Enzymatically dissociated muscle fibers display rapid dedifferentiation and impaired mitochondrial calcium control

Summary Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. By comparing adult mouse skeletal muscle fibers, isolated either by mechanical dissection or by collagenase-induced ECM digestion, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling. RNA-sequencing showed striking differences in gene expression patterns between the two isolation methods with enzymatically dissociated fibers resembling myopathic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria following enzymatic dissociation. Repeated contractions resulted in a prolonged mitochondrial Ca2+ accumulation in enzymatically dissociated fibers, which was partially prevented by cyclophilin inhibitors. Of importance, muscle fibers of mice with severe mitochondrial myopathy show pathognomonic mitochondrial Ca2+ accumulation during repeated contractions and this accumulation was concealed with enzymatic dissociation, making this an ambiguous method in studies of native intracellular Ca2+ fluxes.


INTRODUCTION
The intracellular secondary messenger Ca 2+ is of critical importance for a plethora of cellular functions. Within the cell, Ca 2+ levels are compartmentalized and can differ by orders of magnitude between the cytosol and organelles, such as the sarcoplasmic reticulum (SR). In this context, mitochondrial Ca 2+ levels have received increasing attention in recent years for their important roles in energy homeostasis, oxidative stress, and apoptosis. 1 2+ ] mit ) can stimulate mitochondrial respiration and hence play an integral role in the regulation of cellular metabolism, whereas prolonged and excessive uptake can activate apoptotic and necrotic cell signaling pathways. [4][5][6] Tight control of [Ca 2+ ] mit seems particularly important in skeletal and cardiac muscle cells where cytosolic free [Ca 2+ ] ([Ca 2+ ] cyt ) can reach mM concentrations, and even higher in the vicinity of SR Ca 2+ release sites. 7 Accordingly, an increase in [Ca 2+ ] mit in response to increased [Ca 2+ ] cyt has been observed during in vivo contractions of mouse skeletal muscle, and [Ca 2+ ] mit rapidly returned to the basal level after the end of contraction. 8 In mammals, each muscle fiber is under the direct control of a single branch of an a-motoneuron and hence its cellular activation occurs independently of the activity of neighboring muscle fibers. Thus, studies on isolated single muscle fibers will reflect the function of muscle fibers in vivo and are therefore highly valuable in mechanistic studies of, for instance, the intracellular signaling associated with muscle activation and contraction. Of importance, single muscle fibers are often isolated via collagenase treatment, 23,[36][37][38][39][40] which inevitably disrupts the extracellular niche and ECM-cell interactions. Here, we compare collagenase-dissociated mouse muscle fibers with fibers obtained by high-precision mechanical dissection, 41 and demonstrate that an intact microenvironment is required for maintenance of cellular structure and properly controlled mitochondrial Ca 2+ handling. Specifically, by integrating data obtained with second harmonic generation (SHG) and immune-fluorescence imaging, electron microscopy, and RNA-sequencing, we show that enzymatic fiber dissociation, but not mechanical microdissection, causes altered cellular organization and the deterioration of molecular signatures characteristic of mature muscle fibers. Furthermore, repeated tetanic contractions resulted in a marked increase of basal [Ca 2+ ] mit (i.e., measured at rest and not during an ongoing contraction) in enzymatically dissociated but not in mechanically dissected muscle fibers.

-3 A limited and transient increase in free mitochondrial matrix [Ca 2+ ] ([Ca
A pronounced contraction-induced increase in basal [Ca 2+ ] mit similar to that in enzymatically dissociated fibers has previously been observed in mechanically dissected muscle fibers from a mouse model of severe mitochondrial myopathy, the fast-twitch skeletal muscle fiber-specific mitochondrial transcription factor A knock-out (Tfam KO) mouse, and this increase was implied to have a central role in the disease progress. 42,43 In the second part of the present study we therefore asked whether an aberrant contraction-induced increase in basal [Ca 2+ ] mit in mitochondrial myopathy muscle fibers can be detected in enzymatically dissociated fibers, where basal [Ca 2+ ] mit shows a marked increase already in muscle fibers of healthy mice.

