Heat shock protein A4 ablation leads to skeletal muscle myopathy associated with dysregulated autophagy and induced apoptosis

Molecular chaperones assist protein folding, facilitate degradation of misfolded polypeptides, and thereby maintain protein homeostasis. Impaired chaperone activity leads to defective protein quality control that is implicated in multiple skeletal muscle diseases. The heat shock protein A4 (HSPA4) acts as a co-chaperone for HSP70. Previously, we showed that Hspa4 deletion causes impaired protein homeostasis in the heart. However, its functional role in skeletal muscle has not been explored. We performed a comparative phenotypic and biochemical analyses of Hspa4 knockout (KO) mice with wild-type (WT) littermates. HSPA4 is markedly upregulated in regenerating WT muscle in vivo, and in differentiated myoblasts in vitro. Hspa4-KO mice are marked by growth retardation and increased variability in body weight, accompanied by 35% mortality rates during the peri-weaning period. The surviving Hspa4-KO mice experienced progressive skeletal muscle myopathy, characterized by increased number of muscle fibers with centralized nuclei, heterogeneous myofiber size distribution, inflammatory cell infiltrates and upregulation of embryonic and perinatal myosin heavy chain transcripts. Hspa4-KO muscles demonstrated an accumulation of autophagosome-associated proteins including microtubule associated protein1 light chain 3-II (LC3-II) and p62/sequestosome accompanied by increased number of TUNEL-positive nuclei. Our findings underscore the indispensable role of HSPA4 in maintenance of muscle integrity through contribution in skeletal muscle autophagy and apoptosis, which might provide a novel therapeutic strategy for skeletal muscle morbidities.


Background
Protein homeostasis is maintained via efficient elimination of misfolded protein aggregates by protein quality control (PQC) that utilizes a repertoire of chaperones to recognize misfolded proteins and assist their refolding or facilitate their degradation, if refolding is not possible, Page 2 of 10 Elkenani et al. Journal of Translational Medicine (2022) 20:229 through either the ubiquitin-proteasome system (UPS) or the autophagy-lysosome system [1]. Compared with other cell types, PQC in muscle cell is particularly challenging because muscle proteins are in a dynamic state of synthesis and degradation in response to mechanical stress. Making it worse, muscles are postmitotic, and therefore not able to dilute toxic effect of the protein aggregates by division and, thus, are highly susceptible to misfolded proteins. Maintained PQC is critical for proper skeletal muscle homeostasis, and inefficient PQC leads to accumulation of protein aggregates and eventually to muscular disorders [2].
Autophagy is an evolutionarily conserved and a tightly regulated intracellular process that targets the misfolded proteins and damaged organelles for lysosomal degradation. Basal constitutive autophagy is required for maintaining muscle function [3]. Excess attenuation or augmentation of the autophagy result in muscle morbidities [4][5][6][7].
Heat shock proteins (HSPs) function as molecular chaperones to maintain cellular PQC through mediating efficient protein folding and targeting misfolded protein aggregates for degradation, and therefore have an indispensable role for proper myogenesis [8][9][10][11][12]. Mutations in human HSPs have been identified in patients with muscle myopathy [13][14][15]. HSPA4 belongs to HSP110 family that functions as a co-chaperone for HSP70 [16]. HSPA4 is ubiquitously expressed [17], and has been shown to avert inflammation and apoptosis, protect from oxidative stress and improve survival [18][19][20]. A role of HSPA4 in the cross talk between UPS and autophagy has been proposed, but there was no proof for this hypothesis [21]. Hspa4-knockout (KO) mice showed impaired PQC in the heart, characterized by accumulation of misfolded protein aggregates, and resulting in pathological myocardial remodeling and fibrosis [22]. Given the fundamental importance of PQC in skeletal muscle, we hypothesized that HSPA4 would be a novel regulator in skeletal muscle homeostasis.
Here, we observed that Hspa4-KO mice exhibit decreased survival rates, growth retardation and increased variability in body weight. The aged Hspa4-KO mice develop spinal deformities and kyphosis. We therefore characterized the skeletal muscles in Hspa4-KO mice and showed that HSPA4 deficiency causes skeletal muscle myopathy associated with dysregulated autophagy and enhanced apoptosis.

