Idiopathic inflammatory myopathy human derived cells retain their ability to increase mitochondrial function.

Idiopathic Inflammatory Myopathies (IIMs) have been studied within the framework of autoimmune diseases where skeletal muscle appears to have a passive role in the illness. However, persiting weakness even after resolving inflammation raises questions about the role that skeletal muscle plays by itself in these diseases. "Non-immune mediated" hypotheses have arisen to consider inner skeletal muscle cell processes as trigger factors in the clinical manifestations of IIMs. Alterations in oxidative phosphorylation, ATP production, calcium handling, autophagy, endoplasmic reticulum stress, among others, have been proposed as alternative cellular pathophysiological mechanisms. In this study, we used skeletal muscle-derived cells, from healthy controls and IIM patients to determine mitochondrial function and mitochondrial ability to adapt to a metabolic stress when deprived of glucose. We hypothesized that mitochondria would be dysfunctional in IIM samples, which was partially true in normal glucose rich growing medium as determined by oxygen consumption rate. However, in the glucose-free and galactose supplemented condition, a medium that forced mitochondria to function, IIM cells increased their respiration, reaching values matching normal derived cells. Unexpectedly, cell death significantly increased in IIM cells under this condition. Our findings show that mitochondria in IIM is functional and the decrease respiration observed is part of an adaptative response to improve survival. The increased metabolic function obtained after forcing IIM cells to rely on mitochondrial synthesized ATP is detrimental to the cell's viability. Thus, therapeutic interventions that activate mitochondria, could be detrimental in IIM cell physiology, and must be avoided in patients with IIM.

1. Introduction

Idiopathic Inflammatory Myopathies (IIMs) are the most frequent acquired myopathies observed in clinical practice [1] and represent a heterogeneous group of chronic, subacute, or acute acquired muscle disorders of unknown etiology. They share the common feature of muscle inflammation which leads to generalized weakness. In addition, other organs are frequently involved in IIMs (e.g. skin, joints, lungs, gastrointestinal tract, heart, etc.) and these conditions greatly contribute to morbidity and mortality.

According to the recent classification criteria that consider clinical, histopathological and myositis specific antibodies, IIMs may be subdivided in at least five main subtypes: dermatomyositis (DM), polymyositis (PM), immune- mediated necrotizing myopathy (IMNM), overlap myositis (OM), and sporadic inclusion body myositis (sIBM) [1].

Despite the presence of inflammation as a common feature in IIMs, the underlying pathophysiological mechanisms that determine the wide range of manifestations in IIMs are yet to be fully understood. It is generally hypothesized that the obvious consequence of inflammation must be myofiber damage. However, a growing body of evidence supports the view that other mechanisms might also be involved in the pathogenesis of IIM. This evidence has led some to argue that the cause of muscle dysfunction is far more complex than just immune- mediated inflammation [2]. In fact, a lack of correlation between inflammation and skeletal muscle weakness has been reported [3], suggesting that different intracellular processes can also play a role in IIMs. Furthermore, not all patients have a positive response to immunosuppression, yielding a miscorrelation between inflammation and clinical response [2]. As a result, several non-immune-mediated alterations have been proposed to participate in the pathogenesis of PM and DM muscle cells such as disturbances in oxidative phosphorylation (OXPHOS), impairment of ATP production, altered calcium handling, autophagy, and the unfolded protein response [2-5]. Histological and histochemical abnormalities in muscle biopsies from both DM and PM patients have suggested mitochondrial damage at different levels [6, 7]. An abnormal activity of respiratory complexes I, II, III, IV and the citrate synthase has been shown in muscle homogenates from PM patients [8]. In addition, dysfunction of cytochrome c oxidase (COXc) and succinate dehydrogenase (SDH) has also been described in biopsies from IIM patients [6, 7]. In vivo measurements of muscle metabolism using phosphorus magnetic resonance spectroscopy (31P-MRS) showed decreased levels of phospho-creatine (PCr), ATP and elevated inorganic phosphate (Pi)/PCr ratios during rest, exercise, and recovery. Altered resting values and slowed recovery of high energy phosphates after exercise was interpreted as abnormal mitochondrial function [9, 10]. In fact, impaired mitochondrial respiration rates in DM and PM were described by Cea et al (2002), Newman et al. (1992) [11, 12] and Pfleiderer (2004) [13] by using this imaging technique. Interestingly, Cea and Pfleiderer proposed an impaired blood supply as the main cause for the diminished oxidative metabolism observed in DM and PM patients rather than primary mitochondrial abnormalities. The marked muscle atrophy and impaired muscle function seen in DM and PM patients, even after treatment, have also been associated with a hypothetical metabolic dysfunction [14].

Drawing on current evidence, one could infer that an altered mitochondrial profile in IIMs should be expected. However, to the best of our knowledge, no real time measurement toward determining mitochondrial function in these patients seems to be available. Here, mitochondrial function, metabolic flexibility and its potential role in determining IIM cell viability were studied. We hypothesized that mitochondria from IIM-derived cells would show a diminished oxygen consumption rate (OCR), as well as diminished ATP production. We expected that a metabolic challenge imposed by a change in the carbon source in the growth medium would increase mitochondrial function, with a concomitant improvement in cellular fitness. In fact, OCR increased in IIM derived cells reaching values similar as those observed in control cells, showing a preserved metabolic flexibility. Although, on the contrary of what was expected, the ability of IIM cells to adapt to this metabolic stress did not result in increased cellular fitness and endurance to stress.

2. Materials and methods

2.1 Reagents

All reagents were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Stock solutions of all compounds were prepared in dimethyl sulfoxide (DMSO). Collagenase type 1 was obtained from Worthington (Lakewood, NJ, USA). All antibodies were obtained from Dako (Glostrup, Denmark).

2.2 Muscle biopsy samples and pathological confirmation

Deltoid muscle biopsy specimens from five patients clinically diagnosed with IIM were taken for histological and immunohistological examination (Table 1). Controls were obtained from four age-matched patients who underwent shoulder surgery. This study was undertaken with ethical approval from the “Research Ethics Committee at the Hospital Clínico Universidad de Chile” and is in compliance with the provisions of the Declaration of Helsinki. All patients gave their written informed consent before the surgical procedure for obtaining the muscle biopsy sample.

Table 1

Patient clinical data.

Patient	Age	Gender	Weakness evolution	Total Creatine Kinase (IU/L)	Corticoid use before biopsy	Diagnosis
101	59	M	6 months	2079	No	DM
103	63	M	3 weeks	3969	2 days	DM
104	70	F	6 months	8799	2 days	IMNM
105	70	M	1 month	2395	No	IMNM
109	63	M	12 months	6331	No	IMNM/DM

The confirmation for Idiopathic Inflammatory Myopathy (IIM) was accomplished based on the regular analysis of the biopsy sample (see Materials and Methods). M = male; F = female; IU/L = International Units/liter; DM = dermatomyositis; IMNM = immune- mediated necrotizing myopathy.

2.3 Ethics statement

This study was undertaken with ethical approval from the “Research Ethics Committee at the Hospital Clínico Universidad de Chile” and is in compliance with the provisions of the Declaration of Helsinki. All patients gave their written informed consent before the surgical procedure for obtaining the muscle biopsy sample.