Intact links between ECM and cytoskeleton are necessary to maintain the structural integrity of skeletal muscle fibers
To examine the effects of the extracellular microenvironment on cellular phenotype, we compared mechanically dissected mouse flexor digitorum brevis (FDB) fibers with fibers isolated by conventional enzymatic dissociation using collagenase. Mechanical microdissection permits the isolation of muscle fibers with tendons attached and intact sarcolemma, 41 including the adjacent ECM scaffold with preserved focal adhesion complexes. In contrast, physiological ECM contacts are afflicted by collagenase treatment. We first examined whether the different isolation methods translated into differences in cell morphology. To ll OPEN ACCESS 2 iScience 25, 105654,  iScience Article this end, we combined multi-photon-based SHG imaging and a quantitative morphometry technique to assess possible alterations in the myofibrillar architecture induced by the collagenase treatment. 44 General SHG imaging showed collagen structures attached to mechanically dissected fibers (Video S1), whereas no extracellular collagen was detected on enzymatically dissociated fibers (Video S2). For further analyses, we filtered out the collagen-1 signal and specifically focused on myosin. SHG imaging of single mechanically dissected FDB fibers then revealed a complex myofibrillar architecture with longitudinal ridges and valleys. Enzymatically dissociated fibers, on the other hand, were more homogeneously delineated with almost circular cross-sections and shorter, more evenly extended sarcomeres ( Figures 1A and 1B; Videos S3 and S4).
To further characterize the structural difference between mechanically dissected and enzymatically dissociated fibers, we quantified the cosine angle sum (CAS) in SHG images. CAS represents the summed-up contributions of all projections from local myofibrillar directionality against the main fiber axis and is used as a measure of a fiber's myofibrillar angular alignment. [44][45][46] Notably, CAS was significantly higher (reflecting a more linear and parallel pattern) for enzymatically dissociated than for mechanically dissected fibers, further corroborating the observation of homogeneously striated patterns and parallel myofibrillar alignment in dissociated fibers ( Figure 1C).
We used histochemistry to further characterize the cellular structure and identify possible differences between mechanically dissected and enzymatically dissociated fibers. In alignment with the results from SHG imaging, phalloidin labeling of actin showed distinct longitudinal streaks, reflecting longitudinal ridges and valleys, whereas enzymatically dissociated fibers appeared more homogeneously delineated Figure 1. Disruption of the extracellular matrix results in altered cell shape (A) Representative SHG images from a mechanically dissected (Mech) and an enzymatically dissociated (Enz; using collagenase) mouse FDB fiber. Average data of (B) sarcomere length and (C) cosine angle sum (CAS) in mechanically dissected (n = 5) and enzymatically dissociated (n = 13) fibers. CAS of 1 represents a perfectly linear parallel pattern, whereas CAS = 0 reflects perpendicularly oriented structures. Gray bars = mechanically dissected fibers; black bars = enzymatically dissociated fibers. *p < 0.05 and ***p < 0.001 versus mechanically dissected fibers with unpaired t-test. Data are presented as mean G SEM. Representative images of phalloidin-labeled actin (D) and immunofluorescence staining of desmin (E) and plectin (F) in mechanically dissected and enzymatically dissociated fibers. Scale bars, 20 mm. Experiments were performed 4 h after muscle fiber isolation. iScience Article ( Figure 1D). Antibody staining of the cytoskeletal proteins desmin and plectin showed clear cross-striations in both mechanically dissected and enzymatically dissociated fibers ( Figures 1E and 1F). In agreement with the results of SHG imaging, sarcomere lengths were longer in enzymatically dissociated (1.92 G 0.02 mm, n = 57) than in mechanically dissected fibers (1.73 G 0.03 mm, n = 43; p < 0.001).
3D electron microscopy revealed an enzymatic dissociation-induced reduction in volume of individual mitochondria Next, we evaluated whether the collagenase-induced disruption of the extracellular microenvironment would propagate into alterations in mitochondrial spatial organization and ultrastructure. To this end, we analyzed fibers of both mechanically dissected and enzymatically dissociated fibers using transmission electron microscopy (TEM), and 3D mitochondrial models were constructed with focused ion beam scanning electron microscopy (FIB-SEM). 47,48 TEM analysis of ultrathin sections showed no significant difference in mitochondrial cross-sectional areas between the two groups (Figures 2A-2C). 3D mitochondrial models of similar fiber volumes ($365 mm 3 ) were constructed from a mechanically dissected and an enzymatically dissociated fiber by sequential FIB milling of 30 nm slices, ( Figures 2D-2G). The 3D images look grossly similar in the two fibers with mitochondria preferentially oriented perpendicular to the long axis of the fiber and localized adjacent to the z-discs (Videos S5 and S6), which agrees with the pattern previously observed in glycolytic muscle fibers. 49 The total mitochondrial volume was similar in the two models (20.2 versus 17.1 mm 3 , corresponding to 5.5% versus 4.6% of the total fiber volume). On the other hand, the number of mitochondria was clearly lower (86 versus 140) and their average volume larger in the mechanically dissected (0.235 mm 3 ) than in the enzymatically dissociated (0.122 mm 3 ) fiber.

