1.482 to 4.734)
Complex IV (n = 14)	0.175 (0.523)	-0.851 (-1.367 to -0.334)	1.200 (0.684 to 1.717)

SD, standard deviation; CI, confidence interval.

Relationships between NIRS and permeabilized skeletal muscle fibers

Prior to NIRS collection, 2 participants withdrew due to reported pain/sensitivity evoked by the electrical stimulation parameters when combined with cuff occlusions. Due to technical issues, 6 more participants were unable to provide usable NIRS data [43]. This included 4 individuals whose skin adipose tissue exceeded the penetrating depth of the NIRS device employed, and 2 participants’ whose data were lost due to quality issues encountered either during collection (e.g. muscle spasms) or in processing. The remaining data were found to be free from outliers and normally distributed.

The NIRS recovery profiles for the remaining 12 participants are detailed in Table 2. Comparisons of 9 participants were made for both complex I and IV, while comparisons among 8 participants were made for complex II. No significant correlations were observed between the NIRS recovery rates and the permeabilized skeletal muscle fiber oxidative phosphorylation rates for complex 1 (r = 0.47, P = 0.20), complex II (r = -0.22, P = 0.60, or complex IV (r = -0.37, P = 0.32); additionally, no significant changes were noted when controlling for age, weight, BMI, or TSI (Table 3). Bland-Altman analyses comparing NIRS and permeabilized skeletal muscle fibers were not conducted due to the limited NIRS sample size.

Discussion

This study offers evidence that mitochondrial respiratory capacity as measured with PBMCs may be comparable to the gold standard method utilizing skeletal muscle fibers for persons with SCI. Measurement with NIRS, however, could not be compared with the individual mitochondrial complexes in skeletal muscle fibers and thus its utilization potential in this clinical population could not be confirmed. The current study was designed to examine the feasibility and appropriateness of using less and/or noninvasive technologies when assessing mitochondrial capacity following SCI; as such, only comparisons against established permeabilized muscle fiber techniques were considered. The primary findings revealed high-resolution respirometry values for skeletal muscles and PBMCs oxygen consumption rates to be positively related for complex IV independent of age, weight, BMI, and TSI, while also displaying good agreement. Our hypothesis that mitochondrial capacity in persons with SCI could be reliably assessed with alternatively less invasive techniques is therefore only partially supported.

Mitochondria are critical in maintaining traditional skeletal muscle function and vitality. Deficiencies in their function are present in a variety of neurodegenerative and cardiovascular disease states, as a normal component of cellular senescence in aging, as well as metabolic disorders including T2DM and obesity [1, 48–50]. Oxidative capacity of skeletal muscles, reflected by substrate utilization and oxygen uptake of skeletal muscles, typically decrease as a consequence of aging, reduced physical activity, and injury/disease in rodents and humans [36–38, 51–54]. Decreased oxidative capacity, mitochondrial size and protein concentrations have been implicated in individuals with SCI compared to those who were able-bodied [14, 55–57]. While the pathways responsible for mitochondrial dysfunction in individuals with SCI remain unclear, they may be mediated by several mechanisms including oxidative stress [58], apoptosis [59], and aberrant mitophagy [60]. Establishing the utilization of PBMCs and/or NIRS as alternative methods for providing physiological information regarding mitochondrial health for individuals with SCI has yet to be accomplished, thus the findings of this study may serve to provide additional support for this important foundation.

