Molecular genetics of J-domain protein-related chaperonopathies in skeletal muscle

Abstract

The J-domain proteins (JDPs), or HSP40s, are essential molecular co-chaperones that, in concert with HSP70, play a pivotal role in maintaining protein homeostasis, which is particularly critical in skeletal muscle. In recent years, pathogenic variants in several JDP-encoding genes have been identified as a cause of a growing group of inherited muscle diseases, termed JDP-related myopathies. This review provides a comprehensive overview of the current understanding of the molecular genetics, clinical phenotypes, muscle pathology, and pathomechanisms of myopathies caused by mutations in DNAJB6, DNAJB4, and DNAJB2. These disorders present with a wide spectrum of clinical features, including limb-girdle or distal weakness, and, in some cases, severe early-onset respiratory failure with axial rigidity. Pathologically, they are often characterized by rimmed vacuoles and sarcoplasmic protein inclusions. The underlying molecular mechanisms, while all converging on the disruption of the JDP-HSP70 chaperone system, are driven by distinct molecular events depending on the specific gene and mutation type. While loss-of-function is a primary mechanism for recessive forms of DNAJB4 and DNAJB2 myopathy, a toxic gain-of-function mediated by a dysregulated interaction with HSP70 is emerging as a central pathomechanism for dominant myopathies caused by DNAJB6 and DNAJB4 variants. A dominant-negative effect is proposed for dominant DNAJB2 neuromyopathy. This evolving mechanistic understanding is crucial as it informs the development of targeted therapeutic strategies, moving beyond supportive care. Potential future therapies include gene replacement for loss-of-function disorders, and for gain-of-function diseases, approaches including small molecule inhibitors of the JDP-HSP70 interaction or allele- and isoform-specific knockdown.

INTRODUCTION

The J-domain proteins (JDPs), a large and functionally diverse family of molecular co-chaperones, are also referred to as heat shock protein 40 (HSP40) or DnaJ proteins. This nomenclature has historical roots: the archetypal member, DnaJ, was discovered in Escherichia coli during a genetic screen for mutants with defects in DNA replication, hence the name DnaJ. While subsequently characterized as a molecular chaperone, this original naming convention has persisted. All proteins in this family share a highly conserved, ~70-amino-acid sequence derived from DnaJ known as the J-domain, which gives the family its most common name, “J-domain proteins”¹. Their primary role involves modulating the ATPase activity of heat shock protein 70 (HSP70) chaperones, thereby playing indispensable roles in maintaining cellular protein homeostasis (proteostasis), which encompasses processes such as protein folding, assembly, trafficking, and degradation¹. Skeletal muscle is an organ characterized by high protein synthesis and turnover rates and is frequently subjected to a variety of stresses, including mechanical strain, oxidative stress, and metabolic fluctuations^2–5. Consequently, a robust and efficient chaperone network is of paramount importance for preserving proteostasis and ensuring overall muscle health and function.

The understanding of JDPs in muscle health and disease was significantly advanced by the seminal report in 2012, which identified mutations in DNAJB6, a JDP-encoding gene, as a cause of limb-girdle muscular dystrophy (LGMD)^6,7. This pivotal discovery catalyzed a surge in research interest, leading to the progressive elucidation of the critical physiological and pathological roles of JDPs within skeletal muscle. Subsequently, a growing number of neuromuscular conditions have been classified as ‘chaperonopathies,’ arising from pathogenic variants in genes encoding molecular chaperones or their co-factors, co-chaperones⁸. Among these, mutations in several members of the JDP family, most notably DNAJB6, DNAJB4, and DNAJB2 have been definitively linked to a spectrum of myopathic disorders, including LGMDs and distal myopathies. A thorough understanding of the molecular genetic underpinnings of these JDP-related myopathies is crucial for enhancing diagnostic precision and for the rational development of targeted therapeutic interventions. Therefore, this review aims to provide a comprehensive overview of the current knowledge regarding the clinical phenotypes, genotypic spectrum, and molecular pathogenesis of myopathies caused by mutations in JDP-encoding genes. Furthermore, we will discuss emerging therapeutic strategies relevant to these disorders.

J-DOMAIN PROTEINS: CLASSIFICATION, CLIENT INTERACTION, AND THE HSP70 CHAPERONE SYSTEM

JDPs represent a critical family of co-chaperones that function in concert with HSP70 chaperones to maintain cellular protein homeostasis (Fig. 1A). They play a crucial role in recognizing and binding to a wide array of specific substrate proteins, referred to as “client proteins.” It is thought that JDPs largely determine the specificity of the HSP70 system by targeting it to these diverse clients, which include nascent polypeptides, misfolded proteins, or proteins destined for translocation or degradation¹.

Figure 1. The JDP-HSP70 chaperone cycle and domain organization of JDPs implicated in myopathies.