2.4 Patient consent

Written, informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

After excision, muscle samples were immediately frozen in isopentane previously cooled in liquid nitrogen. Samples were stored in -80°C until biopsy processing. We studied 10 μm tissue sections employing the following techniques: hematoxylin eosin, Gomori trichome, PAS, Oil Red O, NADH, SDH, COX, ATPase 9.4, 4.6, and 4.3. Antibodies were used in the following dilutions: HLA I (1:2000), HLA II (1:500), C5b9 (1:50), and CD68 (1:50).

2.5 Cell cultures

Human primary culture cells were isolated from fresh biopsy samples obtained from the deltoid muscle of IIM patients and age-matched controls, following the guidelines of the research ethics committee at the Hospital Clínico Universidad de Chile. After extracting a biopsy, muscle tissue was immediately placed in standard culture medium (high glucose DMEM + F12; 10% fetal bovine serum). Then, skeletal muscle tissue was mechanically disaggregated and subjected to collagenase treatment for 30 min (0.2% Collagenase in PBS), under gentle agitation. The suspension was spun down at 2500 RPM for 10 min. The resulting pellet was washed with 4 mL of PBS and the supernatant was centrifuged again (2500 RPM for 10 min). Finally, the cellular pellet that included myoblasts and myofibroblasts was plated at 30% - 50% confluence and grown until reaching 80% confluence. Control- and IIM-derived cells were grown in standard culture medium: Dulbecco´s modified Eagle´s medium (DMEM), containing 25 mM glucose and 4 mM glutamine supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 μg/mL).

2.6 Cell culture conditions for metabolic stress

For the generation of cellular subpopulations with different metabolic phenotypes, a fraction of control and IIM-derived cells were maintained in standard culture media (25 mM glucose and 4 mM glutamine). Other fraction of cells was grown in media in which glucose was replaced by 10 mM galactose. In both conditions, cells were maintained in a humidified atmosphere at 37°C and 5% CO2.

2.7 Real-time metabolic analysis

For oxygen consumption measurements, control and IIM-derived cells were studied with an Extracellular Flux Analyzer (Seahorse XFe96; Agilent Technologies™, CA, USA). Briefly, Control and IIM–derived cells were trypsinized and pre-plated for 30 min to enriched cell suspension with myoblasts. Then, 20.000 cells were seeded in each well and the experiment was performed after 24 hrs. (growing conditions: 37°C in 5% CO2). One hour before running the experiment, culture medium was replaced with "assay medium" (DMEM, 1 mM glutaMAX®, 10 mM glucose, pH 7.4). Three points were measured to establish the baseline of the oxygen consumption rate (OCR), and after the sequential injection of oligomycin [1μM], FCCP [0.1 μM] and rotenone+antimycin A [1 μM each]. This protocol allowed us to reveal basal, maximal, ATP-coupled and proton leak-linked respiration. All data were normalized by cell number.

2.8 Intracellular ATP determination

ATP levels were determined with CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, USA) according to the manufacturer's specifications. Control and IIM-derived cells (1x105 cells/mL) were seeded into 96-well plates and grown overnight. To determine the contribution of OXPHOS on total ATP levels, cells were incubated for two hours with 2 μM oligomycin. After exposure, the cells were washed twice with cold-PBS to remove the culture medium and re-suspended in 20 μL PBS.

2.9 Mitochondrial membrane potential (ΔΨm) determination

ΔΨm in Control and IIM-derived cells was determined by flow cytometry using the potentiometric probe tetramethylrhodamine methyl ester (TMRM, Molecular Probe) in non-quenching mode. Cells (1.5 x 105 cells/mL) were treated with Dimethyl sulfoxide (DMSO) or FCCP (0.5 and 1 μM), which was used as positive control, for 30 min. Then, cells were incubated with 5 nM TMRM for 20 min, washed with cold-PBS, collected, re-suspended and fluorescence measured using a FACS Calibur flow cytometer.

2.10 Determination of intracellular Reactive Oxygen Species (ROS) measurements

The generation of intracellular oxidative stress was determined using the dihydroethidium (DHE) probe. Control- and IIM-derived cells were grown in GLU and GAL media, seeded in 12-well plates and allowed overnight to attach. Then, culture media was replaced by a solution containing 5 μM DHE in HBSS and incubated for 20 min in the dark. Afterward, cells were washed, trypsinized, and resuspended in 200 μL of HBSS and measured by FACS Calibur flow cytometer.

2.11 Cell viability assay

Cell viability was evaluated using the ability of live cells to exclude propidium iodide (PI). Control- and IIM-derived cells were submitted to different challenges in 24-well plates treated with PI, collected, washed and run in a FACS Calibur flow cytometer (Becton-Dickinson, San Jose, CA) for quantification of PI incorporation. For “redox stress” conditions, cells were pre-incubated with hydrogen peroxide (H2O2) as an oxidant stimulus [100 uM], 48 hours before reading the experiment.

For comparison, we show the "Δ death cell %", that corresponds to the difference in death percentage in the "experimental medium" (galactose, GAL), minus the percentage of death in "regular medium" (glucose, GLU): Δ = % death in galactose medium—% death glucose medium. In this condition, positive values for Δ death cell % represent cells more prone to die in GAL than in GLU media, whereas negative values represent cells more likely to survive in GAL media than in GLU media.

2.12 Determination of mitochondrial mass

Control and IIM myoblasts were loaded with the cardiolipin-binding probe 10‐N-nonyl acridine orange (NAO). Briefly, 1.5 x 105 cells/mL were incubated with NAO probe, at a concentration of 0.1 μM, for 15 minutes. After that, cells were washed with PBS, collected, re-suspended and fluorescence was measured by flow cytometry (FACS Calibur).

2.13 Statistics

All experiments were performed at least three independent times. Values were expressed as mean ± SEM. For statistical analysis, Mann-Whitney and Wilcoxon tests were performed, as well as two-way ANOVA tests with Bonferroni post-test to determine significance. Significance level was set at p < 0.05 value. For statistical analysis, GraphPad Prism 6 was used.

3. Results

3.1 Control and IIM-derived cell bioenergetic characterization, in high glucose media

Myotubes and myoblasts are traditionally cultured in high glucose growing media (see Materials and Methods), and our first attempts to bioenergetically characterize control and IIM-derived cells were conducted in this scenario. Real time oxygen consumption measurements, as indicators of mitochondrial respiration, revealed that in IIM-derived cells, mitochondria exhibit a significantly lower basal OCR compared to normal cells (Fig 1A and 1B). Sequential injections of Oligomycin, Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) and Rotenone + Antimycin A, revealed that ATP-linked respiration followed the same trend (Fig 1B), with an OCR significantly lower in IIM-derived cells compared with control cells (p<0.05). Interestingly, the proton leak OCR, that represents the oxygen consumption not associated with ATP generation, showed a tendency to be higher in IIM condition (Fig 1B). Consistently, the Respiratory Control Ratio (RCR), which represents the mitochondrial coupling state, was significantly lower in IIM-derived cells (p <0.05) (Fig 1), suggesting an uncoupled OXPHOS. Finally, the non-mitochondrial OCR, showed no differences (Fig 1B). This last parameter showed unexpected high values in both Control and IIM conditions which may reflect the presence of a higher cellular metabolism in myoblasts.

Fig 1

IIM-derived cells show diminished oxygen consumption rate, high mitochondrial membrane potential but similar total ATP levels than control-derived cells.

