Goutam Chandra

Sen Chandra Sreetama

Davi A G Mázala

Karine Charton

Jack H Vandermeulen

Isabelle Richard

Jyoti K Jaiswal
email: jkjaiswal@cnmc.org.

Endoplasmic reticulum maintains ion homeostasis required for plasma membrane repair

Of the many crucial functions of the ER, homeostasis of physiological calcium increase is critical for signaling. Plasma membrane (PM) injury causes a pathological calcium influx. Here, we show that the ER helps clear this surge in cytoplasmic calcium through an ER-resident calcium pump, SERCA, and a calcium-activated ion channel, Anoctamin 5 (ANO5). SERCA imports calcium into the ER, and ANO5 supports this by maintaining electroneutrality of the ER lumen through anion import. Preventing either of these transporter activities causes cytosolic calcium overload and disrupts PM repair (PMR). ANO5 deficit in limb girdle muscular dystrophy 2L (LGMD2L) patient cells compromises their cytosolic and ER calcium homeostasis. By generating a mouse model of LGMD2L, we find that PM injury causes cytosolic calcium overload and compromises the ability of ANO5-deficient myofibers to repair. Addressing calcium overload in ANO5-deficient myofibers enables them to repair, supporting the requirement of the ER in calcium homeostasis in injured cells and facilitating PMR.

......

Introduction

There is an ∼1,000-fold calcium (Ca 2+ ) gradient across the plasma membrane (PM), which is maintained by the barrier function of the PM and by pumping Ca 2+ into the extracellular space or intracellular compartments [START_REF] Jaiswal | Calcium -how and why?[END_REF][START_REF] Mcneil | Plasma membrane disruption: repair, prevention, adaptation[END_REF]. PM injury disrupts this gradient, causing a large and rapid influx of extracellular Ca 2+ , which triggers the PM repair (PMR) response [START_REF] Cooper | Membrane Repair: Mechanisms and Pathophysiology[END_REF][START_REF] Horn | Cellular mechanisms and signals that coordinate plasma membrane repair[END_REF][START_REF] Mcneil | Plasma membrane disruption: repair, prevention, adaptation[END_REF]. How cells undergoing repair buffer this cytosolic Ca 2+ ([Ca 2+ ] c ) excess and how failure to do so affects PMR have not been resolved.

The ER, which constitutes ∼50% of the eukaryotic cell's membrane, is the major reservoir for intracellular calcium. While the resting [Ca 2+ ] c is ∼100 nM, sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pumps [Ca 2+ ] c into the ER and maintains free Ca 2+ concentration in the ER ([Ca 2+ ] ER ) in the range of 100 to 400 µM [START_REF] Palmer | Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs[END_REF][START_REF] Samtleben | Direct imaging of ER calcium with targeted-esterase induced dye loading (TED)[END_REF][START_REF] Verkhratsky | Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons[END_REF]. This Ca 2+ is a required cofactor for the ER-resident chaperones to maintain their optimal activity. ER Ca 2+ homeostasis is essential for a variety of functions, including cell signaling, immune cell development and function, and muscle cell contraction. However, whether the ER plays a role in homeostasis of PM injury-triggered increase in cellular Ca 2+ and its role in PMR have not been investigated. For the ER to play a role in [Ca 2+ ] c buffering, it must sense the increase in [Ca 2+ ] c and buffer it by rapidly sequestering it. SERCA, the pump responsible for Ca 2+ import into the ER, binds Ca 2+ for its import. However, instead of [Ca 2+] c level, SERCA pump is regulated by the binding of multiple small-molecular-weight proteins such as sarcolipin and phospholamban [START_REF] Chen | Primary Active Ca 2+ Transport Systems in Health and Disease[END_REF][START_REF] Stammers | The regulation of sarco(endo)plasmic reticulum calcium-ATPases (SERCA)[END_REF]. Ca 2+ pumping into the ER by SERCA is kept electroneutral through the exchange of imported Ca 2+ with exported H + [START_REF] Møller | The sarcoplasmic Ca2+-ATPase: design of a perfect chemi-osmotic pump[END_REF] and transport of counter-ions such as Chloride (Cl -) into the ER is required to maintain electroneutrality during uptake of large Ca 2+ influx [START_REF] Pollock | Chloride channel blockers inhibit Ca2+ uptake by the smooth muscle sarcoplasmic reticulum[END_REF]. Large [Ca 2+ ] c surge, such as that caused by purinergic signaling, enables Ca 2+ import into the ER by calcium-activated chloride channel (CaCC)-mediated Cl -uptake into the ER [START_REF] Neussert | The presence of bestrophin-1 modulates the Ca2+ recruitment from Ca2+ stores in the ER[END_REF].

The transmembrane protein 16 (TMEM16) family, also known as the Anoctamin (ANO) family of proteins, includes calcium-activated anion channels [START_REF] Berg | Ca2+-activated Cl-channels at a glance[END_REF][START_REF] Huang | International Union of Basic and Clinical Pharmacology. LXXXV: calcium-activated chloride channels[END_REF][START_REF] Whitlock | Anoctamins/TMEM16 Proteins: Chloride Channels Flirting with Lipids and Extracellular Vesicles[END_REF]. ANO5 (TMEM16E), a member of this family that is proposed to be a nonselective ion channel, localizes to the ER and is required for PMR [START_REF] Di Zanni | Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia[END_REF][START_REF] Griffin | Defective membrane fusion and repair in Anoctamin5-deficient muscular dystrophy[END_REF][START_REF] Jaiswal | Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect[END_REF][START_REF] Tsutsumi | The novel gene encoding a putative transmembrane protein is mutated in gnathodiaphyseal dysplasia (GDD)[END_REF]. Thus, lack of ANO5 may disrupt rapid electroneutral exchange of Ca 2+ required for ER Ca 2+ homeostasis. However, ANO5 function has not been examined at the ER, and much of the analyses have focused on its activity at the PM [START_REF] Di Zanni | TMEM16E/ANO5 mutations related to bone dysplasia or muscular dystrophy cause opposite effects on lipid scrambling[END_REF][START_REF] Di Zanni | Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia[END_REF][START_REF] Duran | ANOs 3-7 in the anoctamin/Tmem16 Cl-channel family are intracellular proteins[END_REF]. Recessive mutations in the ANO5 gene are associated with limb girdle muscular dystrophy 2L (LGMD2L) and Miyoshi muscular dystrophy 3, characterized by progressive muscle weakness and degeneration [START_REF] Hicks | A founder mutation in Anoctamin 5 is a major cause of limb-girdle muscular dystrophy[END_REF][START_REF] Sarkozy | ANO5 gene analysis in a large cohort of patients with anoctaminopathy: confirmation of male prevalence and high occurrence of the common exon 5 gene mutation[END_REF][START_REF] Witting | Anoctamin 5 muscular dystrophy in Denmark: prevalence, genotypes, phenotypes, cardiac findings, and muscle protein expression[END_REF]. Defects in PMR are also associated with muscle diseases including, LGMD2B, LGMD2D, Facioscapulohumeral muscular dystrophy, and Duchenne muscular dystrophy, where PMR deficit is linked to altered membrane trafficking and mitochondrial redox signaling [START_REF] Bansal | Defective membrane repair in dysferlin-deficient muscular dystrophy[END_REF][START_REF] Bittel | Membrane Repair Deficit in Facioscapulohumeral Muscular Dystrophy[END_REF]Defour et al., 2014b;[START_REF] Han | Basal lamina strengthens cell membrane integrity via the laminin G domain-binding motif of alpha-dystroglycan[END_REF][START_REF] Vila | Mitochondria mediate cell membrane repair and contribute to Chandra et al[END_REF]. However, cells from muscular dystrophy patients with ANO5 mutations [START_REF] Bolduc | Recessive mutations in the putative calcium-activated chloride channel Anoctamin 5 cause proximal LGMD2L and distal MMD3 muscular dystrophies[END_REF] show poor PMR even when the conventional PMR pathways are unaffected [START_REF] Chandra | Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells[END_REF][START_REF] Jaiswal | Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect[END_REF]. Despite over a dozen unique ANO5 mutations identified, the exact mechanism for poor PMR in ANO5-deficient cells has not been established [START_REF] Penttilä | Eight new mutations and the expanding phenotype variability in muscular dystrophy caused by ANO5[END_REF].

Here, we studied Ca 2+ dynamics during the repair of injured cells and the role of CaCC in regulating this homeostasis. Our work examines whether the ER helps regulate Ca 2+ homeostasis following PM injury and the consequence of failure to buffer [Ca 2+ ] c excess caused by PM injury. We also examined whether ER-mediated calcium homeostasis plays a role in the repair of ANO5-deficient cells. To examine these, we made use of ANO5 patient muscle cells as well as a new ANO5 knockout (KO) mouse model. Our studies with healthy cells identify that CaCCmediated chloride uptake by the ER is required for PMR and that this is compromised in muscle cells of ANO5-deficient patient, as well as in myofibers of ANO5-KO mice. This deficit in [Ca 2+] c clearance results in ER and mitochondrial damage due to Ca 2+ overload, the reversal of which restores PMR ability in ANO5-deficient cells. These findings identify a novel role of the ER in cell survival, shows that regulation of Ca 2+ homeostasis is a key step in the process of PMR, and provides mechanistic insight into the basis for PMR deficit in LGMD2L/Miyoshi muscular dystrophy 3 patient muscles.