Mitochondrial respiration in PBMC and permeabilized skeletal muscle fibers

Recently, potential links between mitochondrial health measured in white blood cells with various disease states and disorders have been explored, highlighting their potential as a reliable and convenient source for assessing mitochondrial bioenergetics in translational research [19, 24, 26, 61–64]. Specifically, PBMCs have been recognized as a potentially sensitive marker for mitochondrial dysfunction in a number of neurodegenerative and pathological disease states [24, 26, 63, 65]. Amongst individual oxygen consumption rates, complex IV in both PBMC and skeletal muscle consistently yielded the highest oxygen consumption rates, followed by complex I and complex II (Table 2). To the best of our knowledge, this is the first study to demonstrate evidence of complex IV in PBMCs as a surrogate for complex IV respiratory rates in skeletal muscle; additionally, no previous assessments of PBMC respiration rates for complex II and IV have been performed. Mean PBMC respiration rates for complex I (Table 2) were comparable to able-bodied subjects in similar studies [21, 66, 67]. In contrast, permeabilized skeletal muscle fiber respiration rates (Table 2) were lower among participants in the current study versus able-bodied populations reported in the literature [20, 21]. These opposing observations could be the result of the differences in mitochondrial respirometry protocols and/or instrumentation implemented between studies, as such making comparisons challenging. Alternatively, this may suggest mitochondrial function is inherently lower in the skeletal muscle fibers for individuals with SCI. However, the use of the robust substrate, TMPD-ascorbate (with corresponding inhibitor to provide a background subtracted rate), can nonetheless provide useful estimates of skeletal muscle oxidative function despite a small sample size. Furthermore, confidence in our low measures for complex I and II respiratory rates is supported by the lack of significance observed in the a posteriori normalization techniques utilized in the current study.

Attenuation in the function of complex I occurs in individuals with several disease states including T2DM [68] and Huntington’s disease [66], as well as ischemia damaged murine cardiac mitochondria [69]. To identify the potential defect in complex I activity in persons with SCI, respiratory enzyme activities of the electron transport chain need to be studied in future work. Respiration rates using combined complex I+II substrates measured in prior studies [19, 20], was not assessed in the current study. In the present study, separate substrate and inhibitors were utilized to localize defects in the electron transport chain. The assessment of complex I+II respiration rates should be considered in a future study to provide a more physiological relevant assessment of maximal mitochondrial respiratory capacity. However, the measure of complex IV rates may provide a downstream rate similar to complex I+II; thus, providing a greater signal to noise ratio in both skeletal muscle and PBMCs.

The general lack of correlation and Bland-Altman agreements for complexes I and II contrasts that of a study showing strong correlation of complex I in a study evaluating peripheral monocytes and permeabilized skeletal muscle mitochondrial function in healthy non-human primates [19], suggesting a potential impact of SCI on complex I function. In prior studies involving elderly adults, respirometry profiles of PBMCs have been shown to be positively associated with markers of physical function and strength; however, comparison between individual mitochondrial complexes were not evaluated [24, 26]. Partial correlations adjusting for age, weight, BMI, and TSI were performed in the current study primarily due to the wide range in the demographic variables. Large demographic ranges were also present in previous work when comparing mitochondrial mass and activity after SCI, trends and significant changes in activities remained consistent independent of demographic factors including age, TSI, and level of injury [9]. The fact that significant positive correlations remained after independently controlling for age, weight, BMI, and TSI, provide additional support for the correlations observed.