Disruption of the cellular microenvironment causes a rapid deterioration of transcriptomic signatures
We used RNA-sequencing to compare the transcriptomic signatures of freshly isolated muscle fiber bundles to those of fibers isolated either by mechanical dissection or by enzymatic dissociation. Notably, we observed striking differences in gene expression patterns depending on the isolation method ( Figure 3A). Principal component analysis revealed that overall changes in expression were more rapid and extensive in dissociated fibers ( Figure 3B). Compared to freshly isolated muscle fiber bundles, 514 genes were differentially expressed in mechanically dissected fibers, whereas more than twice as many (1156 genes) were altered upon enzymatic dissociation (FDR = 0.01). Systematic comparison of expression differences between isolation methods revealed that after 4h, 3,210 genes showed significantly higher levels in mechanically dissected than in enzymatically dissociated fibers and these were enriched in complement signaling, ECM proteoglycans, ephrin signaling and rRNA processing ( Figure 3C). In contrast, only 85 genes showed higher expression in enzymatically dissociated than in mechanically dissected fibers, specifically those involved in glycogen synthesis and gluconeogenesis. Conversely, 24h after isolation, mechanically dissected fibers featured 1,203 significantly upregulated genes mostly related to ECM biogenesis and organization, whereas enzymatically dissociated fibers show upregulation of 2,323 genes significantly enriched in cell cycle, DNA replication and repair ( Figure 3D).
The expression of genes encoding for myosin heavy chain IIX (Myh1), IIB (Myh4), and I (Myh7) was substantially reduced after 4h irrespective of isolation method; after 24h, on the other hand, expression had recovered and was significantly higher in mechanically dissected than in enzymatically dissociated fibers (Figure 4A), whereas the gene encoding for myosin heavy chain IIA (Myh2) was not differentially expressed between the two groups ( Figure S1A). In enzymatically dissociated fibers, we furthermore observed a rapid and significant downregulation of genes involved in mitochondrial fusion (Mfn1, Mfn2 and Opa1), whereas no difference was observed for the fission component Dnm1l (also referred to as DRP; Figure 4B). These findings are consistent with our observations that enzymatically dissociated muscle fibers featured significantly more but smaller mitochondria and suggest that these differences are likely because of reduced mitochondrial fusion rather than increased fission.
The gene expression of critical ECM components, including the abundant muscle collagens Col1a1, Col1a2, Col3a1, and Col6a1, as well as the proteoglycan decorin (Dcn), were almost entirely lost in enzymatically dissociated fibers at both time points, whereas expression remained relatively stable in mechanically dissected fibers ( Figure 4C). Similar effects were observed for the gene expression of gelsolin (Gsn), a calcium-regulated protein involved in the assembly of actin filaments, and myocilin (Myoc), a regulator of iScience Article cytoskeletal function ( Figures 4D and 4E). Expression of the cytoskeletal scaffolding protein plectin (Plec) was also significantly downregulated in enzymatically dissociated fibers after 4h, whereas the expression was increased in both groups at 24 h ( Figure 4F).
Of interest, at 24h the expression of Ppif and Mcu were higher with enzymatic dissociation than with mechanical dissection ( Figures 4G and 4H), whereas the expression of Nclx was lower in enzymatically dissociated fibers ( Figure 4I), hence all three differences acting toward increased mitochondrial Ca 2+ accumulation in enzymatically dissociated fibers. The expression of Mcu regulators showed no iScience Article consistent pattern (Figures S1B-S1F). Taken together, these findings confirm that enzymatic dissociation of muscle fibers results in a rapid deterioration of the mature muscle phenotype, approaching signatures that resemble pathological phenotypes with decreased expression of genes encoding for critical ECM elements and ECM-cytoskeletal signal transducers, as well as an activation of regenerative pathways.    Figure 5C). In mechanically dissected iScience Article fibers, basal [Ca 2+ ] mit was only marginally increased after the repeated tetani irrespective of whether the fibers contracted isometrically or were allowed to shorten freely, which mimics the conditions for enzymatically dissociated fibers. However, a marked increase in basal [Ca 2+ ] mit after the repeated contractions were observed in fibers that were first mechanically dissected and subsequently treated with collagenase, implying that the observed effects critically depend on the collagen-containing microenvironment of the isolated fibers. Of importance, the differences in mitochondrial Ca 2+ accumulation between mechanically dissected and enzymatically dissociated fibers were not because of differences in tetanic [Ca 2+ ] cyt (Figure 5D), which indicates that defective mitochondrial Ca 2+ control rather than general alterations in cellular Ca 2+ handling underlie the aberrant elevation in basal [Ca 2+ ] mit during repeated contractions in enzymatically dissociated fibers.

