Blockage of autophagy causes severe skeletal muscle disruption in a mouse model for myofibrillar myopathy 6

Domain: neuroscience
Published: 2026-04-11
Editorial outcome: minor_revision

Abstract
Myofibrillarmyopathy 6 is a rare, autosomal-dominant neuromuscular disorder caused by an amino acid exchange Pro209Leu in the co-chaperone BAG3, which disrupts muscle protein turnover and causes severe muscle weakness and shortened lifespan. We generated transgenic mice overexpressing the human mutant BAG3P209L-GFP, which rapidly develop skeletal muscle weakness unlike controls expressing BAG3WT-GFP. Here we show that mutant mice exhibit sarcomere breakdown, inflammation, protein aggregates, centralized nuclei and mitochondrial defects in their skeletal muscles, thereby reducing contraction force by ~90%. Omics profiling uncovered impaired protein synthesis, blocked autophagy, impaired mitophagy and loss of sarcomere proteins. Pathway modulation in vitro and in vivo showed autophagy dysfunction as the primary driver for the pathology, while BAG3 knockdown gene therapy markedly restored muscle function in vivo. In summary, this model recapitulates core disease features, revealing how BAG3 aggregates and loss of BAG3 function impair autophagy to drive muscle degeneration.

Introduction
Myofibrillarmyopathies (MFM) are defined by disintegration of myofibrils and aggregation of proteins into intracellular inclusions and manifest as progressive skeletal muscle weakness. While the majority of MFMs are caused by mutations in structural genes, such as TTN or DES, some are caused by mutations in genes encoding protein quality control factors, such as BAG3 (Bcl-2 associated athanogene 3).

BAG3 is a co-chaperone that forms the chaperone-associated selective autophagy (CASA) complex together with HSPB8, HSPA8, and CHIP. CASA is able to bind to misfolded sarcomeric proteins, such as Filamin C, and trigger their degradation by autophagy1. Because of this role of BAG3 as a central node in protein quality control, it may affect many sarcomeric proteins and the maintenance of myocyte function. A point mutation in the BAG3 gene (c.626C>T, p.P209L) causes BAG3P209L-associated myofibrillarmyopathy (MFM6), which is a severe and potentially life-threatening condition. Affected patients experience proximal and generalized muscle weakness that progresses rapidly, polyneuropathy, and sometimes restrictive cardiomyopathy with early onset during childhood2. The primary cause of death in these patients is often respiratory failure due to skeletal muscle weakness, and they have a life expectancy of around 20 years. Histological hallmarks of the disease are the formation of cytoplasmic protein inclusions, disintegration of sarcomeres, and Z-line streaming2.

Several attempts were undertaken to generate a mouse model that mimics the cardiac and skeletal muscle phenotypes of patients. Beginning with the overexpression of human BAG3P209L in mouse cardiomyocytes, some aspects of the cardiac pathology were recapitulated3. Introduction of the homolog mutation (P215L) into mouse Bag3 did not lead to an obvious phenotype even when mice were bred to homozygosity4. A compound line consisting of a Bag3P215L knock-in and a Bag3 knock-out caused a mild skeletal muscle phenotype but without any impairment in muscle strength5. To better mimic the cardiac and skeletal muscle pathologies observed in patients, we developed a novel humanized mouse model for MFM6 in which a fusion protein of human BAG3P209L and eGFP is ubiquitously expressed6.

In the hearts of these mice, the solubility of BAG3 is altered due to the point mutation that renders the protein more prone to aggregation6,7. Accordingly, BAG3 is no longer able to fulfill its function as a co-chaperone in the CASA complex, and its loss of function leads to the accumulation of damaged muscle proteins and consequently to the disintegration of sarcomeres. Besides the severe cardiac phenotype, we noticed that these mice display clear signs of muscle weakness and are therefore an ideal model for studying the pathomechanism underlying MFM6 in skeletal muscle. Since skeletal muscle is a tissue capable of regeneration by tissue-derived stem cells, it is particularly interesting to explore the differences between skeletal and cardiac muscle, as the latter lacks stem cells and exhibits poor regeneration.

The analysis of our mouse model revealed a striking decrease in muscle force and sarcomere disintegration, along with protein aggregate formation and accumulation of autophagy and mitophagy markers. Since skeletal muscle is a regenerative tissue, we observed an increase in protein synthesis in the transgenic mice, but this was not sufficient to compensate for the loss of sarcomere proteins. Additionally, mitochondrial function was severely impaired and mitophagy was blocked, potentially contributing to the skeletal muscle phenotype. Notably, induction of autophagy in vivo and in vitro, but not induction of mitophagy, was able to attenuate the phenotype. Taken together, our mouse model mimics the skeletal phenotype of MFM6 and provides new insights into disease mechanisms mainly caused by a blockage of autophagy and can be rescued by an shRNA-mediated knockdown of BAG3P209L.

Results
To investigate the pathomechanism of BAG3P209L-associated MFM, we generated a humanized mouse model with inducible overexpression of human BAG3P209L fused to eGFP. In addition, to rule out any effects caused by BAG3-eGFP overexpression, we generated a control mouse line overexpressing human BAG3 fused to eGFP6. Both inducible mouse lines were crossed with PGK-Cre transgenic mice to achieve ubiquitous expression of BAG3P209L-eGFP and BAG3WT-eGFP, respectively, and then further bred to homozygosity (homozygous PGK-Cre/CAG-flox-hBAG3P209L-eGFP mice are referred to as CAG-BAG3P209L, and homozygous PGK-Cre/CAG-flox-hBAG3WT-eGFP mice as CAG-BAG3WT).

CAG-BAG3P209L-mice were significantly smaller than their control siblings and exhibited typical signs of muscle wasting, such as rigid spine and malalignment of the hind legs resulting in a swaddling gait (Fig. 1a–c). In contrast, CAG-BAG3WT-mice showed no obvious phenotype and were indistinguishable from non-transgenic littermate control (CTRL) mice (Supplementary Fig. 1a–k). Life expectancy of CAG-BAG3P209L-mice was about 5–8 weeks, as reported before6, due to the restrictive cardiomyopathy leading to heart failure. The mice did not lose weight but grew more slowly than their control littermates (Supplementary Fig. 2a) and CAG-BAG3WT-mice (Supplementary Fig. 1a), indicating growth restriction. In fact, e.g., both the M. soleus and also the EDL were clearly smaller than in controls (Fig. 1d), and statistical analysis of muscle length and weight confirmed significantly shorter and lighter EDL and soleus muscles in 2- and 4-week-old CAG-BAG3P209L-mice (Supplementary Fig. 2b, c). EGFP expression was detected in the muscles of these mice (e.g. soleus muscle and diaphragm), which was not seen in CTRLs (Fig. 1e, f) but was observed in CAG-BAG3WT-mice (Supplementary Fig. 1b). Cross-sections through the EDL, soleus, quadriceps muscles, or diaphragm revealed expression of hBAG3P209L-eGFP or hBAG3WT-eGFP (Fig. 1f, g; Supplementary Fig. 1c). Despite clear differences in muscle size and weight, we found that tibia length at 5 weeks of age was not significantly reduced in CAG-BAG3P209L-mice, indicating muscle atrophy (Supplementary Fig. 2d). To get an impression of the extent of overexpression, we measured the amount of mBag3 and hBAG3 by Western Blot analysis. We found that the total amount of BAG3 protein (the sum of endogenous Bag3 and transgenic BAG3-eGFP) in quadriceps muscle from 4-week-old CAG-BAG3P209L and CAG-BAG3WT mice was 11.36 ± 0.45 times higher in CAG-BAG3P209L mice (Supplementary Figs. 2e) and 4.46 ± 0.04 times higher in CAG-BAG3WT mice (Supplementary Fig. 3a) than in CTRL mice (Supplementary Fig. 2f). To determine whether this increase was due to elevated mRNA levels or accumulation of protein as a manifestation of the phenotype, we next measured the amount of transgenic hBAG3P209L-eGFP and endogenous mouse Bag3 mRNA by qPCR with probes specific for mouse and human BAG3. The relative mRNA-levels of mouse Bag3 normalized to GAPDH were not statistically significantly different (Supplementary Fig. 2g). The same was true for the expression of the human BAG3 transgene mRNA in CAG-BAG3P209L and CAG-BAG3WT-mice (Supplementary Fig. 2g). Since the total mRNA level of BAG3 (mouse Bag3 + human BAG3) induction was only 3.01 ± 1.41 and 3.37 ± 1.77 compared to CTRL mice (Supplementary Fig. 2h), this indicated an accumulation of mBag3/BAG3 protein, which was very prominent in CAG-BAG3P209L mice but not in CAG-BAG3WT mice. However, compared to skeletal muscle, total BAG3 protein was 3.01 ± 0.69 times more abundant in heart muscle from CAG-BAG3P209L-mice (Supplementary Fig. 3a). Also, the ratio of human BAG3-eGFP protein to endogenous mouse Bag3 protein was 3.56 ± 0.20 (Fig. 5n) which was higher than in cardiac muscle, because the amount of mouse Bag3 protein was lower in skeletal muscle (Supplementary Fig. 3b, c). In summary, the increase in total BAG3 mRNA (mouse Bag3 + human BAG3) was significantly lower than the increase in total BAG3 protein in skeletal muscle in CAG-BAG3P209L-mice, which is likely a sign of the pathological phenotype, probably reflecting protein aggregation.

