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 , 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 aggregation-prone 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 . 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 . However, no significant changes in the content of ubiquitinated proteins was found between Hspa4-KO and WT muscles , 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 . 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 . Recently, it has been reported that HSPA4 inactivates NF-κB pathway and therefore inhibits inflammatory signaling . 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 . 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 , 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.