Animals
Male and female Hspa4-KO mice were generated on 129/ Sv genetic background as described previously [17].

Histological analyses
Muscles were collected and either paraffin-embedded, or immediately frozen in isopentane. Sections (6 µm) were stained with hematoxylin and eosin (H&E), and the number of centrally nucleated fibers was counted across 5 separate fields of view from at least three sections of each mouse. TUNEL assay was performed in paraffin-embedded sections using In Situ Cell Death Detection Kit (Roche Diagnostics). After fixation in ethanol-acetic acid, TA sections were treated with proteinase K and permeabilized with 0.5% Triton X-100. The sections were then incubated in the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and nucleotide mixture for 60 min at 37 °C in a dark humid chamber. TUNEL-positive cells were counted in 5-8 random fields/ muscle. For immunofluorescence, frozen sections were permeabilized using 0.2% Triton X-100 in phosphate-buffered saline (PBS), blocked with 5% bovine serum albumin in PBS and incubated with rabbit anti-LC3 (Cell signaling technology). Photomicrographs were captured using a microscope Olympus BX60 fluorescence microscope.

Quantitative real-time polymerase chain reaction (qRT-PCR) and Northern blotting
For real time PCR, cDNA synthesis was carried out with iScript cDNA synthesis kit (Bio-Rad). QRT-PCR was performed on a Biorad iQ-Cycler using SYBR Green Supermix (Bio-Rad). For Northern blot analysis, 20 μg of total RNA samples was size fractionated by electrophoresis, transferred onto nylon membrane (Amersham Bioscience) and hybridized with a 32P-labeled fragments. All the primers used are listed in the Additional file 1: Table S1.

Determination of 20S proteasome activity
Using 20S Proteasome Assay Kit (10,008,041; Biomol), the 20S proteasome assay was carried out in a total volume of 100 μl in 96 well plates. Assays were initiated by addition of 100 μM of fluorescently labeled substrate, succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC), to the protein lysates (50 μg) and incubation at 37 °C. These substrates are cleaved by the proteasome, releasing free AMC which was then measured spectrofluorometrically after one hour at an excitation wavelength of 360 nm and an emission wavelength of 480 nm. Each assay was conducted in duplicates and in the absence and presence of the specific proteasomal inhibitor, lactacystin (20 μM).

Cardiotoxin (CTX) injection
Adult mice were anesthetized with isoflurane and 50 μL of 10 μM cardiotoxin was injected into the left TA muscle. As a control 0.9% saline (vehicle) was injected in the contralateral side. Carprofen was used for post-treatment analgesia. Mice were sacrificed and TA muscles were dissected at various time points after injection.

Statistical analysis
Statistical analysis was carried out using GraphPad Prism 7.0 (GraphPad Software, Inc, California, USA) with twotailed unpaired Student's t-test or one-way ANOVA with Bonferroni post-test correction where appropriate. Kaplan-Meier survival analysis was performed, and a Log-rank test was used to determine significance.

Upregulation of HSPA4 during skeletal muscle regeneration and myoblast differentiation
Western blot analysis revealed that HSPA4 is ubiquitously expressed in different skeletal muscles Additional file 1: Fig. S1). Markedly increased Hspa4 mRNA and protein levels were detected in regenerating TA muscle after CTX injection, which induces muscle degeneration followed by regeneration ( Fig. 1A-C). HSPA4 protein expression was also markedly upregulated in immortalized C2C12 myoblasts after induction of differentiation ( Fig. 1D). Taken together, our data suggest a relevant role of HSPA4 in myogenesis, which prompted us to characterize skeletal muscles in Hspa4-KO mice.