(A) The diagram illustrates the fundamental protein quality control cycle mediated by J-domain proteins (JDPs) and HSP70. A JDP recognizes and delivers a misfolded client protein to an ATP-bound HSP70, stimulating HSP70’s ATPase activity. Upon ATP hydrolysis, HSP70 tightly binds the client, preventing its aggregation and facilitating either its refolding to a native state or its triage for degradation. In JDP-related myopathies where this system is disrupted, several therapeutic strategies (indicated in red) can be envisioned. These include (i) reducing the synthesis of aggregation-prone client proteins (e.g., via siRNA), (ii) modulating the client-JDP interaction, or (iii) inhibiting the JDP-HSP70 interaction, particularly to counteract toxic gain-of-function effects. Created in BioRender. Inoue, M. (2025) https://BioRender.com/ 0mk7efg (B) Domain architecture of the three major classes of human JDPs (Class A, B, and C). All classes share a conserved J-domain (J) responsible for HSP70 interaction and a C-terminal domain (C-term) involved in substrate binding. Class A proteins additionally contain a glycine/phenylalanine-rich domain (G/F) and a zinc-finger-like motif (ZnF). Class B proteins have a G/F domain but lack the ZnF, while Class C proteins possess neither. (C) Schematic representation of pathogenic variants in DNAJB6 and DNAJB4 associated with myopathy. Variants written in black indicate autosomal dominant inheritance, while those in blue indicate autosomal recessive inheritance. All listed DNAJB6 variants affect both major isoforms (DNAJB6a and DNAJB6b), with the exception of the p.Val232Glyfs7 variant, which is specific to the DNAJB6a isoform. The splice-site variant c.236–1_240delGGTGGA has been reported to cause deletions of both p.Gly79_Phe115 and p.Gly79_Cys96.

The defining feature of all JDPs is the highly conserved J-domain, which directly interacts with HSP70 to stimulate its ATPase activity. Beyond the conserved J-domain, JDPs are broadly categorized into three main classes—Class A, Class B, and Class C—based on their additional domain architecture, as schematically represented in Figure 1B. Class A JDPs are characterized by an N-terminal J-domain, an adjacent glycine/phenylalanine (G/F)-rich domain, and C-terminal domains with a zinc-binding motif. Class B JDPs, which include DNAJB6, DNAJB4 and DNAJB2 predominantly implicated in myopathies, also possess the J-domain, G/F-rich domain, and C-terminal domains, but they lack the zinc-binding motif. Class C JDPs are a more heterogeneous group, containing a J-domain but lacking some or all of the other conserved domains found in Class A and B proteins.

This JDP-driven regulation is central to the HSP70 chaperone cycle (Fig. 1A). The process begins when a JDP delivers a bound client protein to an ATP-bound HSP70 molecule. The interaction of the JDP’s J-domain with HSP70 stimulates the hydrolysis of ATP to ADP, which induces a conformational change in HSP70, leading to the stable, high-affinity binding of the client protein. The cycle is completed by nucleotide exchange factors, which promote the exchange of ADP for ATP on HSP70. This regenerates ATP-bound HSP70, which releases the client and readies the system for subsequent rounds of activity⁹.

The precise regulation and execution of this HSP70 chaperone system are fundamental for maintaining cellular proteostasis. Consequently, dysfunction of JDPs, arising from genetic mutations, can disrupt this vital protein quality control pathway, likely leading to the accumulation of pathogenic client proteins and contributing to the development of various human diseases, including the JDP-related myopathies central to this review.

JDP-RELATED MYOPATHIES: GENETIC, CLINICAL, AND PATHOLOGICAL SPECTRUM

Mutations in several JDP-encoding genes cause inherited myopathies. This section will focus on the molecular genetics, clinical phenotypes, and pathology associated with the most prominently implicated JDPs. These include DNAJB6 and DNAJB4, whose pathogenic variants are schematically summarized in Figure 1C, and DNAJB2. Regarding other JDPs, a p.Pro15Ser variant in DNAJB5, for example, has been reported in a family with neuromyopathy¹⁰. However, its definitive role in myopathy remains unclear at present, primarily because it has been identified in only a single family and is found with a relatively high frequency in public database (Minor allele frequency based on 291/1,551,582 alleles in gnomAD) for a rare dominant disorder. Thus, conclusive evidence establishing DNAJB5 as a primary myopathy-causing gene is currently lacking. Genetic and clinical features of JDP-related myopathies are summarized in Table 1.

Table 1.

