1 Antioxidants an d Redox Signal ing PHYSICAL AND FU N C TIONAL CROSS ‐TALK BET W EE N EN DO ‐SA R C O P LA SM IC RETICUL U M AN D MITOCHONDR IA IN SKEL ETA L MUSCLE (D O I: 10 .10 89/ ar s.2 0 1 9 .79 34 ) This paper has been peer ‐r ev iewed an d acc ept ed fo r p u blicat ion, but has yet to und ergo copy editin g and proof co rr ec ti o n . The fi nal published ve rs io n may di ff er from this pr oo f. Invited review article for the Special Forum Issue of “Mitochondrial Metabolism and Mitophagy” in Antioxidant & Redox Signaling
PHYSICAL AND FUNCTIONAL CROSS‐TALK BETWEEN ENDO‐
SARCOPLASMIC RETICULUM AND MITOCHONDRIA IN SKELETAL
MUSCLE
Simona Boncompagni2, Diego Pozzer1, Carlo Viscomi3, Ana Ferreiro4, Ester Zito1
1 Istituto di Ricerche Farmacologiche Mario Negri‐IRCCS, Milan, Italy 2 CeSI‐Met ‐ Center for Research on Ageing and Translational Medicine and DNICS ‐ Dept. of Neuroscience, Imaging and Clinical Sciences; University G. d' Annunzio, Chieti, Italy 3 MRC‐Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK 4 Pathophysiology of Striated Muscles laboratory, Unit of Functional and Adaptive Biology, BFA, University Paris Diderot/CNRS, Sorbonne Paris Cité, Paris, France ‐ AP‐HP, Centre de Référence Maladies Neuromusculaires Paris‐Est, Groupe Hospitalier Pitié‐Salpêtrière, Paris, France Abbreviated title for running head: Association between endo‐sarcoplasmic reticulum and mitochondria Corresponding author: Ester Zito, Istituto di Ricerche Farmacologiche Mario Negri‐IRCCS Via Mario Negri 2, Milano, Italy Tel: +39 0239014480 E‐mail: ester.zito@marionegri.it Word count: 4721 words Reference numbers: 72 references Number of grayscale illustrations: 0 Number of color illustrations: 6 (online 6 and hard copy 0)
2 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correc tion . The fi nal published versi o n may differ from this proof.
Key words: Mitochondria associated membranes; endoplasmic reticulum stress; mitochondria; congenital myopathies; calcium handling proteins; skeletal muscle
3 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correct ion . The fi nal published ve rs io n may differ from this proof. ABSTRACT Significance: The physiological relevance of contacts between the sarcoplasmic reticulum (SR), a specialized domain of the endoplasmic reticulum (ER) in skeletal muscle, and mitochondria is still not clear.
Recent Advances: An extensive close proximity of these two organelles is a late developmental event, which suggests that it does not have an essential function.
Critical Issues: The intimate SR/mitochondria association develops during murine post‐ natal differentiation and the recovery of denervated atrophic muscle, which suggests this is a highly regulated process with a specific function. Analysis of mouse models for muscle diseases suggest that impaired ER/SR‐mitochondria contacts may be due to ER stress and lead to defective bioenergetics and insulin signalling.
Future Directions: Future studies are necessary to identify the molecular determinants weakening insulin signalling upon impairment of ER/mitochondria contacts in skeletal muscles as well as to analyse the distance between SR/ER and mitochondria in muscle diseases associated with ER stress.
4 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correc tion . The fi nal published versi o n may differ from this proof. INTRODUCTION Contacts between the endoplasmic reticulum (ER) and mitochondria are signalling hubs in many tissues, but their importance in skeletal muscle is still questioned. Extensive contacts between these two organelles appear following a late developmental event in mammalian skeletal muscle, and are completely lacking in very fast skeletal muscles such as the toadfish swimbladder muscle where mitochondria are relegated to the periphery (3); this suggests a non‐essential functional role of the ER/mitochondria pairing. On the other hand, the close proximity between the sarcoplasmic reticulum (SR), which is a very extensive specialized sub‐domain of the ER in skeletal muscle, and mitochondria seems to be generated through a regulated process that takes place during post‐natal murine differentiation and the recovery of human denervated atrophic muscle, so this indicates that although not essential, this structural organization is advantageous.