Delayed growth and early mortality in Hspa4-KO mice
Hspa4-KO pups were born from heterozygous breeding pairs at the expected Mendelian ratios (Table 1).
In the first week postnatal, the Hspa4-KO pups were indistinguishable from their Hspa4 +/+ and Hspa4 ± littermates. However, starting at postnatal day 8 (P8), Hspa4-KO pups gained less weight and showed clear growth retardation between P14 and P28 ( Fig. 2A, B), likely the result of decreased milk intake caused by muscle weakness. Serum glucose levels and expression of the key gluconeogenic enzyme, lipid transport related-genes and growth hormone-responsive gene did not show any significant difference between Hspa4-KO and control animals (Additional file 1: Fig. S2). In accordance with stunted growth, Hspa4-KO mice also exhibited generalized dwarfism affecting all organs tested. However, the decrease in muscle mass was much more severe than other organs ( Table 2). About 35% of the Hspa4-KO mice died during the peri-weaning period (Fig. 2C).
After weaning, surviving Hspa4-KO mice normalized their body weight and were generally similar to wildtype (WT) littermates by two months of age (Fig. 2B). By the age of 12 months, Hspa4-KO mice developed spinal deformity in the form of kyphosis (Fig. 2D), indicative of paraspinal muscles weakness [23]. The protein level of HSPA4L and HSPH1, other members of HSP110 family, was not different between WT and Hspa4-KO muscles ruling out any compensatory upregulation of the studied proteins in the Hspa4-KO muscles (Fig. 2E, F).

Skeletal muscle myopathy in Hspa4-KO mice
By the age of 4 months, H&E-stained Hspa4-KO TA muscles exhibited myopathic changes including heterogeneous myofiber size distribution, numerous centrally nucleated fibers (CNF), and inflammatory cell infiltrates ( Fig. 3A-C). The induction of the inflammatory response in Hspa4-KO TA muscles was confirmed at the transcript level by the detection of macrophage-specific markers, Cd68 and F4/80, and interleukins, Il6 and Il1β (Fig. 3D). TA muscles from Hspa4-KO mice experienced induction of embryonic (Myh3) and perinatal (Myh8) muscle myosin heavy chain genes, indicative of ongoing de-and regeneration (Fig. 3E, F). These data suggest that the Hspa4 deletion impairs the integrity of the myofibers resulting in the activation of a regenerative response. Interestingly, induction of Myh8 and Myh3 and increased percentage of CNF were also observed in Hspa4-KO muscles during the peri-weaning period (Fig. 3E, F and Additional file 1: Fig. S3). Myopathy was observed in other examined muscles including soleus, gastrocnemius and paraspinal muscles ( Fig. 3A and Additional file 1: Fig.  S4), indicating a generalized skeletal muscle involvement in Hspa4-KO mice.

Preserved skeletal muscle regeneration in Hspa4-KO mice
Induced HSPA4 expression in muscles following muscle injury forced us to address the requirement of HSPA4 for  normal muscle regeneration and recovery following muscle injury. We monitored skeletal muscle repair in CTXinjected TA muscles of 4-month-old WT and Hspa4-KO mice. One day following CTX injury muscles from both genotypes showed significant fiber degeneration and necrosis, as confirmed by eosinophilic staining and marked mononuclear cell infiltration. By 5 days following injury, degenerating muscle fibers were largely cleared in WT and Hspa4-KO muscles, and replaced by centrally nucleated nascent myoblasts and other mononuclear cells, indicative of the early phase of muscle regeneration. At 15 days of injury, the nascent myofibers in both genotypes were similarly enlarged ( Fig. 4A-C). Complete restoration of the muscle architecture was observed in both genotypes at 30 days post-injury (Fig. 4A). The relative mRNA levels of Pax7, Myod1 and Myogenin, satellite cell-related markers for activation, fusion and differentiation, respectively, were not statistically different between WT and Hspa4-KO muscles at any time point post-injury ( Fig. 4D-F). These data therefore suggest that the ability of Hspa4-KO mice to activate the myogenic program is not markedly altered. Overall, our results denote that increased degeneration, rather than defective regeneration, is the underlying cause for skeletal muscle myopathy in Hspa4-KO mice.