Centralized nuclei and fiber type switch in skeletal muscle from CAG-BAG3P209L mice
For histological analysis of skeletal muscle, we focused on quadriceps femoris muscle and the extensor digitorum longus (EDL) muscle as muscles with a high percentage of fast-twitch fibers, and on M. soleus as a muscle with a high percentage of slow-twitch fibers. Since respiratory muscle failure is a major cause of death in patients, we also examined the diaphragm. Similar to other muscular dystrophies, we found a significant increase in the number of centralized myocyte nuclei in all four muscles compared to controls, indicating a regenerative response (Fig. 1i–p). This was not seen in CAG-BAG3WT-mice (Supplementary Fig. 1d, e). In addition, in skeletal muscle of CAG-BAG3P209L mice there was an accumulation of degenerating muscle cells and general atrophy of muscle fibers, which was evident by their smaller size in cross sections (Fig. 1j, l; Supplementary Fig. 3d). Muscle fibers were infiltrated by interstitial cells, which were likely fibroblasts or immune cells (Fig. 1j and Supplementary Fig. 3d, arrowheads). As fibrosis is a main feature of the phenotype in hearts of CAG-BAG3P209L-mice6, we stained sections of EDL, soleus, and diaphragm muscles for collagen by Sirius Red (Fig. 2a) and quantified the fibrotic area (Fig. 2b–d). There was a significant increase in fibrotic tissue in the EDL, soleus and diaphragm muscles from CAG-BAG3P209L-mice, but not in CAG-BAG3WT-mice (Supplementary Fig. 1f, g), and it was not as prominent as in hearts from these mice6. The number of satellite cells in the EDL and soleus muscles was unchanged in CAG-BAG3P209L-mice compared to controls (Supplementary Fig. 3e–g), indicating no impairment of regeneration due to stem cell pool depletion. Despite using the ubiquitous CAG promoter, BAG3P209L-eGFP was not expressed in satellite cells.

A change in muscle fiber types is often observed in muscular dystrophies that have concomitant neuropathies, as observed in MFM6 patients. Interestingly, we noticed a switch in skeletal muscle fiber types from fast-twitch to slow-twitch types in 4-week-old CAG-BAG3P209L mice (Supplementary. Fig. 4a–d). In both soleus and EDL muscles, the percentage of MHC-I type fibers increased, while the percentage of MHC-IIb fibers decreased (Supplementary Fig. 4e, f). Additionally, MHC-IIb fibers in the EDL muscle of CAG-BAG3P209L-mice had a smaller diameter (Supplementary Fig. 4g), indicating atrophy, while in soleus muscle this was unchanged (Supplementary Fig. 4h). There was no correlation between the level of hBAG3-eGFP expression and fiber type in either soleus or EDL muscle at 4 weeks (Supplementary Fig. 4i, j) of age. The change in the composition of fiber types indicated a neuronal phenotype in our mouse line, which is also observed in MFM6 patients.

Functional impairment and aggregate formation in skeletal muscle from CAG-BAG3P209L mice
Since skeletal muscles were smaller, we wondered whether muscle function was decreased and therefore measured force development in CAG-BAG3P209L-mice and controls. We performed frequency-dependent force measurements (FFR) with isolated EDL and soleus muscles from 4-week-old CAG-BAG3P209L, CAG-BAG3WT, and control mice. Muscles were stimulated with electric pulses at increasing frequencies from 10 to 125 Hz (Fig. 2d–g, Supplementary Fig. 1h, i). In individual muscles, maximal force was reduced by ~3-fold (Fig. 2d, e). To exclude that this was due to different muscle size, the maximal force was normalized to muscle length and weight. In both muscles, the maximal normalized force was significantly (1.5 to 2-fold) decreased in CAG-BAG3P209L-mice. This was especially evident at high stimulation frequencies (Fig. 2f, g), which induce fused tetanic contractions and did not occur in CAG-BAG3WT-mice (Supplementary Fig. 1h, i). Interestingly, the decrease in maximal force negatively correlated with the intensity of CAG-BAG3P209L-eGFP expression, which displayed some degree of variability among the transgenic mice (Supplementary Fig. 4k, l). As maximal force was normalized, it became apparent that the reduced force was due to either structural changes, such as the disintegration of sarcomeres, or to an impairment in energy metabolism, or to both.

Because a hallmark of MFM6 is the formation of cytoplasmic protein inclusions in skeletal muscle2, we performed an immunofluorescence analysis of sections from the EDL and soleus muscles of 4-5-week-old CAG-BAG3P209L-mice. BAG3P209L-eGFP protein formed cross-striated structures in the muscles from control mice (Fig. 3a–d, arrows) and CAG-BAG3WT-mice (Supplementary Fig. 1j, k), while in muscles from BAG3P209L-mice, irregular structures and protein aggregates were found (Fig. 3a–d, arrowheads). Staining for ACTN2 revealed the typical cross-striation of sarcomeric Z-disks, which was disturbed and severely impaired in myofibers from CAG-BAG3P209L-mice (Fig. 3a, b) but not CAG-BAG3WT-mice (Supplementary Fig. 1j). The desmin intermediate filaments displayed abnormalities and a loss of the irregular structure when compared with controls and CAG-BAG3WT-mice (Fig. 3c, d; Supplementary. Fig. 1k), hinting at decreased integrity of the myocyte cytoskeleton, as has been reported in mice carrying a dominant negative desmin mutation8. A potential block of autophagy was assessed by quantification of SQSTM1 and LC3B stainings (Fig. 3e, f). SQSTM1 and LC3B accumulated in small puncta in the quadriceps muscle of CAG-BAG3P209L mice (Fig. 3e), and quantitation revealed a statistically significant increase in both values (Fig. 3f, g). Since protein aggregates accumulated in skeletal myocytes of these mice, the increase in SQSTM1 is due to a blockage of autophagy and not to an increase.

To assess the degree of intercellular damage in skeletal muscle, parts of the quadriceps femoris muscle were dissected from CAG-BAG3P209L and control mice, and ultrathin sections were prepared for ultrastructure analysis by electron microscopy. In the electron micrographs, the skeletal muscles of controls showed a regular arrangement of sarcomeres with typical transverse striation (Fig. 3h), whereas in skeletal muscles from CAG-BAG3P209L-mice, disintegration of sarcomeres with intersarcomeric electron-dense regions (Fig. 3i, arrows) was evident. This is a typical finding in myofibrillarmyopathies and one of the hallmarks of the human pathology9. Various types of aggregates (granulo-filamentous and vesiculated-subsarcolemmal) could be observed. Numerous lysosomal and autophagic vacuoles were also visible, indicating increased autophagy or a blockage of autophagy manifested by an accumulation of autophagosomes (Fig. 3j). Notably, mitochondria were disarranged in skeletal muscle from CAG-BAG3P209L-mice and displayed a variety of shapes and sizes compared to controls (Fig. 3i, arrowheads). Signs of mitophagy, such as remnants of mitochondrial cristae in autophagosomes, were also visible (Fig. 3j, asterisks). Overall, the structural changes resembled those described in patients with MFM6.