Results

The ER facilitates PMR by buffering an injury-triggered increase in [Ca 2+] c Using the free Ca 2+ -sensor Fluo-4, we monitored the change in [Ca 2+ ] c during PMR in healthy human myoblasts. Focal PM injury caused influx of extracellular Ca 2+ , which caused the [Ca 2+ ] c to peak in 6.06 ± 0.45 s, which returned to baseline in 39.52 ± 5.13 s (Fig. 1, A andB). Use of membrane-impermeant FM1-43 dye entry into cells as an indicator of PMR showed that [Ca 2+ ] c influx returns to near baseline as the PM wound closes by 40 s. This leads to the plateauing of FM dye entry, but this cessation of FM dye entry fails to occur when cells are injured in the absence of extracellular Ca 2+ (Fig. 1, C and D). Thus, PM wound closure requires extracellular Ca 2+ entry to trigger repair, but this Ca 2+ is rapidly buffered by an as-yet-unknown mechanism. Because the ER is the major intracellular store for Ca 2+ , we examined whether it helps buffer the injury-triggered increase in [Ca 2+ ] c . For this, we used the Förster (fluorescence) resonance energy transfer (FRET)-based ER luminal Ca 2+ sensor (T1ER; [START_REF] Abell | Parallel adaptive feedback enhances reliability of the Ca2+ signaling system[END_REF]. Following PM injury, ER Ca 2+ levels increased immediately (Fig. 1, F and G), with the fastest increase occurring during the first 30 s of injury, the period when the [Ca 2+ ] c increase was buffered most rapidly (Fig. 1 B). To test whether ER Ca 2+ uptake helps buffer the injury-triggered increase in [Ca 2+ ] c , we blocked ER Ca 2+ uptake using the SERCA pump inhibitor thapsigargin (TG; Fig. 1, H andG). This prevented the injury-triggered increase in [Ca 2+ ] c from returning to baseline (Fig. 1, B andI). Blocking the ER uptake of Ca 2+ prevented PMR, as indicated by continued FM1-43 dye entry in TG-treated injured cells (Fig. 1, D and J), and reduced the number of injured cells managing to undergo PMR (Fig. 1 E). While signal-triggered Ca 2+ release from the ER is well studied, this is not so for the mechanism of [Ca 2+] c -activated Ca 2+ uptake by the ER.

Counter-ion transport is required for ER Ca 2+ uptake during PMR While the SERCA pump is not sensitive to the [Ca 2+ ] c level, import of counter ions such as Cl -into the ER maintains electroneutrality during uptake of large Ca 2+ influx [START_REF] Pollock | Chloride channel blockers inhibit Ca2+ uptake by the smooth muscle sarcoplasmic reticulum[END_REF]. Ca 2+ sensitivity of counter-ion import is due to CaCCs [START_REF] Neussert | The presence of bestrophin-1 modulates the Ca2+ recruitment from Ca2+ stores in the ER[END_REF]. To investigate whether this mechanism enables ER Ca 2+ uptake during PMR, we inhibited CaCCs using 5nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB; [START_REF] Dreinhöfer | Blocking kinetics of Clchannels in colonic carcinoma cells (HT29) as revealed by 5-nitro-2-(3phenylpropylamino)benzoic acid (NPPB)[END_REF]. To monitor NPPB-blocked anion entry into the ER, we used the anion sensitivity of YFP [START_REF] Kuner | A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons[END_REF]. ER-localized YFP (YFP ER ) and RFP (RFP ER ) were expressed in cells and treated with NPPB or vehicle (DMSO). PM injury of vehicle-treated cells led to anion uptake-induced rapid quenching of YFP ER fluorescence, decreasing the YFP ER /RFP ER ratio (Fig. 2, A and B; and Fig. S2). As expected, this was prevented in NPPB-treated cells (Fig. 2, A and B), identifying that NPPB-sensitive Ca 2+ -activated anion channels enable injurytriggered anion influx into the ER. Use of T1ER showed that NPPB treatment also prevented the injury-induced increase in [Ca 2+ ] ER (Fig. 2,C and D). This NPPB-induced block in [Ca 2+ ] ER increase prevented the buffering of the injury-triggered increase in the [Ca 2+ ] c , (Fig. 2, E andF). NPPB treatment led to a dosedependent decrease in PMR, as shown by increased FM1-43 dye entry and a decrease in the number of cells that underwent PMR (Fig. 2, G-I). These results suggest that CaCC activity allows Ca 2+ uptake by the ER and is required for PMR.

ANO5 deficit impairs [Ca 2+ ] ER homeostasis and prevents PMR by [Ca 2+ ] c overload KO of the ANO family member ANO5 (TMEM16E) and cells from patients with mutations in ANO5 both exhibit poor PMR [START_REF] Chandra | Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells[END_REF][START_REF] Griffin | Defective membrane fusion and repair in Anoctamin5-deficient muscular dystrophy[END_REF][START_REF] Jaiswal | Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect[END_REF]. To examine if injured ANO5 patient fibroblasts exhibit altered ER Ca 2+ homeostasis, we monitored [Ca 2+ ] ER following injury. This showed significantly reduced [Ca 2+ ] ER in injured patient fibroblasts (Fig. S1, A andB). As lack of CaCC in the ER has been shown to affect ER calcium homeostasis during purinergic signaling [START_REF] Neussert | The presence of bestrophin-1 modulates the Ca2+ recruitment from Ca2+ stores in the ER[END_REF], we treated patient cells with ATP (50 µM) and found that unlike the healthy cells, ATP failed to increase in [Ca 2+ ] ER in patient cells (Fig. S1, C and D). Similar to fibroblasts, the YFP ER /RFP ER ratio in injured healthy human myoblasts decreased, indicating anion influx in the ER, and this was blocked in ANO5 patient myoblasts (Fig. 3, A and B; and Fig. S2). Using T1ER, we found that the block in ER anion uptake in patient cells occurred concomitantly with the block in ER Ca 2+ uptake in patient cells (Fig. 3, C and D). Further, failure of ER Ca 2+ uptake by injured patient cells correlated with their poor PMR (Fig. 3, E-G). To confirm if failure of ER calcium uptake compromises PMR of patient cells, we examined whether SERCA inhibition (by TG) can worsen the PMR deficit in patient cells. While TG impaired PMR of healthy cells, it did not worsen the repair deficit of patient cells (Fig. 3, E-G). Together, these results indicate that lack of ANO5 prevents PMR by preventing the ER counter-ion influx required for cytosolic [Ca 2+ ] c buffering during PMR.

To determine why failed [Ca 2+ ] c buffering compromises PMR, we examined whether poor buffering of [Ca 2+ ] c disrupts normal PMR responses. Annexins are among the earliest cytoplasmic proteins that respond to injury-triggered [Ca 2+ ] c increase, and their strict temporal response is required for successful PMR [START_REF] Demonbreun | An actin-dependent annexin complex mediates plasma membrane repair in muscle[END_REF][START_REF] Jaiswal | S100A11 is required for efficient plasma membrane repair and survival of invasive cancer cells[END_REF][START_REF] Jaiswal | S100 and annexin proteins identify cell membrane damage as the Achilles heel of metastatic cancer cells[END_REF][START_REF] Sønder | Annexin A7 is required for ESCRT III-mediated plasma membrane repair[END_REF]. We monitored the response of annexin A1 and A2, both of which are implicated in PMR. Both annexins accumulated immediately upon PM injury and cleared within 1 min of injury in healthy cells (Fig. 3, H-J). However, in patient cells, both annexins continued to accumulate for longer, accumulated to a higher level, and failed to clear away from the injury site (Fig. 3, H-J). This demonstrates that poor [Ca 2+ ] c buffering in injured patient cells disrupts the timely PMR response by annexins.

Another response triggered by injury-induced increase in Ca 2+ is mitochondria-mediated PMR [START_REF] Horn | Cellular mechanisms and signals that coordinate plasma membrane repair[END_REF][START_REF] Horn | Mitochondrial fragmentation enables localized signaling required for cell repair[END_REF][START_REF] Horn | Mitochondrial redox signaling enables repair of injured skeletal muscle cells[END_REF][START_REF] Sharma | Use of quantitative membrane proteomics identifies a novel role of mitochondria in healing injured muscles[END_REF]. To examine this response in ANO5-deficient cells, we used the [Ca 2+ ] m sensor mitoCAR-GECO1. Patient cells showed a greater increase in [Ca 2+ ] m at the injury site, which took longer to return to preinjury baseline, resulting in an excessive [Ca 2+ ] m load (Fig. 3, K-M). This [Ca 2+ ] m overload caused large-scale depolarization of mitochondria, as demonstrated by the mitochondrial potential-sensitive dye tetramethylrhodamine, ethyl ester (TMRE; Fig. 3, N andO; and Fig. S2). In contrast to healthy myoblasts, where PM injury caused the mitochondrial potential to transiently increase before returning to preinjury level within the next minute, mitochondria in patient cells were increasingly depolarized following injury (Fig. 3 O). These results show that ER Ca 2+ imbalance in ANO5 patient cells disrupts multiple Ca 2+dependent PMR processes.

ANO5 deficit impairs myofiber PMR, which is addressed by ameliorating [Ca 2+ ] c overload ANO5 is preferentially expressed in muscles (Fig. S3), and its deficit causes muscular dystrophy. To test whether the above findings in patient myoblasts extend to mature muscle fibers, we generated ANO5-KO mouse by deleting exons 10-12 from the ANO5 gene. Laser injury of isolated flexor digitorum brevis (FDB) myofibers showed that compared with the WT myofibers, ANO5-KO myofibers allowed significantly higher FM1-43 dye entry, and nearly twice as many ANO5-KO myofibers failed to repair compared with WT myofibers (Fig. 4, A-C). To test the myofiber repair ability, we also examined this in an independent muscle, the extensor digitorum longus (EDL). Myofibers in intact EDL muscles isolated from 10-mo-old ANO5-KO mice also repaired poorly following laser injury, as indicated by greater FM1-43 dye entry into the myofiber (Fig. 4, D andE). To further confirm the poor repair ability of ANO5-deficient myofibers, we monitored their ability to recover from eccentric exerciseinduced myofiber injury. Isolated EDL muscles from these animals showed that force loss following 10% lengthening contraction (LC) injury was significantly greater for EDL muscles from ANO5-KO mice compared with the age-matched WT mice (Fig. 4 F). Greater LC-induced damage could be due to sarcoplasmic reticulum Ca 2+ imbalance during excitation-contraction coupling. However, neither the level of the sarcoplasmic reticulum Ca 2+ release channel RYR1 nor Ca 2+ homeostasis during myofiber contraction showed any dysregulation in ANO5-KO mice (Fig. S3). Together, the findings above indicate that the force loss following LC contraction was due to poor repair of damaged myofibers. This was confirmed by the enhanced entry of membrane-impermeant dye (procion orange [PO]) in EDL myofibers from LC-injured ANO5-KO muscle as compared with WT muscle (Fig. 4, G andH).

