Actin Polymerization Defects Induce Mitochondrial Dysfunction in Cellular Models of Nemaline Myopathies

1. Introduction

Congenital myopathies are a group of genetic muscle diseases that are classified based on the histopathological features observed on muscle biopsy [1]. Among these disorders nemaline myopathy (NM) was first described as a non-progressive congenital musculoskeletal disorder, characterized by the presence of inclusions in muscle fibers called "nemaline rods" [2]. This pathology contains a wide genetic heterogeneity, since multiple mutations in different genes that produce a similar phenotype have been identified [3]. Its incidence is approximately 1 in 50,000 live births.

The International European Neuromuscular Group classifies NM into six clinical subtypes based on the severity, age of onset, and degree of muscle weakness: severe congenital (neonatal), Amish, intermediate congenital, typical congenital, childhood onset, and adult-onset [4]; Of these, the most common subtype corresponds to the typical congenital subtype [5]. The most common clinical presentation is characterized by an onset in early infancy or childhood with hypotonia or generalized weakness predominantly in facial, axial, and proximal limb muscles. Additional features include skeletal deformities, dysmorphic fascia, arched high palate, and respiratory distress with respiratory tract infections [1]. The natural history of the disease is usually static or very slowly progressive and many of the patients can lead normal lives. Serious clinical complications are almost always secondary to respiratory deficiency and sometimes to cardiological problems. In most cases, cardiomyopathy develops in adulthood, while rarely occurs in childhood [6].

There are 14 known causative genes of NM [7]. Mutations in the ACTA1 and NEB genes, which code for actin 1 alpha (ACTA1) and nebulin (NEB) proteins, respectively, critical components of the sarcomeric thin filament, result in the most prevalent types of NM. More specifically, it is predicted that mutations in the NEB gene account for over 50% of cases of NM, whereas mutations in the ACTA1 gene account for between 15%-25% of cases [8]. NEB, known as the giant actin-binding protein because it has a molecular weight of between 600-800 kDa, plays a very important role in stabilizing and regulating the length of the actin filament [9]. ACTA1 is the actin isoform predominantly found in the thin filaments of skeletal muscles and essential, along with myosin, for muscle contraction [10].

The diagnosis is established from the clinical suspicion, since paraclinical studies such as creatinine kinase levels may be normal or slightly elevated and electromyography may show nonspecific myopathic changes. The diagnosis is confirmed histopathologically by muscle biopsy analysis, in which characteristic rod bodies (nemaline bodies) are found in the sarcoplasm, evidenced by modified Gomori stain [1]. Increasingly the diagnosis is made or confirmed by molecular genetic testing for mutations in the genes known to cause NM.

The etiology of the disease remains unclear. The accumulation of nemaline bodies by itself does not explain the muscle weakness characteristic of the disease and could be only a secondary phenomenon of the main pathogenic process. In fact, no correlation has been observed between the severity of the disease and the degree of accumulation of nemaline bodies [11]. In addition to nemaline bodies, pathological defects have been also observed in skeletal muscle fibers both in mouse models and in patients with NM: abundant, unevenly spaced, and with irregular morphology nuclei; interrupted nuclear envelope; impaired chromatin arrangement; and cytoskeletal disorganization [12]. Due to the important role of the nuclear form and the envelope in the regulation of gene expression, and of the cytoskeleton in maintaining the integrity of the muscle fiber, it is likely that these alterations are responsible for some distinctive features of the disease, such as the disorder of the contractile filaments and the altered mechanical properties.

The disease has also been associated with mitochondrial dysfunction, specifically, with complex I dysfunction or deficiency [13]. One hypothesis proposes that by disrupting the integrity of the thin filaments and, consequently, disrupting normal muscle function, less energy is used and there is a "down-regulation" of complex I activity and, therefore, adenosine triphosphate (ATP) production [13]. In addition, regarding the formation of nemaline rods, numerous rods were observed in some patients with complex I deficiency [14]. It has been seen that the formation of these can be induced in muscle and non-muscle cells in vitro by several different cellular stressors [15]. These data suggest that metabolic alterations during the formation and turnover of sarcomeres may induce the formation of nemaline rods. However, it is still unclear how these two pathological phenomena are related. Furthermore, interactions between mitochondria and the actin cytoskeleton have been linked to essential functions of this organelle [16]. Thus, actin filaments primarily modulate mitochondrial dynamics [17,18], trafficking and autophagy [19], but also mitochondrial biogenesis and metabolism [20]. Therefore, it is reasonable to deduce that the actin polymerization defects in NM such as ACTA1 and NEB mutations may affect mitochondrial function.

Animal and cell models are needed to better understand the mechanisms involved in the development of the disease. Currently, animal, and in vitro models of NM are being explored, specifically mouse and zebrafish models [21]. Respect to cellular models, in addition to in vitro contraction studies of muscle fibers [22], functional studies have been conducted in specific genetic mutations and their proteins in several cell lines, such as NIH3T3 fibroblasts, C2C12 myoblasts or Sol 8 myogenic cells that differentiate into myotubes [23,24]. Most patient mutations studied by cell transfection reveal cellular defects typical of the disease, such as actin aggregates and the formation of nemaline bodies seen in patients' muscle biopsies. On the other hand, regarding human cell lines of the disease, recently, two isogenic lines of induced pluripotent stem cells (iPSC) derived from a patient with severe nemaline myopathy with a dominant heterozygous mutation in the ACTA1 gene have been achieved [25]. Alternatively, patient-derived skin fibroblasts can be easily obtained by small biopsies of the skin in a non-invasive way, have a great capacity for division and harbor the specific mutations of the patients [26].