Loss of structural integrity could lead to cell decay followed by an immune response. Therefore, we screened for both inflammatory markers and programmed cell death markers. There was a significant increase in CD45-positive cells in the quadriceps and both EDL and soleus muscles from CAG-BAG3P209L-mice (Fig. 3k, l, Supplementary Fig. 5a), as a sign of immune cell infiltration and inflammation, which was confirmed by increased staining for acidic phosphatase (Supplementary Fig. 5b). In addition, staining for cleaved caspase 3 revealed a statistically significantly higher number of apoptotic fibers in both EDL and soleus muscles (Supplementary Fig. 5c, d). These apoptotic muscle fibers were likely replaced by fibroblasts, which produced more extracellular matrix proteins, as Gomori Trichrome staining revealed an increase in collagen deposition in CAG-BAG3P209L-mice (Supplementary Fig. 5e).

Staining for the cell cycle marker Ki-67 was performed to obtain first information about possible satellite cell proliferation in the skeletal muscles of CAG-BAG3P209L-mice (Supplementary Fig. 5f). A significant difference was observed between CTRL and CAG-BAG3P209L-mice in the soleus muscle but not in the EDL muscle (Supplementary Fig. 5f, g), indicating increased cell proliferation during regenerative processes in some skeletal muscles of CAG-BAG3P209L-mice. However, the underlying molecular mechanisms causing this chain of events were unclear, so we performed Omics analysis to gain deeper insights.

Massive protein accumulation and damaged mitochondria in CAG-BAG3P209L-mice
We took an unbiased approach and performed a multi-omics analysis of the quadriceps femoris muscle from 5-week-old CAG-BAG3P209L-mice and their matching controls. RNA-Seq analysis, in which a total of 4 CAG-BAG3P209L mice were compared with 5 control mice, revealed 1981 significantly differentially expressed genes, of which 1105 were upregulated and 876 were downregulated (Fig. 4a, Supplementary Tables 1+2). GO-analysis revealed a strong expression of genes associated to protein synthesis and translation in skeletal muscle from CAG-BAG3P209L-mice (Fig. 4b). As mTOR-signaling was among the enriched terms, we hypothesized that this pathway caused the increase in protein synthesis genes. Increased transcription by RNA polymerase II was also evident from the GO-analysis. Conversely, expression of genes involved in catabolic processes such as autophagy and macroautophagy was upregulated, too. Moreover, protein degradation terms such as “ubiquitin-dependent protein catabolic process”, “proteasome-mediated ubiquitin-dependent protein catabolic process”, and “endoplasmic-reticulum-associated protein degradation pathway” were among the enriched GO-terms. Interestingly, hypoxia response gene expression was also upregulated, which could indicate reduced blood flow and thus oxygen supply due to impaired cardiac output in these mice6. These data demonstrated that increased protein synthesis is the response of a highly adaptable tissue to sarcomeric damage caused by impaired autophagy due to the expression of BAG3P209L.

In addition, we found the GO-terms “response to mitochondrial depolarization”, “autophagy of mitochondrion”, and “mitochondrion disassembly”, indicating mitochondrial alterations among the genes that were at least 2-fold upregulated (log2 fold change ≥ 1) (Fig. 4b). This was consistent with the ultrastructural analysis, which revealed irregular shapes of mitochondria in skeletal muscle of CAG-BAG3P209L-mice when compared to controls (Fig. 3g–i).

Among the GO-terms associated with downregulated gene expression were “mitotic cell cycle” and “muscle cell proliferation”, including genes such as AurkB and several cyclins. A detailed analysis of the downregulated GO-term “system development” revealed that “vasculature development” and “endothelium development” were affected. Among the other downregulated GO-terms was a strong signature of angiogenesis genes and “cell migration”. Together this could indicate a decrease in capillarization, which in turn could lead to hypoxia. In fact, the capillary density in the soleus muscle was significantly decreased in CAG-BAG3P209L mice (Supplementary Fig. 5h). Interestingly, neurogenesis and neuronal genes were also among the depleted GO-terms, hinting at a neuronal phenotype that has been described in patients suffering from MFM62. We also observed decreased fatty acid metabolism in skeletal muscle, manifested by depletion of the GO category “fatty acid beta-oxidation” (Fig. 4b), pointing to impaired mitochondrial function.

For proteome analysis, the quadriceps femoris muscle from 5-6 week-old CAG-BAG3P209L-mice was dissected and total protein extracted. Label-free mass spectrometry analysis in data-independent acquisition (DIA) mode identified 3399 protein groups in the total lysate. 971 protein groups significantly increased in abundance (t-test with permutation-based FDR < 0.05, S0 = 0.1) and 68 protein groups significantly decreased in abundance in skeletal muscle of the CAG-BAG3P209L-mice when compared to the controls (Fig. 5a, Supplementary Table 3). In addition, 215 protein groups were found exclusively in the CAG-BAG3P209L mouse muscle lysate, and 4 protein groups in the control lysate (Supplementary Table 4). GO-CC term enrichment analysis of the 1186 protein groups with exclusive or higher abundance in mutant mice showed that these were enriched in proteins associated with the sarcomere, myofibril, and actin cytoskeleton (Supplementary Fig. 6a). Analysis of KEGG pathways further revealed that accumulating proteins were involved in protein synthesis (ribosome, spliceosome, protein processing in the ER), protein degradation (proteasome, phagosome, lysosome, mitophagy), as well as components of mechanosensitive structures (focal adhesions, regulation of actin cytoskeleton, adherens junctions) (Supplementary Fig. 6b). Similar to our previous analysis of heart samples from CAG-BAG3P209L-mice6, components of the CASA-complex such as BAG3, HSPB8, and other heat shock proteins, such as CRYAB, HSPB1, DNAJB2, and DNAJB5 massively accumulated in CAG-BAG3P209L-mice. Thus, expression of BAG3P209L massively perturbs skeletal muscle proteostasis, leading to the accumulation of a large but specific set of proteins in 5–6 week-old mice. This was not observed in skeletal muscle from CAG-BAG3WT-mice (Suppl. Fig. 7).

To verify the changes in the protein quality control system observed in proteomics analysis, we looked at protein expression focusing on alterations in heat shock protein expression, CASA, and autophagy. The quadriceps femoris muscle was dissected from 5 week old CAG-BAG3P209L, CAG-BAG3WT and control mice and protein extracts were prepared for Western Blot analysis (Fig. 5b, Supplementary Fig. 7a). In agreement with the mass spectrometry data, we observed a statistically significant increase in BAG3, CRYAB (Fig. 5c, d) and the small heat shock proteins HSPB7 and 8 (Fig. 5e, f). Expression of HSPB6 showed a consistent trend of increase that did not reach statistical significance due to greater variations in protein abundance (Fig. 5g). The autophagy markers LC3B and SQSTM1, and the CASA-related protein SYNPO2 were significantly increased in muscles from CAG-BAG3P209L-mice (Fig. 5h–j). These protein accumulations, which did not take place in CAG-BAG3WT-mice (Supplementary Fig. 7f–l), suggest cellular stress and altered autophagy, in agreement with the ultrastructural data that showed an increase in autophagic vesicles (Fig. 3i). Together, this indicated a dysregulation of autophagy and a reduced ability to clear non-soluble protein accumulations.

Subcellular fractionation reveals massive protein aggregation with a shift from the soluble to the insoluble fraction
Since the dysregulation of autophagy led to the formation of protein aggregates, we analyzed these in more detail. To distinguish between aggregating proteins and proteins that might accumulate in insoluble forms as a stress response, we further separated protein extracts from skeletal muscle into a soluble and a detergent-resistant insoluble fraction (Fig. 6a). Label-free DIA mass spectrometry identified 3838 protein groups in the soluble and 2539 in the urea-dissolved pellet fractions. Comparison of pellet and soluble fractions showed the expected enrichment of cytoskeletal proteins in the pellet in both wild-type and CAG-BAG3P209L-expressing mice (Supplementary Fig. 8a–c), thereby confirming the method. Compared to the total cell lysate, analysis of the separated fractions identified 1059 additional protein groups, while 1975 of the protein groups were observed in all three proteomes (lysate/soluble/insoluble, Supplementary Fig. 8d). To determine which proteins accumulate in the observed protein aggregates, we compared protein abundance in the insoluble fraction between the two strains. Using the same significance criteria as above (t-test with permutation-based FDR < 0.05) revealed that 961 protein groups increased in abundance in the pellet of CAG-BAG3P209L-mice (Fig. 6b, Supplementary Table 5), and an additional 500 proteins were only observed in the insoluble pellet fraction of the CAG-BAG3P209L-mice but not in the controls (Supplementary Table 6). KEGG term analysis of these 1461 protein groups showed the enrichment of proteins involved in autophagy, mitophagy, mTOR signaling, and mitochondrial processes such as oxidative phosphorylation (Suppl. Fig. 9a). In contrast, only 30 proteins showed decreased abundance in the pellet from CAG-BAG3P209L mice (Supplementary Table 6). In the soluble fraction, 616 protein groups accumulated, while 248 protein groups decreased in abundance in the CAG-BAG3P209L-mice (Fig. 6c, Supplementary Table 7), with 46 protein groups observed exclusively in CAG-BAG3P209L and 3 protein groups in the control mice (Supplementary Table 8). Notably, proteins accumulating in the soluble fraction were mainly associated with protein turnover, including the ribosome, spliceosome, and proteasome (Suppl. Fig. 9b). Comparing the change in protein abundance between the two strains in both pellet and supernatant showed a weak positive correlation (Pearson’s correlation Coefficient r = 0.2, -log10(Pearson p-value) = 13.9) but also revealed that most proteins accumulated significantly more in the insoluble fraction (Fig. 6d).