Dysregulated autophagy in Hspa4-KO muscles
Autophagy is initiated with the sequestration of cytoplasmic components by isolation membrane that expands to form double-membrane vesicles, the autophagosomes, which fuse with endosome/lysosome, followed by lysosomal hydrolysis of sequestered cytoplasmic components [24]. Autophagy can be assessed by detection of the modification of microtubule associated protein1 light chain 3 (LC3) from free mature LC3-I form to membrane-bound lipidated LC3-II form and by assessment of protein level of the autophagy adaptor, p62/sequestosome [25]. Hspa4-KO TA muscles showed marked increased LC3-II and p62 protein levels (Fig. 5A, B). Consistently, immunofluorescence revealed an abundant LC3 puncta in Hspa4-KO muscles (Fig. 5C), suggesting the dysregulated autophagy as a possible cause for skeletal muscle myopathy in Hspa4-KO mice.
Because an impaired proteasome pathway can also lead to an accumulation of p62 protein, we assessed proteasome activity in WT and Hspa4-KO TA muscles. No marked changes were found in chymotrypsin enzyme activity in total homogenates from WT and Hspa4-KO TA muscles (Fig. 5D). Treatment with known 20S proteasome inhibitors, lactacystin, resulted in a significant, but comparable, inhibition of 20S activity in WT and Hspa4-KO muscles (Fig. 5D). Moreover, the expression of atrogenes, MuRF1 and MAFbx, was not different between WT and Hspa4-KO muscles (Fig. 5E), indicating that the proteasome activity is not impaired in Hspa4-KO muscles.

Induced apoptosis in Hspa4-KO muscles
An anti-apoptotic effect of HSPA4 was previously reported [18][19][20]. We examined the apoptosis in the skeletal muscle of Hspa4-KO mice by performing TUNEL assay using TA sections from 4-month-old WT and Hspa4-KO mice. The number of apoptotic nuclei was markedly increased in Hspa4-KO compared to WT muscles (Fig. 6A, B). Additionally, the protein level of antiapoptotic factor BCL-2 and consequently BCL-2/ BAX ratio were markedly decreased in Hspa4-KO TA muscles, suggesting that apoptosis may have exacerbated skeletal muscle myopathy in Hspa4-KO mice.