A study investigating the adaptation of 2-week high-intensity interval training in ten young able-bodied men recently demonstrated that PBMCs do not reflect mitochondrial function of skeletal muscles [21]. Although the results of the current study involving complex I is consistent, the utilization of different study populations in each investigation should be carefully noted. Additionally, Hedges et al. did not examine the individual respiration rates for complex II and complex IV. Following SCI, several profound physiological changes are observed, including long-term negative effects on mitochondrial regulation and activity [70]. While, Hedges et al. enrolled homogenous, able-bodied young male participants, the current study successfully recruited a heterogenous group of individuals with chronic SCI. Absolute maximal oxygen uptake (VO2) peaks of the healthy participants were significantly (2.1–3.6-fold) higher than those typically observed in SCI populations [71, 72]. Unlike for skeletal muscle, the physiologic mechanisms for whether mitochondrial respiratory capacity in less-oxidative tissue (e.g. PBMCs) should increase in response to physical training is less clear. The mitochondrial capacity of PBMCs appear to be less responsive to change in the direction of an increase in respiratory capacity following acute high-intensity training [21], but may be more susceptible to become impaired in chronic diseases states that consist of systemic factors including inflammation, oxidative stress, and/or metabolic syndrome that are evident in individuals with SCI [70, 73, 74]. Therefore, PBMCs may still serve as a useful biomarker of muscle mitochondrial function in individuals with SCI, a pathological population with chronically compromised mitochondrial respiratory capacity compared to their able-bodied counterparts. This is further supported by a recent study revealing that telomeres in PBMCs, a senescence biomarker, from sarcopenic older individuals were shorter relative to non-sarcopenic peers [75]. Since telomeric shortening in PBMCs appears to be reflective of muscle loss, it is possible that chronic inflammatory and metabolic changes influence mitochondrial function within PBMCs [75, 76]. Overall, compared to those who are able-bodied, individuals with SCI have inherent systematic factors that may negatively influence the detection of mitochondrial respiratory capacity. This may provide a potential explanation for the lack of correlation and agreement for mitochondrial complexes I and II in the current cross-sectional study with untrained persons with SCI. However, the robust positive correlations and agreement observed after the addition of TMPD-ascorbate in PMBCs and skeletal muscles in the current study supports that mitochondrial complex IV function in PBMCs is a useful indicator of mitochondrial function in skeletal muscle of persons with SCI. The minimally invasive procedure of obtaining a blood sample to measure mitochondrial function via PBMCs will allow more research groups and clinicians alike to conduct this type of analysis and facilitate maximum benefit of interventions for rehabilitation. Future work investigating the effects of endurance and resistance training on mitochondrial functional improvement over time for persons with SCI is fundamentally necessary to assess the potential to reduce or limit long-term systemic complications. Having convenient access to mitochondrial health will allow providers to adjust type, duration and intensity of exercise intervention in order to maximize health benefits.

Mitochondrial respiration in skeletal muscle fiber measured in NIRS and OXPHOS

The average NIRS recovery rate measured in the current study (0.011 s-1) was consistent with literature reported average values among other SCI groups ranging from 0.008 to 0.017 s-1 [12, 36, 77, 78]. The average recovery rate observed in this investigation fell below previously reported able-bodied cohorts by greater than 60% [20, 36, 37, 77, 79, 80]. This observation is consistent with prior work performed by Erickson et al. [36] comparing the SCI group with able-bodied controls.

Previous findings highly suggested that NIRS is an effective tool to measure overall mitochondrial oxidative capacity. However, the current findings did not support the hypothesis that NIRS could be used to measure mitochondrial complexes similar to what has been reported in the able-bodied population [20]. A lack of significant correlations alone does not imply poor agreement between the NIRS and permeabilized skeletal muscle fiber results as these measures may be somewhat misleading, especially when considering our small heterogeneous sample [45-47]. However, using NIRS to measure oxidative capacity in persons with SCI has proven to be more technically challenging than in able-bodied groups [43]. These challenges resulted in a diminished sample size and the exclusion of Bland-Altman analyses comparing NIRS and permeabilized skeletal muscle fiber techniques. Previous comparisons of these tools in an able-bodied population by Ryan et al. [20] displayed good agreement for complex I (95% CI: 2.43) and complex II (95% CI: 1.53) while complex IV was not explored. Researchers should consider examining the agreement between mitochondrial supercomplex activity and NIRS, as the nature of this noninvasive tool may be better suited to capture global oxidative processes.