This review will analyse the characteristics of the ER, SR and mitochondria, how their contacts are regulated in skeletal muscle, and it will describe what has been learned about the physiological role of these contacts from animal models of muscle diseases. Finally, the thought‐provoking hypothesis that these contacts are impaired in ER stress conditions and this triggers a metabolic impairment of the skeletal muscle will be suggested.
Sarcoplasmic Reticulum: an endoplasmic reticulum specialized for skeletal muscle cells
The endoplasmic reticulum (ER) of eukaryotic cells consists of a network of flattened cisternae and tubule‐like membranes which are continuous with the outer nuclear membrane and distributed throughout the cell (47). The ER plays a variety of functions including storage of intracellular Ca2+, lipid metabolism or protein synthesis and folding (51).
Some of the cellular functions of the ER are fullfilled by distinct domains (6,42,51). For example the rough ER is associated with ribosomes and is the site of protein synthesis. In 1988 Volpe et al. described discrete cytoplasmic organelles, referred to as “calciosomes”, that contained calsequestrin in their lumen and whose membrane was studded with the reticulum calcium pump, SERCA (63). Since calciosomes were intracellular targets for inositol 1,4,5‐trisphosphate (Ins‐P3), it was speculated that the calciosome was a small ER
5 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correct ion . The fi nal published ve rs io n may differ from this proof. domain communicating with the rest of the ER and involved in maintaining luminal calcium homeostasis (32,63).
Muscle fibers contain not only ER but also an extensive myofibril‐associated network of membranes, termed “sarcoplasmic reticulum”, SR. The SR is a fully differentiated domain of the muscle ER (46), dedicated to the uptake, storage and efflux of calcium (23). Analysis of the levels and distribution of well‐known (general house‐keeping) ER proteins indicated that they were present in SR, but at lower levels, suggesting a general reorganization of the ER membranes to give rise to SR during myogenic differentiation (64). Volpe and colleagues showed that during the early stages of muscle differentiation the ER, in which they identified several house‐keeping proteins, enlarged in size and volume, with myofibrils surrounding them like a net, and lost many but not all of the generic ER proteins (64). The "myofibril associated‐ER", conveniently called SR, is just an expanded differentiated version of ER domains or "enlarged calciosome" for adult skeletal muscle cells (47,63,64). The SR origin from ER is clearly demonstrated not only by the fact that like the ER, SR is a network that is continuous throughout the cell (41) but also because it contains some ER proteins (30).
Unlike other cells which often have a quite large amount of cytoplasmic space, skeletal muscle fibers are filled with myofibrils except for the regions under the sarcolemma near nuclei. In these regions and close to the nuclei there are both rough and smooth ER components and Golgi, which are dedicated to protein synthesis, folding and transport. SR is a subdomain of ER specialized to serve muscle contraction, and contains all the proteins necessary for this function (i.e. RyRs, CASQ, SERCA, etc). The SR function in the control of muscle contraction involves two quite distinct actions: one is the uptake and storage of calcium ions, which is mediateds by the calcium ATPase pump, SERCA, distributed over the extensive free SR surface. The second function is the rapid release of calcium during muscle activation thtat occurs at the specialized junctional SR membranes associated with plasmalemma/transverse tubules. To conclude, a skeletal muscle fiber contains three distinct domains of a membrane delimited system, that are part of a continuum with distinct functions: the ER domain, the longitudinal or free SR domain (fSR) and the junctional SR domain (jSR) (Figure1).
6 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correc tion . The fi nal published versi o n may differ from this proof. Sarcoplasmic Reticulum (SR) and Calcium Release Units (CRUs)
Skeletal muscle contraction and relaxation are finely regulated by changes in the intracellular calcium (Ca2+) concentration. The SR is functionally and morphologically organized to support the muscle relaxation‐contraction cycle. This is achieved through specialized SR domains, the jSR and the fSR, which are in direct continuity with each other (Figure 1 and 2).