GO-CC term enrichment analysis of proteins depleted in both soluble and pellet fractions indicated that this group included many sarcoplasmic and sarcomeric proteins, in line with the observed phenotype (Fig. 6e). Notably, 215 proteins accumulating in the pellet fraction (log2(BAG3P209L/control) > 0.25) showed decreased abundance in the soluble fraction (log2(BAG3P209L/control) < -0.25), indicating increased association with the cytoskeleton or aggregation (Fig. 6d). GO-CC term analysis indicated that many of these proteins were of mitochondrial origin (Fig. 6f). Since BAG3 has previously been implicated in regulating mitophagy10, we wondered if this accumulation of mitochondrial proteins in the pellet might coincide with mitophagy markers. Indeed, we observed a strong accumulation of the PARKIN target MFN1 and the mitophagy-regulating Bcl-2 family members BNIP3 and BCL2L13 in the pellet of CAG-BAG3P209L mice (Fig. 6b), but not in the supernatant (Fig. 6c), while citrate synthase (CS), a marker of total mitochondrial protein, showed no significant change between the two genotypes. In addition, the mitophagy receptor PINK1, and several other factors involved in mitophagy (ATF5, ULK1, MFN2, SIRT4, KEAP1) were found only in the pellet fraction of CAG-BAG3P209L mice, but not in controls (Fig. 6G, Supplementary Table 6). In summary, the omics-analysis revealed impaired autophagy and mitophagy, both of which could be responsible for the manifestation of the pathological phenotype.

Functionally impaired mitochondria in skeletal muscles from CAG-BAG3P209L-mice
Proteomic analysis revealed mitochondrial proteins and mitophagy markers as components of the aggregates observed in both immunofluorescence and ultrastructural analysis. The latter illustrated that most mitochondria displayed an irregular shape, were disorganized, and showed signs of mitophagy in skeletal myocytes from CAG-BAG3P209L-mice. These data suggested that mitochondria could play a role in the pathomechanism of the disease, and therefore, we analyzed their biochemical properties and their ability to provide energy. To this end, we isolated intact mitochondria from total mouse hindlimb muscles. As presented in Table 1, oxygraph measurements revealed that the maximum respiration capacity of isolated skeletal muscle mitochondria obtained by uncoupling is significantly lower for both substrate combinations glutamate + malate, which measures respiration without fatty acids and octanoylcarnitine and malate, which examines fatty acid oxidation. Resting state respiration activities (state 4) remained unaltered. Additionally, we observed that the stimulatory effect of creatine on mitochondrial respiration was greatly reduced for both substrate combinations (Table 1). To elucidate the cause of the observed functional alterations of skeletal muscle mitochondria, we further analyzed the enzyme pattern of isolated mitochondria (Table 2). We observed that the activities of all enzymes expressed per protein were significantly decreased. Importantly, the ratio to the marker enzyme citrate synthase was significantly lower for complex I, while the cytochrome c oxidase/citrate synthase ratio was unaltered, suggesting additionally a specific impairment effect of transgene expression on complex I activity and ruling out potential effects of the purification procedure. Since complex I is required for glutamate + malate and also octanoylcarnitine + malate respiration, its functional impairment explains the remarkable reduction in the maximal respiration activities detected in the oxygraphic determinations (Table 1).

In contrast to the decreased enzymatic activities of isolated mitochondria in whole skeletal muscle, mitochondrial accumulation was evident in COX-SDH staining, which was markedly increased in the quadriceps muscle of CAG-BAG3P209L-mice. (Suppl. Fig. 10). This is likely due to an accumulation of damaged and functionally impaired mitochondria as seen in electron micrographs (Fig. 3i), since oxygraphic and enzymatic data of isolated skeletal muscle mitochondria show considerably decreased activities. Most likely, these effects can be attributed to an impairment of mitophagy caused by BAG3P209L overexpression. This is underlined by the upregulation of Pink1, Bnip3l, and Tomm20 seen in the RNA-seq analysis (Supplementary Table 1). Moreover, in the proteomics analysis PINK1, BNIP3, and FUNDC1 were enriched in the detergent-resistant insoluble fraction (Supplementary Tables 5+6), while TOMM22, TOMM40, and TOMM70 were enriched in the soluble fraction (Supplementary Table 7). The observed enrichment of proteins functioning as different mitophagy receptors, like PINK1, BNIP3, and FUNDC1, strongly indicates increased activation of different mitophagy pathways despite insufficient final removal of functionally impaired mitochondria. This can be explained by a block of final execution of mitophagy due to the loss of BAG3 function.

Block in autophagy is the primary cause of the phenotype, while impairment of mitochondria is secondary
Next, we asked whether the observed severe skeletal muscle phenotype was mainly caused by impaired autophagy, mitophagy, or both. To test this, we isolated skeletal myocyte precursor cells from the skeletal muscles of CAG-BAG3P209L mice using an established protocol11. After differentiation into skeletal myocytes, these cells displayed formation of BAG3P209L-positive protein aggregates (Fig. 7a) and could therefore be used to study the effects of inducing autophagy, mitophagy, or mitochondrial biogenesis. We transfected these cells with plasmids carrying expression cassettes for Becn1 to induce general autophagy, Bnip3 to specifically induce mitophagy, or Ppargc1a to induce mitochondrial biogenesis. Successful overexpression of these proteins was confirmed by positive staining for the Myc (BECN1), FLAG (BNIP3), or HA (PPARGC1a) tags fused to the different proteins (Fig. 7a).

Since bafilomycin A1 blocks the fusion of autophagosomes with lysosomes, an increase in autophagic markers like SQSTM1 indicates the level of autophagic flux, while an increase in mitophagy markers such as PINK1 reflects the level of mitophagic flux. There was a statistically significant increase in SQSTM1 in the BECN1-overexpressing cells, which was consistent with a rise in autophagic flux and the release of the autophagic block seen in cells from CAG-BAG3P209L mice (Fig. 7b). This was not observed after induction of mitophagy (BNIP3) or increased mitochondrial biogenesis (PPARGC1a). PINK1 accumulated in BNIP3-overexpressing cells after Bafilomycin A1 treatment, confirming that the induction of mitophagy was successful (Fig. 7c). Interestingly, mitophagy was also induced by BECN1 overexpression, as PINK1 levels decreased (Fig. 7c), which had initially accumulated due to a block in mitophagy. As expected, the level of BAG3P209L-eGFP increased following BECN1 overexpression and Bafilomycin A1 treatment, indicating enhanced autophagic flux and degradation of BAG3P209L-eGFP-positive protein aggregates (Fig. 7d). In contrast, neither the induction of mitophagy through overexpression of BNIP3 nor the promotion of mitochondrial biogenesis via PPARGC1a overexpression affected autophagy, mitophagy, nor the amount of BAG3-eGFP containing protein aggregates (Fig. 7b–e). In addition, cell morphology improved after overexpressing BECN1, as the average size of skeletal myocytes was larger (Fig. 7a) and had a more prominent cross-striation. Unbiased analysis of sarcomere quality using the SarcAsM algorithm12 revealed a statistically significant increase in Z-band length in BECN1 transfected cells compared to CTRL and PPARGC1a transfected cells (Suppl. Fig. 11a). Furthermore, cells transfected with BECN1 showed a tendency toward increased sarcomere length, sarcomere area ratio, and cell area ratio (Suppl. Fig. 11b-d), supporting the morphological observations.