Limitations

The relatively small sample sizes in the current study increases the likelihood for type II error and limits the strength of correlation. The small sample of this very heterogeneous population with multiple factors influencing their physiologic health could also explain the presence of the outliers in the analysis. With outliers included, however, the mean respiration rates were largely unaffected. The presence of greater and variable amounts of intramuscular fat among the biopsy samples [7], in addition to not correcting for auto-oxidation (for both muscle and PBMCs), may have influenced the respiration rates, further complicating the analysis and potentially explaining some of our outliers. However, using inhibitor sensitive rates in the current study was deemed a better alternative approach since rates are more specific than correcting for auto-oxidation [41, 81]. Maximal respiratory capacity, an important parameter for determining mitochondrial dysfunction, was not assessed due to limited muscle biopsy size and quality, and thus should be considered in future work. Unfortunately, a number of methodological issues arose using NIRS, dramatically reducing the sample size and limiting the utilization of Bland-Altman comparisons. Furthermore, capturing repeated NIRS data across multiple days or from the opposite limb may have allowed for establishing test-retest reliability measures; however, due to difficulties with transportation to-and-from the medical center, the length of time necessary for testing, and potential interlimb variability these additional steps were not considered. Including an able-bodied matched cohort would also provide additional strength to the study; however, the design of the current work did not require such an addition as our aim was to compare techniques to assess mitochondrial health in SCI. At the expense of facilitating easily interpretable results, it was necessary to reconcile unit differences between comparisons using Z-score; future studies should explore alternative tools that may not be limited in this fashion. While current high-resolution respirometry techniques employed in this study require small volumes of blood to assess OXPHOS rates, PBMCs naturally contain a lower density of mitochondria when compared to skeletal muscle [65]. Such differences may artificially influence bioenergetic outcomes, resulting in less accurate assessments of mitochondrial capacity, particularly for complex I and II in the current study. Additionally, larger or more homogenous samples may allow researchers to accurately estimate the minimal detectible change values for these noninvasive assessment techniques. Due to limitations in biopsy and blood samples collected, reliability data were not performed in the current study, however, will be considered in future work.

Conclusion

This preliminary study, to the authors’ knowledge, is the first to explore and support the agreement of less invasive clinical strategies for assessing mitochondrial respiratory capacity in persons with SCI using PBMCs, particularly for complex IV. Further studies are warranted to provide a measurement of complex I and II activity using less and noninvasive techniques. At this time, NIRS appears to be challenging to implement for investigating individual mitochondrial complex activities in persons with SCI. Overall, the findings may assist in supporting the implementation of PBMCs as an alternative when assessing mitochondrial health in future SCI clinical trials, especially those evaluating exercise interventions. The mitochondrial health information provided through these less-invasive procedures may ultimately serve as a critical marker for clinical decision-making aiding in driving down rates of the many chronic metabolic comorbidities that typically plague individuals with SCI.

19 Oct 2021

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I appreciated reading your submission and feel that the Reviewers have provided a substantive and fair assessment of your work; that said, I have additional concerns that I would like you all to address in your rebuttal.

1. As muscle samples were acquired from these patients, is there a reason why citrate synthase activity, a well known surrogate of mitochondrial content, was not determined? Or cytochrome oxidase as a surrogate of ETC function? You know that these are widely assayed in metabolic studies performed in people without SCI. I know that these tell the scientist something different than respiratory capacity, but they would likely be associated with some of the outcomes (PBMCs, etc.) presented in this paper.

2. The methods section would benefit from inclusion of a brief Experimental Design section, specifically including text concerning fed state of participants, if they abstained from physical activity for some time before testing, voided their bladder, time of day of testing, etc.

3. Do you have any reliability data for your measures? If so, these would be useful to include in the Methods text.

4. Table 2 seems to suggest that PBMC underestimates many of these outcomes vs. muscle fibers; do you have any explanation for this finding? And is there a reason why paired t-tests were not used to compare these mean values statistically rather than only using Pearson r?

5. Lines 436-439 seem to need a sentence or 2 specifically referring to mitochondrial dysfunction-abnormality in persons with SCI.

6. Were these people with SCI physically active, and if so, it may be worthwhile to report this as improved mitochondrial function would be expected irrespective of their disability status.

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Reviewer #1: The authors analyzed 22 individuals to evaluate whether mitochondrial respiration of PBMCs and NIRS are predictive of respiration of permeabilized muscle fibers after SCI. The results showed positive correlation between PBMC and permeabilized muscles fibers. However, no association was found between NIRS mitochrondrial capacity and respiration.