The jSR is the region closely associated to both sides of the transverse tubules (TTs), that are invaginations of the sarcolemma carrying an electrical signal from the plasmalemma inside the fiber. The jSR‐T tubule association forms the triads or Ca2+ Release Units (CRUs) in many mammalian skeletal muscles. In the myofibers of some mammals (e.g. mouse) CRUs are placed in proximity to the transition between the A‐ and I‐bands of the sarcomere and contain the macromolecular complex involved in excitation‐contraction (EC)‐coupling. The EC‐coupling events link an action potential carried along TTs to SR Ca2+ release via the RyRs, or calcium release channels of the SR. The lumen of the jSR contains CASQ1, a Ca2+ binding protein that in EC coupling plays a dual role as: a) an intraluminal moderate‐affinity (KD of 1mM), high‐capacity Ca2+ buffer and b) a modulator of RyR1‐
mediated SR Ca2+ release by interacting with RyR1 (29,37) (25). The fSR surface has a high density of SERCA that pumps two Ca2+ ions from the cytoplasm to the SR lumen by hydrolysing an ATP molecule and allows Ca2+ refill in the SR lumen after a muscle contraction (20,21,66). Redox regulation of calcium‐handling proteins and excitation‐contraction coupling There are more than ten isoforms of SERCA and three isoforms of RyR in skeletal muscle, the most expressed variants being SERCA1a, SERCA2a and RyR1. The SERCA1 and RyR1 isoforms are both involved in severe skeletal muscle diseases: SERCA1 mutations have been associated with Brody myopathy, and RyR1 mutations with malignant hyperthermia (MH) and core myopathies (CCD) (28) (17).
Given the importance of SERCA pumps and RyRs in Ca2+ metabolism and muscle health, it is not surprising that their activity in skeletal muscle is closely regulated at many levels: for
7 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correct ion . The fi nal published ve rs io n may differ from this proof. example, sarcolipin (SLN) binds to the trans‐membrane domains of SERCA1a and SERCA2a, and inhibits the pump’s affinity for Ca2+. Abnormally high levels of SLN in a mouse model of Duchenne muscle dystrophy are part of the pathogenic phenotype and the partial ablation of SLN improved the muscle pathology by restoring SERCA activity (62).
SERCAs and RyRs contain numerous thiols and are thus redox‐regulated by different chemical modifications. For example, SERCA2 has two cysteines in the L4 ER luminal loop which might be oxidised to form a disulfide bond that impairs SERCA2 activity (35). Selenoprotein N1 (SELENON, formerly SEPN1), a protein whose mutations give rise to SEPN1‐related myopathy and which carries the highly nucleophilic selenocysteine residue, interacts with SERCA2 in a redox‐dependent manner and modulates SERCA‐mediated calcium pumping into the ER/SR. Its activity counteracts the inhibitory and oxidative effects of the protein disulfide oxidase ERO1 on the SERCA pump. In line with impaired SERCA activity in cells lacking SELENON, relaxation times are longer in electrically stimulated fast digitorum brevis (FDB) muscle fibers (39) (48). Similarly, RyR1 activity is also regulated in a redox‐dependent manner; for example, H2O2‐mediated oxidation
generated by SR‐localised NOX isoform 4 affects the redox state of regulatory RyR1 thiols, leading to a leaky RyR1 and affecting muscle performance (56).
ER, ER stress and muscle defects
ER, like its close cognate continuum SR, is a calcium reservoir but also the site of protein folding and lipid synthesis. Calcium dysregulation impairs the activity of calcium‐ dependent chaperones and therefore raises the levels of unfolded proteins and triggers ER stress (Figure 3) (65). ER stress activates a complex signalling response (the ER stress response) initiated by the three proximal sensors of ER stress: inositil‐requiring enzyme 1 (IRE1), a kinase and endoribonuclease that promotes the splicing of X box binding protein 1 (XBP1), which in turn becomes a transcription factor of the genes involved in protein folding and ER‐associated protein degradation (ERAD); activated protein kinase R‐like ER kinase (PERK), which attenuates protein synthesis by phosphorylating eukaryotic initiation factor 2‐alpha (eIF2‐alpha); and activated transcription factor (ATF6), which traffics to the Golgi where it is proteolytically cleaved from its transmembrane domain, and then
8 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correc tion . The fi nal published versi o n may differ from this proof.
migrates to the nucleus where it acts as a transcription factor of chaperones such as BIP/GRP78 and GRP94. An adaptive ER stress response therefore acts by attenuating protein translation and favoring protein folding, and thus helps to re‐establish ER homeostasis (65).