J-domain proteins causing myopathy

Gene	Inheritance	Disease/Phenotype	Typical Age of Onset	Key Clinical Features	Muscle Pathology
DNAJB6	AD	LGMDD1 / Distal myopathy	Adulthood (variable)	Slowly progressive limb-girdle and/or distal weakness	Rimmed vacuoles, myofibrillar disorganization, occasional cytoplasmic inclusions
	AR†	Distal myopathy	Late adulthood	Slowly progressive distal to limb-girdle weakness	Rimmed vacuoles, myofibrillar disorganization, cytoplasmic inclusions
DNAJB4	AR	Myopathy with early respiratory failure / Rigid spine syndrome	Infancy to adulthood	Early respiratory failure, axial rigidity, variable limb weakness	Large cytoplasmic inclusions , rimmed vacuoles, myofibrillar disorganization
	AD†	Distal myopathy	Adulthood	Distal weakness with onset in hand muscles, progressing to involve distal leg and proximal muscles	Large cytoplasmic inclusions, rimmed vacuoles, myofibrillar disorganization
DNAJB2	AR†	dHMN with myopathy	Adulthood	Progressive atrophy and weakness of distal muscle	Neurogenic change with infrequent rimmed vacuoles
	AD†	Neuromyopathy	Late adulthood	Sensorimotor polyneuropathy combined with myopathy, ataxia	Chronic neurogenic changes with rimmed vacuoles, internal nuclei, and fiber splitting

^†indicates cases in one family have reported in the literature so far. AD, autosomal dominant; AR, autosomal recessive; LGMDD1, limb-girdle muscular dystrophy type D1; dHMN, distal hereditary motor neuropathy.

1. DNAJB6-related myopathies

Genetic spectrum and inheritance patterns

Mutations in DNAJB6 are predominantly inherited in an autosomal dominant manner and are a recognized cause of LGMDD1 as well as distal myopathy. Initially, pathogenic variants were almost exclusively identified within a highly conserved region of the G/F-rich domain, encoded by exon 5. This region was considered a mutational hotspot for the gene. These variants are typically missense mutations, such as the frequently reported p.Phe93Leu and p.Pro96Arg, or small in-frame deletions like p.D98del. Splice-site mutations leading to the deletion of the entire G/F domain (e.g., p.Gly79_Phe115del as a result of c.346+5G>A) also occur and often result in severe phenotypes^11–18.

More recently, the mutational spectrum has expanded beyond the G/F domain with the identification of pathogenic missense variants within the N-terminal J-domain, such as p.Ala50Val and p.Glu54Ala. These J-domain mutations also cause dominantly inherited myopathy, typically presenting as distal or proximo-distal myopathy, broadening the understanding of critical functional regions within DNAJB6¹⁹.

Further expanding the genetic complexity, a single case report with functional studies has described a homozygous frameshift mutation (c.695_699del; p.Val232Glyfs*7) in exon 9, uniquely affecting the DNAJB6a isoform, associated with autosomal recessive late-onset distal myopathy²⁰. While this isolated finding suggests the potential for recessive inheritance and isoform-specific pathogenic mechanisms in DNAJB6-related myopathies, confirmation through additional cases is still required. Thus, the known spectrum of DNAJB6 pathogenic variants predominantly involves autosomal dominant missense changes in the G/F or J-domains.

Clinical phenotypes

Dominantly inherited DNAJB6-related myopathies typically manifest as LGMDD1, but a significant portion of patients present with distal predominant weakness. These conditions exhibit considerable variability in age of onset, which can range from early childhood to late adulthood (mean ~30 years), and in the rate of progression²¹. Genotype influences these clinical features; for instance, specific mutations like p.Phe89Ile, p.Phe91Ile/Leu, and p.Pro96Arg/Lue are often associated with an earlier onset compared to p.Phe93Ile/Leu variants, and particular mutations correlate with weakness patterns (e.g., p.Pro96 mutations frequently with distal weakness; J-domain mutations such as p.Ala50Val and p.Glu54Ala also causing distal or proximo-distal forms)^19,21. Disease progression is generally slow, with a median time to loss of ambulation reported around 34 years from onset, a timeframe also impacted by the specific DNAJB6 mutation²¹. Dysphagia is the most common additional symptom. Respiratory and cardiac involvement are less frequent, typically mild, and manifest later in the disease course, although severe respiratory failure has been documented with certain mutations^2,21. Central nervous system involvement is rare, and serum creatine kinase (CK) levels are usually normal or only mildly elevated^2,22.

Muscle pathology

Key histopathological features of DNAJB6 myopathy, often presenting as a myofibrillar myopathy, include the presence of rimmed vacuoles (Fig 2A), occasional sarcoplasmic protein aggregates, and varying degrees of myofibrillar disorganization or sarcomere disruption. These pathological changes are observed across different pathogenic DNAJB6 variants and clinical presentations^13,22.

Figure 2. Representative myopathological features of JDP-related myopathies.

