Chaperones escort other macromolecular structures whilst the latter acquire their proper conformational structure, through processes of (un)folding and (dis)assembly. Molecular chaperones termed heat shock proteins (HSPs) can be induced by heat stress and are grouped according to molecular weight into different families [1]. The heat shock proteins of 70kd (HSPs70) are structurally conserved proteins composed of an N-terminal ATPase domain, a peptide-binding domain and a C-terminal “lid” domain that can capture substrates at their binding site [2]. In humans, the HSPs70 lie encoded within multiple genes and pseudogenes, producing high numbers of mRNA variants and isoforms [3]. Particular HSPs70 may have preferential client proteins and/or co-chaperones allowing them to perform, in addition to their recognized redundant functions, also more unique tasks. The seven main isoforms are termed HSP70-1A, HSP70-1B, HSP70-1T, HSP70-2, HSP70-6, HSC70 and HSP70-14. Part of these display transiently increased expression following cellular insults [4]. The HSPA1A gene produces the most abundant stress-induced isoform, which will be referred to as HSP70 in this study.

The functionality of HSP70 is highly dependent on its localization. Intracellularly, HSP70 is involved in aiding de novo protein synthesis and protein restorative actions in response to cellular damage. HSP70 interacts with its client polypeptides in a process which is stabilized by complex joint interventions with co-chaperones belonging to the DnaJ family, and specific allosteric regulators such as the C-terminus-HSP70-Interacting Protein (CHIP). In addition, HSP70 activity is further fine-tuned through intracellular re-localization [5] and post-transcriptional modifications [6]. Secreted HSP70 on the other hand has a regulatory role through activation of different signalling pathways. Its immunoregulatory role known as ‘chaperokine’ activity is mediated via its interaction with innate immune receptors. HSP70 released into the extracellular milieu activates Toll-like receptors (TLRs) on antigen-presenting cells, facilitating antigen cross-presentation. Downstream effects include upregulation of adhesion molecules, co-stimulatory molecule expression, and cytokine and chemokine release [7] achieved through nuclear factor ĸB (NFĸB)-mediated pathways. A protective effect of HSP70 against apoptosis has also been established. HSP70 can directly associate with apoptotic protease activating factor 1 (APAF-1), preventing recruitment of caspases to the apoptosome complex and blocking the latter’s functional assembly [8, 9].

Chaperones are abundant in skeletal muscle, which is unsurprising as the tissue is highly metabolically active and susceptible to contraction-induced and other damage, which further increases in both acquired and hereditary muscle disorders [10, 11]. Such general upregulation of chaperones in diseased muscle illustrates the tissue’s attempt to restore and regenerate. However, individual studies reported varied regulation and systematic evaluations are few, clouding our understanding of the beneficial potential of these self-protective factors. To elucidate the role of chaperones further, this study zoomed in on the muscular dystrophies (MD). They represent a diverse group of hereditary muscle disorders, with a broad spectrum of severity and common as well as more specific disease features. Duchenne MD is the most frequent and severest form and is, along with milder Becker MD, caused by mutations in the gene encoding the muscle membrane protein dystrophin. Another large subgroup encompasses limb girdle MD (LGMD) characterized by proximal weakness affecting the pelvic and shoulder girdles and caused by more than 30 different genetic defects of both autosomal dominant and recessive inheritance [12].

HSP70 displays a broad spectrum of activity, yet is presumed to display mainly positive effects on muscle fiber recuperation from damage, which makes it a potential target for therapy. Our study aimed to further elucidate its potential role in successful muscle fiber regeneration by investigating the mdx mouse, the most widely used animal model for Duchenne MD. In the time frame between 4 and 12 weeks of age, mdx skeletal muscles experience severe bouts of muscle fiber degeneration and regeneration, yet subsequently evolve to stable disease [13, 14]. The mdx model thus displays a milder skeletal muscle phenoytype and near normal life expectancy, accomplishing more successful skeletal muscle tissue recuperation than human Duchenne MD. In this study, we confront findings in the earliest phase of disease of mdx mice with results of quantification and localization of HSP70 in patients with genetically characterized Becker MD and LGMD due to ANO5 variants. We observed disease stage- and regeneration-dependent HSP70 expression patterns.

HSP70 dysfunction has been firmly associated with reduced capacity of muscle to recover from damage [15]. However, there is little consensus on the importance of HSP70 in disease progression of MD, due to reported age- and individual patient-related differences. Interestingly, dominant LGMD type 1 (LGMDD1) is caused by mutations in the HSP70 co-chaperone gene DNAJB6 and is characterized by protein aggregation and vacuole formation, illustrating the potency of HSP70 to prevent proteinopathy [16]. Earlier, we reported elevated levels of both HSP70 and HSP90 in two 9-year-old patients diagnosed with Duchenne MD, the severest form of MD [10]. In the current study, we analysed muscle from patients with milder Becker MD, and LGMD due to ANO5 defects, and found HSP70 levels to be unchanged compared to normal controls. We confirmed earlier reports of low TLR expression in muscle biopsies of healthy individuals, with TLR4 the most abundant [17], and found their levels did not significantly increase in LGMD. In the mdx mouse model for Duchenne MD, we could show a transient upregulation of HSP70 specifically during the earliest phase of MD at 4 weeks of age. Increased protein levels were not accompanied by increased hspa1a expression, suggesting regulation downstream from transcription. Similarly, hspa1a mRNA levels have been reported unchanged during proliferation and differentiation of C2C12 cells in culture, while protein levels increased 2-fold at day 2 of differentiation [18]. Such increased protein translation unparalleled by increased gene transcription is quite common in stimuli-induced expression [19]. From murine models, the picture is emerging of subtle and transient HSP70 induction in young mice, which vary between studies and disease stages [20, 21]. In addition, HSP70 muscle levels have been proposed a valuable mdx disease severity marker, decreasing with therapeutic response [22]. The elevated HSP70 levels in mdx of the youngest age we reported here, later returning to levels of healthy controls, possibly mark the most active phase of muscle fiber regeneration and degeneration. Such small window of HSP70 upregulation in mdx fits with in vitro studies that determined HSP70 levels increased when cells differentiate from myoblast to myotube [18], a process impaired when cells were treated with HSP70 siRNA [23] further illustrating the positive role HSP70 plays in myogenic differentiation and regeneration.