In this manuscript, we have evaluated whether fibroblasts derived from NM patients carrying ACTA1 and NEB mutations can be useful cellular models for studying disease pathophysiology. The work is based on the hypothesis that both affected proteins, ACTA1 and NEB, may participate in the formation and stabilization of actin filaments, and therefore, alterations of actin polymerization can be visualized in patient-derived fibroblasts. In addition, we examined the consequences of actin polymerization defects on mitochondrial function. Finally, we also evaluated the correction of all pathological alterations by mitochondrial targeting compounds such as linoleic acid (LA) and L-carnitine (LCAR).

2. Materials and Methods

2.1. Reagents

The subsequence antibodies were acquired from Abcam (Cambridge, United Kingdom): Alpha Skeletal Muscle Actin (ab28052), Alpha tubulin (ab7291), NADH:Ubiquinone oxidoreductase subunit A9 (NDUFA9) (ab14713), cytochrome c oxidase subunit IV (COXIV) (ab14744), ATP synthase F1 subunit alpha (ATP5F1A) (ab1478), Voltage-depebdent anion channel 1 (VDAC1) (ab14734) and Superoxide Dismutase 2 (SOD2) (ab68155). The next antibodies were purchased from Invitrogen (Thermo Fisher Scientific (Whaltham, MA, United States)): Rho (A, B, C) (PAI-338), Rho-associated protein kinase 1 (ROCK1) (PA5-22262), Phospho-ROCK1 (PA5-36763) and NADH:Ubiquinone oxidoreductase core subunit S1 (NDUFS1) (PA5-22309). RhoA (8789S), Vimentin (D21H3) and Glutathione Peroxidase 4 (GPX4) (52455S) were purchased from Cell Signaling (Danvers, MA, United States). Antibodies for beta actin (MBS448085) and Mitochondrially Encoded NADH:Ubiquinone oxidoreductase core subunit 1 (Mt-ND1) (6888S) were purchased from MyBioSource (San Diego, CA, United States). Cytochrome b-c1 complex subunit 2 (UQCR2) was supplied from US Biological (Salem, MA, United States). The following antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, United States): dynamin-related protein 1 (DRP1) (sc-32898), succinate dehydrogenase complex iron-sulfur subunit B (SDHB) (sc-271548) and Superoxide Dismutase 1 (SOD1) (sc-101523). Optic atrophy type 1 (OPA1) (HPA036926) was acquired from Sigma-Aldrich (San Luis, MO, United States). Novus Biologicals (Centennial, CO, United States) provided Mitochondrially Encoded cytochrome c oxidase subunit 2 (Mt-CO2) (NBP1-778220). Trypsin, Dimethyl Sulfoxide (DMSO), Saponin, Tris-Base, 4ʹ,6-Diamidino-2-phenylindole (DAPI), tetramethylethylenediamine (TEMED), Dulbecco's Modified Eagle Medium (DMEM) 4.5 g/l and 1 g/l glucose, L-glutamine, Pyruvate (Gibco), Penicillin:Streptomycin 10,000:10,000 (Gibco), Fetal Bovine Serum (FBS) (Gibco), Mowiol 4-88 Mw (Sigma Chemical Co. (St. Louis, MO, United States)). Bovine Serum Albumin (BSA) (Santa Cruz Biotechnology (Santa Cruz, CA, United States)). PierceTM BCA Protein Assay Kit (Fisher Scientific, Waltham, MA, USA). Rho Activation Assay Kit (Rf. 8820) (Cell Signaling Technology (Massachusetts, United States)). Rhodamine Phalloidin Reagent (Abcam (Cambridge, United Kingdom)). Acrylamide 37.5:1 solution, Clarity TM Western ECL substrate, electrophoresis buffer (TGS), sodium dodecyl sulfate (SDS), Triton X-100, blot buffer (TG), Tween-20, DC Protein Assay Reagent A, B, S, (Bio-Rad Laboratories Inc. (Hercules, CA, USA)). 2-Isopropanol, Ethanol, Methanol, Sodium Chloride (NaCl), Ammonium Persulfate (APS), Glacial Acetic Acid, Potassium Hydroxide (KOH), Potassium Chloride (KCL) (Panreac (Barcelona, Spain)), Protease Inhibitor Cocktail (Roche (F. Hoffmann-La Roche Ltd, Basel, Switzerland). L-carnitine (sc-205727), Y27632 (sc-3536) and Paraformaldehyde (PFA) (sc-25326B) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Sigma-Aldrich (San Luis, MO, United States) supplied Linoleic acid (L1376).

2.2. Patients and cell culture

Four lines of fibroblasts derived from patient skin biopsies from the Pediatric Department of Hospital Universitario Virgen del Rocío, Sevilla, Spain were used. Two controls lines of primary human skin fibroblasts were purchased from ATCC. Patient 1 (P1) presents a heterozygous pathogenic variant c.133G>T (p.Val45Phe) in ACTA1 that causes a missense variant. The second patient (P2) is also heterozygous carrying changes in position c.760A>T (p.Asn254Tyr) in ACTA1. The third patient (P3) presents heterozygous pathogenic variants c.10321A>C (p.Thr3441Pro) and c.13669C>T (pArg4557*) in NEB. The fourth patient (P4) presents heterozygous pathogenic variants c.24407_24410dup (p.Leu8137Phefs*18) c.8425C>T (p.Arg2809*) in NEB.