(A) Representative OCR profile plot in control and IIM-derived cells. (B) Oxygen consumption rate (OCR) in control and IIM-derived cells from biopsy samples showed a significant decrease of ATP-linked OCR and a marked tendency to decrease basal and maximal OCR when compared to controls. Proton leak and non-mitochondrial (non-mito) OCR exhibited no changes. (C) Respiratory Control Ratio (RCR) was significantly lower in IIM derived cells than in control. (D) Total ATP levels measured in control and IIM-derived cells showed no differences in basal conditions (DMSO) or after treatment with oligomycin (Oligo). (F) Mitochondrial membrane potential (ΔΨm) determined by TMRM in non-quenching mode showed that IIM-derived cells have a higher ΔΨm compared with control derived cells. Original FACS traces showing ΔΨm measure in (F) Control and (G) IIM-derived cells treated with FCCP (0.5 and 1 μM) exhibiting the expected ΔΨm depolarization. Data shown represent the mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p< 0.001, n.s. not significant.

To complement the bioenergetic analysis, measurements of total intracellular ATP levels and ΔΨm were performed. Both control and IIM-derived cells showed similar levels of total basal ATP, as well as a minimal drop on ATP levels after ATP synthase inhibition with oligomycin (Fig 1D). This result suggests that in standard, high glucose culture conditions, both control and IIM-derived cells rely mostly on glycolysis to synthesize their ATP, as oligomycin, an inhibitor of mitochondrial ATP synthase did not decrease ATP levels as expected. Regarding the ΔΨm, an increased TMRM incorporation was observed in IIM-derived cells compared with controls (p<0.001) (Fig 1E). This finding was indicative of mitochondrial hyperpolarization that can be dissipated by two different FCCP concentrations, as well as the normal ΔΨm in normal cells (Fig 1F and 1G).

3.2 IIM mitochondria are metabolically flexible and sensitive to oxidative stress

Generally, muscle derived cells are cultured in high glucose media which favors glycolytic metabolism over oxidative metabolism even in the presence of oxygen (“Crabtree effect”). Thus, to truly reveal mitochondrial functional state under a condition where mitochondrial function is a determinant of cell viability, we changed the carbohydrate availability using a culture medium with galactose (GAL) instead of glucose (GLU) (15). IIM- and control-derived cells were cultured during seven days in GAL-medium, where GAL metabolization by glycolysis yields no net ATP production, forcing cells to rely on mitochondria [15, 16]. Surprisingly, OCR measurement showed that IIM-derived cells were able to increase their mitochondrial function to the same level as normal-derived cells when grown in GAL-media. Basal OCR did not show differences between control and IIM-derived cells in GAL-media (Fig 2A). Conversely, ATP-linked OCR, which was significantly lower for IIM grown in GLU-media in comparison with the control, did increase to the same level as the control in GAL-media (Fig 2B).

Fig 2

A metabolic challenge reveals functional mitochondria in IIM-derived cells and a mitochondrial-dependent sensitivity to ROS-mediated cell death.

Control and IIM-derived cells were grown for seven days either in glucose (GLU) or galactose (GAL) media. Oxygen consumption rate (OCR) show that both Control and IIM-derived cells were able to; (A) increase basal and (B) ATP linked OCR when cultured in GAL. (C) Total ATP levels in control and IIM-derived cells showed negligible differences in basal conditions (DMSO) and a significant drop when treated with the ATP synthase inhibitor oligomycin (Oligo) (1 μM) for 2 h. (D) Mitochondrial mass was determined using the cardiolipin fluorescence label NAO. No differences were found between Control and IIM-derived cells after seven days in GAL media (E and F) Reactive oxygen species (ROS) levels were measured using DHE. In both growing media (GLU and GAL) IIM derived cells showed increased ROS levels when compared with control cells.

Determination of total intracellular ATP levels revealed that both IIM and control-derived cells in GAL media rely on mitochondria for ATP synthesis, since the inhibition of the ATP synthase with oligomycin significantly reduced the levels of ATP (Fig 2C). Taken together, these results suggest that mitochondria retain the potential to reach full functionality in IIM cells independent of the growing culture medium, and that the decrease observed in mitochondrial function in GLU-media may correspond to an adaptive response undergoing the aforementioned “Crabtree effect”. Nonylacridine orange (NAO) staining show no differences between control and IIM cells (Fig 2D) suggesting no differences in mitochondrial mass. To strengthen this point, we labeled biopsy samples from control and IIM patients with an antibody against the outer mitochondrial protein VDAC and we determined its expression by quantifying the number of pixels per area in confocal microscope images. As shown in the S1A Fig, no difference in the distribution nor in the expression were found between normal and IIM samples. In addition, using an antibody cocktail we determined the expression of the electron transport chain complexes by Western blot in biopsy samples from control and IIM patients. As shown in S1B Fig, no changes in the expression of complex I, III, IV and V were observed between control and IIM samples. Due to technical reasons complex II was not identified in any of our samples. These results strengthen the idea that no difference in mitochondrial mass exists between normal and IIM cells. More experiments are necessary to confirm this point.

Over-functioning mitochondria are a potential source of ROS, which are a known mediator of skeletal muscle cell damage [17, 18]. Thus, we decided to explore whether IIM-derived cells generate more ROS levels in basal conditions. Compared with control cells, IIM-derived cells showed elevated ROS levels in both conditions, GLU- and GAL-media (Fig 2E and 2F).

Finally, we decided to test IIM-cell sensitivity to death under an additional oxidative challenge. To this end, we determined cell viability in control and IIM-derived cells grown in GLU and GAL-media, challenged with hydrogen peroxide (H2O2) used as an oxidant stimulus. As shown in Fig 3A, IIM-derived cells reach the highest death values in GLU and GAL culture media, both in basal and the “challenged” condition (H2O2). For comparison, we decided to show the percentage delta of cells that died in GAL minus GLU medium. This result corresponds to the “percentage of death in the GAL-media” minus the “percentage of death in GLU-media” (see Materials and Methods). As shown in Fig 3B, the metabolic stress imposed by the change from GLU to GAL-media displays no perceptible difference between control and IIM cells. Interestingly, when a redox stress (100 μM H2O2 for 48 h) was applied, IIM-derived cells displayed an increase in the delta percentage of cell death, which suggests that derived the activity of mitochondria renders IIM-derived cells more prone to oxidative damage. In contrast, control-derived cells showed negative values which in turn suggests that fewer cells die in GAL-media when mitochondria are working to supply the majority of ATP.

Fig 3

IIM-derived cells are more susceptible to metabolic stress showing increased ROS-mediated cell death.

Control and Idiopathic Inflammatory Myopathy (IIM)-derived cells were grown for seven days either in glucose (GLU) or galactose (GAL) media. Cell viability was measured with and without an acute redox stress induced by 100 μM H2O2. (A) IIM-derived cells reach the highest death values in every one of the four different conditions analyzed. The mean values for cell death also showed a tendency to be higher for the IIM condition, mainly in GLU media. (B) The delta of cell death (i.e. Δ = % death in galactose medium—% death glucose medium) was not significant for control and IIM-derived cells under “metabolic stress only”. After an acute redox stress, the delta of cell death was significantly higher in IIM-derived cells than in controls. Data shown represent: (A) minimum, maximum and mean values of three independent experiments, (B) the mean ± SEM of three independent experiments: *p<0.05, **p<0.01, ***p<0.001, n.s. not significant.