We expanded these experiments in vivo by inducing autophagy in 3-week-old CAG-BAG3P209L mice through daily i.p. injection of 8 mg/kg rapamycin for 14 days. Rapamycin is known to induce autophagy by inhibiting the mTOR signaling pathway13. Grip strength and motor coordination, measured by the Rota-Rod device, improved on day 7 and day 14 under rapamycin treatment compared to untreated or vehicle-treated mice (Suppl. Fig. 11e, f). The same was true for voluntary movements (Suppl. Fig. 11g). The amount of LC3B II protein was significantly increased in the quadriceps muscles of rapamycin-treated CAG-BAG3P209L mice after 14 days of rapamycin treatment (Suppl. Fig. 11h), indicating enhanced autophagy. Together with the in vitro data, we conclude that the phenotype in the skeletal muscle of CAG-BAGP209L mice is caused by a blockage of autophagy and that the impairment and accumulation of mitochondria are secondary to this.

Improved skeletal muscle strength after AAV-mediated knockdown of hBAG3P209L
Gene therapy offers a promising approach for treating monogenetic inherited diseases. As an experimental approach, we developed a knockdown strategy targeting the human BAG3P209L-eGFP fusion protein. The construct consisted of an expression cassette with an U6 promoter driving the expression of either a hBAG3-specific shRNA or a scrambled shRNA along with a CMV-promoter for mCherry expression as an indicator of successful transduction. For delivery of the shRNA expression construct, we utilized an adeno-associated virus (AAV), which was injected into the jugular vein of P15 mice. The transduction rate was determined by counting the mCherry-positive skeletal muscle myocytes, while the effect on BAG3P209L-eGFP expression was monitored by measuring the total eGFP intensity in sections from skeletal muscles (Suppl. Fig. 12a, b). Both AAVs displayed a high transduction rate of approximately 80% (Supplementary Fig. 7b), but only the shRNA AAV was able to visibly reduce BAG3P209L-eGFP expression (Fig. 8a–c). Additionally, the number of BAG3P209L-eGFP containing protein aggregates was reduced, and a regular sarcomere structure was obvious after staining for ACTN2, DES, and FLNC (Suppl. Fig. 12c–e). Western Blot analysis confirmed the immunofluorescence findings (Suppl. Fig. 13a-n) and showed a reduction in BAG3 expression (by ~60%, Fig. 8c) and autophagy markers (Suppl. Fig. 13i, m). Centralization of nuclei in skeletal muscles decreased significantly in the shRNA-treated group in the quadriceps muscle (from 35.9 ± 4.1 to 7.0 ± 3.8% of fibers) together with a reduction in granular areas (Fig. 8d, e).

Fibrosis was markedly reduced after BAG3 shRNA-treatment (from 23.8 ± 3.7 to 6.8 ± 0.8% of fibers in quadriceps muscle, Fig. 8f, g). Most strikingly, force measurements following AAV treatment revealed a significant increase in normalized force development in both soleus and EDL muscles (Fig. 8h, i), demonstrating a functional improvement (EDL muscle from 4 to 102 mN/mm2 and soleus muscle from 53 to 301 mN/mm2 at 100 Hz). Also, both the soleus and EDL muscles exhibited a significant improvement in muscle length and weight (Suppl. Fig. 13n–q). In summary, we demonstrate a significant improvement in the pathological phenotype using this proof-of-concept gene therapy approach for myofibrillarmyopathies.

Methods
Animal procedures
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The reporting of animal experiments abides by the ARRIVE guidelines. All procedures performed in studies involving animals were following the ethical standards of the institution at which the studies were conducted and were approved by the responsible governmental authorities (Landesamt für Natur, Umwelt und Verbraucherschutz, NRW, Germany) (animal protocols #81-02.04.2021.A068 and 81-02.04.2019.A062). Mice are housed in a ventilated animal house (15 air changes per hour) in laboratory rooms under artificial lighting (12h light/12h dark), constant ambient temperature (22°C), constant humidity (approximately 50%) with access to food and clean water ad libitum. Mice are fed standard laboratory animal feed (V1534-300 From ssniff Spezialdiäten GmbH, Germany) and maintained in approved laboratory cages 15 cm high and at least 400 cm2 in size (5 individuals per breeding cage). Animals are not housed individually because, as preliminary studies have shown, this generates very severe stress, which may lead to their premature death. In order to provide mice with a pathological phenotype with appropriate conditions, the cage equipment is adapted to their current condition (appropriate bedding, food fed directly into the cage, areas providing shelter, appropriate attractants). In addition, periodic health evaluations by qualified persons is performed. Mice were killed by cervical dislocation.

Control mice (CTRL) were defined as siblings of homozygous PGK-Cre/CAG-flox-hBAG3P209L-eGFP or PGK-Cre/CAG-flox-hBAG3WT-eGFP mice with either WT, PGK-Cre, CAG-flox-hBAG3P209L-eGFP or CAG-flox-hBAG3WT-eGFP genotype. Mice from the homozygous (PGK-Cre/CAG-flox-hBAG3WT-eGFP) strain did not display any obvious phenotype despite the strong hBAG3WT-eGFP expression in their skeletal muscles.

Generation of the CAG-flox-hBAG3P209L-eGFP and the CAG-flox-hBAG3WT-eGFP mouse lines has been described before6.

The transgenic mice were bred in a mixed genetic background (129S6/SvEvTac x C57BL/6N x CD-1). For genotyping of the Tg(CAG-flox-hBAG3WT-eGFP) and Tg(CAG-flox-hBAG3P209L-eGFP) transgenic mice genomic DNA of tail tips was isolated and PCR was performed using the primers CAG2-fw and BAG3-h-spez.tev, oIMR9021 common rev and oIMR9020 wt fw, and loxP-hygro-genotyp-BAG3 fw and BAG3 human-spez rev1 (see Supplemental Information Table 1 for primer information).

Rapamycin/vehicle or saline injection
The experiments were conducted on 16 male 3 weeks old CAG-BAG3P209L mice with a strong phenotype, manifested by severe myofibrillarmyopathy and cardiomyopathy and 8 male 3 weeks old non-transgenic littermates, constituting the control group (CTRL).

A dose of rapamycin of 8 mg/kg b.w., suspended in 0.9% NaCl and 2% ethanol at concentration of 0.5 mg/mL (547 µM), was used for experiments. During adaptation period, the mice were habituated to the administration procedure in order to minimize stress that could affect the obtained results. In the course of injection, the mouse was gently restrained (lifting the loose skin on the back of the neck) and a small-gauge needle (27G) was inserted at a 45° angle in the lower right quadrant of the abdomen, avoiding the cecum. The vehicle and saline were administered in a similar manner.

Sarcomere analysis multitool SarcAsM
To analyze sarcomere organization, we utilized SarcAsM, an AI-powered software designed for the automated, multiscale assessment of sarcomeres in microscopy images of myocytes immunostained for the Z-band protein ACTN2. For each myocyte, represented as a Z-stack of images, sarcomere and Z-band lengths were measured in each individual slice. These slice-by-slice measurements were then aggregated to calculate the mean and standard deviation for the entire cell, providing a quantitative measure of sarcomere and Z-band morphology. In a typical Z-stack, our analysis identified and measured an average of 540 distinct Z-bands (defined as structures >0.2 µm in length) and 400 sarcomere vectors, which represent the length and orientation of sarcomeres at different positions within the cell. To quantify the density of organized sarcomeric structures, we calculated the sarcomere area ratio. This was determined by creating maximum intensity projections of both the cell area mask and the sarcomere mask predicted by SarcAsM, and then computing the ratio of the sarcomere mask area to the cell mask area. Additionally, we assessed the cell area ratio, which represents the proportion of the total image area occupied by cells.

Measurement of locomotor activity in actometers
The animals’ locomotor activity was measured using actometers (Opto Varimex, Minor-Columbus, USA). The actometers consisted of four plexiglass walls measuring 43 × 43 × 20 cm. When the animal moved, a photocell recorded each interruption of the infrared beam, which was then counted by a digital counter. The animals’ locomotor activity was recorded for 15 min and expressed as the number of horizontal movements (performed in the horizontal plane). Measurements were taken at a fixed time between 4.00 p.m. and 6.00 p.m. This test allowed us to determine the effect of rapamycin on improving the locomotor activity of CAG-BAG3P209L mice. The measurement was performed three times: at point “0”, after 7 and 14 days of rapamycin administration.