Control values were represented as mean±SD of three control lines. Fibroblasts were grown in Dulbecco's modified Eagle's medium DMEM (Gibco™, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Gibco™, ThermoFisher Scientific, Waltham, MA, USA), and 100 mg/ml penicillin/streptomycin. Fibroblasts were cultured at 37°C and 5% CO₂. Experiments were performed with less than 12 passage fibroblasts cultures.

2.3. Immunoblotting

Western blotting was performed using standard methods. After protein transfer, the membrane was incubated with various primary antibodies diluted 1:1000 and then with the corresponding secondary antibody coupled to horseradish peroxidase (HRP) at a 1:10000 dilution. The Immun-Star HRP substrate kit (Biorad Laboratories Inc., Hercules, CA, USA) was used to identify particular proteins.

2.4. Real-time quantitative PCR (qPCR)

ACTA1 and NEB gene expression in fibroblasts was analysed by qPCR using mRNA extracts. Using TrizolTM (Invitrogen, Carlsbad, CA, USA), mRNA was extracted in accordance with the manufacturer's instructions. To obtain complementary DNA (cDNA), RNA was retrotranscribed using the Iscript cDNA synthesis Kit (Bio-Rad, Hercules, CA, United States). TB Green^TM Premix Ex Taq^TM (Takara Bio Europe S.A.S., Saint-Germain-en-Laye, France) was used for qPCR. CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) was used to detect accurate quantification of gene expression. ACTA1 primers were 5’-CCATTTATGAGGGCTACGCG-3’ (Forward primer) and 5’- CAGCTTCTCCTTGATGTCGC-3’ (Reverse primer) amplifying a sequence of 158 nucleotides. NEB primers were 5’- GAAACCAGACCACAGCCTTG-3’ (Forward primer) and 5’-TAGGGCATCTTTCACCGTGT-3’ (Reverse primer) amplifying a sequence of 224 nucleotides. Human α Tubulin was used as a housekeeping control gene and the primers were 5’-GCAGCATTTGTAGCAGGTGA-3’ (Forward primer) and 3’- GCATTGCCAATCTGGACAC -5’ (reverse primer). All primer pairs were previously validated by PCR, followed by gel electrophoresis, confirming correct product amplification.

2.5. Active RhoA assay

Fibroblasts were grown on 75 cm² flasks in DMEM containing 10% FBS. According to the manufacturer’s recommendations, media were removed, and cells were washed twice using ice-cold PBS 1X and the cell lysate was collected using Lysis/Binding/Wash buffer supplemented 1X protease inhibitor cocktail provided by the Active Rho Detection Kit (Cell Signaling Technology, Danvers, MA, USA, Cat. 8820). After centrifuging at 16000 g at 4°C for 15 min, lysate protein concentration was determined using BCA Protein Assay Kit and a 1 mg/ml lysate in 1X Lysis/Binding/Wash buffer was prepared. After making the cell lysate stock, protein then was incubated with GST Rhotekin-RBD affinity beads at 4°C for 1 h with gentle rocking. Bound proteins (Active RhoA form) were washed and prepared with SDS Sample Buffer and then, Western blotting analysis was carried out according to the previously mentioned protocol.

2.6. Immunofluorescence microscopy

Fibroblasts were grown on 1 mm width (Goldseal No. 1) glass coverslips for 24–48h in DMEM containing 20% FBS. Cells were rinsed once with PBS 1X, fixed in 4% PFA for 5 min at room temperature and permeabilized in 0.1% saponin for 5 min. Then, cells were blocked with blocking solution (1% BSA in PBS 1X). For immunostaining, glass coverslips were incubated with primary antibodies diluted 1:100 in blocking solution, overnight at 4°C. Unbound antibodies were removed by washing the coverslips with PBS 1X (three times, 5 min). The secondary antibody, a FITC-labelled goat anti-mouse antibody, or a tetramethyl rhodamine goat anti-rabbit (Molecular Probes), diluted 1:400 in blocking solution, were added and incubated for 2h at room temperature. Coverslips were then rinsed with PBS 1X for 5 min, incubated for 1 min with PBS 1X containing DAPI (1 μg/ml), and washed with PBS (three 5-min washes). Finally, the coverslips were mounted onto microscope slides using Mowiol aqueous mounting medium and analysed using a DeltaVision system (Applied Precision; Issaquah, WA) with an Olympus IX-71 microscope (Olympus Corporation, Shinjuku, Tokyo, Japan) as well as the colocalization.

2.7. Immunofluorescence staining of cytoskeletal F-actin

Briefly, fibroblasts were seeded on 1 mm coverslips (Goldseal No. 1) for 24–48 h in DMEM containing 10% FBS. Next day after reaching to 50% confluency, cells were fixed with 4% PFA for 10 minutes at room temperature and permeabilized with 0.1% Saponin for 15 minutes. Then, cells were washed with PBS 1X twice and incubated with Rhodamine-Phalloidin, a high affinity F-actin probe conjugated to red-orange fluorescent dye, at 1 μg/ml for 30 minutes. After incubation, cells were washed with PBS 1X three times and incubated with DAPI at 1 μg/ml for 5 min to nuclei staining. Subsequently, cells were washed three times with PBS 1X for 5 minutes. Finally, the coverslips were mounted on microscope slides with Mowiol aqueous mounting medium. After staining, images were taken by a DeltaVision system with an Olympus IX-71 fluorescence microscope with a 40× oil objective. Images were analysed by Fiji-ImageJ software.