However, sustained activation of the PERK pathway and of its effector, the transcription factor C/EBP homologous protein (CHOP), may induce a maladaptive response to ER stress that triggers dysfunction and eventually apoptosis. This may also be due to the two downstream targets of CHOP: endoplasmic oxidoreductin (ERO1α, henceforth ERO1), which produces H2O2 and may promote the hyperoxidation of the ER lumen, and GADD34,
a phosphatase involved in the dephosphorylation of eIF2‐alpha that reactivates the pace of protein translation (Figure 3) (38) (69).
The mouse models carrying a deletion in each of the three proximal sensors of ER stress (IRE1, PERK and ATF6) demonstrate the importance and specific roles of the three related branches in skeletal muscle. Muscle‐specific PERK ablation reduces skeletal muscle mass and force, which suggests that the PERK pathway is important in mantaining muscle homeostasis (26). However, given the double‐edged nature of the PERK pathway, CHOP deletion improves the pathological skeletal muscle phenotype in exercise‐intolerant PGC1‐ alpha muscle knock‐out (KO) mice and also ameliorates impaired diaphragm force in a SELENON KO mouse model, indicating that the PERK pathway may be maladaptive in muscle‐related pathological conditions (48) (68). ATF6‐alpha KO mice are exercise‐ intolerant and show high levels of ER stress in skeletal muscle after running, which suggests that the ATF6 branch of the ER stress response is important for preserving muscle integrity during ER stress (68). There are no data concerning the IRE1 branch in skeletal muscle physiology.
Mitochondria structure and function
Mitochondria are organelles surrounded by an outer mitochondrial membrane (OMM) and an inner mitochondrial membrane (IMM), which is folded to form the mitochondrial cristae where the respiratory complexes involved in the oxidative phosphorylation (OxPhos) system are located. The OxPhos converts the energy derived from the nutrients into Adenosine Triphosphate (ATP). The efficiency of this oxidative metabolism critically depends on the Krebs cycle for the generation of NADH and FADH2, which feed electrons
9 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correct ion . The fi nal published ve rs io n may differ from this proof. to the electron transport chain. Electron flow is coupled with the translocation of protons across the IMM, generating a transmembrane electrochemical gradient, which is finally exploited by ATP synthase to generate ATP.
Oxidative metabolism is controlled at multiple levels: for example, in this context, it is worth citing that calcium ions cross the OMM through a voltage‐dependent anion channel (VDAC) (55) and the IMM through the mitochondrial calcium uniporter (MCU) (5,16), and activate several key metabolic enzymes, including pyruvate dehydrogenase, alpha‐ ketoglutarate, isocitrate‐dehydrogenase, as well as complexes I, III, IV and V of the electron transport chain. Mitochondrial calcium therefore is an important regulator of ATP production (27).
Mitochondria are highly dynamic organelles that adapt their shape and distribution to the metabolic conditions of the cells. In post‐mitotic tissues, such as skeletal muscle, which have limited ability to dilute damaged mitochondria among dividing cells, mitochondrial dynamics determined by fusion/fission and mitophagy is a very important means of preserving mitochondrial function. The core machinery of mitochondrial dynamics consists of highly regulated GTPases involved in the fission and fusion. Fission is carried out by the Dynamin‐related protein 1 (Drp1) and Dynamin2 (Dnm2) and fusion by Mitofusins 1 and 2 (Mfn1 and Mfn2) on the OMM and Opa1 on the IMM. However, numerous additional proteins take part in these processes (58).
Mitochondrial fusion gives rise to interconnected mitochondrial networks and leads to greater cell oxidative capacity, whereas increased mitochondria fission gives rise to isolated mitochondria and less efficient mitochondrial respiration, which are easily eliminated by mitophagy (7,67). Mitophagy is triggered as a consequence of several processes, such as cellular differentiation, mitochondrial damage and after fertilization to eliminate paternal mtDNA (45). For each of these processes, mitochondria are flagged to mitophagy by specific proteins. For instance, NIX/BNIP3L mediates mitophagy in reticulocyte differentiation, BNIP3 in hypoxia‐induced mitophagy, PINK1 and Parkin in the presence of mitochondrial damage. Once mitochondria have been flagged on the OMM with one of these proteins, they are recognized by the adaptor proteins NDP52 and OPTN that target them to the autophagic machinery and finally to the lysosomes for elimination
10 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correc tion . The fi nal published versi o n may differ from this proof. (45). These observations have led to the hypothesis that mitochondria dynamics regulate mitochondrial function in response to metabolic demand (71). Mitochondrial activity and metabolism are clearly dependent on mitochondrial biogenesis, which is boosted, among the other triggers, also by physical activity. Seminal studies have shown that more active muscles have greater mitochondrial content and activity than sporadically working muscles, and that runners have a larger percentage of oxidative, slow‐twitch skeletal muscle fibres and more succinate dehydrogenase activity than sedentary controls. PGC‐1 is a co‐transcriptional master regulator of the oxidative metabolism induced by muscle exercise, which positively regulates mitochondrial biogenesis and mitochondrial oxidative metabolism genes (36).