Taken together, our results indicate that: (1) in IIM, mitochondria retain their ability to increase oxidative function in order to meet cellular ATP demands and (2) the increase in oxidative mitochondrial function may have a detrimental effects on IIM cell viability.

4. Discussion

It has been proposed that in IIM, the inflammatory local response is not the only factor responsible for the pathophysiology of this illness [2]. There is evidence that points to mitochondrial malfunction also being responsible, proposing that mitochondria are more likely to produce high levels of ROS and a decrease in ATP synthesis capacity [2–12, 18]. In this work, we show for the first time that in human derived IIM myoblasts, mitochondria are not dysfunctional and retain their ability to adapt and respond to environmental signals, at a cost that renders cells more prone to death after a specific, oxidative, insult.

IIM-derived cells grown in a high glucose medium exhibited a lower OCR profile and ΔΨm hyperpolarization compared to control-derived cells. These results were not followed by decreases in ATP levels (Fig 1), probably due to the “Crabtree effect” that maintains adequate ATP levels in both types of cells under these culture conditions. The Crabtree effect is characterized by the inhibition of respiration by high concentrations of glucose or fructose. In this condition, ATP is generated primarily by glycolysis and not by mitochondria. Fig 2 shows that when we “forced” mitochondria to work by substituting galactose for glucose in the media, both control and IIM-derived mitochondria were able to increase their respiration rates. We interpret this result as a demonstration of the IIM cell ability to adapt successfully to a metabolic stress condition. Nonyl acridine orange (NAO) labeling of cells in culture, in addition to the labeling of the outer mitochondrial protein VDAC and Western blot of the electron transport chain complexes I, III, IV and V on biopsy samples from control and IIM patients, suggests that no changes in mitochondrial mass between normal and IIM samples exist. NAO binds cardiolipin, which is highly concentrated in the mitochondria independent of ΔΨm [19], however under certain circumstances it accumulates in the mitochondria in a ΔΨm-dependent fashion [20, 21]. To confirm that the mitochondrial mass is similar between control and IIM samples, further experiments measuring the mitochondrial/nuclear DNA ratio are necessary.

Interestingly, IIM-derived cells showed a tendency to die in higher proportions than controls as a result of this metabolic stress. However, we should keep in mind that this trend only reached significance after adding a redox stimulus (Fig 3). This observation suggests that higher mitochondrial metabolic rates in IIM-derived cells increase their susceptibility to die. Mechanistically, it has been proposed that activation of endoplasmic reticulum stress in IIM leads to ROS production. Such increased ROS production would occur through increased calcium transfer to mitochondria [18], as well as an increase in the expression levels of nitric oxide synthase (NOS). As known, NOS is an enzyme that is highly related to increased ROS generation and necrosis [22]. Elevated ROS levels have been found in biopsy samples from DM patients. Likewise, higher levels of H2O2 have been described in skinned fibers [17]. In the current research, we observed an increase in ROS levels in IIM-derived cells in both GLU- and GAL-media (Fig 2E and 2F). However, to be sure about the role of ROS, further experiments using molecular and/or pharmacological intervention of the redox homeostatic system are necessary.

The potential role of “non-immune-mediated" cellular processes in the pathophysiology of IIM is a relatively new area of research. Mitochondrial alterations were considered early in the history of IIM pathophysiology; however, cellular research in this field has been insufficient until recently. Histochemical evidence performed mainly on DM and PM muscle tissue suggested mitochondrial abnormalities as a mechanism that acts on the pathophysiology of IIM [6-8]. Our observations in this study added to the earlier idea of damaged mitochondria in IIM. More precisely, our findings seemed to reveal that in IIM-derived cells, mitochondria is not dysfunctional and can still respond to metabolic stress (which in this particular case was the replacement of GLU by GAL) by increasing their respiration (Fig 2). This feature suggests that IIM-derived cells can still sense and transduce the availability of nutrients and energy to adapt to environmental changes. Previously, Robinson et al. (1992) took advantage of this kind of stress by testing the ability of patient’s fibroblasts to survive a challenge, which consists of growing cells in galactose medium as the main source of carbon compounds [23]. They postulated this strategy as a test procedure to detect certain types of oxidative defects. Namely, they observed that cells with severe oxidative defects experienced increased susceptibility to death when cultured in galactose media as their sole source of carbohydrates. Similarly, in our research we tested that notion after allowing an adaptation time frame, studying whether IIM cells were more susceptible to dying in GLU or GAL media. As seen in Fig 3, IIM cells tended to die in higher proportions than controls as a result of metabolic stress. This trend became even more pronounced once a redox stress (H2O2) was applied. The IIM cell death rate was significantly increased for cells cultured in GAL media. We interpret this finding, which we believe to be one of the main contributions, as an increased susceptibility of IIM cells to perish when mitochondria were forced to increase their oxygen consumption. The aforementioned approach has also been used to test whether different toxic compounds can affect mitochondria in cultured cells. Dott et al. [24] demonstrated that mitochondria from L6 myoblasts cultured in galactose media were more susceptible to classic mitochondrial toxins. Furthermore, they demonstrated slower proliferation and increased OXPHOS capacity for L6 cells cultured in galactose media compared to cells cultured in glucose.

For the purpose of the present research, testing the mechanisms underlying the higher cell death rates of IIM cells grown in GAL medium was beyond our scope. Two recent studies that focused on IIM are worth mentioning. Both studies pointed to increased ROS levels as significant contributors to the pathophysiology of the disease. Increased amounts of ROS (measured as DHE) in biopsy samples from DM patients, as well as higher H2O2 production in skinned fibers were described by Meyer et al. [17]. These researchers postulated a role for ROS in the pathophysiology of the disease, suggesting a direct damage to mitochondria, inducing their malfunction. In the same study, an experimental mouse model with autoimmune myositis confirmed increased ROS levels in quadriceps and gastrocnemius muscles. Supplementation of NAC (an antioxidant) to mice resulted in reduced ROS levels and preserved grip strength. A reduction in mitochondrial respiration was also observed in permeabilized fibers [17]. Lightfoot et al. [18] also proposed ROS as an important mediator in IIM pathophysiology. The review hypothesized about the mechanisms that could explain the increased ROS in IIM cells: they suggested that endoplasmic reticulum stress had the potential to increase calcium influx into mitochondria, thus increasing mitochondrial ROS production. Although our results also revealed increased ROS levels in IIM-derived cells (Fig 2E and 2F), we did not investigate ROS differences between GLU and GAL media. We recognize that in the absence of such a measure, we cannot hypothesize the existence of a possible relationship between the increase in ROS levels and the increase in the mortality rate in GAL media.

Our observations, made for the first time in primary human skeletal muscle cells, showed that mitochondria from IIM patients were able to exhibit plasticity, showing the capacity to increase their functional status when necessary (i.e. the replacement of glucose by galactose), as determined by oxygen consumption measurements. This “mitochondrial flexibility” seemed to suggest that this organelle is still capable of detecting and transducing environmental signals, in order to maintain the relation between mitochondrial function and cellular signaling. Nonetheless, the fact that higher death rates were observed for IIM cells suggested that the processes required for adaptation might ultimately be detrimental to the survival of cells. This finding shed light on the role that mitochondria might play in the weakness and atrophy seen in these patients.