In order to evaluate the state of actin filaments, the percentage of cells with correct actin polymerization and the length of actin filaments present in each cell were examined. To estimate the percentage of cells with correct actin polymerization for each patient, three counts of 100 cells per sample were performed; the values of these counts were compared among themselves and between the different samples. Cells with correct actin polymerization were considered those cells that presented actin filaments similar to those presented by control cells. The length of actin filaments present in each cell were measured by Fiji-ImageJ software. To execute this, the filaments of 30 cells of each cell line were measured in triplicate. The actin filament length values represented in the figures are expressed as the average of the measured filament lengths.

2.8. Bioenergetics and Oxidative Stress Analysis

Mitochondrial respiratory function of control and NM fibroblasts were measured using mito-stress test assay by XF24 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA). Cells were seeded at a density of 15000 cells/well in XF24 cell culture plates in 150 μl growth medium (DMEM medium containing 20% FBS) and placed in 37°C incubator with 5% CO₂. After 24h incubation, growth medium from each well was removed, leaving 50 μl of media. Then, cells were washed twice with 1 ml of pre-warmed assay medium (XF base medium supplemented with 10-mM glucose, 1-mM glutamine, and 1-mM sodium pyruvate; pH 7.4) and 450 μl of assay medium (500 μl final) was added. Cells were incubated in 37°C incubator without CO₂ for 1h to allow pre-equilibrating with the assay medium. Mitochondrial functionality was evaluated by sequential injection of four compounds that affect bioenergetics. The final concentrations of injections were 1 μM oligomycin, 2 μM FCCP (carbonyl cyanide-4-trifluoromethoxy-phenylhydrazone), and 2.5 μM rotenone/antimycin A. The best concentration of each inhibitor and uncoupler, as well as the optimal cells seeding density, were determined in preliminary assays. A minimum of five wells per treatment were utilized in any given experiment. This assay allowed for an estimation of basal respiration, ATP production, maximal respiration, and spare respiratory capacity parameters.

2.9. Mitotracker staining. Analysis of Mitochondrial Network

Cells were stained with MitoTracker Red CMXRos (100 nM, 45 min, 37°C), a mitochondrial potential-dependent dye, and examined by fluorescence microscopy. The amount of mitochondrial fragmentation was evaluated using the Fiji software. For each experimental condition, a total of 100 cells collected in three different experiments were used to calculate the number of small, rounded mitochondria per cell. Length or ratio between the major and minor axis of the mitochondrion, degree of circularity and percentage of rounded/tubular mitochondria were considered.

2.10. Measurement of intracellular reactive oxygen species (ROS) generation

Following the manufacturer’s instructions, fibroblasts mitochondrial superoxide generation was measured using 5 μM MitoSOX™ Red. Previously, cells were seeded and grown on coverslips until reaching 80% of confluency. Mitochondrial localization of MitoSOX™ Red signal was confirmed in combination with MitoTracker™ Deep Red FM staining (100 nM, 45 min, 37°C), an in vivo mitochondrial membrane potential-independent probe. Cells' nuclei were stained with 1 μg/ml DAPI. After staining, images were taken by a DeltaVision system with an Olympus IX-71 fluorescence microscope with a 40× oil objective. Images were analysed by Fiji-ImageJ software.

2.11. Statistics

Statistical analysis was conducted in accordance with our research group's previous description [27]. In situations where there were few events (n<30), we employed non-parametric statistics that do not make any distributional assumptions [28]. In these cases, multiple groups were compared using a Kruskal-Wallis test. We used parametric tests when the number of events was greater (n>30). In these instances, a one-way ANOVA was used to compare multiple groups. Statistical analysis were conducted using the GraphPad Prism 9.0 (GraphPad Software, San Diego, CA). The information is presented as the mean±SD values or as an example from three independent experiments. p-values of less than 0.05 were considered significant.

3. Results

3.1. NM fibroblasts present alterations in actin alpha1 (ACTA1) and nebulin (NEB) expression levels.

First, we analysed ACTA1 and NEB protein expression levels in control and NM patients derived fibroblasts (P1 and P2 harboring ACTA1 mutations; and P3 and P4 carrying NEB mutations). As shown by immunofluorescence microscopy in Figure 1A,B, ACTA1 expression levels were markedly reduced in P1 and P2 cells, while NEB (Figure 2A,B) expression levels were reduced in P3 and P4. Interestingly, ACTA1 immunostaining colocalized with actin filaments in control cells but not in mutant cells.

Reduced expression levels of ACTA1 were confirmed by Western blotting analysis (Figure 3A,B). Low protein expression levels of mutant proteins were associated with a reduction in ACTA1 transcript levels in P1 and P2 mutant fibroblasts (Figure 3C). Moreover, NEB transcripts levels in P3 and P4 were confirmed suggesting a decrease of gene expression of affected genes or an increase of mutant transcripts degradation in NM cells (Figure 3C,D).

3.2. NM patient-derived fibroblasts show defects in actin filament polymerization.

Cell staining with Rhodamine-Phalloidin showed that fibroblasts derived from NM patients have marked defects in the polymerization of actin filaments. Thus, mutant fibroblasts P1 and P2 harboring ACTA1 mutations and P3 and P4 harboring NEB mutations displayed unstructured actin filaments of shorter length than control fibroblasts (Figure 4A,B). In addition, the analysis revealed that the percentage of NM fibroblasts with correct actin filament polymerization was much lower than in control cells (Figure 4C). P1 fibroblasts showed the more severe defect in actin filament polymerization (only 7% of P1 fibroblasts showed a correct actin polymerization). Interestingly, expression levels of β-actin (component of microfilaments) were up-regulated both in ACTA1 and NEB mutant fibroblasts (Supplementary Figure S1A,B).