(A, B) DNAJB6-related myopathy. (A) Modified Gomori trichrome (mGT) stain of a muscle biopsy showing a small angular fiber with rimmed vacuoles (arrow). (B) Dual immunofluorescence reveals sarcoplasmic aggregates that are immunoreactive for both DNAJB6 (red) and the RNA-binding protein TDP-43 (green). Panel B is adapted from Harms et al., Ann Neurol, 2012. (C, D) Autosomal recessive DNAJB4-related myopathy. (C) Hematoxylin and eosin (H&E) stain showing myofibers with large cytoplasmic inclusions (arrowheads) and rimmed vacuoles (arrow). (D) Immunohistochemistry demonstrates desmin-positive sarcoplasmic accumulation in affected fibers. Panels C and D were kindly provided by Dr. Aurelio Hernández Lain. (E, F) Autosomal dominant DNAJB4-related myopathy. (E) mGT stain showing large, centrally located cytoplasmic inclusions (arrowheads). (F) Dual immunofluorescence demonstrates that these large inclusions are immunoreactive for both DNAJB4 (red) and desmin (green). Panel F is adapted from Inoue et al., Acta Neuropathol, 2023. Scale bars: (A, B, F) 50 μm; (C, D) 20 μm; (E) 100 μm.

The protein aggregates in DNAJB6 myopathy are typically complex in their composition. Immunohistochemical studies have revealed that these inclusions frequently contain the DNAJB6 protein itself, other chaperone proteins, and components of the Z-disc (Fig. 2B)^6,7,13,22. Notably, RNA-binding proteins, particularly TDP-43, are also commonly found within these aggregates. The co-sequestration of these diverse proteins, especially those involved in protein quality control and RNA processing, strongly suggests that a significant disruption of cellular proteostasis is a central element in the pathogenesis of DNAJB6-related myopathies.

2. DNAJB4-related myopathies

Pathogenic variants in the DNAJB4 gene have been linked to distinct forms of myopathy with both dominant and recessive inheritance patterns, underscoring the critical role of DNAJB4 in skeletal muscle proteostasis.

Genetic spectrum and inheritance patterns

DNAJB4-related myopathies exhibit both autosomal dominant and autosomal recessive modes of inheritance, contingent upon the specific genetic variant and its functional impact.

An autosomal dominant form of DNAJB4 myopathy is caused by a heterozygous missense variant, c.270T>A (p.Phe90Leu), located in the G/F-rich domain of DNAJB4. This phenylalanine at position 90 is significant as it corresponds to the Phe93 residue in the homologous protein DNAJB6, where mutations are also known to cause myopathy.

Autosomal recessive DNAJB4 myopathy results from biallelic variants. The spectrum of these recessive variants is diverse, encompassing loss-of-function (LOF) alterations such as nonsense (stop-gain) mutations (e.g., p.Arg183*, p.Arg259*, p.Lys286*), frameshift mutations, and exon deletions (e.g., an exon 2 deletion). These typically lead to diminished protein stability or the absence of functional DNAJB4 protein. Homozygous missense variants have also been identified, some residing within the J-domain (e.g., p.Lys35Asn, p.Arg61Gly, p.Arg25Gln) and others in the C-terminal domain (e.g., p.Leu262Ser). While certain missense variants like p.Leu262Ser result in an unstable protein, others such as p.Arg25Gln, p.Lys35Asn, and p.Arg61Gly in the J-domain can be stable yet exhibit a loss of function in cellular or yeast-based functional assays. Heterozygous carriers of these recessive variants are reported to be clinically unaffected^23,24.

Clinical phenotypes

The clinical presentations of DNAJB4-related myopathies differ notably between the dominant and recessive forms.

Dominant DNAJB4 myopathy, associated with the p.Phe90Leu variant, characteristically presents as a late-onset distal myopathy, with symptoms typically emerging in the third to fifth decade of life. Initial manifestations often include asymmetric handgrip weakness and atrophy of thenar and hypothenar muscles, which may progress to involve distal leg muscles and, eventually, proximal musculature. In advanced stages, respiratory insufficiency can develop²⁵.

Recessive DNAJB4 myopathy is frequently marked by early respiratory failure and significant axial muscle involvement, such as rigid spine syndrome and neck stiffness, with an age of onset ranging from infancy to adulthood. Additional clinical features can include dysphagia, ankle contractures, scoliosis, and cardiac dysfunction. Limb muscle weakness may not be the primary or most pronounced initial symptom^23,24.

A notable genotype-phenotype correlation has been identified in this recessive form. In contrast to what is typically observed in recessive diseases, biallelic missense variants within the J-domain are associated with a more severe clinical course and earlier onset than biallelic loss-of-function variants (e.g., nonsense or frameshift mutations). This severe phenotype can include higher mortality and, in some cases, brain imaging abnormalities. This observation suggests that these J-domain missense mutations may induce a pathogenic mechanism beyond a simple loss of function, possibly involving a toxic effect²⁴.