Mitochondria distribution in skeletal muscle fibers
The intensity of skeletal muscle activity varies widely, and different muscle fibers have evolved different biophysical properties to appropriately respond to different functions. To meet metabolic demand during contraction and relaxation, skeletal muscle fibers require large amounts of ATP, mostly supplied by mitochondria (43). Slow‐twitch (type I) fibers are highly oxidative and have the highest mitochondrial content compared to fast‐twitch (type II) fibers. In these fibers three sets of mitochondria with different orientation and myofibrillar disposition are clearly distinguishable (Figure 4). The majority of mitochondria appear in longitudinal sections as round/oval paired profiles symmetrically positioned in the inter‐myofibrillar spaces at the I‐band of the sarcomere on either side of the Z line adjacent to the CRU or triads (triadic mitochondria; Figure 4). Triadic mitochondria are structurally tethered to the jSR (Figures 1 and 4) (9) and often continuous with a smaller subset of mitochondria, positioned longitudinally at the A–band of the sarcomere between myofibrils (longitudinal mitochondria; Figure 4). Longitudinal mitochondria also appear structurally anchored to the free SR (Figure 1). A third set of mitochondria, smaller and with a more variable profile, accumulate at the fiber periphery under the plasmalemma (subsarcolemmal mitochondria; Figure 4). Subsarcolemmal mitochondria also show some continuity with the single, extended network formed by the first two sets, although several studies sustain that they are part of a different subpopulation given their distinct morphological and biochemical properties (1,44) (57). Type II fibers have almost exclusively triadic mitochondria while longitudinal mitochondria are less frequently found
11 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correct ion . The fi nal published ve rs io n may differ from this proof. as well as clusters of subsarcolemmal mitochondria that are usually very small in size when they are present (Figure 4). Mitochondrial connections with the ER/SR The first description of a close association between mitochondria and ER dates back to the 1970s (54) although their functional relevance for the transfer of phospholipds and Ca2+ emerged only in the ‘90s. Nowadays, mitochondria‐ER contact sites are considered central hubs for the exchange of ions and lipids between the two compartments and for a large number of signalling pathways, including those related to mitochondrial dynamics, mitochondrial DNA replication, apoptosis and autophagy. Over the last few years, considerable progress has been made in identifying the nature of tethers that keep the ER and mitochondria together (see (14) for an extensive review). The physical properties of the linkers determine the gap distance and the extension of the contact sites. For instance the presence of ribosomes, IP3Rs or RyRs pose physical constraint to the minimal distance between the two compartments. This distance is critical to ensure efficient exchange of Ca2+ and lipids between the compartments, as well as the recruitment of apoptotic and autophagy machineries to the contact sites (49). In murine skeletal muscles, both ER and SR are closely associated to mitochondria (12) (Figure 5 ) and about a quarter of the outer surface of mitochondria is very close to the jSR and to the CRUs. The connection between mitochondria and SR is maintained by a tether about 10 nm long (9); that was first seen by electron tomography in fractioned rat‐liver mitochondria (13). Electron microscopy indicates that the OMM is on an average of 130 nm from the site of RyR‐mediated Ca2+ release (22). These measurements support the concept of short‐term formation of high [Ca2+] microdomains that functionally allow efficient Ca2+ uptake by mitochondria (50). Although mitochondrial Ca2+ uptake is likely to be minimal, it has been reported in myotubes and skeletal muscle fibers after RyR1‐mediated Ca2+ release (22). In addition, skeletal muscle mitochondrial Ca2+ uptake occurs during electrical stimulation and exposure to caffeine, and persists in a subpopulation of mitochondria (presumably those tightly juxtaposed to CRUs) even in the presence of fast Ca2+ chelators (53).