Discussion
Here, we identified a previously unexplored role of HSPA4 in skeletal muscle homeostasis using Hspa4-KO mice. Previous reports have linked HSPA4 with several morbidities and mortality [17][18][19][20][21][22]. However, its role in skeletal muscle remains unknown. Our results demonstrated that HSPA4 is ubiquitously expressed in all muscles tested including fast twitch (TA and gastrocnemius) and slow twitch (soleus) muscles. Furthermore, HSPA4 expression is induced in regenerating WT muscles upon CTX-induced muscle injury and in myoblast upon differentiation, highlighting a potential role of HSPA4 in myogenesis.
Our results showed that HSPA4 is crucial for normal survival and growth. Hspa4-KO mice show growth retardation associated with 35% mortality rates within the first 4 weeks of life, which is likely due to skeletal muscle affection. Although cardiac structure and function are not deteriorated at the peri-weaning stage in Hspa4-KO mice [22], we cannot exclude acute decompensated myocardial function, beside skeletal muscle myopathy, as a possible underlying cause of early death in Hspa4-KO mice. Hspa4-KO mice that survive the first month of life develop a progressive myopathy, characterized by centrally nucleated myofibers, heterogeneous myofiber size distribution and inflammatory cell infiltrates, associated with defective autophagy and increased apoptotic cell death.
The UPS and autophagy are the major proteolytic systems of the cell that have a crucial role in the removal of protein aggregates. As one of the post-mitotic tissues, the highly dynamic skeletal muscle is particularly vulnerable to dysfunctional organelles and aggregationprone proteins. In this regard, it is not surprising that dysregulated activity of the autophagy and/or UPS is implicated in a variety of myofiber degeneration and muscle weakness [5,26]. Several molecular chaperones  and co-chaperones, including HSPA4, play a role in the cross-talk between UPS and autophagy to maintain cellular protein homeostasis [21,27,28]. Autophagy is markedly dysregulated in Hspa4-KO muscles as shown by accumulation of LC3-II protein. Thus, it is tempting to speculate that perturbed autophagy contribute to the muscle abnormalities in Hspa4-KO mice. However, increased LC3-II protein level can occur due to either induction of early or inhibition of late autophagy. We therefore examined the p62 protein level to clarify the effect of Hspa4 deletion on autophagy. The protein p62 is a specific target of the autophagy degradation. Thus, intracellular accumulation of this protein is indicative of insufficient autophagy [29]. Indeed, an increased p62 protein level was detected in Hspa4-KO muscle despite the increase of LC3-II, suggesting a late block in autophagy occurring after autophagosome formation, and involves autophagsome/ lysosome fusion or lysosomal degradation. However, autophagy is a highly dynamic and complex process, and therefore accurate assessment of the autophagy flux using lysosomal inhibitors, such as bafilomycin or chloroquine, among others, is necessary to confirm our assumption. Collectively, these data suggest that HSPA4 may have a beneficial role in the muscle via maintaining proper autophagy.
P62 is an autophagy receptor of ubiquitinated proteins that interact simultaneously with LC3 and promote the degradation of ubiquitinated protein aggregates [29]. However, no significant changes in the content of ubiquitinated proteins was found between Hspa4-KO and WT muscles [22], suggesting that Hspa4 deletion in skeletal muscle does not impair the degradation of ubiquitinated proteins, despite of the accumulation of p62. Consistently, Hspa4-KO muscles did not exhibit perturbed UPS activity, as evidenced by comparable proteasome activity and atrogenes expression to that in WT muscles.
Autophagy is an essential protective mechanism against apoptotic cell death [30]. Moreover, anti-apoptotic effect of HSPA4 has been previously reported [18][19][20]. Our results consistently revealed a significantly higher proportion of TUNEL-positive nuclei, downregulation of anti-apoptotic BCL-2 in the Hspa4-KO muscles, indicating that increased apoptosis, probably due to impaired autophagy, may be one of the reasons for the skeletal myopathy observed in Hspa4-KO muscles.
The transcription factor nuclear factor κB (NF-κB) is a key mediator of inflammation through induction of various pro-inflammatory cytokines, including interleukins and a large number of inflammatory genes, including macrophages-related markers [31]. Recently, it has been reported that HSPA4 inactivates NF-κB pathway and therefore inhibits inflammatory signaling [19]. Consistently, we showed here that the expressions of Il1b and Il6 as well as Cd68 and F4/80 are increased in Hspa4-KO muscles, suggesting an overall inflammation, possibly due to augmented NF-κB activity. However, a comprehensive analysis of inflammation in our mice is needed to support this hypothesis.
Several genes are associated with inherited skeletal muscle myopathies, and the list is still expanding [32]. Although Hspa4 mutations have not yet been linked to any muscle morbidities in human, the myocardium of Hspa4-deficient mice experiences pathological remodeling and fibrosis [22], which highlights the importance of HSPA4 for striated muscle integrity, and suggests that HSPA4 may be a promising therapeutic candidate for skeletal muscle myopathy. It remains to be addressed whether myopathy patients with genetically unknown cause carry Hspa4 mutations. We therefore propose that genetic screening by Hspa4 gene sequencing could identify novel mutations and expand the spectrum of myopathy-associated genes in patients with inherited skeletal muscle myopathies and/or pediatric heart diseases.
A full body HSPA4 ablation might have some limitations. Deletion of Hspa4 during whole life span might affect embryogenesis and thus influence the myogenesis. Moreover, our mouse model experiences global Hspa4 deletion in all cell types and possible unexplored functions of HSPA4 might therefore influence the outcome. Therefore, rescue study to address the ability of targeted HSPA4 expression using viral-mediated gene delivery with adeno-associated viral (AAV) vectors or non-viral nanoparticles delivery approach to correct the muscle phenotype in Hspa4-KO mice is warranted. Moreover, generation and characterization of muscle-specific Hspa4-KO mice are required to rule out the possibility of secondary effects.
In conclusion, we demonstrate that the deletion of HSPA4 in skeletal muscle leads to a progressive generalized myopathy, highlighting the critical role of HSPA4 in regulating the genetic repertoire required for the appropriate maintenance of skeletal muscle integrity. Furthermore, these findings support the investigation of HSPA4 as a novel therapeutic target for the amelioration of many inherited muscle diseases with impaired autophagy.