1. Line 289. Outliers were excluded based on studentized residuals of 2.5. the threshold of 2.5 seems quite liberal. How many data points were removed? Didn’t these data points provide information or the model was not a good one?

2. Only parametric test (T-test, linear regression and correlation) were implemented. What distribution were the data? Whether they satisfy the model assumptions?

3. Line 323. No significant difference for age,….. Please provide the information about statistical approach implemented for these participant demographics comparison in statistical analysis section.

Reviewer #2: Overview

Mitochondrial dysfunction likely contributes to the etiology of T2DM, obesity, and cardiovascular disease in those with SCI. Mitochondrial function is typically measured from permeabilized muscle fibers attained via muscle biopsy, a technique that is difficult to perform in those with SCI. Therefore, the purpose of this study was to examine the validity of two other potential methods of assessing mitochondrial function, mitochondrial respiration of peripheral blood mononuclear cells (PBMCs) and near-infrared spectroscopy (NIRS). Mitochondrial respiration was measured from permeabilized muscle fibers and PBMCs and mitochondrial capacity was measured by NIRS in 21 individuals with complete or incomplete SCI. A positive, significant correlation was found between PBMC and permeabilized muscle fibers for mitochondrial complex IV, but no relationship was found for NIRS. This study provides some evidence that PBMCs can be used to assess mitochondrial function using a minimally invasive procedure in those with SCI.

Specific comments

Lines 68-70 How does SCI result in mitochondrial dysfunction?

Line 71 It is easy to see why the measurement of mitochondrial function is important to measure to assess patient prognosis, but it would be good to know more about its relevance during initial diagnosis shortly following injury. How would the assessment of mitochondrial function in those with SCI be used by clinicians to treat their patients more effectively?

Lines 76-83 It is difficult to follow the relevance of the previous research results mentioned here. It is easy to understand that mitochondrial function decreases with age, but this occurs in those without SCI as well. The effects of mitochondrial function on substrate use are certainly applicable here, but there is a plethora of evidence that heavy reliance on carbohydrate occurs in those with SCI during voluntary exercise, a finding with that is more generalizable than acute electrical stimulation. In this section, it would be good to know more precisely how mitochondrial dysfunction in those with SCI leads to an increased risk for T2DM, obesity and cardiovascular disease.

Line 244 When were the NIRS measurements taken relative to the other measures and were subjects tested in an overnight fasted state?

Line 406 “Data” is plural. Thus, follow it with “were” rather than “was” here and throughout.

Line 419 While the authors adequately explain the likely mechanisms for their primary findings, they do little to address the applicability of the data. Under what context might a clinician use a measurement of mitochondrial respiration of PBMCs to alter the treatment of their patients? It is interesting that a minimally invasive technique may adequately estimate mitochondrial function in those with SCI, but these results have little meaning if the reader cannot see how they would actually be applied to improve the lives of those with SCI.

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9 Feb 2022

Additional Editor Comments:

Q1. As muscle samples were acquired from these patients, is there a reason why citrate synthase activity, a well known surrogate of mitochondrial content, was not determined? Or cytochrome oxidase as a surrogate of ETC function? You know that these are widely assayed in metabolic studies performed in people without SCI. I know that these tell the scientist something different than respiratory capacity, but they would likely be associated with some of the outcomes (PBMCs, etc.) presented in this paper.

Response: Thank you for your comment and suggestion. We absolutely agree that citrate synthase serves as a surrogate for mitochondrial content. The activity of citrate synthase (CS) is measured by mitochondrial enzyme activity assays which is beyond the scope of our research question in the current manuscript. Mitochondrial enzyme assays performed analyzing skeletal muscle biopsy samples is actually being explored in other work we have in the pipeline where we are measuring CS activity as a surrogate for mitochondrial mass. The focus of our current submission is non-invasive strategies to assess mitochondrial function. From our understanding, we are unaware of how to obtain and measure CS content though PBMCs and NIRS testing.