Muscle pathology

Muscle pathology in DNAJB4-related myopathies can share overlapping features between the autosomal dominant and some autosomal recessive forms, although variability, particularly in the prominence of inclusions, has been noted in recessive cases. A key characteristic often observed is the presence of rimmed vacuoles and relatively large cytoplasmic inclusions within affected myofibers (Fig. 2C, E)^23–25. The cytoplasmic inclusions commonly contain Z-disc-related proteins such as desmin and myotilin (Fig. 2D). In the dominant form caused by the p.F90L missense variant, DNAJB4 protein itself is also found within these inclusions (Fig. 2F). Ultrastructural examination reveals common alterations such as the accumulation of dense granulofilamentous or woolly/sand-like materials within these inclusions, alongside Z-disc streaming. While the inclusions themselves contain degenerated sarcomeric material, Z-discs and sarcomeric structures immediately surrounding these aggregates can appear relatively preserved^23–25.

3. DNAJB2-related myopathies/neuromyopathies

Pathogenic variants in the DNAJB2 gene (also known as HSJ1), which encodes the JDP cochaperones DNAJB2a and DNAJB2b, predominantly cause autosomal recessive peripheral axonal neuropathies. However, two families involving muscle tissue, including a distinct autosomal dominant neuromyopathy, have been reported^26,27. Most recessive DNAJB2 variants lead to LOF. Myopathy is an uncommon feature in these recessive disorders but has been described as a rimmed vacuolar myopathy in conjunction with distal hereditary motor neuropathy (dHMN) in a patient homozygous for the c.184C>T (p.Arg62Trp) missense variant²⁶. More recently, a heterozygous stop-loss variant in DNAJB2 (c.832T>G, p.(*278Glyext*83)), resulting in a C-terminal extension specifically of the DNAJB2a protein isoform, was identified as the cause of a late-onset (fifth to sixth decade) autosomal dominant neuromyopathy²⁷. Clinically, this dominant form presented with a progressive combination of sensorimotor polyneuropathy and myopathic features, including significantly elevated serum CK levels. Muscle biopsy in this dominant case revealed severe chronic neurogenic changes alongside clear myopathic alterations such as fiber splitting, internalized nuclei, and rimmed vacuoles, but without significant accumulation of p62, TDP-43, or DNAJB2/DNAJB6 proteins²⁷.

MOLECULAR PATHOMECHANISMS OF JDP-RELATED MYOPATHIES

Mutations in genes encoding JDPs disrupt cellular proteostasis through diverse molecular mechanisms, including LOF, dominant-negative effects, or toxic gain-of-function (GOF), ultimately leading to muscle degeneration. This section will primarily focus on the pathomechanisms underlying DNAJB6-related myopathy, the most extensively studied among JDP-opathies, and will integrate relevant findings for DNAJB4 where applicable.

Disrupted Chaperone Function and Client Processing

One key aspect is the altered chaperone activity of mutant DNAJB6. Wild-type DNAJB6, particularly its cytoplasmic isoform DNAJB6b, possesses a potent Hsp70-independent capacity to suppress the aggregation of misfolded proteins, such as polyglutamine-expanded huntingtin^7,19,28. While early studies by Sarparanta et al., utilizing cell-based assays (e.g., filter-trap assays on lysates from 293 cells), suggested that LGMDD1-associated DNAJB6b mutations (e.g., p.Phe93Leu, p.Phe89Ile) significantly impaired this protective anti-aggregation function in cellulo⁷, more recent work by Abayev-Avraham et al. using purified recombinant proteins (in vitro) demonstrated that several DNAJB6 disease mutants surprisingly retain intrinsic aggregation-prevention activity comparable to wild-type against both polyQ and TDP-43 substrates²⁸. This apparent discrepancy highlights that while the intrinsic ability of mutant DNAJB6 to bind misfolded substrates in vitro may be preserved, its overall protective capacity within a complex cellular context could be compromised due to altered interactions with other cellular components, particularly HSP70²⁸.

The pathomechanism appears intricately linked to substrate conformer-specific defects in client processing. This concept, exploring how chaperones interact differently with misfolded proteins of the same sequence but distinct three-dimensional structures (conformers), was investigated by Stein et al.²⁹. Using a yeast model with a Sis1-DNAJB6 G/F domain chimera, their work revealed that LGMDD1-analogous mutations differentially impaired the processing of specific amyloid conformations (strains) of yeast prions, suggesting that the G/F domain is crucial for recognizing and handling particular substrate structures²⁹. This was further supported by Bhadra et al., who showed that LGMDD1-analogous G/F domain mutations in the DNAJB6 yeast homolog Sis1 altered client (Rnq1) aggregate structure, impacted Sis1 dimerization, and modified the Hsp70 ATPase cycle and substrate processing in a client-conformer specific manner. These mutant Sis1 proteins were also defective in refolding denatured luciferase and showed impaired binding to both substrate and Hsp70 (Ssa1)³⁰.