In summary, our evidence suggests that boosting mitochondrial function in IIM, as is done in other muscle-related diseases, could have a detrimental effect on skeletal muscle health. Future research should focus on unraveling the balance between ROS generation and ROS scavenging to develop new therapeutic strategies for this complex group of diseases.

5. Conclusion

Mitochondria of IIM-derived cells are functional and can adapt and respond to a metabolic challenge. However, a concomitant effect is that cells tend to become more susceptible to cellular insults that result in increased death.

Mitochondrial content is similar between control and IIM patients.

(A) Human skeletal muscle biopsies obtained from controls and IIM patients were labeled with a specific antibody against the outer membrane mitochondrial protein VDAC. Equally sized "Regions Of Interest” (ROIs) were analyzed with Image J, and the area was expressed in pixel units. No differences were observed between controls and IIM patients. Controls n = 3; Patients n = 4. (B). Mitochondrial complexes (I, III, IV and V) were analyzed by Western Blot in tissue samples from human skeletal muscle biopsies obtained from control and IIM patients. No differences were observed. Controls n = 3; IIM n = 3. Mean ± SEM.

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17 Jul 2020

PONE-D-20-20169

Idiopathic Inflammatory Myopathy Human Derivate Cells retain their ability to increase mitochondrial function

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript, “Idiopathic Inflammatory Myopathy Derivative Cells retain their ability to increase mitochondrial function” by Basualto-Alarcon et al. investigates mitochondrial function, ATP production, and ROS generation in myoblasts derived from patients suffering from some form of idiopathic inflammatory myopathy (IIM), comparing them to healthy controls. The authors demonstrate that both basal and ATP-linked oxygen consumption rates (OCR) are decreased in IIM myoblasts, which is associated with uncoupled, hyperpolarized mitochondria. Furthermore, the authors show that changing the growth conditions of IIM myoblasts from high glucose to galactose can rescue the defects in mitochondrial function; however, these myoblasts are more susceptible to cell death. The strongest aspect of this study is the translational relevance of using human myoblasts from patients actually suffering from these diseases. However, the novelty is a bit lacking due to the previously cited publications (References 15 and 16) indicating galactose improves mitochondrial function in differentiated myoblasts from diabetic patients and C2C12 mouse myoblasts, respectively. While the possible translational importance is high, there are a number of instances where the authors overstate some results based on the data provided. There is also some contradiction between how the results are interpreted and then covered in the discussion. Suggestions to improve are included below.

1) Realistically, this work should all be done in differentiated myoblasts, or at least the authors could give a comparison between undifferentiated and differentiated myoblasts. Presumably, the undifferentiated cells are much more glycolytic (as the authors cover regarding the Crabtree effect in cell culture with high glucose), and once differentiated would rely on mitochondrial respiration more. At that point, the authors might see more significant effects with galactose supplementation (similar to Ref 15) than they did with the undifferentiated cells (i.e. less death, ATP differences, ROS changes).

2) The authors argue that the myoblasts are more or less glycolytic without ever showing any measure of glycolysis. As the Seahorse was used for OCR measurements, the authors could also include ECAR graphs to show changes in glycolytic flux in the presence of glucose or galactose in control versus IIM myoblasts. The utilization of 2-DG, or even the seahorse glycolytic stress test (Glucose/Galactose-Oligomycin-2DG), coupled with the OCR data would provide a great deal of information regarding how glycolysis is being affected in these cells.

3) The results and discussion seem to disagree a bit regarding the actual functional capacity of IIM myoblast mitochondria. In the results, the argument seems to be that mitochondria in IIM cells retain their ability to function during stress but can only do so in the presence of galactose where they are forced to not rely on glycolysis. However, in the discussion, as well as the fact that the IIM myoblasts are more prone to die during metabolic/redox stress, the authors indicate that when IIM cells are forced to utilize their mitochondria, it enhances their susceptibility to cell death. Thus, it would seem that regardless of substrate, mitochondria of IIM patients are severely compromised, which should be a more prevalent focal point of this study.

4) Along the lines of point 3, the inclusion of some indicator of mitochondrial number/mass would be useful (i.e. mtDNA to nuclear DNA ratio, citrate synthase, MitoTracker/TOMM20 staining). All of the OCR is normalized to cell number, so it would be good to show whether or not the number of mitochondria is altered in IIM myoblasts. Regarding normalization, it might be better to normalize the OCR measurements to total protein or some other better indicator of final cell number post-assay. The authors mention in the methods that 20,000 cells were plated, but the graphs are all normalized to 106 cells, how was this number determined? This should be detailed in the materials and methods section. Similar to this point, do the IIM myoblasts grow at the same rate as the normal healthy controls? Based on their decreased metabolic capacity, it would seem possible that they grow slower and thus total cell number during the assay may be lower than control. Inclusion of cell counts or MTT data might solidify this point.

5) Figure 1 – The authors should include the actual BOFA curves as well as the bar graphs for Panel A. Also, why is the non-mitochondrial oxygen consumption rate so high in both the control and IIM myoblasts? Most OCR curves in the literature usually have relatively low non-mitochondrial contribution.

6) Figure 2 – Would the fact that the IIM myoblasts produce the same amount of ROS regardless of whether or not they are grown in glucose or galactose-containing media not indicate that the ROS is coming from non-mitochondrial sources? This possibility is briefly mentioned in the discussion but may confound the interpretation that the increased metabolic capacity afforded by galactose is increasing ROS levels due to increased mitochondrial function. Perhaps some indicator of the source of ROS (i.e. changes in MitoSox fluorescence) would help tease out where ROS is generated in the different conditions. Also, did the authors measure membrane potential with TMRM in the presence of galactose? It would be interesting to see if the hyperpolarization observed in IIM cells is decreased upon the provision of galactose (since the cells are more prone to death but have rescued OCR).

7) Figure 3 – While the interpretation of viability as a percent difference between media conditions is an interesting way to represent the data, it makes it a bit difficult to ascertain how many cells are actually alive versus dead. The authors should include a standard viability graph (i.e. %death or %viable cells) for each condition, then could include the assessment of percent change based on glucose or galactose.

8) The discussion could use some editing for proper English.

Reviewer #2: The study by Basualto-Alarcon examined mitochondrial function and adaptation in skeletal muscle-derived cells from idiopathic inflammatory myopathy patients during metabolic challenges, in this study modeled through glucose deprivation. Their conclusion is well supported by the data and the limitations of the study well raised and argued in the discussion section.

Therefore I have no further comment

**********

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Reviewer #2: No

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4 Sep 2020

Regarding our work entitled “Idiopathic Inflammatory Myopathy Human Derived Cells retain their ability to increase mitochondrial function” (PONE-D-20-20169) we thank the reviewers for their comments and the opportunity to improve our manuscript. We have revised the manuscript and addressed the issues that have been raised to the best of our abilities under the difficulties imposed by the COVID-19 pandemia.