12 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correc tion . The fi nal published versi o n may differ from this proof.
Although an extensive close proximity between CRUs and mitochondria appears in mammals (24), it is completely lacking in some very fast‐acting muscles such as the toadfish swimbladder muscle where mitochondria are relegated to the periphery of myofibers (3). This structural evidence suggests that the close association between the two organelles is not essential but, as it is acquired as a developmentally regulated event during post‐natal differentiation in mice and the recovery of human denervated atrophic muscle, is functionally advantageous (24).
Over the last few years considerable progress has been made in identifying the nature of tethers that keep the ER and mitochondria together. Mitofusin 2 (MFN2) is linked to both the OMM and the ER membrane (15) and, through its C‐terminal domain, forms homo‐ and hetero‐typic complexes with other molecules of MFN2 and MFN1 (which is only localised on the OMM) in order to tether the ER/SR to mitochondria. Quantitative electromicroscopy upon acute MFN2 knockdown in the fast digitorum brevis (FDB) muscle have shown that the SR is detached from mitochondria and that their tethering therefore depends on MFN2. Furthermore, functional measurements of calcium levels show that mitochondrial calcium uptake in MFN2 knock‐down muscle fibres decreases during repetitive tetanic stimulation, suggesting that mitochondria are not only more distant from the SR but also take up less calcium (2). Interestingly, it has been shown that muscles in the conditional knock‐out of MFN2 (with ablation of 80% of skeletal muscle MFN2) are insulin resistant, and that insulin‐dependent AKT phosphorylation is blocked in isolated MFN2‐deficient muscle, thus suggesting decreased insulin signalling. A second major finding of this study was that MFN2 deficiency also triggers ER stress in skeletal muscle which, together with mitochondrial dysfunction, is an important driver of insulin resistance (31). It is however unclear whether the lack of MFN2 weakens the insulin signal simply by altering the distance between the SR and mitochondria, or whether it is part of a more complex scenario in which the lack of MFN2 alters the SR/mitochondria distance, which then triggers an ER stress that impairs the insulin (52).
An early report suggested that the cytoplasmic region of PERK is involved in the formation of MAMs (mitochondria‐associated membranes) in mouse embryonic fibroblasts, and this is independent of the activity of PERK as a kinase and mediator of the ER stress response (61). However, the stress response may be involved in shaping the distance between the
13 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correct ion . The fi nal published ve rs io n may differ from this proof.
ER and mitochondria by inducing CHOP and downstream ERO1, which oxidises and hyperactivates the IP3‐mediated calcium release that induces mitochondrial fragmentation (34). Unfortunately, the distance between the ER and mitochondria was not measured in the muscle‐specific PERK KO model, thus leaving a doubt whether PERK tethers the SR to mitochondria in skeletal muscle (26).
Interestingly, H2O2 nanodomains, probably generated by the electron transport chain,
have been detected in the contacts between ER and mitochondria. They might redox‐ regulate IP3R‐ and RyR1‐dependent calcium flux, suggesting that mitochondrial activity may also regulate calcium release from the SR (10).
The SR‐mitochondrial tethers provide a structural framework for bi‐directional SR‐ mitochondrial signalling in adult striated muscle, in which both organelles can influence each other’s function. However, signalling between mitochondria and SR in skeletal muscle is not only due to their physical interactions, but also to the complex co‐regulation of the genes involved in mitochondria and SR homeostasis. In fact, a seminal study showed that the PGC‐1, a master regulator of mitochondrial oxidative metabolism, also acts as a co‐ activator of the ATF6‐alpha mediator of the ER stress response in promoting the transcription of chaperones and enabling skeletal muscle to adapt to physical exercise (68).
MUSCLE MODELS WITH AN ALTERED DISTANCE BETWEEN THE SR AND MITOCHONDRIA
What follows is a description of the phenotype of mouse models of muscle disease that have an altered interaction between the SR and mitochondria, the phenotypic analysis of which provide clues to the physiological role of SR/mitochondria connections.
RyR1
RyR1Y522S heterozygous knock‐in mice, carrying a mutation in RyR1 (orthologous to the Y524S mutation identified in human patients) that leads to malignant hyperthermia (MH) and central core disease (CCD), are viable and reproduce the human phenotype of MH when challenged with a volatile anesthetic or heat stress (19). Electron microscopy analysis of RyR1Y522S/WT muscle fibres at different stages shows that mitochondrial damage and changes in SR/mitochondria contacts are already evident at 2 months of age (Figure
14 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correc tion . The fi nal published versi o n may differ from this proof.