NM patient-derived fibroblasts did not show alterations in the level of expression of other cytoskeletal proteins. Thus, the analysis of expression of vimentin (component of the intermediate filaments), and α-tubulin (component of microtubules) did not reveal significant differences in mutant fibroblasts compared to control fibroblasts (Supplementary Figure S1A,B).

3.3. RhoA/ROCK pathway is overactivated in NM fibroblasts

As the RhoA/ROCK pathway plays an essential role in actin polymerization [27,28], we next examined the expression levels of Rho A, Active RhoA, ROCK1 and phosphorylated ROCK1. NM mutant cells P1, P2, P3 and P4 showed increased activation of active RhoA and phosphorylated ROCK1 indicating overactivation of the RhoA/ROCK pathway (Figure 5A,B). Interestingly, control cells treated with Y-27632, a ROCK inhibitor, mimicked the actin polymerization defects found in NM cells (Supplementary Figure S2A,B), suggesting that RhoA/ROCK pathway overactivation is a compensatory mechanism to increase actin polymerization in ACTA1 and NEB mutations.

3.4. NM fibroblasts display alterations in mitochondrial bioenergetics and network morphology.

Since actin cytoskeleton and the correct actin filament polymerization is essential for mitochondria function [16], we further investigated mitochondrial bioenergetics in NM fibroblasts. For that purpose, mitochondrial bioenergetics parameters were examined in control and NM fibroblasts (Figure 6). Fibroblasts derived from patient P1, P2, P3 and P4 showed reduced basal, maximal, and spare respiration, and decreased mitochondrial ATP production compared to the control cells. This reduced respiratory ability is consistent with the presence of a marked mitochondrial dysfunction.

After labeling mitochondria with MitotrackerTM Red CMXRos and examining them using fluorescence microscopy, we proceeded to investigate the morphology of the mitochondrial network. Representative images of mitochondrial morphology are shown in Figure 7A. A total of 100 cells were quantified for each condition (Figure 7B). The mitochondrial network of NM fibroblast P1, P2, P3 and P4 showed the presence of depolarized and fragmented mitochondria. Mitochondrial fragmentation was evaluated calculating the percentage of rounded and tubular mitochondria. We observed a marked reduction in tubular mitochondria of NM cells compared to control cells (Figure 7C). As positive control of mitochondrial membrane potential depolarization, control cells were treated with 100 µM CCCP (carbonyl cyanide m-chlorophenylhydrazone) for 4 hour as positive control.

As the actin cytoskeleton plays an essential role in the regulation of mitochondrial dynamics [16], we next evaluated the expression levels of DRP1 and OPA1, the main proteins involved in mitochondrial fission and fusion processes, respectively. NM mutant cells P1, P2, P3 and P4 showed increased expression levels of DRP1 and reduced levels of OPA1 in comparison with control cells, indicating an imbalance between mitochondrial fission and fusion processes (Figure 7D,E). These results agree with the fragmented mitochondrial network (Figure 7A) and the reduced percentages of tubular mitochondria and increased percentages of rounded mitochondria (Figure 7C) presented by NM cells compared to control cells.

Mitochondrial dysfunction in NM cells was also confirmed by evaluating the expression levels of mitochondrial proteins (Figure 8A). Thus, expression levels of mitochondrial protein subunits NDUFA9 (Complex I), NDUFS4 (Complex I), mtND1(Complex I), SDHB (Complex II), UQCRC2 (Complex III), mtCOX2 (Complex IV), COX4 (Complex IV), ATP5A (Complex V) and VDAC1 were significantly downregulated in NM mutant cells respect to control cells.

3.5. NM patient- derived fibroblast present increased ROS and alterations in antioxidant enzymes expression levels

As mitochondrial dysfunction is related to an increase in oxidative stress and reactive oxygen species (ROS) production, we also studied the mitochondrial ROS generation by MitoSOX™ and the protein expression levels of antioxidants enzymes. Thus, NM cells showed a significative mitochondrial ROS overproduction compared to control cells (Figure 9A,B). Furthermore, the protein expression levels of cytoplasmic superoxide dismutase (SOD1), mitochondrial superoxide dismutase (SOD2) and glutathione peroxidases (GPX4) were also reduced in NM fibroblasts, suggesting that the enzymatic antioxidant system is downregulated in mutant NM cells (Figure 9C,D).

3.6. Supplementation with linoleic (LA) acid and L-carnitine (LCAR) restores actin polymerization.

After an initial pharmaceutical screening aiming to identify commercial supplements able to restore actin polymerization patterns in mutant cells, we selected two well-known mitochondrial boosting compounds: LA and LCAR. Treatment with 5 µM LA and 10 µM LCAR for 7 days significantly improved actin filaments polymerization in NM cells (Figure 10A). Thus, supplementation with both compounds separately and in combination significantly increased both the percentage of cells with correct actin polymerization and the length of the actin filaments in P1, P2, P3 and P4 fibroblasts (Figure 10B,C). LA and LCAR concentrations were chosen considering the efficiency in recovering actin polymerization in dose-response curve assays (Supplementary Figures S3–S6).

3.7. Supplementation with LA and LCAR improves mitochondrial bioenergetics.

We further examined the effect of LA and LCAR supplementation on mitochondrial function in NM fibroblasts using the bioenergetic profile provided by the Seahorse analyzer. Thus, mitochondrial bioenergetic parameters were examined in control and NM fibroblasts P1 (Figure 11A) and P3 (Figure 11B) treated and untreated with LA and LCAR. Supplementation with LA and LCAR individually or in combination were able to correct the reduced basal, maximal, and spare respiration, as well as mitochondrial ATP production in both ACTA1 (P1) and NEB (P3) mutant fibroblasts.