The findings above show that excess [Ca 2+ ] c is the cellular deficit that leads to poor PMR of ANO5-deficient muscle cells. Muscle diseases associated with [Ca 2+ ] c overload benefit from SERCA1 overexpression [START_REF] Goonasekera | Mitigation of muscular dystrophy in mice by SERCA overexpression in skeletal muscle[END_REF][START_REF] Mázala | SERCA1 overexpression minimizes skeletal muscle damage in dystrophic mouse models[END_REF]. To assess the potential for such a therapeutic approach, we first examined SERCA1 protein expression. Compared with WT mice, muscles in ANO5-KO mice express higher levels of SERCA1 (Fig. 4, I andJ). Thus, as an alternate to SERCA overexpression, we hypothesized that pharmacologically sequestering the acute increase in [Ca 2+ ] c may improve the repair ability of ANO5-deficient muscle fibers. As a proof of principle for this therapeutic approach, we employed a potent Ca 2+ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid (BAPTA), that can be loaded into live cells in its acetoxy-methyl ester form (BAPTA-AM). Acute (20-min) treatment of ANO5-KO myofibers with BAPTA-AM efficiently reduced injury-triggered increase in [Ca 2+ ] c to levels comparable to WT myofibers (Fig. 4, K andL). BAPTA-AM treatment did not perturb the peak [Ca 2+ ] c increase following PM injury (Fig. 4 significantly improved ANO5-KO myofiber PMR, as shown by reduced FM dye entry and reduction in the number of myofibers that fail to repair to the same level as in WT muscles (Fig. 4, A-C and P-R). These findings show that lack of ANO5 compromises myofiber PMR due to poor [Ca 2+ ] c buffering following myofiber injury, addressing which could be a therapeutic approach for LGMD2L.

Discussion

Our effort to understand how cells repair from injury has uncovered a novel role of ER in facilitating the PMR in injured cells. PM injury causes a rapid increase in [Ca 2+ ] c , which is efficiently cleared during successful PMR. We find that ER provides the site for the uptake of the injury-triggered cytosolic Ca 2+ increase.

Pharmacologically blocking the activity of the ER calcium pump by TG leads to [Ca 2+ ] c overload in the injured cells and results in poor repair. We also identify that ER-mediated clearance of the [Ca 2+ ] c in the injured cells requires the CaCC ANO5. Upon being activated by the injury-triggered increase in [Ca 2+ ] c , ANO5 enables the transport of anion into the ER. The anions pumped into the ER then serve as the counter ion for facilitating sequestration of Ca 2+ pumped into the ER by SERCA activity. Such a role has been shown for the ER-localized CaCC protein bestrophin 1 in retinal pigment epithelial cells [START_REF] Neussert | The presence of bestrophin-1 modulates the Ca2+ recruitment from Ca2+ stores in the ER[END_REF]. Here, we show an analogous role of ANO5 in muscle cells, which is consistent with its localization at the ER [START_REF] Chandra | Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells[END_REF][START_REF] Tsutsumi | Molecular cloning and characterization of the murine gnathodiaphyseal dysplasia gene GDD1[END_REF][START_REF] Tsutsumi | The novel gene encoding a putative transmembrane protein is mutated in gnathodiaphyseal dysplasia (GDD)[END_REF]. This role of ANO5 in PMR is supported by our studies using the CaCC inhibitor NPPB, which causes injury-triggered Ca 2+ overload and poor PMR phenotype, in healthy cells. Together, the above findings show that during PMR, SERCA and CaCC both work in concert to enable Ca 2+ -activated [Ca 2+ ] c clearance by the ER. Blocking either of these transporters in healthy cells causes [Ca 2+ ] c overload upon PM injury and compromises PMR. Using ANO5-deficient patient myoblasts and fibroblasts as models for genetic deficit of ER-resident CaCC, we find that failure of ER calcium uptake in these cells disturbs annexin reparative response following injury and causes [Ca 2+ ] m overload. Together, these deficits lead to excessive mitochondrial depolarization and dysfunction. Disruption of these repair process leads to poor PMR due to the failure of ER to uptake calcium. Further, using the ANO5-KO mouse model, we establish that the role of ANO5 in membrane repair is conserved in skeletal myofibers. These results support the findings in an unrelated ANO5-KO mouse model, where a myofiber sarcolemmal repair deficit was observed [START_REF] Griffin | Defective membrane fusion and repair in Anoctamin5-deficient muscular dystrophy[END_REF]. While mitochondrial abnormalities were reported in this ANO5-KO mouse model [START_REF] Griffin | Defective membrane fusion and repair in Anoctamin5-deficient muscular dystrophy[END_REF], how ANO5 deficiency causes this defect was not resolved. We find that reduction in mitochondrial number and function in myofibers of mdx mice (an animal model of Duchenne muscular dystrophy) results in poor PMR [START_REF] Vila | Mitochondria mediate cell membrane repair and contribute to Chandra et al[END_REF]. In ANO5-deficient cells, cytosolic Ca 2+ overload leads to excessive mitochondrial Ca 2+ accumulation, resulting in loss of mitochondrial membrane potential in injured cells, which affects multiple calcium-dependent PMR processes. Key among them is the mitochondria, which, in injured ANO5 patient cells, gets overloaded with Ca 2+ and loses membrane potential. Mitochondrial depolarization and dysregulated calcium homeostasis lead to altered mitochondrial redox signaling required for actin-mediated PM wound closure and this mitochondriamediated PMR response is compromised in several diseases [START_REF] Boehler | Mitochondrial dysfunction and role of harakiri in the pathogenesis of myositis[END_REF][START_REF] Debattisti | Dysregulation of Mitochondrial Ca 2+ Uptake and Sarcolemma Repair Underlie Muscle Weakness and Wasting in Patients and Mice Lacking MICU1[END_REF][START_REF] Horn | Mitochondrial fragmentation enables localized signaling required for cell repair[END_REF]. As mitochondrial pathology in ANO5-deficient cells is downstream of primary deficit in ER calcium handling by the injured cells, it raises the possibility that addressing mitochondrial deficit may offer therapeutic benefits. Calcium influx triggered by the PM injury is a widely recognized prerequisite for the activation of PMR. However, our study highlights the Goldilocks-like nature of calcium during PMR, such that an increase in [Ca 2+ ] c that is either too little or too much increase is detrimental to PMR. We previously observed that excessive [Ca 2+ ] c increase affects ER [START_REF] Chandra | Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells[END_REF]) and here we find that this also dysregulates the annexindependent vesicle-mediated PMR and cytoskeleton-mediated PM wound closure through mitochondrial signaling. Thus, just as a lack of Ca 2+ increase, extended and excessive Ca 2+ increase following PM injury impairs PMR. Ca 2+ excess does so by protracted and excessive response by Annexins and mitochondria, which represent the PMR machineries involved in membrane and cytoskeletal remodeling. These findings also help explain the reports of mitochondrial abnormalities [START_REF] Griffin | Defective membrane fusion and repair in Anoctamin5-deficient muscular dystrophy[END_REF], and aberrant annexin response (Foltz et al., 2020 Preprint) in skeletal muscles in another ANO5-KO mouse model.

Similar to the ANO5 deficit in LGMD2L, poor sarcolemmal repair is also a feature of LGMD2B, which is caused by a lack of the muscle protein dysferlin [START_REF] Bansal | Defective membrane repair in dysferlin-deficient muscular dystrophy[END_REF][START_REF] Bashir | A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B[END_REF]. However, unlike dysferlin-deficient muscle cells, where the PMR deficit is caused by poor lysosomal exocytosis (Defour et al., 2014b), ANO5-deficient cells show normal injurytriggered lysosomal exocytosis [START_REF] Jaiswal | Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect[END_REF]. Thus, the cellular basis of poor PMR in ANO5-deficient cells appears to be distinct from the poor repair of dysferlin-deficient cells. This is supported by the failure of increased ANO5 expression to rescue muscle pathology in dysferlin-deficient mice [START_REF] Monjaret | The phenotype of dysferlin-deficient mice is not rescued by adenoassociated virus-mediated transfer of anoctamin 5[END_REF]. In this study, we show that the functional role of ANO5 in muscle cell is to facilitate Ca 2+ homeostasis during PMR. This role of ANO5 is supported by our findings that reversing the [Ca 2+ ] c overload in ANO5-deficient injured skeletal myofibers improves their repair. It also provides a potential pharmacological strategy for addressing the cellular deficit in muscular dystrophy caused by the ANO5 deficit.

In summary, our study adds ER to the list of organelles that are required for efficient PMR and identifies the basis for poor PMR in ANO5-deficient muscles. It describes the mechanism for calcium-activated ER calcium homeostasis and makes use of patient cells and a mouse model to establish that calcium dysregulation leading to dysfunction in mitochondrial and annexinmediated repair responses leads to poor PMR [START_REF] Boehler | Mitochondrial dysfunction and role of harakiri in the pathogenesis of myositis[END_REF][START_REF] Debattisti | Dysregulation of Mitochondrial Ca 2+ Uptake and Sarcolemma Repair Underlie Muscle Weakness and Wasting in Patients and Mice Lacking MICU1[END_REF][START_REF] Horn | Mitochondrial fragmentation enables localized signaling required for cell repair[END_REF][START_REF] Horn | Mitochondrial redox signaling enables repair of injured skeletal muscle cells[END_REF][START_REF] Vila | Mitochondria mediate cell membrane repair and contribute to Chandra et al[END_REF]. Lack of ANO5 causes excessive mitochondrial Ca 2+ overload and loss of membrane potential, leading to PMR deficit. Our findings identify the requirement of the ER in PMR and show that a deficit in this process contributes to disease, uncovering a new aspect of the ER in maintaining healthy cell physiology and cell survival.

Materials and methods

Cells and treatments

LGMD2L fibroblasts were used as described previously [START_REF] Jaiswal | Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect[END_REF]. Myoblasts were generated as described previously [START_REF] Chandra | Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells[END_REF] and were cultured in Skeletal Muscle Cell Basal Media (PromoCell) containing 15% FBS, 5% fetal calf serum, and all the necessary supplements (human EGF, basic FGF from human, fetuin, insulin, and dexamethasone) as described previously [START_REF] Chandra | Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells[END_REF]. Transfections were performed in OPTI-MEM (Invitrogen) using Lipofectamine LTX for 4 h followed by addition of full growth media and growing the cells for 24-48 h.