Grip strength measurement
This test was conducted using a device that measures the grip strength of rodents. The mouse, held by the base of its tail, was first directed toward a metal cylinder, on which it rested its front paws, and then grabbed a rod used to measure grip strength with its hind paws. When the mouse grabbed the rod with its hind paws, it was pulled away from the grip until it lost its hind paw support. When the mouse released the grip, the maximum force value was recorded on the digital display of the control device. The measurement was performed three times: at point “0”, after 7 and 14 days of rapamycin administration.

The rota-rod test
Motor coordination was assessed using a rota-rod treadmill (Yamato Instruments Corporation, India). Before the proper measurement, the mice were subjected to a habituation procedure. At first, they were placed on a stationary rod for 120 s. Then, they were placed three times on a rod rotating at a speed of 3 rpm in a clockwise direction. The relevant measurement included the average of three trials of the time spent on a rod rotating at a speed of 30 rpm. The maximum duration of a single trial was 120 s. The measurement was performed three times: at point “0”, after 7 and 14 days of rapamycin administration.

Ex vivo skeletal muscle force measurement
Mice were killed by cervical dislocation. Dissection of hindlimbs was performed in electrolyte solution permanently gassed with carbogen. Removing The skin was removed from the hindlimbs and the distal common end tendon of the gastrocnemius and soleus muscles were detached from the calcaneus. By elevating the entire muscle bundle in a proximal direction, the soleus muscle was exposed with its tendon of origin. This tendon of origin was separated from the bone and the soleus muscle was separated from the gastrocnemius muscle in the region of the end tendon. Fixation of the hind legs with the dorsal side up, to remove the extensor digitorum longus (EDL) muscle. After removal of the connective tissue and ligaments in the area of the knee joint, the tendon of origin of the EDL was cut directly at the bone. The soleus muscle and the EDL muscle were clamped at their tendons into the Myostation of the Myotronic device to determine the force of the skeletal muscle. The muscles were set to their optimal muscle length and were in a permanently carbogen-gassed electrolyte solution during the measurement. To analyze the relationship between stimulation frequency and muscle force development in mN, stimulation was first performed every 3 min at 10 Hz (soleus muscle) or every 5 min at 10 Hz (EDL), followed by a pause of 3 (soleus muscle) or 5 min (EDL muscle) and then a second stimulation at 25 Hz, followed by 50 Hz, 75 Hz, 100 Hz and the final frequency of 125 Hz. After completion of the measurements, the length of the purely muscular part of the muscle was determined and the non-muscle parts were removed so that the muscle could be weighed.

Histology, immunofluorescence staining, and microscopy
Skeletal muscles were dissected and fixed in 4% paraformaldehyde (PFA) at 4°C for 4 h. The fixed tissue was equilibrated in 20% sucrose solution and then properly oriented and embedded in Tissue Tek® O.C.T Compound. 10 µm thick cryosections were cut on a cryotome CM3050S (Leica). Sirius red and Fast green staining was performed using standard histology protocols. For staining of fixated cells and skeletal muscle sections the following antibodies were used (for 2 h at RT in 0.2% Triton X in PBS, 5% donkey serum or overnight at 4°C in 0.2% Triton X in PBS, 5% donkey serum): ACTN2 (1:400, Sigma-Aldrich), BAG3 (1:250, Proteintech, recognizing both human and mouse BAG3), desmin (1:100, DAKO), FLNC (1:1000, Biogene), SYNPO2 (1:100, Invitrogen), cCasp3 (1:50, Cell Signaling Technology), CD45 (1:400, Merck Millipore), BF-F3 (1:400, Developmental Studies Hybridoma Bank (Iowa, USA)), BA-F8 (1:400, Developmental Studies Hybridoma Bank (Iowa, USA)), SC-71 1:400 (Developmental Studies Hybridoma Bank (Iowa, USA)), LC3B (1:300, Cell Signaling), SQSTM1 (1:300, Progen), Pax7 (1:50, Developmental Studies Hybridoma Bank (Iowa, USA)), Ki67 (1:200, Dako), WGA (1:1000, Vector Laboratories). After washing, secondary antibodies conjugated to Alexa Fluor 488, Cy3 or Alexa Fluor 647, respectively (1:400, Jackson ImmunoResearch), diluted in 1 µg ml−1 Hoechst (Thermo Scientific, 33342) in PBS were applied for 1 h at room temperature. Sections were mounted with coverslips and imaged using an inverted fluorescence microscope (Axiovert 200, Carl Zeiss) with filters for DAPI, GFP, Cy3, and Cy5, and ×25, ×40, and ×60 Plan Apochromat water objectives, an ebx75 light source and an AxioCam MRm digital camera. The editing of the pictures was performed with the Axiovision Rel. 4.8 Software (Zeiss).

Protein isolation
Tissues from PGK-Cre × CAG-BAG3(P209L)-eGFP-flox mice and controls were prepared with 8 M-urea buffer to extract the proteins within. Due to the instability of urea, the urea buffer always had to be freshly prepared. 50 mg of tissue was mixed with 500 µl of urea buffer and homogenized with a pestle. Protein concentration was determined using the PierceTM BCA Protein Assay Kit (Thermo Scientific, (Waltham, USA)). Quantification of protein concentration of each sample was determined using a microplate reader (Tecan Spark Reader, Cat. No. 30086376) with a measurement below 562 nm.

Protein isolation from quadriceps muscle for enzyme-linked immunosorbent assay (ELISA)
The mice were administered a lethal intraperitoneal dose of pentobarbital anesthesia at a dose of 120 mg/kg b.w., and then the quadriceps muscles were collected. To minimized discomfort to the animal during this procedure, the mice were additionally anesthetized with isoflurane (2.5%, flow rate 0.5 L/min) immediately before the injection. Quadriceps muscles were taken from both hindlimbs, and the muscles were cleaned of fat and connective tissue. The collected tissues were frozen using liquid nitrogen and then stored at −80°C until further analysis. Next, the quadriceps muscles were divided into pieces weighing 100 mg each and placed in microcentrifuge tubes together with an equal mass of zirconium silicate beads (0.5 mm; Next Advance Inc., ZROB05). Then, T-PER Tissue Protein Extraction Reagent was added in a volume ratio of 2:1 to the volume of the tissue (max. 0.6 ml of the homogenization buffer). The tube was sealed and placed in a Bullet BlenderTM, the speed was set to 8, and the time was set to 4 min.

Western blot analysis and enzyme-linked immunosorbent assay (ELISA)
Specific proteins were separated according to their size in a denaturing SDS-polyacrylamide gel. 30 µg of the samples were mixed with the appropriate amount of 4× Laemmli Sample Buffer (Bio-Rad, cat# 161-0747) and then denatured at 99°C for 10 min. The 4–20% Mini-PROTEAN® TGX Stain-Free™ Protein gel (BioRad) was placed in the gel electrophoresis chamber and filled with 10× Tris/Tricine/SDS Running Buffer (BioRad, cat# 1610732). The first well of the polyacrylamide gel was filled with molecular weight marker (BioRad, cat# 1610376), and the remaining wells were successively filled with the prepared samples. After size-dependent separation of the proteins using the SDS-polyacrylamide gel, they were blotted to a polyvinylidene fluoride (PVDF) membrane (BioRad, cat# 1704274) at 25 volts for 10 min using the Trans-Blot Turbo Transfer System (BioRad). Immunostaining was preceded by blocking the nonspecific binding sites by incubating the membrane in 5% skim milk powder in TBS-T for 1 h. The primary antibody was also prepared in 5% skim milk powder in TBS-T (Bag3 1:5000, CRYAB 1:5000, HSPB6 3:5000, HSPB7 1:1000, HSPB8 1:1000, SQSTM1 1:1000, LC3B 1:1000, SYNPO2, ß-actin 1:2000, GFP 1:1000, mCherry 1:1000) or in 5% BSA in TBS-T (PINK1 1:1000). Incubation of the membrane with the first antibody was performed overnight at 4°C. The membrane was then washed three times with 6 ml of TBS-T each time, with TBS-T removed directly the first time, followed by two five-minute incubations. The secondary antibodies (HRP, horseradish peroxidase or fluorescence-coupled) were diluted in 5% skim milk powder/BSA in TBS-T and incubated with the membrane for 1 h at room temperature. The membrane was then washed five times with TBS-T for 5 min each time. The PierceTM Chemiluminescent Reagent (ECL Western Blotting Substrate) from Thermo Scientific was used to detect HRP-coupled secondary antibodies. The membrane was treated for 1 min with a 1:1 mixture of the peroxide solution and the luminol enhancer solution of the ECL system. The ChemiDocTM MP imaging system from BioRad was used for detection. Analysis of the acquired images was performed using BioRad’s Image Lab software, normalizing the relative protein expression of each sample to β-actin or total loaded protein.