Reviewer comments:

Reviewer #1: The manuscript, “Idiopathic Inflammatory Myopathy Derivative Cells retain their ability to increase mitochondrial function” by Basualto-Alarcón et al. investigates mitochondrial function, ATP production, and ROS generation in myoblasts derived from patients suffering from some form of idiopathic inflammatory myopathy (IIM), comparing them to healthy controls. The authors demonstrate that both basal and ATP-linked oxygen consumption rates (OCR) are decreased in IIM myoblasts, which is associated with uncoupled, hyperpolarized mitochondria. Furthermore, the authors show that changing the growth conditions of IIM myoblasts from high glucose to galactose can rescue the defects in mitochondrial function; however, these myoblasts are more susceptible to cell death. The strongest aspect of this study is the translational relevance of using human myoblasts from patients actually suffering from these diseases. However, the novelty is a bit lacking due to the previously cited publications (References 15 and 16) indicating galactose improves mitochondrial function in differentiated myoblasts from diabetic patients and C2C12 mouse myoblasts, respectively. While the possible translational importance is high, there are a number of instances where the authors overstate some results based on the data provided. There is also some contradiction between how the results are interpreted and then covered in the discussion. Suggestions to improve are included below.

Response: We thank the reviewers for acknowledging the strength of our work. Certainly, it was a challenge to work with patient derived samples.

Based on feedback from reviewer #1, we found that we made the mistake of not clearly communicating our main observation, which is not that galactose improves mitochondrial function, but that, independent of the growing medium, IIM mitochondria are able to sense and adapt to environmental stress at the cost that renders IIM cells more prone to suffer oxidative damage and to die. Until today, all the publications regarding mitochondria in IIM suggest mitochondrial dysfunction, which is actually not the case.

1) Realistically, this work should all be done in differentiated myoblasts, or at least the authors could give a comparison between undifferentiated and differentiated myoblasts. Presumably, the undifferentiated cells are much more glycolytic (as the authors cover regarding the Crabtree effect in cell culture with high glucose), and once differentiated would rely on mitochondrial respiration more. At that point, the authors might see more significant effects with galactose supplementation (similar to Ref 15) than they did with the undifferentiated cells (i.e. less death, ATP differences, ROS changes).

Response: We agree with the reviewer that it would be interesting to compare myoblast and myotubes. Unfortunately, in our hands, the differentiation of normal and IIM derivative cells was extremely slow, which made it cost-inefficient and impossible for us to maintain in the long run. Thus, we decided to perform the experiments on myoblasts in the light of previous work that shows that myoblast and myotubes present similar respiration patterns (Olah et al., 2015, Suman et al., 2018, Hoffmann et al., 2018). Thus, we believe similar results will be expected in myotubes and the conclusions will remain the same. We present here some of aformentioned evidence:

● Olah et al. PLoS ONE 10(7):e0134227. 2015. (doi:10.1371/journal.pone.0134227).

● Suman et al. Human Molecular Genetics, 27:2367 – 2382. 2018

(doi:10.1093/hmg/ddy149).

● Hoffmann et al. Scientific Report 8: 737. 2018. (doi.org/10.1038/s41598-017-18658-3).

2) The authors argue that the myoblasts are more or less glycolytic without ever showing any measure of glycolysis. As the Seahorse was used for OCR measurements, the authors could also include ECAR graphs to show changes in glycolytic flux in the presence of glucose or galactose in control versus IIM myoblasts. The utilization of 2-DG, or even the seahorse glycolytic stress test (Glucose/Galactose-Oligomycin-2DG), coupled with the OCR data would provide a great deal of information regarding how glycolysis is being affected in these cells.

Response: We apologize for our writing style, which may have led to a confusion about our experiment’s interpretation. Regarding a hypothetical glycolytic derived ATP production in control and IIM cells in high glucose conditions, we proposed this as a possible explanation, in light of our results showing that oligomycin addition, didn’t diminish “total ATP levels (figure 1C)”. As reviewer #1 suggests, the glycolytic Seahorse stress test would have contributed to clarify this point, however, at the time we discarded to perform this experiment as it was not our main objective. Right now, given the current global epidemic situation, we were not allowed to go in the lab to perform the suggested experiments. Nevertheless, we had gone through the manuscript and changed some of our expressions, as well as added a sentence explaining our interpretation. These changes are in red in the new version of the manuscript.

The new sentence reads as follows (the additional wording is in red):

“This result suggests that in standard, high glucose culture conditions, both control and IIM-derived cells could rely mostly on glycolysis to synthesize their ATP, since oligomycin, an inhibitor of mitochondrial ATP synthase, did not decrease ATP levels as expected for a cell that produces its ATP through the oxidative pathway.”

The ECAR measurement done in parallel with the OCR does not show differences between Control and IIM cells, but we know that this is inconclusive and the glycolytic seahorse stress maneuver is the one that will reveal the detail of the glycolytic function in these cells. Nevertheless, we included here one typical ECAR graph obtained from our measurement.

3) The results and discussion seem to disagree a bit regarding the actual functional capacity of IIM myoblast mitochondria. In the results, the argument seems to be that mitochondria in IIM cells retain their ability to function during stress but can only do so in the presence of galactose where they are forced to not rely on glycolysis. However, in the discussion, as well as the fact that the IIM myoblasts are more prone to die during metabolic/redox stress, the authors indicate that when IIM cells are forced to utilize their mitochondria, it enhances their susceptibility to cell death. Thus, it would seem that regardless of substrate, mitochondria of IIM patients are severely compromised, which should be a more prevalent focal point of this study.

Response: We agree with the reviewer that there seems to be a disconnection between the description of the results section and the discussion regarding the status of mitochondria in IIM. The main take home message from our work is that mitochondria in IIM show less respiration not because they are dysfunctional, but because they adapt their function to improve survival. This adaptation was experimentally revealed by changing glucose for galactose. We amended the manuscript and reinforced this point throughout the discussion.

Results section:

Taken together, these results suggest that mitochondria retain the potential to reach full functionality in IIM cells independently of the growth culture medium, and that the decrease we observed in mitochondrial function in GLU-media may correspond to an adaptive response undergoing the aforementioned “Crabtree effect”.

Discussion section:

It has been proposed that in IIM, the inflammatory local response is not the only factor responsible for the pathophysiology of this illness (2). There is evidence that points to mitochondrial malfunction also being responsible, proposing that mitochondria are more likely to produce high levels of ROS and a decrease in ATP synthesis capacity (2-12, 18). In this work, we show for the first time that in human derived IIM myoblasts, mitochondria are not dysfunctional and retain their ability to adapt and respond to environmental signals, at a cost that renders cells more prone to death after a specific, oxidative, insult.

Our observations in this study corroborated and added to the earlier idea of damaged mitochondria in IIM. More precisely, our findings seemed to reveal that in IIM-derived cells, mitochondria could still respond to metabolic stress (which in this particular case was the replacement of GLU by GAL) by increasing their respiration (fig. 2). This feature suggests that IIM-derived cells can still sense and transduce the availability of nutrients and energy to adapt to environmental changes.

The IIM cell death rate was significantly increased for cells cultured in GAL media. We interpret this finding, which we believe to be one of the main contributions, as an increased susceptibility of IIM cells to perish when mitochondria were forced to increase their oxygen consumption.

Our observations, made for the first time in primary human skeletal muscle cells, showed that mitochondria from IIM patients were able to exhibit plasticity, showing the capacity to increase their functional status when necessary (i.e. the replacement of glucose by galactose), as determined by oxygen consumption measures.

4) Along the lines of point 3, the inclusion of some indicator of mitochondrial number/mass would be useful (i.e. mtDNA to nuclear DNA ratio, citrate synthase, MitoTracker/TOMM20 staining). All of the OCR is normalized to cell number, so it would be good to show whether or not the number of mitochondria is altered in IIM myoblasts.