Toxic Gain-of-Function through Dysregulated HSP70 Interaction

A significant emerging mechanism for DNAJB6 myopathy is a toxic GOF mediated through its interaction with HSP70. Substantial evidence for this model was provided by Bengoechea et al.³¹, who demonstrated that LGMDD1-mutant DNAJB6b exhibited increased association with HSP70 in cellular and mouse models. Notably, they showed that inhibiting this DNAJB6-HSP70 interaction with small molecules remobilized HSP70 at the Z-disc, improved muscle strength, and ameliorated myopathology in LGMDD1 mouse models, suggesting that the aberrant HSP70 interaction is central to toxicity³¹.

Further molecular insights into this GOF mechanism were provided by Abayev-Avraham et al.²⁸. They demonstrated that wild-type DNAJB6 possesses an autoinhibitory mechanism where a helical element within the G/F domain regulates J-domain interaction with HSP70. Their NMR and biochemical data revealed that LGMDD1 mutations disrupt this autoinhibition, rendering the J-domain constitutively accessible for HSP70 binding²⁸. Consequently, mutant DNAJB6 can bind to HSP70 and constitutively stimulate its ATPase activity in a client-independent manner. This high-affinity, non-productive interaction is hypothesized to deplete cellular HSP70 levels, thereby disrupting global proteostasis and inducing toxicity. This model was strongly supported by their findings that abrogating the DNAJB6-HSP70 interaction rescued the disease phenotype in C. elegans models²⁸. A comparable HSP70-dependent toxic GOF is also proposed for the dominant p.Phe90Leu variant in DNAJB4, which enhances TDP-43 aggregation in cell models in a manner that is reversed by an additional H32Q mutation abolishing HSP70 interaction²⁵.

Altered Protein Stability and Dominant Effects

The altered stability of mutant DNAJB6 may also contribute to its dominant pathogenic effect. In cell culture experiments, LGMDD1-mutant DNAJB6b (p.Phe93Leu and p.Phe89Ile) exhibited a significantly increased half-life (i.e., reduced turnover and increased stability) compared to the wild-type protein. Moreover, the mutant protein appeared to extend this stabilizing effect to co-expressed wild-type DNAJB6b, potentially leading to an overall increase in dysfunctional or toxic DNAJB6 oligomeric complexes⁷. This contrasts with many recessive DNAJB4 LOF variants, which result in unstable and rapidly degraded or absent DNAJB4^23,24. However, the relationship between stability and function is complex, as some recessive DNAJB4 missense variants in the J-domain (e.g., p.Arg35Gln) can be stable or even show increased stability compared to wild-type, yet remain functionally impaired²⁴.

Impairment of Chaperone-Assisted Selective Autophagy (CASA)

The impairment of specific protein quality control pathways, such as Chaperone-Assisted Selective Autophagy (CASA), is implicated in the pathology of some JDP-related myopathies. The CASA pathway is a specialized form of autophagy crucial for maintaining sarcomeric integrity, particularly under mechanical stress. This machinery involves a core complex of the co-chaperone BAG3, the small heat shock protein HSPB8, and the E3 ubiquitin ligase CHIP (also known as STUB1), which cooperate with HSP70 to recognize mechanically unfolded or damaged client proteins, such as filamin C, and target them for autophagic degradation³². Evidence suggests that DNAJB6 is functionally linked to this pathway. DNAJB6 localizes to the Z-disc and has been shown to interact with key CASA components, including BAG3, HSPB8, and CHIP/STUB1⁷. Furthermore, patient muscle biopsies consistently show myofibrillar disorganization, autophagic vacuoles (rimmed vacuoles), and the co-accumulation of DNAJB6 with these CASA-related proteins within aggregates, indicative of a dysfunctional protein degradation system^6,7,13,22. While these findings suggest that DNAJB6 dysfunction could impair CASA-mediated Z-disc maintenance, direct evidence establishing DNAJB6 as a canonical component of the CASA complex is still needed.

Pathomechanisms of DNAJB4-Related Myopathies: Loss-of-Function

Recessive DNAJB4 myopathy is predominantly caused by LOF variants, a mechanism distinct from the toxic gain-of-function seen in dominant forms of JDP myopathy. The spectrum of these recessive LOF variants includes nonsense mutations, frameshift mutations, and specific missense variants in the C-terminal domain, including p.Leu262Ser. Consistent with a LOF mechanism, Western blot analysis of skeletal muscle and fibroblast samples from patients with the p.Lys286Ter nonsense variant and the p.Leu262Ser missense variant showed markedly reduced or absent levels of DNAJB4 protein²³. The stability of these mutant proteins was further investigated using a tetracycline-regulatable expression system in cultured cells. This assay confirmed that after halting gene expression, the p.Lys286Ter and p.Leu262Ser mutant proteins were degraded significantly more rapidly than wild-type DNAJB4, demonstrating that these variants lead to protein instability²³.