The increase in basal [Ca 2+ ] mit in enzymatically dissociated fibers is mediated via MCU-and Ppif-dependent pathways
To investigate the molecular mechanisms underlying the aberrant increase in basal [Ca 2+ ] mit in enzymatically dissociated cells, we used the ruthenium red subcomponent Ru360 to inhibit MCU-mediated mitochondrial Ca 2+ entry. 50 Although Ru360 specifically inhibits MCU in experiments on isolated mitochondria, its limited plasma membrane permeability limits its use in intact cell systems. 51 Therefore, enzymatically dissociated FDB fibers were first microinjected with Ru360 and subsequently superfused with Ru360 for 30 min before commencing the repeated tetanic stimulation. Notably, Ru360 treatment significantly decreased, but did not abrogate, the tetanic stimulation-induced increase in [Ca 2+ ] mit ( Figures 6A and 6B). iScience Article Of interest, Ppif was upregulated in enzymatically dissociate fibers (see Figure 4G), and we have previously shown marked increases of PPIF/Ppif in mitochondrial myopathy patients and mice. 43 The cyclic endecapeptide calcineurin inhibitor cyclosporin A (CsA) binds to PPIF/Ppif (cyclophilin D) 52,53 and counteracts mPTP opening. 34,35,54-57 CsA has previously been shown to attenuate the increase in [Ca 2+ ] mit in mouse mitochondrial myopathy muscle fibers exposed to repeated tetanic stimulation as well as in ischemic rabbit cardiomyocytes. 42,43,58 In enzymatically dissociated fibers exposed to 25 repeated tetani, CsA significantly blunted the increase in basal [Ca 2+ ] mit ( Figures 6A and 6B), and the magnitude of reduction was similar to that observed with Ru360. Measurements of [Ca 2+ ] cyt showed no effect of CsA either at rest or during tetanic stimulation ( Figures 6C-6E).
The above-described data suggest an important role of Ppif in mitochondrial Ca 2+ control. To further investigate this possibility, we used the novel, specific cyclophilin inhibitor, NV556. 59 Figures 7C-7E). Thus, these results support a model in which the isolation of muscle fibers from their native microenvironment causes dysregulation of cellular organization and a partly Ppif-dependent Ca 2+ accumulation in the mitochondrial matrix. Enzymatic dissociation masks mitochondrial Ca 2+ handling defects in mitochondrial myopathy muscle fibers In the second part of the study, we assessed whether a contraction-induced aberrant increase in basal [Ca 2+ ] mit in mitochondrial myopathy muscle fibers will escape detection in enzymatically dissociated fibers. First, we used fibers from Tfam KO mice that display important hallmarks of severe mitochondrial myopathy. 60 In agreement with previous results, 42  iScience Article mice were alive (p = 0.015; z-test; Figure 9C). These data agree with previous results obtained with CsA treatment 43 and further support the critical role of Ppif in mitochondrial Ca 2+ control. As such, they show that pharmacological inhibition of mitochondrial Ca 2+ accumulation improves outcomes in a mouse model of lethal mitochondrial myopathy.
Intrigued by the fact that the aberrant increase in basal [Ca 2+ ] mit in Tfam KO muscle fibers eluded detection in enzymatically dissociated fibers, we performed experiments on another mouse model with defective mitochondria; that is, mice deficient of stem-loop interacting RNA binding protein (SLIRP). 61 Despite a 50-70% reduction in the steady-state levels of mtDNA-encoded mRNAs, Slirp KO mice appear largely healthy with only minor ($5%) reduction in body weight. 61 The [Ca 2+ ] cyt -frequency relationship was studied in FDB fibers mechanically dissected from Slirp KO mice and wildtype littermates by producing brief contractions at 1 min intervals. Slirp KO fibers displayed lower [Ca 2+ ] cyt than wildtype fibers at high stimulation frequencies (120-150 Hz) and during tetanic stimulation in the presence of caffeine (5 mM), which facilitates SR Ca 2+ release and hence provides an estimate of the total amount of Ca 2+ stored in the SR ( Figure 9D). 62 These results indicate a reduced SR Ca 2+ storage capacity in Slirp KO muscle, which agrees with previous results obtained in Tfam KO fibers. 42,43 Thus, a decreased SR Ca 2+ storage capacity, which has been attributed to a decreased concentration of the SR Ca 2+ buffering protein calsequestrin 1, 42 is a common feature in muscle fibers of mice with two completely different genetically engineered mitochondrial defects.