Response: We agree with the reviewer that information regarding mitochondrial mass in control and IIM cells would add interesting data, as well as allow a better analysis of our functional experiments. We conducted different strategies directed to measure mitochondrial mass. Control and IIM myoblasts were labeled with 10-N-Nonyl acridine orange (NAO), which had been extensively used to determine mitochondrial mass thanks to its ability to bind cardiolipin (Septinus M, et al., 1985). As shown in the new figure 2D, no NAO label differences were observed between control and IIM myoblasts in galactose medium, which suggests no mitochondrial mass differences.

In addition, patient and control biopsies were labeled with the specific outer mitochondrial membrane protein, VDAC and the pixel intensity analyzed. As shown here for the reviewers, no differences were observed between control and IIM biopsies, regarding pixel intensities. This particular data is part of a different manuscript which is the reason we are not including it in this paper, but we share it.

Septinus et al., Histochemistry. 1985; 82(1):51-66)

Patients and controls did not differ for VDAC

area. Biopsies from controls and patients were

analyzed by immunofluorescence for VDAC channel. Equally sized "Regions Of Interest” (ROIs) were analyzed with Image J, and the area was expressed in pixel units.

No differences were observed for controls and patients. Controls n=3; Patients n=4.

4.1) Regarding normalization, it might be better to normalize the OCR measurements to total protein or some other better indicator of final cell number post-assay. The authors mention in the methods that 20,000 cells were plated, but the graphs are all normalized to 106 cells, how was this number determined? This should be detailed in the materials and methods section.

Response: As the reviewer points out, we seeded 20,000 cells per well for Seahorse experiments. Although, we decided to normalize our results, by a value of 106 cells, for the following reasons:

- Normalizing by 106 cells is a way to standardize the OCR measurements along with the respiration measurements obtained by other techniques of respiration, i.e. Clark-type electrode. Those experiments use a large number of cells, and are normalized by a million of cells (106 cells), thus, in an attempt to homogenize the numbers of our experimental system, we multiplied the numbers obtained from 20,000 cells by a 50-factor. In this way, our results can be easily compared with other data from other equipment.

- As different cell types may have a different optimal number for the “number of seeded cells” to measure OCR, then having a number that allows normalization, helps us to compare different cell types, under different conditions.

- Moreover, in our laboratory we have previously described the changes in mitochondrial respiration using XFe96 technology expressing the data in pmol O2/min/106 cells in previous works (for example, supplementary figure 7g, Urra et al. 2018.

Urra et al., 2018, Scientific Report; 8(1):13190.

4.2) Similar to this point, do the IIM myoblasts grow at the same rate as the normal healthy controls? Based on their decreased metabolic capacity, it would seem possible that they grow slower and thus total cell number during the assay may be lower than control. Inclusion of cell counts or MTT data might solidify this point.

Response: This is a good point. In order to avoid this bias, oxygen consumption measures were always done 12 hours after seeding the cells, which is a time frame in which neither control nor IIM cells are able to divide.

5) Figure 1 – The authors should include the actual BOFA curves as well as the bar graphs for Panel A. Also, why is the non-mitochondrial oxygen consumption rate so high in both the control and IIM myoblasts? Most OCR curves in the literature usually have relatively low non-mitochondrial contribution.

Response: We thank reviewer # 1 for his/her suggestion. We incorporated this new graph in figure 1, which now shows a representative OCR profile for control and IIM cells (fig 1A).

Regarding the non-mitochondrial OCR, it is true that the values are higher than those we usually observe in our experiments, but is not totally rare. We find in the literature a work in primary old and young skeletal muscle cells where a high non-mitochondrial OCR is observed (Pala et al., 2018). We believe this is caused by a high cellular metabolism and as it is present in both control and IIM cells we don’t expect this to change/modify the main conclusions of our work. Also, there is a small possibility that the aliquot of rotenone and antimycin A (AA) we used was not in perfect conditions, causing only a partial inhibition. Again, this will not change the main conclusions of our work.

Pala et al., 2018; Journal of Cell Science 131(14):jcs212977.

6) Figure 2 – Would the fact that the IIM myoblasts produce the same amount of ROS regardless of whether or not they are grown in glucose or galactose-containing media not indicate that the ROS is coming from non-mitochondrial sources? This possibility is briefly mentioned in the discussion but may confound the interpretation that the increased metabolic capacity afforded by galactose is increasing ROS levels due to increased mitochondrial function. Perhaps some indicator of the source of ROS (i.e. changes in MitoSox fluorescence) would help tease out where ROS is generated in the different conditions. Also, did the authors measure membrane potential with TMRM in the presence of galactose? It would be interesting to see if the hyperpolarization observed in IIM cells is decreased upon the provision of galactose (since the cells are more prone to death but have rescued OCR).

Response: This is a very interesting observation made by the reviewer. Unfortunately, our ROS measurements were done in separate time frames. This makes it impossible to compare our glucose-containing media data with our galactose containing-media data. It would be good to perform these experiments in parallel and compare, but unfortunately we cannot go to the lab due to the covid-19 situation. We recognize this as a weakness of our work and we mention this in the discussion section. Also, measuring the mitochondrial membrane potential is a great suggestion that we cannot perform right now. Nevertheless, we believe our work is still very relevant information to the scientific community.

7) Figure 3 – While the interpretation of viability as a percent difference between media conditions is an interesting way to represent the data, it makes it a bit difficult to ascertain how many cells are actually alive versus dead. The authors should include a standard viability graph (i.e. %death or %viable cells) for each condition, then could include the assessment of percent change based on glucose or galactose.

Response: We have added a new figure 3A to show the results in an easier way. We kept the original figure as figure 3B.

8) The discussion could use some editing for proper English.

Response: We are sorry for any inappropriate use of the English language. A native English speaker has looked over and corrected our work.

Reviewer #2: The study by Basualto-Alarcon examined mitochondrial function and adaptation in skeletal muscle-derived cells from idiopathic inflammatory myopathy patients during metabolic challenges, in this study modeled through glucose deprivation. Their conclusion is well supported by the data and the limitations of the study well raised and argued in the discussion section.

Therefore I have no further comment

Response: We appreciate the reviewer’s comments.

Submitted filename: Respond to reviewers comments 083020.CBA_al.docx

Click here for additional data file.

22 Sep 2020

PONE-D-20-20169R1

Idiopathic Inflammatory Myopathy Human Derived Cells retain their ability to increase mitochondrial function

PLOS ONE

Dear Dr. Cardenas

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The authors have addressed major concerns of the manuscript and have thus improved the quality of the paper, however, a few points still need to be reviewed.

1- The authors have provided data to show that mitochondrial mass is similar between both Controls and IMM using NAO probe. Using this probe, the authors have to mitigate their interpretation of the data for a few reasons which are:

-NAO accumulates in mitochondria in a potential-dependent manner. The authors having stated that mitochondria are hyperpolarized in IMM, there's a high possibility that the dye distribution is different between CTL and IMM.

-Cardiolipin are also found in peroxisomes

I suggest the authors use other indicators of mitochondrial mass such as mtdna/nuclear dna ratio in all conditions (glucose and galactose). Indeed, several studies had shown an increase in mitochondrial turnover in galactose, I believe the paper would be greatly improved to show that the observed changes are not attributed to changes in mitochondrial mass.