Improvement of mitochondrial bioenergetics in mutant cells under LA and LCAR supplementation was also accompanied by a significant restoration of mitochondrial membrane potential and mitochondrial network morphology (Figure 12A). Furthermore, supplementation also restored the reduction in the percentages of tubular mitochondria found in NM cells compared to control cells (Supplementary Figure S7).

In addition, LA and LCAR treatment notably increased mitochondrial protein expression levels (Figure 13 and Supplementary Figures S8–S11) and significantly reduced mitochondrial ROS production in NM cells (Supplementary Figure S12), thus confirming the improvement of mitochondrial function.

3.8. Supplementation with LA and LCAR corrects RhoA/ROCK pathway overactivation.

Next, to assess the effect of LA and LCAR on RhoA/ROCK pathway NM fibroblasts, we determined the expression levels of Total RhoA, active Rho A and downstream proteins (ROCK1 and pROCK1) in P1 and P3 NM fibroblast (Figure 14A,B).

Results showed that LA and LCAR individually or in combination reduced the activation of RhoA and key essential downstream proteins such as pROCK1 both in ACTA1 (P1) and NEB (P3) fibroblasts.

4. Discussion

Nemaline myopathy (NM) encompasses a large spectrum of rare genetic myopathies characterized by hypotonia, weakness and depressed or absent deep tendon reflexes. Histology examination of muscle biopsy typically shows the presence of nemaline rods. In this work, we explored the pathophysiological alterations in NM using patient-derived fibroblasts patients carrying ACTA1 and NEB mutations. Mutant fibroblasts manifest alterations in actin filament polymerization associated with mitochondrial dysfunction. In addition, we identified two compounds, linoleic acid (LA) and L-carnitine (LCAR) that restored actin polymerization and corrected bioenergetics deficiency in both ACTA1 and NEB mutant fibroblasts.

Actin is a widely distributed, highly conserved protein that plays a key role in the cytoskeleton and is involved in a variety of biological processes. In addition to providing structural support for the cell, actin plays a role in cell migration, cell division and organelle trafficking [29,30]. Actin's role was first described in muscle contraction in 1942 by A. Szent-Györgyi [31]. Actin is found in two different forms: G-actin, which is the globular monomeric actin that polymerizes to form filamentous F-actin [32].

Six different actin isoforms are expressed in a higher mammal [33]. A group of structurally related genes with highly homologous nucleotide sequences that share a common precursor code for different isoforms of actin [34]. Actin isoforms differ by four amino acid residues localized at positions 1, 2, 3, and 9 of the N-terminus [33]. Six human actin genes - α-skeletal (ACTA1), α-cardiac (ACTC1), α-smooth muscle (ACTA2), γ-smooth muscle (ACTG2), β-cytoplasmic (ACTB), γ-cytoplasmic (ACTG1) - are localized on the different chromosomes [35]. The seventh actin isoform, β-actin-like protein 2 (ACTBL2), has been recently identified. It is a member of the non-muscle actin class, along with γ- and β-cytoplasmic actins, but its expression is extremely low [36]. Cytoplasmic actins are expressed in mammalian cells in various proportions [37].

Conversely, nebulin (NEB) is a very large filamentous protein ranging in size from 600 to 900 kDa that is a crucial part of the thin filament in skeletal muscle. Because of its size and the difficulty in obtaining nebulin in its native state from muscle, its functions are still largely unknown [38].

In our work, we have demonstrated for the first time that both actin 1 alpha (ACTA1) and nebulin (NEB) are expressed in dermal fibroblasts allowing the investigation of pathological alterations induced by mutant proteins in an easy-to-use cellular model. In addition, both proteins indeed participate in actin filament polymerization in dermal fibroblasts since both mutant proteins ACTA1 and NEB caused defects in actin polymerization in NM cells.

One interesting finding in our work is that NM patient-derived fibroblasts exhibited alterations in mitochondrial network morphology associated with down-regulation of the expression levels of mitochondrial proteins and deficient mitochondrial bioenergetics suggesting a marked mitochondrial dysfunction. Mitochondria are organelles that undergo dynamic changes through fission (division into two or more independent organelles) and fusion (formation of a single structure) events, biogenesis, and mitophagy (clearance of damaged organelles) [39,40]. These processes regulate mitochondrial morphology, number, and turnover, respectively, referred to as mitochondrial quality control [41]. Mitochondrial fusion and fission cycles are adaptive changes controlled by dynamin-related protein 1 (DRP1), mitofusin 1 (MFN1), mitofusin 2 (MFN2), and optic atrophy protein 1 (OPA1) in mammalian cells [42]. Mitochondrial fusion increases mitochondrial metabolism, while mitochondrial fission contributes to mitochondrial apoptosis and is considered a prerequisite for mitophagy [43]. Mitophagy removes selectively old or dysfunctional mitochondria through its sequestration and engulfment for the subsequent lysosomal degradation [44]. Different stresses, such as oxidative damage, hypoxia, mitochondrial depolarization or mitochondrial DNA damage enhance mitophagy [45,46]. Accumulating evidence reveals that mitophagy is required to maintain skeletal muscle plasticity by controlling mitochondrial biogenesis turnover, and mitochondrial proteostasis [47,48], in order to increase mitochondrial activity and remodeling during early myogenic differentiation [49,50,51].