Animals

WT mice (C57BL/6) were obtained from Jackson Laboratory. Methods involving animals were approved by the institutional animal care and use committee. All the animal procedures were performed in accordance with the approved guidelines. Animals were maintained in a facility accredited by the American Association for Accreditation of Laboratory Animal Care. 10-moold male and female animals were used for all experimental procedures. Construction of the targeting vector for ANO5-KO mice and generation of these mice was performed at the Genoway. The positive embryonic stem cell (ES) clones were injected into C57BL/6J blastocysts that were reimplanted into foster mothers to generate chimeric mice. Germline transmission was obtained through the breeding of male chimeras with females transgenic for cytomegalovirus-Cre recombinase, which permits the excision of the neo cassette. The Cre transgene was segregated by a first cross on C57BL/6 background; the resulting heterozygous mice were backcrossed for 10 generations on the C57BL/6 (mice were from Charles River) and then interbred. For genotyping, genomic DNA from mouse tail was extracted and amplified using KAPA2G Fast HotStart Genotyping PCR Kit (KK5621; KAPA Biosystems), with the following primers chosen in the ANO5 gene: Flp.F, 59-TCTTCATGGGGATCTGGGGTGAGT A-39, Flp.R, 59-CCCTAGAACTACATAATCTTGGTGTGGTGGTAG-39, and 49683cre.F, 59-ATTCCTGAGAATATGTGTAATTGTGGC Ca 2+ and Cl -measurement assays For measuring the [Ca 2+] c , myoblasts and fibroblasts cultured on coverslips were incubated with DMEM without FBS containing 10 µM Fluo-4-AM (Life Technologies) for 20 min at 37°C and 5% CO 2 . After washing with prewarmed cell imaging medium (CIM), cells were laser injured as described before (Defour et al., 2014a). To monitor Ca 2+ influx, injured cells were imaged at an interval of four to six frames/second for the 3 min after injury using IX81 microscope (Olympus America) custom equipped with a CSUX1 spinning disc confocal unit (Yokogawa Electric). Images were collected using a 60× oil-immersion objective (1.45 NA). Images were acquired using an Evolve 512 high-performance electron multiplying charge-coupled device (EMCCD) camera (Teledyne Photometrics) at 1 Hz. Image acquisition and laser injury was controlled using Slidebook 6.0 (Intelligent Imaging Innovations). For every image, the kinetics of Ca 2+ influx was expressed by measuring the cellular Fluo-4-AM emission, which responds to the free cytosolic Ca 2+ . For measuring the free Ca 2+ in the ER, we used FRET-based biosensor, T1ER [START_REF] Abell | Parallel adaptive feedback enhances reliability of the Ca2+ signaling system[END_REF]. Myoblasts were transfected with T1ER plasmid DNA 24 h before the cells were injured (in presence of 2 mM Ca 2+ ). Pseudocolored images representing fluorescence intensity were generated using Slidebook 6.0. For measuring the mitochondrial Ca 2+ , we used the mitochondrial Ca 2+ sensor CAR-GECO1 as described previously [START_REF] Wu | Improved orange and red Ca 2 ± indicators and photophysical considerations for optogenetic applications[END_REF]. To monitor ER Cl -levels, cells were transfected with KDEL-YFP and KDEL-RFP plasmids 24 h before the assay. Calciumactivated chloride influx into the ER was measured for 2 min following PM injury by measuring the ratio of YFP and RFP fluorescence [START_REF] Jayaraman | Mechanism and cellular applications of a green fluorescent protein-based halide sensor[END_REF][START_REF] Kuner | A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons[END_REF].

Laser injury assays

Cells cultured on coverslips were transferred to and incubated in CIM/PBS buffer with 1 mg/ml FM1-43 dye (Life Technologies) and placed in a Tokai Hit microscopy stage-top ZILCS incubator (Tokai Hit) maintained at 37°C. For laser injury, a 1-to 5-µm 2 area was irradiated for 10 ms with a pulsed laser (Ablate!; 3i Intelligent Imaging Innovations). Cells were imaged using IX81 microscope (Olympus America). The change in FM dye intensity (ΔF/F where F is the intensity at the start of imaging) was used to quantify cell membrane repair kinetics. Images were acquired at 2-s intervals, and data shown in the plots represent frames that are 10 s apart. For myofiber injury, muscles were surgically isolated from euthanized WT or ANO5-KO mice in Tyrode's solution, and laser injury was performed using the microscope and laser injury settings as described previously (Defour et al., 2014a;[START_REF] Leikina | Annexin A1 Deficiency does not Affect Myofiber Repair but Delays Regeneration of Injured Muscles[END_REF] in the Tyrode's buffer containing 1.33 mg/ml FM1-43 dye. For isolating FDB myofibers, we used a method previously described [START_REF] Mázala | SERCA1 overexpression minimizes skeletal muscle damage in dystrophic mouse models[END_REF]. Briefly, FDB muscles were dissected from deeply anaesthetized animals, and single muscle fibers were obtained by collagenase digestion with type 2 collagenase (Worthington) in minimal essential medium with 10% FBS and 1% penicillin-streptomycin (Invitrogen). After incubation at 37°C in a CO 2 incubator (95% O 2 /5% CO 2 ), single muscle fibers were obtained by trituration with fire-polished glass pipettes. FDB fibers were maintained in minimal essential medium with 10% FBS at 37°C, 95% O 2 /5% CO 2 overnight and used for experiment in the next day. The kinetics of repair was determined by measuring the cellular FM1-43 fluorescence. FM1-43 intensity (F/F 0 , where F 0 is the original value at time 0) was used to quantify the kinetics of cell membrane repair.

Western blotting

Cells were lysed with RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Thermo Fisher Scientific). Proteins transferred to nitrocellulose membranes were probed with the indicated antibodies against SERCA1, vinculin, RyR1, and β-actin (all from Santa Cruz Biotechnology). Primary antibodies were followed by the appropriate HRP-conjugated secondary antibodies (Sigma-Aldrich) and chemiluminescent Western blotting substrate (Thermo Fisher Scientific; GE Healthcare). The blots were then processed on the Bio-Rad ChemiDoc Touch Imaging System.

Measurement of mitochondrial membrane potential

Myoblasts were incubated with the potentiometric dye TMRE for 15 min at 37°C and washed three times in CIM, and then we performed laser injury. These cells were previously transfected with Tom20-GFP, and the ratio of fluorescence intensities at 561 nm and 488 nm was considered as an index of mitochondrial membrane potential.

Contraction-induced myofiber sarcolemmal injury

After anesthetizing the mice with a mixture of ketamine and xylazine, EDL muscles from the right hindlimb of C57BL/6 and ANO5-KO mice were carefully exposed, and 6-0 silk sutures were firmly attached to proximal and distal tendons. The EDL was dissected and placed in a bath containing buffered mammalian Ringer's solution (137 mM NaCl, 24 mM NaHCO 3 , 11 mM glucose, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgSO 4 , 1 mM NaH 2 PO 4 , and 0.025 mM tubocurarine chloride) at 25°C and bubbled with 95% O 2 /5% CO 2 to stabilize the pH at 7.4. In the bath, the distal tendon was securely connected to a fixed bottom plate, and the proximal tendon was attached to the arm of a servomotor (800A in vitro muscle apparatus; Aurora Scientific). The vertically aligned EDL muscle was flanked by two stainless steel plate electrodes. Using single 0.2-mm square simulation pulses, the muscle was adjusted to the optimal muscle length (L o ) for force generation. At optimal length, with isometric tetanic contractions 300 ms in duration at frequencies up to 250 Hz separated by 2 min of rest intervals, the maximal force was determined. Specific force, defined as maximal force normalized for the muscle cross-sectional area, was calculated as the maximal force/(muscle mass × (density of muscle tissue × fiber length) -1 ). The fiber length was based on the fiber to muscle length ratio of 0.45. The muscle tissue density is 1.056 kg/liter. To study contraction-induced sarcolemmal injury, EDL muscles were subjected to nine LCs with 10% strain at a velocity of two fiber lengths per second. Each contraction was separated by a 1min rest interval. LC-induced force deficits were expressed as PO and muscle fiber membrane damage PM injury following LC-induced injury was assessed as before [START_REF] Vila | Mitochondria mediate cell membrane repair and contribute to Chandra et al[END_REF] using PO dye, which is excluded from myofibers with intact sarcolemma. Immediately after the LC assay, muscles were trimmed of tendons, blotted, weighed, and then incubated while held at L o in a 0.2% PO solution at room temperature for 30 min, washed in Ringer solution, and quickly frozen in isopentane prechilled with liquid nitrogen.

Frozen cross sections of 10-µm thickness were cut, mounted with fluorescent mounting medium (DAKO), and viewed under a fluorescent microscope to identify the presence of PO. Fields, containing the majority of the muscle cross sections were photographed and scaled under identical conditions at ×10 magnification using Olympus BX61 widefield microscope. By thresholding for unstained muscle, we identified fibers that showed no uptake of PO from fibers that showed PO uptake. From each muscle cross section, the area occupied by PO-labeled fibers ranging from minimal to maximal dye uptake was measured and expressed as a percentage of the complete muscle section area. PO-labeled fibers at the edges of the sections were considered as artifact and were excluded from analysis.

Statistical analysis

For membrane repair analysis, the data are presented as average values for all myoblasts or myofibers used for that analysis. These average values were compared with each other using an unpaired t test with Welch's correction performed using Graph Pad Prism software. When the data were not normally distributed or failed the equal variance test, the Mann-Whitney rank sum test was used.

Online supplemental material Fig. S1 shows that ANO5 deficit reduces ER calcium uptake following purinergic stimulation. Fig. S2 shows monochrome images of data presented in the main figures. Fig. S3 shows ANO5 expression and the effect of its deficit on the ER Ca 2+ -handling machinery.

Figure 1 .