DISCUSSION
In vitro cell studies constitute essential tools for phenotype characterization, as well as for drug development. However, an increasing body of evidence highlights the fact that isolating cells by enzymatic dissociation and subsequently studying them for several days entails the rapid loss of adult cellular phenotypes, iScience Article which confounds result interpretation and impairs the translation of findings. 63 Enzymatic dissociation of cells disturbs integrin-mediated cell adhesion, which provide dynamic connections between the ECM and the intracellular cytoskeleton that are essential for the control of cell structure, including the morphology and function of mitochondria. [19][20][21] In this study, we provide a link between enzymatic disruption of the ECM, altered cell structure, and defective control of basal [Ca 2+ ] mit by comparing enzymatically dissociated muscle fibers with muscle fibers isolated by mechanical dissection, which leaves the immediate ECM intact.
Enzymatic dissociation resulted in a loss of structural integrity and drastically reduced expression of genes encoding for structural proteins, such as collagens and matrix proteoglycans. Moreover, FIB-SEM revealed subtle differences in the mitochondrial 3D network with a higher number of mitochondria with individually lower volumes in enzymatically dissociated fibers, hence supporting an important role of the ECM in orchestrating the dynamic balance between mitochondrial fusion and fission. [64][65][66] In contrast, genes associated with cell cycle and DNA replication were significantly induced in enzymatically dissociated compared to mechanically dissected fibers. These fundamental alterations were paralleled by a downregulation of markers of skeletal muscle maturation, such as genes for myosin heavy chains, in enzymatically dissociated fibers.
Although the exact link between ECM and mitochondrial phenotypes remains elusive, our results pinpoint interesting candidates for further investigations. Gene expression of the actin regulator gelsolin was maintained in mechanically dissected fibers, whereas it was among the most downregulated genes in iScience Article enzymatically dissociated fibers. Gelsolin is inactive in the absence of Ca 2+ ; however, on Ca 2+ binding, gelsolin undergoes conformational changes, and the resulting activated domains participate in the severing and capping of actin filaments. [67][68][69] Moreover, gelsolin has been shown to inhibit the cellular stressinduced increase in mitochondrial membrane permeability and loss of mitochondrial membrane potential in a Ca 2+ -and CsA-dependent manner, 70 i.e. acting on processes that involve Ppif. 34,35,54-57 Another tentative candidate is plectin, for which gene expression was downregulated at 4 h in enzymatically dissociated fibers. Plectin is an intermediate filament-associated protein that acts as a cytoskeletal scaffold connecting myofibrils, mitochondria and junctional complexes at the plasma membrane. 71 We did not detect any major differences in plectin (or desmin) immunofluorescence staining between mechanically dissected and enzymatically dissociated fibers (see Figures 1E and 1F), which indicates that the cytoskeleton remained overall organized. However, subtle structural changes would not have been detected in these experiments and genetic mutations in the PLEC gene are associated with muscular dystrophy, characterized by the detachment of mitochondria from sarcoplasmic reticulum and mitochondrial clustering. 72,73 Moreover, assessment of the myofibrillar structure with SHG imaging and phalloidin labeling showed a loss of distinct longitudinal ridges and valleys in enzymatically dissociated fibers, and these fibers also had longer sarcomeres than mechanically dissected fibers (see Figures 1A-1D). These results indicate that on isolation from their native microenvironment, the internal muscle fiber tension is reduced in enzymatically dissociated fibers, which might cause a rapid dedifferentiation of the adult muscle fiber phenotype. Our findings thus corroborate previous studies showing that ECM has a central role in the skeletal muscle differentiation process, 74 and resemble observations in other tissues, such as liver, brain, and heart. [75][76][77][78] Combined, these results argue for the importance of maintaining the physiological niche in ex vivo experiments of cellular function. ] mit observed in enzymatically dissociated fibers indicates a defective control of mitochondrial Ca 2+ fluxes, i.e. a defect also observed in mechanically dissected muscle fibers of mitochondrial myopathy mouse models (see Figure 9). Notably, in the in vivo study of Rudolf et al., 8 [Ca 2+ ] mit returned to the basal level within 10 s even after a long (2.5 s) tetanic contraction (their Figure 5K), which is in accordance with the return of basal [Ca 2+ ] mit to the pre-contraction level within 1 min after 25 tetani in mechanically dissected WT fibers (see Figure 9E), whereas basal [Ca 2+ ] mit remained markedly elevated 5 min after 25 tetani in enzymatically dissociated WT fibers (see Figures 7B and 9F).