6). These initial alterations will eventually lead to formation of cores, which are areas lacking both functional mitochondria and CRUs and showing sarcomere disorganization (9). At the molecular level, the Y522S mutation triggers SR Ca2+ leakage, and increases RyR1 channel sensitivity to activation by voltage and agonists (17,18). Therefore, it has been proposed that chronic elevated resting cytoplasmic Ca2+ levels result in excessive production of oxidative species, triggering both MH crises and mitochondrial damage leading to development of cores in RYR1Y522S/WT mice (19). Anti‐oxidant treatment was indeed successfully used to lower the sensitivity of RYR1Y522S/WT muscle fibers to heat and a recent investigation shows that a chronic antioxidant treatment with N‐acetylcysteine (NAC) of RYR1Y522S/WT mice was able to: a) lower oxidative stress; b) lower mitochondrial damage and core formation; and c) improve muscle function (40).
RyR1I4895T heterozygous knock‐in mice express a mutation orthologous to I4898T in human RyR1, which is associated with a form of CCD in patients. Unlike Y522S, the RyR1I4895T mutation decreases SR Ca2+ release (72) and in slow‐twitch muscle of heterozygous mice causes the formation of mitochondrial‐deficient areas (8)(Figure 6). The net loss of mitochondria or their possible migration from the inter‐myofibrillar space at the I‐band and relocation in large inter‐myofibrillar and/or sub‐sarcolemmal clusters suggests disruption of mitochondrial/CRUs association possibly due to reduced Ca2+‐signaling. Consistently with the fiber‐type specific effects of the I4895T mutation, fast twitch fibers do not show mitochondrial alteration but an enlargement (or swelling) of junctional and free SR which appear filled with an electron dense material, that is quite likely to be CASQ (Figure 6) (8).
In addition, in a more recent investigation the RyR1I4895T/WT mutation has been associated with ER stress and a reduced distance between the SR and mitochondria (33).
Although these two findings on the distance between SR and mitochondria of RyR1I4895T/WT muscle seem contradictory they could be two sequential events; namely, first the SR/mitochondria contacts increase (33) and consequently a possible alteration in mitochondria triggers loss or migration of mitochondria (8). It is important to note that chronic treatment of RYR1I4895T/WT knock‐in mice with the chemical chaperone 4‐phenyl butyric acid (4‐PBA) improves muscle strength by decreasing ER stress, thus suggesting that ER stress is a pathogenic defect involved in impaired muscle strength; unfortunately,
15 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correct ion . The fi nal published ve rs io n may differ from this proof.
whether this treatment also re‐establishes the correct distance between the ER and mitochondria has not been investigated, thus leaving uncertainty about the real contribution of altered SR/mitochondria distance to the pathological muscle phenotype of RYR1I4895T/WT knock‐in mice.
SELENON
Mutations in the human SELENON gene are associated with autosomic recessive SELENON‐ / SEPN1‐related myopathy, and with insulin resistance in a subgroup of these patients. SELENON activates the ER calcium pump SERCA2 in a redox‐dependent manner, and thus counteracts its ERO1‐alpha‐dependent inactivation. In line with the existence of a mechanism by means of which SELENON regulates SERCA activity, experiments using electrically stimulated FDB muscle fibres devoid of SELENON have shown longer relaxation times, thus suggesting that SELENON might modulate SERCA in muscle as well as in cell lines (39,48). This could explain the reported increase of cytosolic Ca2+ and reduced Ca2+ content in the SR lumen (a ER‐stress trigger) in patient‐derived myotubes devoid of SELENON (4). Although milder than the phenotype of patients with SEPN1‐related myopathy, the SELENON KO mouse leads to diaphragmatic weakness consistently with the human phenotype of respiratory impairment (48) (11).
This weakness is associated with increased levels of BIP, CHOP and ERO1, and attenuated protein translation in the diaphragm, thus suggesting a concomitant chronic ER stress and maldaptive ER stress response. The fact that the genetic ablation of CHOP completely restores the diaphragmatic defect in SELENON KO mice indicates that this maladaptive response is the pathogenic cause of the muscle dysfunction.