The level of LC3 II protein was determined in homogenates from quadriceps muscles and hearts using the ELISA method (cat. number: MBS3806182; MyBioSource, Inc.; San Diego, USA).

Isolation and transfection of primary myoblasts from mice
Primary myoblasts were isolated from hindlimb muscles (tibialis anterior, gastrocnemius, soleus, quadriceps, and extensor digitorum longus) of 6-week-old mice following euthanasia by cervical dislocation. Muscles were aseptically excised, cleared of fat and connective tissue, repeatedly washed, and finely minced under sterile conditions. Tissue was enzymatically digested with collagenase II at 37°C for 50 min, followed by mechanical dissociation, sequential filtration (70 µm, then 30 µm mesh), and centrifugation. Cells were resuspended in myoblast growth medium supplemented with bFGF and seeded onto 10% Matrigel-coated 100-mm culture dishes, maintained at 37°C and 5% CO₂. Fibroblasts were depleted by repeated pre-plating on uncoated dishes until a myoblast purity of >95% was achieved. Cells were maintained below 75% confluency, with medium changes every 2 days. For differentiation, myoblasts were cultured to 75–80% confluency, after which growth medium was replaced with myotube differentiation medium, refreshed every 48 h. Myoblast alignment and fusion were observed within 24–36 h, with initial myotube formation after ~48 h and further maturation over 72–96 h. After 6 days, myotubes exhibited advanced maturation with clearly visible cross-striations. Mature myotubes were transiently transfected with plasmids carrying expression cassettes for Becn1, Bnip3, or Ppargc1a using Lipofectamine 3000 (Thermo Fisher). On the day of transfection, the culture medium was replaced with fresh differentiation medium without penicillin/streptomycin (500 µl per well). For each well of a 24-well plate, 1 µg plasmid DNA was diluted in 25 µl Opti-MEM reduced-serum medium (Thermo Fisher Scientific) and mixed with 1 µl P3000 (Thermo Fisher Scientific) reagent. In parallel, 1.5 µl Lipofectamine 3000 (Thermo Fisher Scientific) was diluted in 25 µl Opti-MEM and incubated for 5 min at room temperature. DNA and Lipofectamine solutions were combined and incubated for 10–15 min at room temperature to allow complex formation. The transfection mixture was added dropwise to the cells, which were then incubated at 37°C and 5% CO₂. After 17 h, transfection was terminated by replacing the medium with fresh differentiation medium. At 48 h post-transfection, cells were either fixed with 4% paraformaldehyde or treated with 300 nM Bafilomycin A1 for 5 h, followed by a 1 h incubation in differentiation medium prior to fixation. BECLIN-HA WT was a gift from Kunliang Guan (Addgene plasmid # 46993; http://n2t.net/addgene:46993; RRID: Addgene_46993). AAV-CMV-Flag-PGC1a-6His was a gift from Connie Cepko (Addgene plasmid # 67637; http://n2t.net/addgene:67637; RRID: Addgene_67637). Myc-Bnip3FL was a gift from Joseph Gordon (Addgene plasmid # 100796; http://n2t.net/addgene:100796; RRID: Addgene_100796).

Allele-specific BAG3 expression-analysis
RNA was isolated from quadriceps muscles of 4 weeks old CAG-BAG3WT, BAG3P209L or non-transgenic sibling control mice using the RNeasy Plus Micro Kit (#74034, Qiagen) according to manufacturer’s instructions. The RNA-integrity was measured with the Bioanalyzer 2100 and RNA 6000 Nano Kit (#5067-1511, Agilent). Only samples with RNA-integrity number (RIN) above 6.4 were used for further experiments. 500 ng of RNA of each sample were transcribed to cDNA in 20 µl mixtures using the SuperScript VILO Master Mix (#11755500, Thermo Fisher) according to manufacturer’s instructions. For allele-specific BAG3 expression analysis an SNP Genotyping TaqMAN qPCR-Assay (Assay-ID: C_160294934_10, SNP-ID: rs121918312, Thermo Fisher) from was used which includes two highly allele-specific fluorescent-labeled probes for BAG3 WT and BAG3 P209L (BAG3 c.626C/T). In separate mixtures TaqMAN probes for murine Bag3 (Assay-ID: Mm00443474_m1 Bag3, Thermo Fisher) and murine Gapdh (Assay-ID: Mm99999915_g1, Thermo Fisher) were used as references. The qPCR was prepared in 20 µl format and technical duplicates using 25 ng of cDNA, 10 µl TaqMAN Master-Mix for gene expression (#4369016, Thermo Fisher) from, either 1 µl of SNP Genotyping TaqMAN probes or 1 µl of each of the TaqMAN probes against murine Bag3 and murine Gapdh. H2O was added until 20 µl for each mixture. The qPCR was performed on the CFX Opus 96 Real-Time PCR System from Bio-Rad using following run-parameters: Initial denaturation 95°C 10 min; 40 cycles: Denaturation 95°C 15 s, annealing/polymerization 60°C 1 min. For further analysis the thresholds of the two fluorophore-channels had to be set to the same value by hand to allow allele-specific comparisons. Relative expression was calculated using RE = 2−(ΔCq) while ΔCq = Cq BAG3-of-interest − Cq mGAPDH.

Fractionation of mouse muscle tissue
Mouse quadriceps muscles were used for the proteomics analysis (n=4 for each genotype). Each muscle sample was cut into 100 thin sections of 5 µm thickness in a cryotome at −20°C. The thin sections were taken in 2 mL tubes containing 1.4 mm and 2.8 mm zirconium oxide beads and weighted. 1× Triton X–100 buffer (Cell Signaling Technology) containing HALT™ protease and phosphatase inhibitors (Thermo Scientific) at a ratio of 1:100 (v/v) was added at an amount of 30 µL per milligram of the sample. The samples were homogenized using a Percellys 24 tissue homogenizer (Bertin instruments). The homogenization was done in 3–5 cycles, each cycle consisting of homogenization at 5800 rpm for 15 s followed by resting on ice for 120 s. About 50 µL of the lysed sample was collected in a microcentrifuge tube, and a 100 mM Tris–HCL buffer (pH 7.9) containing 4 M urea, and HALT protease and phosphatase inhibitors at 1:100 (v/v) ratio was added. The remaining sample was centrifuged at 20,800 × g for 15 min, and Triton X-100 soluble supernatant was transferred to a new microcentrifuge tube. The remaining pellet was washed with Triton buffer, centrifuged, and transferred to the rest of the supernatant and stored at −80°C. The remaining Triton X–100 resistant pellets were solubilized using the above-mentioned urea buffer at a volume equal to 70% of the initial Triton X–100 buffer. The samples were stored at −80°C until further processing.

Proteomics sample preparation
About 20 µg of the proteome sample from the total lysate, the pellet and the supernatant fraction (n=4 biological replicates for each condition and each of the two genotypes) were carbamidomethylated by incubating with 10 mM Dithiothretol (DTT) at 37°C for 30 min followed by incubating with 50 mM Chloroacetamide for 30 min in the dark at room temperature. After that the reaction was quenched by adding 50 mM DTT. The proteome was purified using SP3 beads as described earlier16, dissolved in 100 mM HEPES-NaOH buffer of pH 7.5 and 2.5 mM CaCl2 and digested with trypsin at a ratio of 1 µg trypsin to 100 µg proteome overnight at 37°C, shaking. Digested peptides were desalted using self-packed StageTips following the protocol as described by Rappsilber et al.17. The eluted peptides from the StageTips were dissolved in HPLC-water with 0.1% formic acid and analyzed using LC-MS/MS setup.

