From proteostatic control to collapse: exploring the ribosome-associated quality control – integrated stress response axis in Myofibrillar myopathy 13

Background. The small heat shock protein B8 (HSPB8) is an essential chaperone for proteostasis maintenance, particularly within striated muscles and the nervous tissue. Acting in complex with the cochaperone BAG3 and selected HSPA family members bound to the E3-ubiquitin ligase STUB1, HSPB8 promotes the degradation of misfolded proteins via chaperone-assisted selective autophagy (CASA). Frameshift mutations in the third exon of HSPB8 cause Myofibrillar myopathy 13 by leading to the production of aberrant proteins with a modified C-terminal region and a C-terminal extension that drives cytoplasmic HSPB8 aggregation. Analyses on patient-derived fibroblasts and iPSCs harbouring a heterozygous HSPB8 frameshift mutation revealed a selective downregulation of mutant allele expression, as well as the absence of the elongated protein isoform under basal conditions. The mutant protein becomes barely detectable only upon stress induction, suggesting the existence of quality control mechanisms selectively targeting mutated HSPB8 and conferring protective haploinsufficiency. In this framework, our study explores the interplay between the ribosome-associated quality control system and the integrated stress response (RQC-ISR). We propose that the RQC-ISR axis exerts a proteostatic function that is compromised under conditions of elevated HSPB8 expression and/or stress, facilitating mutant protein accumulation and driving disease progression. Methods. A HEK293T-based transient overexpression system was first exploited to assess the potential impact of HSPB8 frameshift mutations on the RQC-ISR axis. The factors of interest were analysed by western blot, filter trap assay and immunofluorescence. Subsequently, human myogenic differentiation 1-driven differentiation of patient-derived iPSCs to myotubes was performed to generate a more physiological cell model for disease investigation. Results. Experimental evidence supports the involvement of the RQC-ISR axis in HSPB8 frameshift mutant-driven pathology. Frameshift HSPB8 overexpression drives the transcriptional upregulation of RQC factors LTN1 and NEMF, while RQC weakening induced by LTN1 or NEMF knockdown alters mutant HSPB8 aggregate levels and distribution. The increased prevalence of small, dispersed HSPB8 aggregates suggests a reduced coalescence of peripheral microaggregates into the aggresome. Moreover, HSPB8 frameshift mutants alter the subcellular distribution of both the RQC factor VCP and the ISR master regulator ATF4, promoting their aggregation at the aggresome. This correlates with decreased VCP and ATF4 solubility and a tendency toward reduced ATF4 nuclear translocation. The preliminary characterization of iPSC-derived myotubes demonstrates the upregulation of HSPB8 expression relative to iPSCs, concomitant with the detectable presence of the elongated mutant isoform under unstressed conditions. Conclusions. Our findings identify a functional crosstalk between RQC and HSPB8 that promotes the targeting of defective ribosomal products to the aggresome. This proteostatic node likely underlies the protective haploinsufficiency observed in patient-derived iPSCs and fibroblasts, but becomes compromised upon frameshift HSPB8 overload. Consistent with the established coupling between RQC dysfunction and ISR activation, our data support a coordinated response between protein quality control and stress signalling in this context. Notably, excessive accumulation of mutant HSPB8 appears to attenuate ISR activation, thereby further impairing cell ability to respond to stress. Future studies on the newly-established in vitro myotube model will help clarify RQC-ISR dynamics and better identify the molecular mechanisms underlying Myofibrillar myopathy 13.