The lack of SELENON alters the distance between the ER and mitochondria, and sensitises skeletal muscle to the ER stress‐dependent consequence of lipotoxicity in mice that compromises insulin sensitivity and muscle strength similarly to that observed in patients with SEPN1‐related myopathy (60).
RyR1 and SELENON mice are two models of altered calcium transients in skeletal muscle in which, regardless of whether they are reduced or enhanced, defects in SR‐ ER/mitochondria contacts are associated with muscle defects and ER stress. The overlapping phenotypes of the SELENON KO and MFN2 KO mouse models suggest that an
16 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correc tion . The fi nal published versi o n may differ from this proof.
increased distance between mitochondria and the ER in muscle might underlie both the defective bioenergetics and insulin sensitivity of skeletal muscle.
Although the molecular determinants weakening insulin signalling in SELENON and MFN2 KO mice still need to be identified, both models suggest that an altered distance between the ER and mitochondria correlates with insulin resistance. Interestingly, disrupted ER‐ SR/mitochondria interaction is an early event preceding muscle mitochondrial dysfunction and insulin resistance in mouse models of obesity and type II diabetes, which suggests that impairment of ER‐SR/mitochondria interaction might cause insulin resistance. These latter models do not have any genetic defects in proteins involved in tethering the two organelles, but they display ER stress, indicating that ER stress per se may be involved in the disruption of ER‐SR/mitochondria interactions (59).
CONCLUDING REMARKS
An extensive association between mitochondria and SR has only been found in mammalian skeletal muscles. It is a late event and therefore it is not essential in development, however it must offer some advantages. An altered distance between ER, SR and mitochondria is a common feature of mouse models of myopathies due to different genetic defects. However it is not clear whether this altered distance is caused by a direct impairment in the structural proteinaceous tethers joining the two organelles or ‐more likely‐ a consequence of the related ER stress. Strategies aimed at relieving ER stress in these models will help clarify this point, as well as the contribution of altered contacts between ER and mitochondria to the phenotype of the myopathies. Future studies are necessary to identify the molecular determinants weakening insulin signalling upon impairment of ER/mitochondria contacts in skeletal muscles as well as to analyse the distance between SR/ER and mitochondria in muscle diseases associated with ER stress.
Acknowledgments
This study was supported by a Cure CMD/AFM Telethon grant, My first AIRC grant and Ricerca Finalizzata Ministero della salute to E.Zito and by a Cure CMD grant to A. Ferreiro. We are indebted to Prof. Clara Franzini‐Armstrong for useful hints and fruitful discussion of the manuscript.
17 Antioxidants and Re d o x Signaling PH YS IC AL AND FU NCTIONA L CROSS ‐TALK BET W EEN EN DO ‐SA R C O P LA SM IC RETICULUM AN D MITOCHO N DRIA IN SKELETA L MUSCLE (DOI: 10 .10 89/ ars.2 0 1 9 .79 34 ) This paper ha s been peer ‐r ev iewed an d ac ce p te d for pu blicati o n, bu t has yet to u nderg o co p yeditin g and proof correct ion . The fi nal published ve rs io n may differ from this proof. List of Abbreviations ATF6= Activating transcription factor 6 ATP= Adenosine triphosphate BIP= Binding Immunoglobulin Protein CASQ= Calsequestrin CCD= Central core disease CHOP=C/EBP homologous protein DHPR= Dihydropyridine receptor EM= Electron microscopy ER= Endoplasmic reticulum ERO1= Endoplasmic oxidoreductin 1 FDB= Fast digitorum brevis GRP94= 94kDa glucose‐regulated protein H2O2= Hydroperoxide IMM= Inner mitochondria Membrane IRE1= Inositol‐requiring enzyme 1 MAM= Mitochondria associated membranes MFN2=Mitofusin 2 MH= Malignant hyperthermia NOX4=NADPH oxidase 4 OMM=outer mitochondria membrane OxPhos= Oxidative phosphorilation PERK= Protein kinase RNA‐like endoplasmic reticulum kinase JSR=Junctional sarcoplasmic Reticulum FSR= Free or longitudinal sarcoplasmic Reticulum RyR= Ryanodine receptor SELENON= Selenoprotein N1 SERCA= Sarcoplasmic‐endoplasmic reticulum calcium ATPase SR= Sarcoplasmic Reticulum
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