Cytoskeletal components, particularly microtubules and F-actin, work collaboratively to regulate mitochondrial processes of fission/fusion, mitophagy and morphology in response to extracellular stressors [52]. Mitochondrial motility is also dependent on the coordinated action of cytoskeletal elements, especially microtubules and F-actin, which distribute and anchor the organelles to the proper locations within the cell [53]. Specifically, it has been suggested that alterations to the cytoskeleton can affect mitochondria, resulting in functional modifications in the organelle. The primary role of mitochondria in cells is energy production, which is closely correlated with the regulation of their morphology, organization, and distribution by the cytoskeleton [54]. Thus, cytoskeletal abnormalities of actin polymerization in NM can subsequently lead to impairments in mitochondrial respiration, thereby accelerating disease progression.

The process of mitochondrial fission involves the formation of ring-shaped DRP1 oligomers on the outer membrane of the mitochondria, which are then constricted by hydrolysis [55]. In order to facilitate the subsequent recruitment of dynamin-2, which coordinates membrane scission, DRP1 rings tighten and constrict mitochondria. [56]. Nevertheless, in order to reduce their cross-sectional diameter, mitochondria must first go through a pre-constriction step because they are frequently thicker than DRP1 rings. Endoplasmic reticulum (ER) tubules are wrapped and tightened around mitochondria to produce this pre-constriction. Growing evidence points to actin polymerization as the primary force behind the ER's bending around the mitochondria [57,58].

In addition, F-actin at Mito/ER contacts may prime DRP1 to form functional oligomers on the outer membrane of the mitochondria [59]. In vitro GTPase assays show that DRP1 activity is greatly enhanced in the presence of actin filaments [59]. Taken together, these observations suggest that filamentous actin acts as an important regulator of inner and outer mitochondrial membrane fission.

Moreover, the actin cytoskeleton controls the movement of mitochondria in simple eukaryotes like budding yeast [60] and is necessary for the accurate inheritance of mitochondria during cytokinesis [61]. Microtubules in metazoans are responsible for coordinating long-distance mitochondrial movement, but the actin cytoskeleton is also involved in regulating mitochondrial distribution, coordinating anchoring, and coordinating short-distance mitochondrial motility [52].

In addition, actin cytoskeleton plays an important role on mtDNA expression and maintenance [62]. The mammalian mitochondrial DNA genome (mtDNA) contains 37 genes organized in compact DNA:protein complexes called nucleoids [63,64], whose expression requires a high degree of coordination with the nuclear genome [65]. In yeast, the ERMES (ER-mitochondria encounter structure) complex regulates the stability and organization of mtDNA in nucleoids in an actin-dependent manner [66]; in mammals, MERCs (Mitochondria Endoplasmic Reticulum Contact Sites) are spatially-linked to mitochondrial nucleoids, regulating their distribution, division and active transportation by the microtubules [67]. Furthermore, recent super-resolution microscopy-based studies probed the presence of β-actin-containing structures inside the mitochondrial matrix [68].

Furthermore, β-actin-deficient human cells were more susceptible to stress brought on by a decrease in mitochondrial membrane potential (ΔΨ), as well as altered mtDNA mass and nucleoid organization [69], indicating a regulatory function in mtDNA transcription and quality control. In addition to actin, myosin II has been linked to isolated mitochondrial nucleoids, and its silencing results in mtDNA alterations [62]. These evidences support the role of actin and actin-binding proteins in mitochondrial nucleoid segregation and mtDNA transcription and maintenance, likely through formation of a “mitoskeleton” network supporting mtDNA inheritance.

Additionally, the complete activation of metabolic pathways that subsequently control mitochondrial function depends on actin filaments. For example, direct binding of glycolytic enzymes like glyceraldehyde phosphate dehydrogenase or aldolase to F-actin can activate them [70]. Interestingly, in brain mitochondria actin regulates the retention of cytochrome c between respiratory chain complexes III and IV by its direct association with both complexes, and inhibition of actin polymerization with cytochalasin b enhanced mitochondrial respiration through increased complex IV activity [71]. Recently, proteomic analysis using NEB [72] and ACTA1 [73] mice models revealed perturbations in several cellular processes, including mitochondrial dysfunction and changes in energetic metabolism and stress-related pathways. Abnormal mitochondrial distribution, reduced mitochondrial respiratory function, increased mitochondrial membrane potential, and abnormally low ATP content were all revealed by structural and functional studies.

In our work we have also identified two compounds, LA and LCAR, that improving mitochondrial bioenergetics were able to restore actin polymerization.

LA is a carboxylic acid composed of 18 carbon atoms and three cis double bonds (18:2ω6), and is an essential fatty acid indispensable to the human body and therefore, it must be ingested through diet [74,75]. Pharmacological studies have shown that LA has a wide range of pharmacological effects such as anti-metabolic syndrome, antiinflammatory, anticancer, antioxidize, neuroprotection, and the regulation of the intestinal flora [76,77,78]. LA is a major component of cardiolipin (CL), which is a specific inner-mitochondrial membrane phospholipid that is important for optimal mitochondrial function including respiration and energy production [79]. Furthermore, this phospholipid participates in morphology and stability of mitochondrial cristae, fission and fusion-mediated mitochondrial quality control and dynamics, mitophagy, mitochondrial biogenesis and protein import, and multiple mitochondrial steps of the apoptotic process [79]. Due to its high content of unsaturated fatty acids and its location in the IMM near to electron transport chain (ETC) complexes, the main sites of reactive oxygen species (ROS) production, CL is particularly prone to peroxidation, an event that may affect many CL-dependent reactions and processes [80,81,82]. In addition, the accumulation of oxidized CL in the OMM serves as important signaling platform during the apoptotic process, resulting in the opening of the mitochondrial permeability transition pore (mPTP) and in the release of cytochrome c (cyt c) from mitochondria to the cytosol [83,84].