In addition to intracellular protein salvage activities, extracellular HSP70 may have immune modulatory effects which could be relevant to the inflammatory components of MD [24]. A large biomarker study identified HSP70 among the proteins significantly elevated in Duchenne MD sera [25]. Our study found unchanged HSP70 levels in Becker MD and LGMD sera. It is proposed that circulating HSP70 may activate immune cell responses via TLRs on antigen-presenting cells [15]. Circulating HSP70 levels correlated with markers of inflammation in neurological disease [26], and TLR activation has been implicated in MD-associated muscle damage [27]. Muscle is a possible source of circulating HSP70, as challenged muscle cells may induce chaperone expression with regeneration and in response to pro-inflammatory cytokines, releasing HSP70 when they become injured. In addition, muscle cells represent facultative antigen presenting cells through induction of MHC-I (and II). While HSP70 levels were unchanged, we did observe prominent regulation of two proteins involved in apoptotic processes. Fitting with an earlier study in Duchenne MD and FSHD [28], pro-caspase 3 was elevated while the levels of the protective factor catalase were significantly decreased, pointing to nonspecific apoptosis activation in muscle disease.

While we await proliferation of genetic therapies aimed to restore the underlying molecular defect, current treatment available to MD patients mostly aims to alleviate symptoms. Since genetic approaches will not be able to cure all patients in the foreseeable future, further development of alternative supportive approaches remains relevant. Of note, chaperone expression studies harbour yet again strong arguments to advocate for physical exercise to promote health. HSP70 levels decline with disuse [29], athletes exhibit 2.8-fold higher basal serum levels than sedentary age-matched controls [30], and levels significantly increase after high-intensity exercise [31]. In addition, reduced circulating HSP70 has been observed in old age [32, 33] possibly explaining the decline in the body’s resilience to counteract damage [34]. Higher circulating HSP70 levels reported in young Duchenne MD patients compared to healthy age-matched controls diminishing with advancing age [25] could therefore point to an accumulating inability to resolve progressive muscle damage. It has been established that mdx muscle benefits from enhancing HSP70 [35, 36]. Stimulating chaperone activity thus could represent a therapeutic strategy to be explored further for MD, and scientific evidence has emerged appointing protective effects to HSP70 agonists. The pharmacological HSP70 inducer BGP-15 improved dystrophic pathology in mdx treated between ages 4 to 9 weeks, yet interestingly not at a later stage of the disease progression [34, 35], again pointing to a therapeutic ‘window of opportunity’ early in dystrophinopathy. The question remains whether this might translate in an advantageously larger therapeutic timeframe for human Duchenne, which exhibits more constant deterioration of muscle function, or whether or not other MD patient subgroups could benefit.

This descriptive study of chaperone expression in muscular dystrophies found disease stage-associated HSP70 variations without overt activation of HSP70/TLR immune-regulatory pathways. Therapeutic interventions stimulating HSP70 function could therefore hold potential to improve patients’ quality of life in an attempted to attenuate loss of muscle mass and improve repair and regeneration, while we await gene therapies to become available to more patients.

BDP designed the research study. GC, CM, JLDB and BDP performed the research. GC, CM, JLDB and BDP analyzed the data. BDP wrote the manuscript. All authors revised the manuscript and read and approved the final version of the manuscript.

Human experimentation adhered to ethical and privacy regulations, all patients consented to participate to the study of which procedures had been approved by the Ghent University Hospital Ethics Committee (B670201836756, B670201938779). Animal experiments were conducted at the certified specific pathogen free animal facility of Ghent University (ECD17/130), approved by the Animal Ethics Committee of Ghent University, faculty of Medicine and Health Sciences (ECD19/77, ECD19/110) in accordance with European guidelines (Directive 2010/63/EU).

We thank Jana Zschüntzsch and Jens Schmidt from the Neurology department of Göttingen University Medical Center for providing laboratory facilities and expert advice on transcription analyses. Jan L. De Bleecker is a member of the European Reference Network (ERN) for Rare Neuromuscular diseases (Euro-NMD).

CM and BDP received funding from the Association Belge contre les Maladies neuroMusculaires (ABMM) aide à la research grants 2019 and 2020. JDB received research sponsoring from CSL Behring and manages private donations made available to the Fund for neuromuscular disorders UGhent GE461. CM and BDP received funding for research mobility from the ERN European Joint Programme on Rare Diseases (EJP-RD) and from U4 for the OSMYO research network.

The authors declare no conflict of interest.