Rudolf et al. showed a transient increase in [Ca
Our results imply that the aberrant, prolonged elevation of basal [Ca 2+ ] mit in enzymatically dissociated fibers is Ppif-dependent as the excessive increase in basal [Ca 2+ ] mit after the repeated tetanic contractions was significantly decreased by the cyclophilin inhibitors CsA and NV556. In general terms, basal [Ca 2+ ] mit depends on the balance between mitochondrial Ca 2+ entry and extrusion, and the Ca 2+ buffering capacity. Ppif is an integral part of the elaborate mPTP protein complex, 34 which may open in different conductance modes: a high-conductance state that causes collapse of the mitochondrial membrane potential, extrusion of Ca 2+ and peptides that trigger apoptosis and ultimately cell death; a low-conductance state not accompanied by severe mitochondrial depolarization that allows additional Ca 2+ to enter the mitochondrial matrix when [Ca 2+ ] cyt is increased. 58 We did not detect any mitochondrial depolarization in enzymatically dissociated fibers during repeated contractions (see Figure 8A), which means that the driving force for Ca 2+ was in the direction from the cytosol to the mitochondrial matrix; thus, the increase in basal [Ca 2+ ] mit might involve Ppif-dependent opening of mPTP in the low conductance mode. Moreover, CsA has been reported to increase mitochondrial Ca 2+ buffering, 79 and together with opening of the mPTP in its lowconductance mode, this provides a tentative mechanism underlying the lessened contraction induced increase in [Ca 2+ ] mit in enzymatically dissociated fibers. On the other hand, we are not aware of any results indicating Ppif-depent effects on mitochondrial Ca 2+ extrusion, and our results show a slow decline of basal [Ca 2+ ] mit after repeated tetani both with and without Ppif inhibition (see Figure 7B). Notably, the excessive increase in basal [Ca 2+ ] mit in enzymatically dissociated fibers after repeated tetanic stimulation was observed already 4 h after cell isolation, hence too soon for important changes in protein levels to develop. Thus, the excessive mitochondrial Ca 2+ accumulation would be a direct consequence of altered mitochondrial function caused by disrupted ECM and intracellular cytoskeleton. Nevertheless, changes in gene expression 24 h after muscle fiber isolation indicate an additional enzymatic dissociation-induced long-term shift toward increased [Ca 2+ ] mit ; that is, the expression of Mcu and Ppif was higher and the expression of Nclx was lower in enzymatically dissociated than in mechanically dissected fibers, which would promote mitochondrial Ca 2+ influx and limit Ca 2+ extrusion. Of interest, previous studies showed that myopathies in mice deficient in the ECM protein collagen VI could be counteracted by CsA 32 and the non-immunosuppressive Ppif inhibitor alisporivir (also called Debio 025). 33 Furthermore, our finding that pharmacological inhibition of aberrant mitochondrial Ca 2+ accumulation improved survival in the Tfam KO mouse model of lethal mitochondrial myopathy supports a scheme where the adverse effects of ECM perturbations are mediated, at least in part, via impaired Ppif-dependent fine-tuning of cytosolic-mitochondrial Ca 2+ fluxes, thus providing a molecular explanation for prolonged survival previously reported when Tfam KO mice were treated with CsA, 43 as well as treatment with the more specific cyclophilin inhibitor NV556 used in the present study.
The presented findings highlight multiple important implications for the study of cell biological phenomena in vitro. Firstly, our data emphasize the importance of using cell culture systems that preserve the immediate ECM to ensure that results faithfully reflect in vivo processes. Secondly, enzymatic digestion rapidly changes the molecular phenotypes and functionality of cells. For instance, recent studies have shown redundant activation of muscle stem cells isolated from adult skeletal muscle with standard enzymatic dissociation protocols, which has important consequences for the use of these cells as quiescent controls. 80,81 Thirdly, dissolving the microphysiological niche around cells can result in perturbations that resemble pathological phenotypes observed in mitochondrial disease, providing further evidence for an intricate interplay between cellular structure, Ca 2+ fluxes, metabolism, and function. Specifically, the pathognomonic mitochondrial Ca 2+ accumulation during repeated contractions of muscle fibers in mitochondrial myopathy would be missed in experiments performed on enzymatically dissociated cells.
Thus, enzymatically dissociated cells should be avoided as an experimental paradigm for the study of diseases that potentially involve altered mitochondrial Ca 2+ signaling.
In conclusion, disruption of the organotypic niche results in the loss of structural integrity of muscle fibers accompanied by impaired control of mitochondrial Ca 2+ . The molecular link between the processes involves a Ppif-dependent mitochondrial Ca 2+ accumulation resembling that observed in mitochondrial myopathies. Our results support a central role of mitochondrial Ca 2+ as a critical mediator that connects the native extracellular microenvironment to the maintenance of normal cellular structure and function.