Q2. The methods section would benefit from inclusion of a brief Experimental Design section, specifically including text concerning fed state of participants, if they abstained from physical activity for some time before testing, voided their bladder, time of day of testing, etc.

Response: Participants were enrolled in the trial as a part of an ongoing clinical trial looking at the effect of 2-different paradigms of electrical stimulation. The current study was conducted with participant data at the baseline before being enrolled in any intervention. Participants fasted for 10-12 hours and abstained from any level of physical activities 2-3 days prior to the intervention; additionally, our participants were motor complete injury and characterized by a very low-level of physical activity [Line 152]. After measuring basal metabolic rate (6.00-6.30 AM), blood was collected for measuring biomarkers, insulin sensitivity and glucose tolerance. Participants were then admitted to the minor procedure room around 12.00-1.00 to conduct muscle biopsy as explained in the trial. We’ve added a “study design” sub-section in the methods to provide additional clarity [Lines 164-170].

Q3. Do you have any reliability data for your measures? If so, these would be useful to include in the Methods text.

Response: We would like to thank the reviewer for this comment. Because of IRB constraints and the acquired sarcopenia in persons with SCI, we were only limited to small muscle tissue as well as the amount of blood work necessary to conduct the trial. Additionally, the NIRS testing and set up process may take additional 2-3 hours to conduct particularly when adjusting to the necessary settings, which is considered labor-intense and requires additional time commitment from our subjects. However, we are fully aware of the significance of conducting future reliability study on these measures and we have acknowledged this point as one of the limitations of the study [Lines 595-596].

Q4. Table 2 seems to suggest that PBMC underestimates many of these outcomes vs. muscle fibers; do you have any explanation for this finding? And is there a reason why paired t-tests were not used to compare these mean values statistically rather than only using Pearson r?

Response:

We believe that the amount of the cells in the drawn blood, especially in persons with SCI, is lower and may require increased volume to reflect the true mitochondrial respiration. Additionally, persons with SCI characterized by systematic inflammation that is likely to impair PBMC respiration. This point was stated in the discussion section [Lines 521-522 and Lines 523-525]. Previous trials that addressed similar research questions (Tyrell et al, 2016 Ref#19, Hedges, et al 2019 Ref #21, Ryan et al.2014, Ref #20) have used both Pearson r and Bland-Altman analysis.

Paired t-test analysis was performed and is detailed in the methods section [Lines 327-329] as well as in the results section [Line 383]. No significant biases were measured using the paired t-tests. Comparisons made using paired t-tests to assess for biases between each of the normalized complexes revealed no significant differences (I: mean difference: -0.10, P = 0.73; II: mean difference: -0.12, P = 0.79; I: mean difference: 0.17, P = 0.23).

Q5. Lines 436-439 seem to need a sentence or 2 specifically referring to mitochondrial dysfunction-abnormality in persons with SCI.

Response: Thank you for your comment. We have added a sentence to clarify this point [Lines 436-438]

Q6. Were these people with SCI physically active, and if so, it may be worthwhile to report this as improved mitochondrial function would be expected irrespective of their disability status.

Response: The subjects with SCI were not physically active. All of our participants were motor complete and were characterized by very low level of physical activity [Lines 155-158]. We have incorporated this in the methods section [Line 152].

________________________________________

Reviewer #1: The authors analyzed 22 individuals to evaluate whether mitochondrial respiration of PBMCs and NIRS are predictive of respiration of permeabilized muscle fibers after SCI. The results showed positive correlation between PBMC and permeabilized muscles fibers. However, no association was found between NIRS mitochondrial capacity and respiration.

Q1. Line 289. Outliers were excluded based on studentized residuals of 2.5. the threshold of 2.5 seems quite liberal. How many data points were removed? Didn’t these data points provide information or the model was not a good one?

Response: In an attempt to retain as much of the original data as possible, we elected to utilize a threshold of 2.5 which encapsulates over 98% of all normalized data. The effects and number of these outliers were varied across individuals and time points (as detailed in the cited paper Ref#43: Ghatas MP et al., 2019), but generally did not allow for a good fit of the normal physiologic monoexponential curves. For a detailed review and justification of this methodological approach please see the aforementioned publication.