 1 Figure 1. [Ca 2+] c homeostasis is required for PMR. (A) Healthy human myoblasts were labeled with Fluo-4 to monitor [Ca 2+] c following focal PM injury (arrowheads) by laser in the presence (control) or absence (-Ca 2+ ) of extracellular Ca 2+ . Pseudocolored images of the cell at different time points before and following injury are shown. Injury sites are marked with arrowheads. (B) Quantification of averaged kinetics for Fluo-4 intensity change in cells injured in the presence (control) or absence (-Ca 2+ ) of extracellular Ca 2+ or in the presence of SERCA inhibitor (1 mM TG). Plot shows mean ± SEM. n = 33 (control), 19 (-Ca 2+ ) and 29 (TG) cells; #, P < 0.0001. (C) Images showing FM1-43 dye entry into human myoblasts before and 60 s after focal laser injury in the presence (control) or absence (-Ca 2+ ) of extracellular Ca 2+ . (D) Quantification of FM1-43 dye entry kinetics in healthy myoblasts in the presence or absence of extracellular Ca 2+ or treated with 1mM TG in presence of Ca 2+ . Plot shows mean ± SEM. n = 16 (control), 25 (-Ca 2+ ) and 16 (TG) cells.; #, P < 0.0001. (E) Percentage of cells in C and D that successfully repaired. Plot shows mean ± SD. #, P < 0.0001. (F) Images before and 60 s after injury of human myoblasts expressing FRET-based ER calcium sensor T1ER, injured with (control) or without (-Ca 2+ ) extracellular Ca 2+ . (G) Quantification of normalized T1ER FRET ratios in cells injured as indicated. Plot shows mean ± SEM. n = 20 (control), 9 (-Ca 2+ ) and 17 cells (TG); #, P < 0.0001. (H-J) Images of human myoblasts labeled with T1ER (H), Fluo-4 (I), and FM1-43 (J) and treated with TG (1 mM) to inhibit SERCA. Images show the same cell before (preinjury) and 60 s (or indicated time points) after injury. Scale bars, 10 µm. Arrowhead indicates site of laser injury.

  M) but instead worked by reducing the time taken for the increase in [Ca 2+ ] c to return to baseline, allowing the overall injury-triggered [Ca 2+ ] c load to return to WT levels (Fig. 4, N and O). This amelioration of [Ca 2+ ] c overload labeling of human myoblasts treated with vehicle (control) or NPPB. n = 23 (control) and 24 (NPPB) cells; #, P < 0.0001. (G and H) Images (G) and plots (H) showing FM1-43 dye entry into healthy myoblasts treated with vehicle (control) or NPPB (at indicated doses) and focally injured by laser (site marked with arrowheads). (I) Percentage of myoblasts that repaired from PM injury after treatment with indicated NPPB doses. n = 16 (control), 19 (NPPB 10 µM), 12 (NPPB 50 µM), and 18 (NPPB 100 µM) cells; #, P < 0.0001. Error bars represent SD for the plot in I and SEM for all other plots. Scale bars, 10 µm. Chandra et al. Journal of Cell Biology 5 of 12 Role of the ER in plasma membrane repair https://doi.org/10.1083/jcb.202006035

Figure 3 .

 3 Figure 3. ANO5 is an ER-resident ion channel that regulates [Ca 2+ ] c during PMR. (A and B) Pseudocolored images (A) and quantification (B) of the kinetics of change in fluorescence of Cl --sensitive YFP and Cl --insensitive RFP fluorescence targeted to ER in healthy and ANO5-deficient patient myoblasts. n = 18 (healthy control) and 15 (patient) cells. (C and D) Pseudocolored images (C) and plot (D) showing quantification of T1ER FRET ratios before and 60 s after injury of healthy and patient cells. n = 20 (healthy) and 19 (patient) cells. (E and F) Images (E) and plots (F) showing FM1-43 dye entry in healthy and patient cells treated or untreated with the SERCA inhibitor TG. n = 16 (healthy), 31 (healthy, TG), 33 (patient), and 45 (patient, TG) cells. (G) Percentage of myoblasts in E and F that repaired following PM injury after indicated treatments. (H) Images before and 60 s after injury. (I and J) Plots showing average kinetics of annexin A1-GFP (AnxA1-GFP; I) and (J) annexin A2-RFP (AnxA2-RFP) accumulation at the repair site (marked by boxes) of healthy and patient myoblasts coexpressing annexin A1-GFP and annexin A2-RFP. n = 22 (healthy) and 18 (patient) cells. (K) Pseudocolored images of healthy and patient myoblasts expressing the mitochondrial calcium sensor CAR-GECO1 imaged during the course of PMR. (L and M) Plots showing quantification of normalized CAR-GECO1 fluorescence in healthy and patient myoblasts during PMR. n = 26 (healthy) and 18 cells (patient). A.U, arbitrary units. (N and O) Healthy and patient myoblasts were colabeled using Tom20-YFP (green) and the mitochondrial potential-sensitive dye TMRE (red). (N) Images of cells before and 20 s after injury. (O) Schematic and plot for quantification of the TMRE/YFP ratio in mitochondria in the injury-distal region of the cell (indicated by dotted line). n = 7 (healthy) and 15 (patient). Scale bars, 10 µm. Plots show mean ± SD; @, P < 0.05; $, P < 0.001; #, P < 0.0001 by Mann-Whitney test or unpaired t test with Welch's correction. Arrowheads indicate site of injury.

Figure 4 .

 4 Figure 4. ANO5-KO impairs myofiber repair and is improved by preventing [Ca 2+ ] c overload. (A-E) Images (A and D) and plots (B and E) showing entry of FM1-43 dye in isolated FDB myofibers (A-C) or intact EDL muscles (D and E) of 10-mo-old ANO5-KO and parental WT mice focally injured by a 10-ms laser pulse. (C) Fraction of injured myofibers that failed to repair. n = 11 FDB myofibers in each group. n = 17 (WT), 19 (KO) EDL myofibers. Arrowheads point to the site of laser injury. (F) EDL muscles from 10-mo-old WT and ANO5-KO mice were subjected to mechanical injury by a series of LC injuries. After each round of injury, the muscle contractile force at isometric tetanic stimulation was measured. n ≤ 7 animals in each group. (G) Images showing cross sections of the EDL muscles following eccentric injury, marking myofibers labeled with the cell-impermeant PO dye following LC injury. Scale bar, 50 µm. (H) Plot showing quantification of PO labeling. (I) Western blots showing SERCA1 levels in bicep muscles from 10-mo-old WT and ANO5-KO mice. (J) Plot showing quantification of SERCA1 expression normalized to Vinculin. n = 4 mice in each group. (K) Pseudocolored images of Fluo-4-labeled isolated FDB myofibers of WT or ANO5-KO mice injured before or following incubation in the cytoplasmic Ca 2+ chelator BAPTA-AM (10 µM). (L) Plots showing kinetics of change in Fluo-4 fluorescence. (M) Plot showing peak Fluo-4 fluorescence. (N) Time for the peak Fluo-4 fluorescence to reduce by 75%. (O) Area under the Fluo-4 kinetics plot for the various groups of myofibers injured. n = 14 (WT, DMSO), 13 (ANO5-KO, DMSO), 13 (ANO5-KO + BAPTA-AM). (P and Q) Images (P) and plot (Q) showing FM1-43 dye entry into isolated FDB myofibers from ANO5-KO mice following treatment with DMSO or BAPTA-AM. (R) Quantification of the fraction of injured myofibers that failed to repair. n = 11 myofibers in each group. Scale bars for A, D, K, and P, 10 µm. Plots in B, E, L, and Q show mean ± SEM. Rest of the plots show mean ± SD. @, P < 0.05; $, P < 0.001; #, P < 0.0001, Mann-Whitney or unpaired t test with Welch's correction.

  Chandra et al. Journal of Cell Biology 9 of 12 Role of the ER in plasma membrane repair https://doi.org/10.1083/jcb.202006035 percentage of first contraction. The values were represented as mean ± SD.

Downloaded from http://rupress.org/jcb/article-pdf/220/5/e202006035/1411452/jcb_202006035.pdf by INSERM user on 19 March 2021

Acknowledgments

We thank Dr. Ibrahim Mahjneh (University of Oulu, Oulu, Finland) and Dr. Vincent Mouly (Sorbonne Universités, Paris, France) for the patient cell line, Tobias Meyer (Stanford Medicine, Stanford, CA) for the gift of pcDNA-T1ER (plasmid 47928; Addgene), and Robert Campbell (University of Alberta, Edmonton, Alberta, Canada) for the gift of cytomegalovirus-mito-CAR-GECO1 (plasmid 46022; Addgene). We thank Dr. Anamaris Colberg-Poley and members of our laboratory for helpful comments during the course of this work.

I. Richard acknowledges financial support from Association Française contre les Myopathies. J.K. Jaiswal acknowledges financial support from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (grant R01AR055686) and the National Institute of Child Health and Human Development (grant U54HD090257).

The authors declare no competing financial interests.Author contributions: G. Chandra and J.K. Jaiswal designed the study. G. Chandra performed experiments through help from S.C. Sreetama with cellular studies, D. Mázala with myofiber calcium measurements, and J.H. VanderMeulen with muscle physiology studies. I. Richard and K. Charton generated and genetically characterized the KO mouse model. J.K. Jaiswal conceived the study, acquired funding, helped with data analysis, and wrote the manuscript together with G. Chandra, and with help from all authors.

Submitted: 6 June 2020 Revised: 11 December 2020 Accepted: 27 January 2021

Supplemental material


Characterisation of interfacial adhesion in hemp composites after H2O2 and non-thermal plasma treatments

Keywords: interfacial adhesion, fragmentation tests, hemp/epoxy composites, hydrogen peroxide, plasma

Interface optimisation for continuous hemp reinforcements in epoxy resin is a current challenge for the development of biocomposites. A chemical treatment based on hydrogen peroxide and a physical one using a non-thermal plasma have been tested to optimise interface adhesion, by varying several parameters. FTIR analysis and FE-SEM observations have shown the effects of the treatments on chemical and morphological aspects of the treated yarns. Tensile tests on hemp yarns have allowed the selection of the treatment parameters leading to the best strength. Fragmentation tests results showed that the two treatments lead to a decrease in the fragment lengths and thus, an enhancement of the Interfacial Shear Strength (IFSS) values in comparison with the untreated yarn. This is confirmed by the micro-CT observations of the debonding lengths in the vicinity of each yarn fragment extremity. Finally, the plasma treated samples exhibit a better interface adhesion quality (IFSS=44.7±4MPa) than the chemically treated ones (IFSS=24.2±4MPa), which are better than the non-treated ones (IFSS=13.5±4MPa).