Specifically, CL and its LA content are known to be positively associated with cytochrome c oxidase (COX) activity [85] thereby making this lipid molecule an essential factor related to mitochondrial health and function. Structural studies have shown that CL is important for stabilization (i.e., the assembly of complex subunits) of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), complex II (CII), complex III (CIII), and complex IV (CIV) [86,87,88,89]. On the other hand, a decrease in CL levels by low dietary intake of LA promotes the disassembly of these mitochondrial OXPHOS complexes [90]. In contrast, LA-enriched diet increased mitochondrial CL, and OXPHOS protein levels [91]. Additionally, it was recently discovered that cardiolipin is necessary for the respiratory chain to organize into supramolecular assemblies [92]. Furthermore, incubation of cells with different concentrations of LA led to a dose- and time-dependent increase of cardiolipin levels [93].

It is conceivable that LA supplementation by increasing cardiolipin levels may positively impact the activity of a variety of mitochondrial proteins and enzymes, including the electron transport chain (ETC) and oxidative phosphorylation (OXPHOS) complexes, thus increasing mitochondrial function and dynamics. Furthermore, LA stimulates mitochondrial biogenesis signaling by the upregulation of PPARγ coactivator 1α (PGC-1α) in C2C12 cells [94].

In addition of its cardiolipin dependent actions, LA also promotes an increase of fascin expression, an actin crosslinker globular protein that generates actin bundles built of parallel actin filaments, which mediate formation and stability of cellular protrusions including microspikes, stress fibers, membrane ruffes, and filopodia [95]. In this respect, LA is found to induce membrane ruffling and phagocytic cup formation along with cytoskeletal rearrangement in microglia [96]. Furthermore, LA has many effects on cytoskeleton proteins in general and actin filaments in particular. For instance, it has been reported that LA enhances cell migration by altering the dynamics of microtubules and the remodeling of the actin cytoskeleton at the leading edge [97]. Moreover, LA may activate the RhoA/ROCK pathway and therefore, may facilitate actin polymerization. Thus, it has been shown that LA increased Intercellular Adhesion Molecule 1 (ICAM-1) expression and phosphorylation of ROCK and Myosin Phosphatase Target Subunit 1 (MYPT-1), a distal signal of ROCK [98].

On the other hand, LCAR, a well-known dietary supplement which is needed for the translocation of fatty acids into the mitochondrial compartment for β-oxidation [99,100] and has a role in carbohydrate metabolism possesses this potential to raise the mitochondrial biogenesis, increasing various mitochondrial components’ gene expression and maintains their function via supplying their respective substrates and protecting them against insults including the toxic products’ or reactive radicals’ accumulation [101,102]. Therefore, LCAR as natural compounds that can enhance cellular energy transduction may have therapeutical potential in NM. Studies in recent years have demonstrated the protective effects of LCAR treatment on mitochondrial functions [102]. Consistent with this hypothesis, a premature boy with a congenital form of nemaline myopathy due to mutation in the ACTA1-gene showed decreased LCAR levels in the eighth week of life. After sufficient oral LCAR substitution he improved gradually [103]. Furthermore, LCAR ameliorates congenital myopathy in a tropomyosin 3 de novo mutation transgenic zebrafish [104]. Furthermore, LCAR significantly reduced statin-induced myopathy [105] and skeletal muscle atrophy in rats [106]. In addition, LCAR improved exercise performance in human patients with mitochondrial myopathy [107] and may prevent age-associated muscle protein degradation and regulate mitochondrial homeostasis [108].

As actin polymerization is dependent of ATP levels. During cycles of actin assembly and disassembly, actin monomers polymerize into filaments in an adenosine triphosphate (ATP)-bound state, and the polymerization of actin is followed by the irreversible hydrolysis of ATP to adenosine diphosphate (ADP) and phosphate (6).

Thus, in eukaryotic cells, the actin cytoskeleton's turnover between its monomeric and filamentous forms is a significant energy drain. It is not surprising that lower ATP levels slow actin polymerization since ATP is essential for actin polymerization. Reduced ATP levels most likely cause an abundance of ADP-G-actin versus ATP-G-actin. The lower actin polymerization rate observed in the ATP-deficient cells may be explained by the fact that ADP-G-actin polymerizes at a substantially slower rate than ATP-G-actin [109,110]. Actin filament assembly and dynamic behavior are mediated in large part by binding with ATP and ATP hydrolysis.

In addition, RhoA activity which regulates actin polymerization decreased in parallel with the concentration of ATP and GTP during depletion by mitochondrial inhibitors recovered rapidly when cells were returned to normal culture conditions [111], suggesting that ATP and GTP levels modulate RhoA activation.

In this regard, mitochondrial dysfunction in NM cells may cause ATP deficiency and aggravate the impairment of actin polymerization. LCAR and/or LA supplementation may boost mitochondrial ATP formation and consequently may facilitate actin filament formation by increasing actin monomers bound to ATP and RhoA activation by increasing GTP levels.

5. Conclusions

In conclusion, our findings demonstrate that fibroblasts derived from patients with NM are useful cellular models to study the disease and allow evaluating the effectiveness of pharmacological compounds. Furthermore, we confirm the close relationship between actin cytoskeleton and mitochondria function. Supplementation with LA or/and LCAR was able to restore actin polymerization and correct mitochondrial dysfunction in NM cells. Further studies are required to assess the benefit of both compounds in sarcomere organization and skeletal muscle contraction. Patient-derived cellular models may complement ACTA1 and NEB mouse and zebrafish models and enable the evaluation of genomic or pharmacological therapies.