This primary LOF mechanism contrasts sharply with dominant DNAJB6 myopathy, for which myopathy-causing LOF variants like nonsense mutations have not been reported. Consistent with its critical role, a complete knockout (KO) of DNAJB6 in mice results in embryonic lethality³³. In contrast, some recessive DNAJB4 missense variants within the J-domain (e.g., p.Arg25Gln, p.Lys35Asn, p.Arg61Gly) present a different scenario; these mutant proteins are stable, yet cellular and yeast-based assays show they are functionally impaired^23,24. The fact that biallelic LOF variants in DNAJB4 cause a recessive myopathy and that the full Dnajb4 KO mouse is viable with a distinct muscle phenotype underscores a specific, critical role for DNAJB4 in skeletal muscle that provides a valuable opportunity to study the fundamental functions of this JDP in muscle proteostasis.

Pathomechanisms in DNAJB2-Related Neuromyopathies

Pathogenic variants in DNAJB2 cause neuromuscular disorders through distinct mechanisms. Recessive DNAJB2 variants, which predominantly cause peripheral neuropathies with rare myopathic features (e.g., p.Arg62Trp), are generally considered to act via a LOF mechanism. In contrast, the autosomal dominant neuromyopathy caused by the DNAJB2a p.(*278Glyext*83) stop-loss variant involves a more complex mechanism. The mutant DNAJB2a protein mislocalizes to the endoplasmic reticulum, undergoes rapid proteasomal degradation, and appears to accelerate the turnover of wild-type DNAJB2a. This leads to a significant reduction of both DNAJB2a and DNAJB2b protein isoforms in patient muscle, suggesting a combined mechanism of haploinsufficiency and a dominant-negative effect²⁷.

Insights from Animal Models of JDP-Related Myopathies

Animal models have been essential in defining the pathogenesis of JDP-related myopathies. Early functional studies were critical; for instance, one of the seminal discovery papers utilized zebrafish to provide the first in vivo evidence that the identified mutations were pathogenic, causing distinct muscle defects and implicating the cytoplasmic functions of the DNAJB6b isoform in the disease process⁷. This finding was powerfully substantiated by the subsequent development of a transgenic mouse by Weihl’s group, which overexpressed the mutant DNAJB6b-Phe93Leu isoform specifically in muscle³⁴. This transgenic model was pivotal as it faithfully recapitulated an aggressive form of the human LGMDD1 phenotype, including progressive muscle weakness and myopathology with protein aggregates, providing robust support for a toxic GOF mechanism. Subsequently, the same group developed a more physiologically relevant knock-in mouse model expressing an LGMDD1 mutation (Phe90Ile) at endogenous levels³¹. This knock-in model also developed a progressive myopathy, confirming that the disease was not an artifact of protein overexpression. These models then became instrumental for therapeutic development. The seminal study by Bengoechea et al. utilized both the transgenic and the knock-in mice as key preclinical platforms. They demonstrated that a small molecule inhibitor (JG231) targeting the aberrant interaction between mutant DNAJB6 and HSP70 could successfully improve muscle strength in both models and ameliorate pathology in the transgenic model. This work, progressing from zebrafish to sophisticated mouse models, provided a direct and compelling link between a specific molecular mechanism and a viable therapeutic strategy.

The study of DNAJB4 myopathy has benefited from a similar progression of animal models, which have highlighted key differences from its homolog, DNAJB6. A crucial insight came from the Dnajb4 KO mouse, which, unlike the embryonic lethal Dnajb6 KO³³, is viable but develops a distinct myopathy. The Dnajb4 KO mouse exhibits kyphosis, muscle weakness, and significant diaphragm atrophy, effectively modeling the early respiratory failure seen in patients with recessive LOF mutations²³. This model definitively proved that a simple loss of DNAJB4 function is sufficient to cause myopathy.

Furthermore, a knock-in mouse model carrying the dominant p.Phe90Leu. Dnajb4 variant was generated and shown to reproduce the late-onset human phenotype²⁵. A major contribution from studying both the knock-in and KO models was the elucidation of a mechanism for muscle-specific vulnerability. The models revealed that the soleus and diaphragm muscles, which have the highest endogenous Dnajb4 expression, were the most severely affected, providing a compelling explanation for the selective muscle involvement seen in the human disease²⁵. The progression of research on DNAJB6 and DNAJB4 models showcases the power of these systems in validating disease causality and advancing targeted therapies.

THERAPEUTIC PERSPECTIVE

To date, no specific, mechanism-based therapies have been approved for any JDP-related myopathy. Broader therapeutic approaches, such as inducing a general heat shock response to upregulate chaperones (e.g., with arimoclomol) or enhancing protein degradation pathways like autophagy, have been considered for various myopathies with protein aggregation. However, these strategies have thus far shown limited success in clinical trials for related disorders such as inclusion body myositis³⁵, suggesting that a more tailored, mechanism-based approach will likely be necessary for treating JDP-opathies³⁶.