2-Authors suggest that forcing IMM cells to rely on mitochondrial synthesized ATP renders the cells more prone to death following acute Oxidative insult. The study would be strengthened by inhibition of ROS production, to further establish a direct role of ROS production in the IMM cells sensitivity to death, particularly in galactose medium culture.

3-Authors have shown graph of ECAR measurement which do not show differences between Control and IMM. I believe these were done in high glucose medium. It would be interesting to show these ECAR measurements in Galactose medium.

**********

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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22 Oct 2020

Responses to Reviewers’ Comments

Regarding our work entitled “Idiopathic Inflammatory Myopathy Human Derivate Cells retain their ability to increase mitochondrial function” (PONE-D-20-20169) we want to thank reviewer 1 for accepting our amended manuscript and reviewer 2 for acknowledging that the quality of our work has greatly improved and that all his major concerns have been addressed.

Reviewer comments:

Reviewer #2: The authors have addressed major concerns of the manuscript and have thus improved the quality of the paper, however, a few points still need to be reviewed.

1- The authors have provided data to show that mitochondrial mass is similar between both Controls and IMM using NAO probe. Using this probe, the authors have to mitigate their interpretation of the data for a few reasons which are:

-NAO accumulates in mitochondria in a potential-dependent manner. The authors having stated that mitochondria are hyperpolarized in IMM, there's a high possibility that the dye distribution is different between CTL and IMM.

-Cardiolipin are also found in peroxisomes

I suggest the authors use other indicators of mitochondrial mass such as mtdna/nuclear dna ratio in all conditions (glucose and galactose). Indeed, several studies had shown an increase in mitochondrial turnover in galactose, I believe the paper would be greatly improved to show that the observed changes are not attributed to changes in mitochondrial mass.

Response: We thank the reviewer for this insightful comment. We agree that NAO is not the best probe to determine mitochondrial mass. Conflicted results have been published regarding its use (REF, siy no). Related to our experiments, the fact that the measurements with NAO are similar in control and IMM cells, despite IMM cells being hyperpolarized may suggest that; 1- IMM cells have way less mitochondria but given the high mitochondrial membrane potential, accumulated so much dye that the quantification is similar to control, or 2- NAO binds mainly to cardiolipin and there is no difference in mitochondrial mass.

As mentioned by the reviewer the best methods to quantify mitochondrial mass is by performing a mitochondrial DNA/nuclear DNA ratio. This is a technique that we want to implement in our lab but unfortunately is not working at the moment. In the current situation, we have no access to patients’ samples to culture cells and do the required experiments. All the health personnel is working on covid-19 related tasks and all other projects are on hold. We have some frozen material, but the cells were not able to grow. The only material we have available are biopsy samples. We performed Western blots of the mitochondrial complexes in biopsy samples from normal and IIM patients. In agreement with the previous analysis of VDAC performed on biopsy samples from control and IIM patients, no differences in the expression of complex I, III, IV and V were found between the samples. This data has been added as supporting figure 1. Altogether, our data suggest that no difference in mitochondrial mass is observed between normal and IIM conditions, however, we understand that better experiments can be done to show this with less doubt. We made a statement regarding this in the discussion. We add the following sentences (red) to the results and the discussion section;

Results

…” Nonylacridine orange (NAO) staining show no differences between control and IIM cells (fig. 2D) suggesting no differences in mitochondrial mass. To strengthen this point, we labeled biopsy samples from control and IIM patients with an antibody against the outer mitochondrial protein VDAC and we determined its expression by quantifying the number of pixels per area in confocal microscope images. As shown in the supporting figure 1A, no difference in the distribution nor in the expression were found between normal and IIM samples. In addition, using an antibody cocktail we determined the expression of the electron transport chain complexes by Western blot in biopsy samples from control and IIM patients. As shown in supporting figure 1B, no changes in the expression of complex I, III, IV and V were observed between control and IIM samples. Due to technical reasons complex II was not identified in any of our samples. These results strengthen the idea that no difference in mitochondrial mass exists between normal and IIM cells. More experiments are necessary to confirm this point.

Discussion

…” We interpret this result as a demonstration of the IIM cell ability to adapt successfully to a metabolic stress condition. Nonylacridine orange (NAO) labeling of cells in culture, in addition to the labeling of the outer mitochondrial protein VDAC and Western blot of the electron transport chain complexes I, III, IV and V on biopsy samples from control and IIM patients, suggests that no changes in mitochondrial mass between normal and IIM samples exist. NAO binds cardiolipin, which is highly concentrated in the mitochondria independent of ΔΨm (19), however under certain circumstances it accumulates in the mitochondria in a ΔΨm-dependent fashion (20,21). To confirm that the mitochondrial mass is similar between control and IIM samples, further experiments measuring the mitochondrial/nuclear DNA ratio are necessary.

2-Authors suggest that forcing IMM cells to rely on mitochondrial synthesized ATP renders the cells more prone to death ffollowing acute Oxidative insult. The study would be strengthened by inhibition of ROS production, to further establish a direct role of ROS production in the IMM cells sensitivity to death, particularly in galactose medium culture.

Response: We agree with the reviewer. The utilization of antioxidants or other means to inhibit ROS would strengthen our conclusion. Unfortunately, we don’t have cells to perform this experiment and given the covid-19 pandemic, all the health personnel that could help us with the samples, including Dr Bozan, are now working to contain covid-19. In the discussion we have stated that experiments with antioxidants are necessary to prove the role of ROS in the cell death observed.

…” In the current research, we observed an increase in ROS levels in IIM-derived cells in both GLU- and GAL-media (fig. 2E and 2F). However, to be sure about the role of ROS, further experiments using molecular and/or pharmacological intervention of the redox homeostatic system are necessary.

3-Authors have shown graph of ECAR measurement which do not show differences between Control and IMM. I believe these were done in high glucose medium. It would be interesting to show these ECAR measurements in Galactose medium.

Response: As mentioned by the reviewer, the ECAR measurements we share with the reviewers in our first respond were indeed performed in high glucose. Here we added a representative ECAR of cells growth in galactose. As shown in the figure 1 of this respond, no differences in ECAR are observed between control and IIM cells grown in galactose medium. Further experiments using the glycolysis stress kit are necessary to get more details about glycolysis in these cells. However, the lack of differences observed in these measurements suggest that no differences will be found.

Nevertheless, the main focus of this manuscript is on the mitochondrial function and its unexpected deleterious effect on IIM cells.

We understand that we have not been able to fully respond to the observation of reviewer 2, not because we didn’t consider his/her input, but because of the catastrophe caused by covid-19, which has basically stopped all research in our country. The main substrate for our research are the samples from patients, to which we have no access now. All health personnel, including authors of this manuscript are now fighting covid-19 and non-medical personnel are not considered necessary and therefore not allowed access. The basic researchers involved in this project cannot go to the hospital. We have tried to respond to best of our ability. We also modified the discussion to reveal the weakness of our work. Nevertheless, as is, we believe our work offers new insight into a problem poorly studied and Plos One will give us the exposure we need.

Submitted filename: second Respond to reviewers comments 101520_al.docx

Click here for additional data file.

3 Nov 2020

Idiopathic Inflammatory Myopathy Human Derived Cells retain their ability to increase mitochondrial function

PONE-D-20-20169R2

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6 Nov 2020

PONE-D-20-20169R2

Idiopathic Inflammatory Myopathy Human Derived Cells retain their ability to increase mitochondrial function

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