Characterisation of interfacial adhesion in hemp composites after H2O2 and non-thermal plasma treatments R. Barbière 1 , F. Touchard 1 , L. Chocinski-Arnault 1 , E. Fourré 2 , E. Leroy 3 , J. Barrault 3 e-mail : romain.barbiere@ensma.fr e-mail : fabienne.touchard@ensma.fr (corresponding author) e-mail : laurence.chocinski@ensma.fr e-mail : elodie.fourre@univ-poitiers.fr e-mail : eleroy@valagro-rd.com e-mail : joel2.barrault@gmail.com

Introduction

The environmental consciousness as well as the European legislation has encouraged academic and industrial research to develop eco-friendly, sustainable and recyclable composite materials, often referred to as "green composites". The interest in natural plant fibres as reinforcement in polymer matrix has grown quickly in the last decade. Industries as automotive, shipbuilding and construction have started the manufacturing of products using natural fibres [START_REF] Gowda | Polymer matrix-natural fiber composites: An overview[END_REF][START_REF] Girijappa | Natural Fibers as Sustainable and Renewable Resource for Development of Eco-Friendly Composites: A Comprehensive Review[END_REF][START_REF] Gholampour | A review of natural fiber composites: properties, modification and processing techniques, characterization, applications[END_REF][START_REF] Flax | Linum usitatissimum L.) fibre reinforced polymer composite materials: A review on preparation, properties and prospects[END_REF]. However, these biocomposites are mainly used for secondary or tertiary structures, because of their poor mechanical properties, which depend on the individual mechanical properties of the matrix and fibres as well as on the fibre/matrix interfacial bonding. Interfacial adhesion between the fibres and the matrix plays a fundamental role in the mechanical characteristics of the composite [START_REF] Zhou | Interface and bonding mechanisms of plant fibre composites: An overview[END_REF][START_REF] Latif | Surface treatments of plant fibers and their effects on mechanical properties of fiber-reinforced composites: A review[END_REF][START_REF] Perrier | Mechanical behaviour analysis of the interface in single hemp yarn composites: DIC measurements and FEM calculations[END_REF]. The factors affecting the interfacial bonding between the fibre and matrix are the mechanical interlocking, the molecular attractive forces, the electrostatic adhesion and the chemical bonds. However, the naturally hydrophilic plant fibres are not inherently compatible with polymers, which are comparatively more hydrophobic [START_REF] Perrier | Influence of water on damage and mechanical behaviour of single hemp yarn composites[END_REF]. In addition to the pectin and waxy substances in plant fibre acting as a barrier to interlock with nonpolar polymer matrix, the presence of numerous hydroxyl groups hinders its operative reaction with the matrix [START_REF] Mohit | A comprehensive review on surface modification, structure interface and bonding mechanism of plant cellulose fiber reinforced polymer based composites[END_REF]. Therefore, the modification of the surface characteristics of plant fibre is essential in order to formulate a composite with superior interfacial bonding and effective stress transfer throughout the interface [START_REF] Bartos | Alkali treatment of lignocellulosic fibers extracted from sugarcane bagasse: Composition, structure, properties[END_REF]. Two types of methods can improve the fibrematrix interface: chemical and physical ones. Among the chemical ones, the most common treatments are using liquid ammonia, permanganates, isocyanates or consist in silane coupling, esterification and graft copolymerization [START_REF] Kabir | Chemical treatments on plant-based natural fibre reinforced polymer composites: An overview[END_REF]. Commonly used physical methods are thermotreatment, electric discharge, corona or plasma treatment. In particular, non-thermal plasma process allows surface modification without the use of chemicals, nor solvents. It is a fast, environmentally friendly, nontoxic and dry process [START_REF] Ventura | Effects of hydrothermal aging on the water uptake and tensile properties of PHB/flax fabric biocomposites[END_REF]. For this method, an ionised gas is created by applying enough energy to reorganise the electronic structure of the species present in the gas phase. This kind of treatment can be useful to activate the fibre surface and enhance the interfacial adhesion, as it has been shown, for example, in flax/polyethylene composites [START_REF] Enciso | Influence of Low Pressure Plasma Treatment on the Durability of Thermoplastic Composites LDPE-flax/coconut under Thermal and Humidity Conditions[END_REF].

A number of studies have been carried out on the effects of different fibre surface treatments on the properties of natural fibre composites [START_REF] Zhou | Interface and bonding mechanisms of plant fibre composites: An overview[END_REF][START_REF] Werchefani | Effect of chemical and enzymatic treatments of alfa fibers on polylactic acid bio-composites properties[END_REF][START_REF] Tita | Chemical modification of sugarcane bagasse and sisal fibers using hydroxymethylated lignin: Influence on impact strength and water absorption of phenolic composites[END_REF]. In particular, concerning hemp composites, the effect of NaOH treatments [START_REF] Troëdec | Influence of chemical treatments on adhesion properties of hemp fibres[END_REF][START_REF] George | Characterization of chemically and enzymatically treated hemp fibres using atomic force microscopy and spectroscopy[END_REF], silane treatments [START_REF] Moonart | Effect of surface treatments and filler loading on the properties of hemp fiber/natural rubber composites[END_REF] or alkalisation treatments [START_REF] Mazzanti | Reinforcing mechanisms of natural fibers in green composites: Role of fibers morphology in a PLA/hemp model system[END_REF][START_REF] Dayo | The influence of different chemical treatments on the hemp fiber/polybenzoxazine based green composites: Mechanical, thermal and water absorption properties[END_REF] have been studied. Other treatments based on vanillin alcohol [START_REF] Touchaleaume | Polyhydroxybutyrate/hemp biocomposite: tuning performances by process and compatibilisation[END_REF] or on the deposition of reactive monomers by plasma [START_REF] Brunengo | Chemical modification of hemp fibres by plasma treatment for eco-composites based on biodegradable polyester[END_REF] have also been tested. Most of the studies concern short fibre or mat composites [START_REF] Tanasa | Modified hemp fibers intended for fiber-reinforced polymer composites used in structural applications-A review. I. Methods of modification[END_REF]. However, in those composites, the fibre orientation is random and, consequently, mechanical properties are not as high as in continuous fibre composites. There is a lack of data concerning the interface optimisation for continuous hemp reinforcements.

In the present study, continuous hemp reinforcement in epoxy matrix is used. Hemp (Cannabis sativa L.) is one of the oldest crops and is now considered as one of the most eco-friendly industrial fibres [START_REF] Manaia | Industrial Hemp Fibers: An Overview[END_REF]. The aim of this work is to enhance the interface adhesion quality in hemp/epoxy composites. For this purpose, two different types of treatments are applied to hemp yarns. The first one consists in a chemical treatment using hydrogen peroxide and the second treatment is physical, using a non-thermal plasma. For each of these treatments, different parameters are tested and the chemical and morphological effects on hemp yarns are analysed by FTIR (Fourier Transform Infrared) spectroscopy and FE-SEM (field-emission gun scanning electron microscopy). The untreated and treated hemp yarns are tested in tensile loading in order to compare their strengths.

The aim is to find a good compromise between the effectiveness of the treatment and the preservation of the yarn mechanical properties. Fragmentation tests on single yarn hemp/epoxy composite specimens are performed and interfacial shear strength (IFSS) values are determined after measuring the obtained fragment lengths. It allows the characterization of the yarn/matrix interface adhesion quality at the relevant scale, without the influence of other neighbouring yarns.

Moreover, this type of interface test not only allows to check the adhesion quality but also the mechanical properties of the chosen yarn-matrix couple [START_REF] Seghini | Interfacial adhesion assessment in flax/epoxy and in flax/vinylester composites by single yarn fragmentation test: Correlation with micro-CT analysis[END_REF]. A complete analysis of the interfacial debonding mechanism is also carried out using X-ray micro-CT.

Materials and methods

Tested materials

Hemp yarns were provided by Lin et L'autre (France). They were made of bundles of hemp fibres twisted together with a mean twisting angle around 11° and a linear density of about 83tex, as determined in a previous study [START_REF] Guillebaud-Bonnafous | Experimental and numerical investigation of the interface between epoxy matrix and hemp yarn[END_REF]. The untreated hemp yarns were used as reference and noted "Ref". The tested hemp yarns were considered cylindrical; their mean diameter was measured using an optical microscope and was found to be equal to 308±25µm.

The single yarn composite specimens were manufactured with resin injection moulding technique.

The used resin is the EPOLAM 2020 epoxy resin from Axson. A special mold was developed to elaborate composite samples with a single hemp yarn aligned in the loading direction. The specimens were dogbone shaped, with a gauge section of 15mm long, 3mm wide and 2mm thick (Fig. 1). They were cured with the following cycle: 24h at ambient temperature, 3h at 40°C, 2h at 60°C, 2h at 80°C and 4h at 100°C, to achieve a cross-linkage as complete as possible. The final glass transition temperature measured by DSC (Differential Scanning Calorimetry) was 89 ± 2°C [START_REF] Barbière | Fatigue behaviour and interfacial optimization of a woven hemp/epoxy composite: moisture effect[END_REF].

Hydrogen peroxide treatments

At first, hemp yarns were dried under vacuum at 60°C during 24h. Then they were immersed in a basic solution of hydrogen peroxide during 15 minutes at room temperature. Three different treatments with various concentrations of H2O2 (weight %) were used: 10%, 30% and 50%, noted H-T10, H-T30 and H-T50 respectively. Hemp yarns were then washed in distilled water and finally dried. Two different types of drying sequence were applied. For each treatment, hemp yarns were either dried under vacuum at 60°C during 24h, or dried in oven at 103°C during 24h. These two drying sequences were noted S1 and S2 respectively. Six different H2O2 treatments were thus obtained: H-T10S1, H-T10S2, H-T30S1, H-T30S2, H-T50S1 and H-T50S2.

Non-thermal plasma treatments

For the non-thermal plasma treatment, the reactor that was used had a cylindrical configuration (Fig. 2). The first electrode consisted in a stainless steel coiled filament, fitting to the inner wall of the glass cylinder (4cm diameter). The second electrode was a copper sheet (Advance tape) taped around the external wall of the cylinder. The total height of the plasma surface in the cylinder was 10cm. The electrodes were connected to a high voltage generator (A2E Technologies Enertronic) providing bipolar pulses. The signals were measured via two high voltage probes (Lecroy, PPE20KV-CC) and a current probe (Stangenes Industry 60MHz) connected to an oscilloscope (Lecroy WaveSurfer, 64Xs-A, 600MHz). Different parameters were tested for hemp yarn treatment, via the chemical nature of the gas (air or O2; flow rate: 100ml.min -1 ), the consumed energy (15 or 25W) and the duration of the treatment (2 to 10min). The yarns were placed around the filament coil and three different plasma treatments were compared: a treatment in air atmosphere with a power of 25W (noted Pl-T1), a treatment also in air atmosphere but with a power of 15W (noted Pl-T2) and a treatment in pure oxygen with a power of 25W (noted Pl-T3).