Limitations of the study
We show clear morphological and functional differences between enzymatically dissociated and mechanically dissected fibers, and we attribute these to disruption of the extracellular matrix with collagenase treatment. However, there are inevitably other methodological differences between the two groups, such as, the strain imposed by tendons and connective tissue during contractions of mechanically dissected fibers, which are absent in enzymatically dissociated fibers. To deal with this, experiments were performed where mechanically dissected fibers were allowed to shorten freely during contractions (i.e., without mechanical stress via the tendons) or where mechanically dissected fibers were subsequently treated with collagenase and contractions performed under the same conditions as enzymatically dissociated fibers. Nevertheless, we cannot exclude that the observed differences between mechanically dissected and enzymatically dissociated fibers involved other methodological aspects; for instance, mechanically dissected fibers were not exposed to the trituration process.
Our results imply that contraction-induced mitochondrial Ca 2+ accumulation in enzymatically dissociated fibers occurs partly via a Ppif-dependent pathway. Cyclophilin inhibitors act on Ppif to inhibit opening of mPTP. In situations of severe cellular stress, mPTP enters a high-conductance state. In the present study, however, the acute stress is relatively mild, and the high-conductance state is not entered. We propose that the cyclophilin inhibitors then act by inhibiting opening of a low-conductance state or by increasing the mitochondrial Ca 2+ buffering capacity, but further experiments are required to clarify the exact details of their action.

DECLARATION OF INTERESTS
The laboratory of H.W. received financial support from NeuroVive Pharmaceutical AB (current name: Abliva AB). V.M.L. is CEO and shareholder of HepaPredict AB, co-founder and shareholder of PersoMedix AB, and discloses consultancy work for EnginZyme AB.  iScience Article buffer. All samples were processed using Pelco Biowave Pro + microwave tissue processor (Ted Pella) according to, 85 with minor modifications: no Ca 2+ was used during the fixation and contrasting steps with lead aspartate were omitted to reduce overstaining. Samples were trimmed with a glass knife and 70 nm ultrathin sections were picked up on Cu-grids and examined with the TEM Talos L120C (FEI, currently Thermo Fischer Scientific) operating at 120 kV. Micrographs were acquired with a Ceta 16M CCD camera using TEM Image & Analysis software ver. 4.17 (Thermo Fisher Scientific).
For focused ion beam scanning electron microscopy (FIB-SEM), a small cube of the sample was cut and glued on an SEM stub with epoxy and silver glue. In the SEM chamber, the specimen was coated with a 5 nm thin layer of platinum to reduce charging. Specimens were imaged using Scios DualBeam SEM and the 'Auto slice and view 4' software system (Thermo Fisher Scientific); the electron beam operated at 2 kV and 0.2 nA and was detected with T1 in-lens detector. A 1 mm protective layer of platinum was deposited on the selected area before milling. FIB milling thickness was set to 30 nm and each slice was imaged with pixel sizes 3.55 3 3.55 nm (for mechanically dissected fiber) and 3.74 3 3.74 nm (for enzymatically dissociated fiber). Images were further processed using the ImageJ plugins 'Linear Stack Alignment with SIFT' and 'Multistackreg' (https://imagej.nih.gov/ij/) and the mitochondrial network was reconstructed and analyzed in a final volume of 365 mm 3 (7.9 3 4.5 3 10.3 mm). Identified mitochondrial volumes were modeled and measured using the IMOD software package ver. 4.9.13. 82