Based on our current understanding of the pathomechanisms, several potential intervention points can be envisioned for a more personalized therapeutic strategy (Fig. 1)³⁷. These include: (i) reducing the level of specific aggregation-prone client proteins (substrate reduction), (ii) modulating the client-JDP interaction, and (iii) inhibiting the JDP-HSP70 interaction.

For dominant myopathies driven by a toxic GOF, such as those caused by DNAJB6 and some DNAJB4 variants, modulating the aberrant JDP-HSP70 interaction is a promising, albeit still proof-of-concept stage, therapeutic strategy. The seminal work by Bengoechea et al. demonstrated that small molecule inhibitors of this interaction could rescue the toxic effects of LGMDD1-mutant DNAJB6 in cellular and mouse models³¹.

Strategies that reduce the level of the mutant protein are also central to treating GOF disorders. These include allele-specific knockdown using antisense oligonucleotides or RNA interference. In protein aggregate myopathies, including those caused by mutant Myotilin, preclinical studies demonstrated that RNAi-based strategies could successfully reduce aggregate formation and improve muscle function in mouse models of LGMD1A, suggesting therapeutic promise for similar approaches in JDP-related myopathies³⁸. However, critical issues remain. First, disease-relevant client proteins of JDPs are not yet fully identified. Without a clear understanding of these client proteins, therapeutic strategies aimed at reducing client protein abundance or disrupting client-JDP interactions cannot be reliably developed. Second, RNAi strategies carry risks of off-target effects and must carefully assess whether reducing the protein levels below a certain threshold might negatively affect cellular homeostasis^38,39. Thus, rigorous preclinical validation to confirm target specificity and safety remains essential.

A more refined approach has been explored for DNAJB6, where the DNAJB6b isoform appears to be the primary pathogenic driver. Findlay et al. demonstrated that isoform-specific knockdown of DNAJB6b using morpholinos was feasible in vitro and in vivo. This approach corrected the proteomic signature in LGMDD1 model myotubes³³. This strategy offers a potential path to reduce the toxic protein while preserving the potentially necessary DNAJB6a isoform.

In contrast, disorders driven primarily by LOF mechanisms, such as many cases of recessive DNAJB4 myopathy, require fundamentally different therapeutic strategies. For these conditions, gene replacement therapy using adeno-associated virus (AAV) vectors to restore functional DNAJB4 protein represents a viable option, although key challenges for AAV-based therapies—including muscle-specific delivery efficiency, immune responses to viral capsids and therapeutic proteins, and the persistence of therapeutic gene expression⁴⁰—must be overcome to achieve durable benefit. In addition, the existence of J-domain missense variants in recessive DNAJB4 myopathy that may mediate toxicity through a GOF mechanism underscores the importance of understanding the precise molecular mechanism for each specific variant before designing therapies²⁴. Ultimately, the identification of key pathogenic client proteins will also be critical for developing substrate reduction therapies and for a deeper understanding of the disease process.

CONCLUSION AND FUTURE PERSPECTIVE

In recent years, the understanding of JDP-related myopathies has matured from initial gene discoveries to a more sophisticated appreciation of their complex and distinct pathomechanisms. Pathogenic variants in DNAJB6, DNAJB4, and DNAJB2 are now known to disrupt skeletal muscle proteostasis through distinct LOF, dominant-negative, or GOF pathways, leading to a wide spectrum of clinical phenotypes. A central theme, particularly for DNAJB6 and DNAJB4 myopathies, is the emergence of a GOF mechanism centered on a dysregulated interaction with HSP70. Notably, the existence of both dominant GOF and recessive LOF variants in DNAJB4 presents a unique opportunity to dissect the distinct consequences of these opposing mechanisms, using DNAJB4 myopathy as a powerful comparative model.

Despite this progress, key questions remain. The muscle-specific nature of these diseases, despite the ubiquitous expression of the causative JDPs, is still not fully understood. The precise identities of the critical client proteins whose mishandling initiates the pathogenic cascade also remain largely unknown. Future research should focus on leveraging advanced proteomic techniques to identify these clients and further characterizing the unique cellular vulnerabilities of muscle tissue. The continued use of sophisticated disease models will be essential for dissecting these molecular pathways and for the preclinical evaluation of promising therapeutic strategies, including gene replacement for LOF disorders and JDP-HSP70 interaction inhibitors or isoform-specific knockdown for GOF disorders. Ultimately, a deeper mechanistic understanding, combined with comprehensive natural history studies and biomarker development, will be paramount to bringing targeted, mechanism-based therapies for these devastating muscle diseases to clinical reality.