FTIR

Fourier Transform Infrared (FTIR) spectra of untreated and treated hemp yarns were recorded. For the chemical treatments, a Thermo 6700 FTIR spectrometer (resolution: 2cm -1 , 16 scans) equipped with a deuterated-triglycine sulfate (DTGS) detector was used. For the plasma treatments, a Perkin Elmer FTIR (Spectrum One) spectrometer was used, in the range of 4000-650cm -1 . The spectra were normalised for comparison. Each yarn was analysed at three different positions after each treatment in order to evaluate the homogeneity of the treatment.

Microscopy

A morphological investigation of the surface of treated and untreated hemp yarns was carried out using a field-emission gun scanning electron microscope (FE-SEM) JEOL JSM-7000F. All specimens were sputter coated with gold prior to FE-SEM observation. After fragmentation tests, the fragment lengths were measured by using a Leica MZ95 microscope.

Micro-CT

A micro-CT analysis was performed using an UltraTom CT scanner manufactured by RX Solution (France). The system consists in a Hamamatsu micro focus sealed X-ray tube operating at 20-150kV/0-500 µA, within a maximum power of 75W. X-rays, generated by the source, diverge at an angle providing a cone-beam. Various geometric magnifications can thus be obtained by moving the sample close to the source to provide high resolution mode or close to the detector to provide low resolution measurements. The generator and the detector are also mobile to cover a large range of magnification. A 1.5µm resolution was used in this work, with an accelerating voltage of 65kV and a beam current of 144µA. The image acquisition time was about 6h per specimen. The 3D reconstruction was performed using an algorithm based on the filtered back-projection procedure for Feldkamp cone beam geometry.

Tensile testing

Tensile tests were performed on single hemp yarns according to ASTM C-1557. Each yarn was mounted on paper frames with a central window of 10mm long. Hemp yarns were tested using an INSTRON 1195 machine with a load cell of 500N and a crosshead speed of 0.1mm/min. At least ten tests were performed for each type of yarn.

Fragmentation tests

The investigation of the interfacial adhesion of hemp yarns in epoxy resin was achieved via fragmentation tests. When applying a tensile loading to single hemp yarn composites, the load is transferred through the matrix into the yarn by means of shear stress at the interface. Yarn failure occurs when this transferred stress reached the tensile strength of the yarn. The yarn will continue to fracture into shorter length fragments as the load increases, until the yarn fragments are so small that the tensile stresses induced in the yarn can no longer reach the yarn tensile strength. At this point, a state of saturation is reached and the fragmentation process ceases. The fragmentation tests were performed using the INSTRON 1195 machine with a load cell of 500N. Tests were carried out at a rather low loading rate (0.01mm/min) in order to reach the fragmentation saturation level before the specimen failure. The loading phase was stopped if the specimen failed, or when the fragmentation saturation level was achieved, which, in the present case, was defined as occurring when no new yarn break appeared during a subsequent strain increase by 0.5%, as frequently reported in literature [START_REF] Joffe | Interfacial shear strength of flax fiber/thermoset polymers estimated by fiber fragmentation tests[END_REF]. 80 grit sand papers were used in the jaws to improve clamping. After testing, fragment lengths were measured using an optical microscope and a statistical analysis was performed.

Results and discussion

Chemical and morphological effects on hemp yarns

Peroxide is a chemical compound with the functional group ROOR containing the divalent ion bond O-O. In contrast to oxide ions, the oxygen atoms in the peroxide ion have an oxidation state of -1. It is inclined to decompose to free radicals of the form RO . , and then the RO . can be grafted onto the cellulose macromolecule polymer chain by reacting with the hydrogen groups of plant fibre, as explained by Zhou et al. [START_REF] Zhou | Interface and bonding mechanisms of plant fibre composites: An overview[END_REF]. In order to investigate the effect of the hydrogen peroxide treatments on the chemical composition of hemp yarns, Fourier Transform Infrared (FTIR) analysis was performed. As examples, FTIR spectra in Fig. 3 show the results for the untreated (reference) hemp yarn and the hemp yarns after H-T10S2 (10%, 103°C) and H-T50S2 (50%, 103°C) treatments.

Untreated hemp yarn consists of alkane, esters, aromatics, ketone and alcohol belonging to cellulose, hemicellulose and lignin [START_REF] Seghini | Effects of oxygen and tetravinylsilane plasma treatments on mechanical and interfacial properties of flax yarns in thermoset matrix composites[END_REF]. Signals between 3600 and 3000cm -1 are consistent with stretching vibration of OH bonds of cellulose and hemicellulose (Table 1). Fig. 3 shows that this part of the spectrum reveals the presence of hydrogen bonding in the intra and inter molecular cellulose network. Similar effects can be observed over IR bands in the 2935-2862cm -1 domain (C-H bond stretching) and in the 1125-895cm -1 domain (which characterises the polysaccharidic nature of fibres). The hydrogen peroxide activation modified the infrared signals. Overall, a sharpening of the main infrared bands can be observed. It is due to the fact that the peroxide treatment led to the removal of lignin, waxy substances and impurities, the 50% H2O2 concentration being slighter more beneficial to oxidation than the 10% one. The main change is observed on the band at 1735cm -1 .

This can be attributed to the formation of carbonyl and/or carboxylic bonds (from the oxidation treatment). It is also worth mentioning that this band is more important for the H-T50S2 sample than for the H-T10S2 one. Similar results were obtained for the drying sequence S1. FTIR results were confirmed by FE-SEM analysis, which revealed the surface morphology of the untreated (reference) and hydrogen peroxide treated hemp yarns. Fig. 4 allows the comparison of the three different H2O2 concentrations, 10%, 30% and 50%, for the drying sequence S2. The higher the H2O2 concentration, the cleaner the hemp fibres due to the leaching out of waxes, gums and pectic substances. The fibrillar structure of the yarn with the individual ultimate fibres is revealed for the strongest treatment H-T50S2, even if there is still some remaining external substances (Fig. 4c).

For the non-thermal plasma treatment, different durations were tested. For 2min duration, an inhomogeneous oxidation of the yarn surface was detected by FTIR analysis at different locations along the yarn. For 10min duration, a degradation of the yarn was observed, with a color change to dark brown. Finally, an intermediate treatment time of 5min was chosen and different gas flow chemical natures and power values were tested. FTIR spectra for Pl-T1 (air, 25W), Pl-T2 (air, 15W) and Pl-T3 (O2, 25W) treatments are presented in Fig. 5. Oxidation is visible on every spectrum with the appearance of a band at 1735cm -1 , corresponding to a C=O stretching bond. In terms of band intensities, the strongest oxidation was obtained after 5min of air plasma at 15W (Fig. 5A) and O2 plasma at 25W (Fig. 5C).. The band at 1635cm -1 corresponds to OH band from water (Table 1). FE-SEM observations on plasma treated hemp yarns showed no clear differences with the untreated yarn, demonstrating that there is no significant modification of the yarn surface morphology after this type of treatment.

Tensile strength of treated and untreated hemp yarns

The aim of chemical and physical treatments applied on hemp yarns is to enhance the interfacial adhesion between yarns and epoxy matrix. However, these treatments can also have consequences on the yarn mechanical properties. A compromise has to be done between the treatment efficiency and the residual mechanical properties of the yarns. Indeed, the composite laminates made of treated yarns should have good interfacial adhesion quality together with high mechanical characteristics. Therefore, tensile tests were realised on single hemp yarns and a first selection between the different treatments was performed thanks to the comparison of the tensile strength values (σu) between treated and untreated yarns (Table 2). First results concern the chemically treated hemp yarns. As it can be seen in Fig. 6, despite the experimental dispersion, the global tendency shows that the drying sequence S2 (i.e. in oven at 103°C during 24h) leads to slightly higher mean values than the drying sequence S1 (i.e. under vacuum at 60°C during 24h). Moreover, all the treatments allow to obtain higher mean σu values than the one measured for untreated hemp yarns. The maximum σu value is reached for the H-T10S2, i.e. the treatment with the lowest H2O2 concentration level. Therefore, H-T10S2 samples were selected for the fragmentation tests.

The same analysis was performed on plasma treated hemp yarns. Fig. 7 shows the results obtained for the three different plasma tests. The plasma treatments with the highest power value (25W) lead to a decrease in the tensile strength of the hemp yarns, independently of the gas flow chemical nature. The hemp yarns with the plasma treatment Pl-T2 (air, 15W) are the only ones to exhibit a mean σu value equivalent to the one of the untreated hemp yarns. This treatment is also the most ecological one, using less energy and being under air atmosphere. Therefore, this plasma treatment has been selected for fragmentation tests.

Interfacial shear strength

Fragmentation tests were performed on single hemp yarn/epoxy samples for the three selected types of yarns: untreated ones (reference), H2O2 treated yarns (H-T10S2) and plasma treated yarns (Pl-T2). An example of fragmentation test is shown in Fig. 8. By looking carefully at the tensile curve, it is possible to observe a slight decrease in the stress at each fragmentation occurrence (Fig. 8b). At the end of the test, after the sample failure, several fragments are visible in the gauge length (Fig. 8c). Each fragment length, lf, is then measured by optical microscopy on all the tested samples. The critical fragment length value can thus be determined by using Eq. 1 [START_REF] Ohsawa | Temperature dependence of critical fiber length for glass fiber-reinforced thermosetting resins[END_REF]:

=

Eq. 1

It allows plotting the Weibull distribution of critical fragment lengths for each tested configuration (Fig. 9). Despite the dispersion, results show that the samples with the treated hemp yarns exhibit lower lc values than the untreated ones. Moreover, samples with the plasma treatment show lower lc values than samples with the H2O2 treatment (Fig. 9).

In order to compare the adhesion quality of the different treatments, the IFSS (Interfacial Shear Strength) values have been calculated using Eq. 2 based on the Kelly and Tyson law [START_REF] Seghini | Interfacial adhesion assessment in flax/epoxy and in flax/vinylester composites by single yarn fragmentation test: Correlation with micro-CT analysis[END_REF][START_REF] Kelly | Tensile properties of fibre-reinforced metals: Copper/tungsten and copper/molybdenum[END_REF]:

= × × ×
Eq. 2 where is the representative critical fragment length determined by Weibull analysis, is the yarn ultimate tensile force extrapolated at a length equal to the critical fragment length lc and determined thanks to the Weibull parameters [START_REF] Zafeiropoulos | On the use of single fibre composites testing to characterise the interface in natural fibre composites[END_REF], and the average hemp yarn diameter. Results are reported in Table 3. Results show that treatments lead to an increase in the IFSS values in comparison with the untreated material. The plasma treatment seems to be the most efficient with the greatest increase in the adhesion quality (44.7MPa vs 13.5MPa for the untreated sample). This high IFSS value is directly linked to the decrease in the fragment length of more than fifty percent compared to the untreated single yarn composite (Table 3).