RNA-sequencing
RNA sequencing by poly-A capture was performed using >10 ng RNA input material. Image analysis, base calling and quality checks were performed using the RTA3.4.4 pipeline and Bcl2fastq (v2.20) conversion software (Illumina). Quality control, removal of genomic contaminants and ribosomal RNA, UMI-based read deduplication, transcript assembly and quantification were conducted using the Stringtie nf-core/ rnaseq package (https://nf-co.re/rnaseq/usage). Genes with an average number of fragments per kilo base per million mapped reads (FPKM) > 0.5 across all samples were analyzed using Qlucore Omics Explorer (Lund, Sweden). Differential gene expression analysis was conducted using DESeq2 and multiple testing correction was applied using the Benjamini-Hochberg procedure with false discovery rates (FDRs) % 5%. Pathway enrichment analysis was conducted based on the PANTHER gene family classification system using the WebGestalt toolbox. 83 Gene expression values (FPKM) of all samples are presented in Table S1. Note that confocal imaging was always performed at rest and not during an ongoing contraction due to fiber movement. Confocal images were analyzed using ImageJ and data are expressed as F/F 0 , i.e., the ratio of the fluorescence intensity after and before the repeated contractions, respectively.

Confocal measurements with fluorescent indicators
For measurements of [Ca 2+ ] mit , fibers were incubated in 5 mM rhod-2 in the membrane permeable AM form (Invitrogen). Rhod-2 was excited with 531 nm light and the emitted light was collected through a 585 nm long-pass filter.
For measurements of the mitochondrial membrane potential (Dj m ), fibers were incubated in 1 mM TMRE (Invitrogen). TMRE was excited at 531 nm and the emitted light was collected through a 605 nm longpass filter. Approximately 30 min after completion of the 25 repeated tetani, fibers were exposed to 1 mM FCCP (Sigma-Aldrich) to significantly depolarize the mitochondria and confocal images were obtained.
For measurements of mitochondrial ROS production, fibers were loaded with 5 mM MitoSOX Red (Invitrogen

Ru360, CsA and NV556 experiments
Enzymatically dissociated FDB fibers were loaded with rhod-2 to measure [Ca 2+ ] mit as described above. To investigate the potential sites of Ca 2+ entry into mitochondria, enzymatically dissociated fibers were exposed to either Ru360 (Calbiochem), CsA or NV556 (Abliva AB, Lund, Sweden). Ru360 was first injected into the fibers and they were subsequently superfused with 10 mM Ru360 throughout the experiment. CsA (1.6 mM; Novartis) or NV556 (5 mM) was applied to the fibers for 5 min before and during the 25 repeated tetani.

Single fiber [Ca 2+ ] cyt measurements
Intact single FDB fibers were mechanically dissected. 41 Aluminum or platinum clips were attached to the tendons and the fiber was mounted in a chamber between an Akers 801 force transducer (Kronex Technologies, Oakland, CA, USA) and an adjustable holder and subsequently superfused by Tyrode solution (see above) at room temperature. The fiber length was adjusted to obtain maximum tetanic force. The fiber was stimulated with supramaximal electrical pulses (0.5 ms in duration) delivered via platinum electrodes placed along the long axis of the fiber. The steady-state [Ca 2+ ] cyt -frequency relationship was obtained by stimulating fibers for 350 msat 15-150 Hz every 1 min; 150 Hz contractions were also produced in the presence of 5 mM caffeine to assess the SR Ca 2+ storage capacity.
We used the relatively high-affinity fluorescent indicator indo-

QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical analyses were performed with SigmaPlot 13 (Systat Software Inc, CA). Student's paired, unpaired t-tests, one-way ANOVA, or z-test were used as appropriate. Two-way repeated measures ANOVA was used to determine differences between two groups of repeatedly stimulated fibers. The Holm-Sidak post-hoc analysis was used when ANOVA showed a significant difference between groups. Significance was assumed for p< 0.05. Data are presented as mean G SEM.