Q2. Only parametric test (T-test, linear regression and correlation) were implemented. What distribution were the data? Whether they satisfy the model assumptions?

Response: Our data were all normally distributed prior to analysis as determined using Q-Q plots with outliers removed first as determined using box plot analysis. Mean differences calculated for paired t-tests were all normally distributed as determined using Q-Q plots) [Lines 358-359]. All other paired t-test assumptions were met. Analysis of residual plots showed constant variance among our correlation analyses. All other correlation assumptions were met.

Q3. Line 323. No significant difference for age,….. Please provide the information about statistical approach implemented for these participant demographics comparison in statistical analysis section.

Response: Independent t-tests were performed. We have added this to the statistical analysis section [Lines 314-315].

________________________________________

Reviewer #2: Overview

Mitochondrial dysfunction likely contributes to the etiology of T2DM, obesity, and cardiovascular disease in those with SCI. Mitochondrial function is typically measured from permeabilized muscle fibers attained via muscle biopsy, a technique that is difficult to perform in those with SCI. Therefore, the purpose of this study was to examine the validity of two other potential methods of assessing mitochondrial function, mitochondrial respiration of peripheral blood mononuclear cells (PBMCs) and near-infrared spectroscopy (NIRS). Mitochondrial respiration was measured from permeabilized muscle fibers and PBMCs and mitochondrial capacity was measured by NIRS in 21 individuals with complete or incomplete SCI. A positive, significant correlation was found between PBMC and permeabilized muscle fibers for mitochondrial complex IV, but no relationship was found for NIRS. This study provides some evidence that PBMCs can be used to assess mitochondrial function using a minimally invasive procedure in those with SCI.

Specific comments

Q1. Lines 68-70 How does SCI result in mitochondrial dysfunction?

Response: Exploring the effects of chronic SCI on mitochondrial function is essentially the question we are trying to uncover through our current work. We have updated these lines in order to clarify that mitochondrial dysfunction may be related with having chronic SCI [Lines 68-70]. We have recently published two reviews [Gorgey A, 2018 (Ref# 70) and Goldsmith et al., 2021 (PMID:33891156)] that highlighted how SCI impacts mitochondrial health. Based on the initial observation (O’Brien et al. 2017 (Ref#44); Sumrell et al. 2018 (PMID: 30169541), we have noticed that increasing visceral adipose tissue (VAT) is associated with mitochondrial dysfunction. We further noticed that increasing VAT is accompanied with increasing biomarkers of inflammation (TNF-alpha). We have then hypothesized that increasing VAT may have lead to increasing circulating inflammation that causes mitochondrial dysfunction (Goldsmith et al. currently under review). Additionally, we cannot rule out the effects of ROS on mitochondrial function and health; which may be mediated by increasing systematic inflammation.

Q2. Line 71 It is easy to see why the measurement of mitochondrial function is important to measure to assess patient prognosis, but it would be good to know more about its relevance during initial diagnosis shortly following injury. How would the assessment of mitochondrial function in those with SCI be used by clinicians to treat their patients more effectively?

Response: Mitochondrial evaluation has been implemented in many neurologic and cardiovascular conditions. Evaluating mitochondrial function may provide integrative measurements of oxidative capacity which include physiological temperatures, endogenous oxygen delivery systems, and preservation of the mitochondrial reticulum (Willingham et al 2017 Ref#15). The lower cost and portability of NIRS makes the methodology more readily available to researchers and healthcare professionals and increases the potential for integration into clinical practice. These tools may ultimately conveniently guide clinicians to develop and implement strategies to enhance mitochondrial oxidation capacities similar to electrically evoked resistance exercise or delivering pharmaceutical intervention (i.e. testosterone, anti-oxidant medications or MitoQ). Obtaining a better understanding of the cellular response of skeletal muscles in chronic SCI may help clinicians better formulate a therapeutic and/or rehabilitative regimen for improving the long-term health and quality of life for individuals with SCI (Gorgey et al, 2021 Ref #13, Gorgey et al., 2020 Ref#16). We’ve added a few lines in the introduction, discussion, and conclusion to address this point [Lines 83-90, 530-538, 607-610]. Lines 530-538 in the discussion section, suggests that mitochondrial health data in potentially reducing long-term systemic complications.