After fragmentation tests, a morphological investigation of the fracture surface has been performed using the FE-SEM. Fig. 10 shows micrographs obtained on single yarn composites with untreated and treated hemp yarns. Two treatments are compared: the H2O2 treatment H-T10S2 (10%, 103°C)

and the plasma treatment Pl-T2 (air, 15W). For the H2O2 treatment, it can be seen in Fig. 10a that many elementary hemp fibres have been pulled out from the matrix at the moment of failure in the reference sample. Fewer fibres have been pulled out from the matrix in the case of the treated hemp yarns (Fig. 10b andc), which confirms the better interfacial adhesion after the treatments.

Moreover, for the plasma treatment, Fig. 10c shows that many fibres are still well-bonded to the matrix and have broken in the matrix failure plane. It confirms the better adhesion quality obtained with the plasma treatment.

Interfacial debonding

Another parameter allowing the comparison of the interfacial adhesion quality is the debonding length, characterising the interfacial decohesion which occurs around each yarn failure. Fig. 11a presents a micrograph, acquired with polarised light, of a single untreated hemp yarn/epoxy composite after fragmentation test and before sample failure. As it can be seen in Fig. 11b, the debonding length values, ld, can be easily measured as the length of the black area around each yarn failure. Therefore, the debonding length values were determined by optical micrography observations around each fragmentation in the tested samples for the three configurations: with untreated yarns (reference), with Pl-T2 (Plasma, air 15W) treated yarns and with H-T10S2 (H2O2, 10% 103°C) ones. The obtained Weibull distributions are plotted in Fig. 12. Results in Fig. 12 prove that the debonding lengths present the same ranking as for the critical fragment lengths in Fig. 9. For the two selected treatments, the debonding phenomenon is lower than for the untreated case. The lowest values are obtained for the single yarn composite samples made with plasma treated hemp yarns. Representative values of debonding lengths obtained by Weibull analysis for each configuration are given in Table 3.

Micro-CT observations were also performed on fragmented samples for each type of treatment. An example is given in Fig. 11 for an untreated sample. Thanks to the very fine micro-CT resolution (1.5µm for 1 voxel), it was possible to visualise the small black lines corresponding with the interfacial debonding between the hemp yarn and the matrix (Fig. 11c). By looking closely at the yarn cross-section in the sample, it appears that the yarn/matrix interface in the healthy zone of the gauge length (i.e. far from the yarn failure) is undamaged (Fig. 11e), while damage is present all around the yarn in the debonding zone near the yarn fragment extremity (Fig. 11d). By using the micro-CT scans, it is also possible to build a 3D reconstruction of the damage near the yarn failure zone (Fig. 11f). It allows a real representation of the debonding phenomenon occurring at the yarn/matrix interface in such a composite material.

In Fig. 13, observations made by micro-CT in the (x,y) plane are presented for untreated yarn, H-T10S2 and Pl-T2 treated yarns. Focus is done on a yarn failure zone, allowing the measurement of the corresponding debonding length. These micro-CT observations show that the same average values of the interfacial debonding zone is obtained by the two methods, X-ray micro-CT and optical micrography, and the same classification can be made: the debonding phenomenon is more significant for the untreated hemp yarn than for the H2O2 treated one, which is more pronounced than for the plasma treated yarn. These results confirm that IFSS values determined by fragmentation tests on single yarn composites are directly linked with debonding lengths at the yarn/matrix interface. The higher the IFSS value, the shorter the interfacial debonding and thus, the better the adhesion quality at the interface. Therefore, the comparison between untreated and treated hemp/epoxy composites shows that hydrogen peroxide and non-thermal plasma treatments enhance the interfacial adhesion quality, the plasma method being more efficient than the chemical one. As the plasma method is the most "ecological" one, without chemicals nor solvents, these are promising results for the optimization of plant fibre composites.

Conclusion

The use of plant fibre composites for engineering applications has considerably increased in the recent years, especially for the transportation domain. Understanding the interface and bonding mechanisms of these composites is a key issue to maximise their industrial applications. This paper deals with the interface optimisation of continuous hemp reinforcements in epoxy resin using two different types of treatment. The first one consists in a chemical treatment based on hydrogen peroxide and the second one is a physical treatment using a non-thermal plasma. For these two methods, different parameters were studied: the H2O2 concentration and the drying sequence for the hydrogen peroxide treatment; the duration, the chemical nature of the gas phase and the consumed energy for the plasma treatment. FTIR analysis and FE-SEM observations have shown the effects of both treatments on chemical and morphological aspects of the hemp yarns. Tensile tests on hemp yarns with and without treatments have allowed the selection of the parameters leading to the best tensile strength values. Single hemp yarn composite samples were then made with epoxy resin using an original specific mould, allowing to align the yarn in the longitudinal axis direction.

Fragmentation tests were performed with three types of yarns: non-treated hemp yarns, chemically treated ones with a weight concentration of H2O2 of 10% and a drying sequence in oven at 103°C during 24h, and plasma (15W) treated yarn in air atmosphere during 5min. Results show that the two treatments lead to a decrease in the fragment lengths, thus enhancing the Interfacial Shear Strength (IFSS) values in comparison with the untreated yarn, the plasma treatment being the most effective. This was confirmed by micro-CT observations. Debonding lengths values measured around each fragmentation in the reference samples are higher than those measured in the H2O2 treated samples, which are themselves higher than those measured in the plasma treated ones.

The next step will be to apply these treatments at the fabric scale, in order to manufacture woven hemp/epoxy composites with treated reinforcement. If this up-scale step is quite easy for the H2O2 treatment, it has still to be developed for the non-thermal plasma one.

Table 1: Main infrared vibration bands of plant fibres (from [START_REF] Célino | Qualitative and quantitative assessment of water sorption in natural fibres using ATR-FTIR spectroscopy[END_REF]).

Wave number (cm

Fig. 1 .

 1 Fig. 1. a) Mould used for sample manufacture. b) Specimen geometry for the single yarn

Fig. 2 .

 2 Fig. 2. Scheme of the non-thermal plasma reactor.

Fig. 3 .

 3 Fig. 3. Infrared spectra of the different hemp yarns: untreated (reference) and hydrogen peroxide

Fig. 4 .Fig. 5 .Fig. 6 .

 456 Fig. 4. SEM micrographs of hydrogen peroxide treated hemp yarns for three different H2O2

Fig. 7 .

 7 Fig. 7. Tensile strength values of untreated (reference) and plasma treated hemp yarns, for the three

Fig. 8 .

 8 Fig. 8. Example of fragmentation test on a single hemp yarn/epoxy composite. a) full tensile curve,

Fig. 9 .Fig. 10 .

 910 Fig. 9. Cumulative distribution of critical fragment lengths measured on single hemp yarn/epoxy

Fig. 11 .Fig. 12 .Fig. 13 .

 111213 Fig. 11. Microscopic pictures of a single untreated hemp yarn/epoxy composite after fragmentation

Fig. 1 .Fig. 2 .

 12 Figure captions:

Fig. 3 .

 3 Fig. 3. Infrared spectra of the different hemp yarns: untreated (reference) and hydrogen peroxide

Fig. 4 .

 4 Fig. 4. SEM micrographs of hydrogen peroxide treated hemp yarns for three different H2O2

Fig. 5 .Fig. 6 .

 56 Fig. 5. Infrared spectra after 5 minutes of plasma treatment: A) Pl-T2: air, 15W; B) Pl-T1: air, 25W;

Fig. 7 .

 7 Fig. 7. Tensile strength values of untreated (reference) and plasma treated hemp yarns, for the three

Fig. 8 .

 8 Fig. 8. Example of fragmentation test on a single hemp yarn/epoxy composite. a) full tensile curve,

Fig. 9 .

 9 Fig. 9. Cumulative distribution of critical fragment lengths measured on single hemp yarn/epoxy

Fig. 10 .

 10 Fig. 10. SEM micrographs of single yarn composite fracture surfaces after fragmentation test. a) for

Fig. 11 .

 11 Fig. 11. Microscopic pictures of a single untreated hemp yarn/epoxy composite after fragmentation

Fig. 12 .

 12 Fig. 12. Cumulative distribution of debonding lengths measured by optical micrography on single

Fig. 13 .

 13 Fig. 13. Micro-CT images of single hemp yarn/epoxy composites after fragmentation tests for a)

Table 2 :

 2 Experimental tensile strength values of single hemp yarns for untreated (reference), hydrogen peroxide treated and plasma treated yarns.

	Hemp yarn	u (MPa)	Dispersion (MPa)
	Untreated	Reference	219	53
		H-T10S1	246	44
		H-T10S2	287	47
	Hydrogen peroxide	H-T30S1	235	50
	treated	H-T30S2	278	42
		H-T50S1	255	52
		H-T50S2	267	43
		Pl-T1	191	54
	Plasma treated	Pl-T2	217	48
		Pl-T3	187	51

Table 3 :

 3 Yarn ultimate tensile force, representative critical fragment length, IFSS value and representative debonding length for untreated and treated hemp yarn/epoxy samples.

	Hemp yarn treatment	(N)	(mm)	IFSS (MPa)	(mm)
	Untreated (reference)	18.4 ± 3.5	3.2 ± 0.70	13.5 ± 4	0.46 ± 0.07
	H-T10S2	27.5 ± 3.5 2.35 ± 0.15	24.2 ± 4	0.37 ± 0.04
	Pl-T2	23.7 ± 3.5	1.4 ± 0.15	44.7 ± 4	0.30 ± 0.05

Acknowledgements: This work was partially funded by the French Government program "Investissements d'Avenir" (EQUIPEX GAP, reference ANR-11-EQPX-0018) and by the CPER FEDER project of Région Nouvelle Aquitaine. It pertains to the French Government program "Investissements d'Avenir" (EUR INTREE, reference ANR-18-EURE-0010).