Q3. Lines 76-83 It is difficult to follow the relevance of the previous research results mentioned here. It is easy to understand that mitochondrial function decreases with age, but this occurs in those without SCI as well. The effects of mitochondrial function on substrate use are certainly applicable here, but there is a plethora of evidence that heavy reliance on carbohydrate occurs in those with SCI during voluntary exercise, a finding with that is more generalizable than acute electrical stimulation. In this section, it would be good to know more precisely how mitochondrial dysfunction in those with SCI leads to an increased risk for T2DM, obesity and cardiovascular disease

Response: The reviewer has brought several excellent points. Persons with SCI are considered an excellent model of premature aging. We have noticed in our earlier work that following the age of 40; there is a dysfunction in both mitochondrial density and activity. Dysfunction in mitochondrial function will lead to decrease fatty acid oxidation and will result in both accumulation of intra and inter-muscular adipose tissue that are likely to cause insulin resistance and then development of type II diabetes.

We totally agree with the reviewer that persons with SCI relies primarily on carbohydrates and upper extremity voluntary exercise favors carbohydrate utilization. Even with acute bout of NMES in the no-trained muscle, we have noticed reliance on carbohydrate utilization that may highlight mitochondrial dysfunction (Gorgey and Lawrence, 2016 (Ref#10)).

However, when we performed 12 weeks of resistance training, we have noticed that increase in fat utilization at low-intensity FES cycling compared to carbohydrate utilization; suggesting enhancement of mitochondrial machinery at low-intensity exercise in persons with SCI (Gorgey et al. 2021 (Ref# 13)).

Please refer to our review (Gorgey A et al. 2018; EJAP (Ref #70)) that discussed all these important factors in detail.

Q4. Line 244 When were the NIRS measurements taken relative to the other measures and were subjects tested in an overnight fasted state?

Response: Subjects underwent an overnight fast and NIRS testing was conducted 3-4 days after the muscle biopsy procedure. For example, subjects typically came in on Thursday for the biopsy and NIRS testing was performed on either Monday or Tuesday morning. We have added this information in lines 163-170.

Q5. Line 406 “Data” is plural. Thus, follow it with “were” rather than “was” here and throughout.

Response: Thank you for this suggested correction. We have updated this throughout the manuscript.

Q6. Line 419 While the authors adequately explain the likely mechanisms for their primary findings, they do little to address the applicability of the data. Under what context might a clinician use a measurement of mitochondrial respiration of PBMCs to alter the treatment of their patients? It is interesting that a minimally invasive technique may adequately estimate mitochondrial function in those with SCI, but these results have little meaning if the reader cannot see how they would actually be applied to improve the lives of those with SCI.

Response: Thank you for your comments. Please see response from Question 2.

Submitted filename: Rebuttal Letter_13DEC2021 R1.docx

Click here for additional data file.

24 Feb 2022

Assessment of mitochondrial respiratory capacity using minimally invasive and noninvasive techniques in persons with spinal cord injury

PONE-D-21-26520R1

Dear Dr. Gorgey,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Todd A. Astorino, Ph.D FACSM

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I thank you Dr. Gorgey for an effective and substantive rebuttal to my comments as well as those of the Reviewers. The paper was improved and in its current form, is of sufficient quality to merit publication in this Journal. Best of luck in your future work and take care.

Reviewers' comments:

2 Mar 2022

PONE-D-21-26520R1

Assessment of mitochondrial respiratory capacity using minimally invasive and noninvasive techniques in persons with spinal cord injury

Dear Dr. Gorgey:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Todd A. Astorino

Academic Editor

PLOS ONE