Mass spectrometry data acquisition
The peptides were separated using an Ultimate 3000 RSLC nano chromatography system (Thermo Fisher) operated in two-column setup. First, the peptides were loaded onto a short 2 cm µPAC reverse phase trap column (Thermo Fisher) using a loading pump buffer of 2% acetonitrile and 0.05% TFA. The peptides from the trap column were eluted and separated in a 50 cm µPAC reverse phase analytical column (Thermo Fisher) with an acetonitrile gradient of 2 to 30% eluent B (Eluent A—HPLC water, 0.1% FA, Eluent B—acetonitrile, 0.1% FA) over a period of 90 min. The whole LC method for each sample was about 120 min including the washing and the equilibration phases.

Peptides eluted from the column were directly introduced into an Impact II Qq-ToF-MS (Bruker) using a captive spray ion source as described18. The instrument was operated in data independent acquisition (DIA) mode, with acquisition of MS1 spectra from 400 m/z to 2200 m/z followed by MS2 spectra of ions in 32 mass windows of 25 m/z from 400 m/z to 1200 m/z. Each mass window had an overlap of 0.5 m/z on either side to compensate for the inefficiency of the quadrupole. The MS was operated at 15 Hz resulting a cycle time of 2.2 s.

Proteomics data analysis
The acquired data was converted into mzML format using proteowizard19 with the recommended settings for DIA-NN analysis. The mouse reference proteome was downloaded from Uniprot20 (release 2022_05). A FASTA database listing contaminants commonly observed for mouse tissue was downloaded from the github (https://github.com/HaoGroup-ProtContLib) and the BAG3P209L-EGFP protein sequence was also added to the search. DIA-NN21 version 1.8 was used for peptide identification with the following settings: Methionine excision and carbamidomethyl were set as fixed modifications, enzyme was set to strictly trypsin with up to 1 missed cleavage, and variable modification was set to 1 and methionine oxidation was set a variable modification. The MS1 and MS2 mass error were set to 20 ppm with a scan window of 4. Deep learning-based spectra and RT prediction was switched on. The analysis mode was set to double pass mode and quantities matrices were switched on. All other settings were default. Data obtained from DIA-NN were further analyzed using Perseus22 version 1.6.15.0. The protein groups matrix was uploaded to Perseus and the values were log2 transformed. After removing contaminants and non-unique genes, the matrix was filtered for valid values. Proteins found exclusively in samples of one genotype (quantified in at least 3 of the 4 replicates) were analyzed separately. For proteins found in both control and the mutant mice, significantly changing proteins were identified with an FDR controlled. GO and KEGG term enrichment analyses were done using cytoscape23 and stringDB24. In cytoscape ClueGo was used with standard settings and only significant pathways with a Bonferroni stepdown corrected p-value ≤ 0.05. Boxplots were created using Raincloud Shiny app25.

Electron microscopy
A standard EM protocol was used in this study, briefly, stretched quadriceps femoris muscle samples from WT and CAG-BAG3P209L-mice (n=3 per genotype) were fixed in 4% paraformaldehyde, 15% saturated picric acid and 0.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 overnight at 4°C. After rinsing in PBS, sections were treated with 0.5% OsO4, washed and counterstained with uranyl acetate, dehydrated via ethanol series and embedded in Durcupan resin (Fluka, Switzerland). Ultrathin sections were prepared (Ultracut S; Leica, Germany) and adsorbed onto glow-discharged Formvar-carbon-coated copper grids. Ultrastructural analysis was performed in a Zeiss LEO 910 electron microscope and images were taken with a TRS sharp eye CCD camera (TRS Systems, Moorenweis, Germany).

Isolation of skeletal muscle mitochondria
Mouse skeletal muscle mitochondria were prepared by rapidly removing the hindlimb muscles and transferring them into ice-cold isolation medium (180 mM KCl, 10 mM EDTA-Na2, pH 7.4). Muscles were minced with scissors, trimmed clean of visible fat and connective tissue, and placed in 30 ml of isolation medium supplemented with trypsin (1 mg per 1 mg of tissue). After 30 min, the tissue was homogenized using a motor-driven Teflon-glass Potter homogenizer. The homogenate was centrifuged at 800 × g for 6 min. The supernatant was decanted and centrifuged at 9000 × g for 10 min at 4°C. The final mitochondrial pellet (M-fraction) was resuspended in medium containing 0.2 M sucrose and 20 mM HEPES, pH 7.2

Determination of substrate oxidation rates
Mitochondrial oxygen consumption was measured at 25°C using an Oroboros oxygraph (Anton Paar, Austria) in a medium containing 10 mM KH2PO4, 60 mM KCl, 60 mM Tris, 110 mM mannitol, 5 mM MgCl2, 0.5 mM EDTA, pH 7.4 and 10 mM glutamate (glu) + 5 mM malate (mal) or 1 mM octanoylcarnitine (OC) + 5 mM malate (mal) as respiratory substrates. The respective additions were 10 mM creatine (dry powder), 1 µM carboxyatractyloside (CAT) or 2 µM of the uncoupler TTFB. The concentration of mitochondria was 0.3 mg protein/ml.

Mitochondrial enzymatic measurements
We measured activity of the mitochondrial enzymes NADH-CoQ oxidoreductase, cytochrome c oxidase (COX), as well as citrate synthase using standard methods. All measurements were performed at 30°C with a dual wavelength spectrophotometer (Aminco DW-2000, SLM Instruments, Rochester, NY, USA). Shortly, the activity of rotenone-sensitive NADH-CoQ1 oxidoreductase was measured in a reaction medium containing 50 mM KCl, 1 mM EDTA, 10 mM TRIS-HCl (pH 7.4), 1 mM KCN, 100 µM CoQ1, and 150 µM NADH. The assay was initiated by addition of the sample and the velocity of NADH oxidation was monitored. After 2 min of registration, 20 µM rotenone was added to determine the rotenone-insensitive NADH oxidation rate. The activity of mitochondrial NADH-CoQ oxidoreductase is the difference between the total NADH oxidation rate and the rotenone-insensitive NADH oxidation rate. The cytochrome c oxidase activity was measured in 0.1 M potassium phosphate buffer (pH 7.4), 0.02% lauryl maltoside and 200 µM reduced cytochrome c. The reaction was started with sample addition and the oxidation of ferrocytochrome c was monitoring at the wavelength pair 510/535 nm (ε red-ox = 5.9 mM−1 cm−1).

Tissue RNA-seq analysis
For RNA-Seq experiments, DNA-free total RNA was isolated from quadriceps muscle from 4 CAG-BAG3P209L and 5 control mice (5 weeks old) using the RNeasy Kit (Qiagen) including on-column DNAse digestion. RNA quality was analyzed by an Agilent Bioanalyzer (Agilent). For library preparation the Trio RNA-Seq Library Preparation kit for mouse (TECAN) was used, starting with 50 ng of total RNA. Six PCR cycles were used for library amplification and libraries with an average fragment size of 322 bp were sequenced on a NextSeq 500 in paired-end mode (75 bp, Illumina). For bioinformatics analysis, we used the Galaxy platform (Freiburg Galaxy Project26). RNA sequencing reads were mapped using RNA STAR27 followed by counting reads per gene by using featureCounts28. Differentially expressed genes were identified by DESeq229. For data visualization, normalization and cluster analysis heatmap2 and Volcano plot (Freiburg Galaxy Project28) was used. Gene ontology analysis was performed by ClueGO by using the KEGG pathway and GO-term databases with a significance interval for pathways of p < 0.05. p-Values were corrected for multiple-testing by the Holm-Bonferroni method.

Injection of AAV/rh10
AAV/rh10-mCherry-U6-h-BAG3-shRNA (RNAi) and AAV/rh10-mCherry-U6-scrmb-shRNA (control) were used for gene therapy (Vector BioLabs). P15 CAG-BAG3P209L mice were injected into the left jugular vein with a total volume of 100 µl PBS containing 1 × 1011 virus particles of either RNAi or control AAV/rh10. Mice were euthanized 3 weeks later (P37) and analyzed.

Statistical analysis
Data are depicted as mean ± SEM. Normal distribution of data was determined by D’Agostino & Pearson omnibus normality test, while the Levene test was used to assess the homogeneity of variance. Depending on these results, analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for normally distributed data. If the assumptions of normality and homogeneity of variance were not met, the nonparametric Kruskal-Wallis test was applied, followed by Dunnett’s test for multiple comparisons. Unpaired t-test (two-sided) was used to test for significance for two independent groups. All statistical analyses were conducted using GraphPad Prism V9.0.1 software. P-values < 0.05 were considered statistically significant. Statistically significant differences are indicated by exact P-values. All measurements were taken from distinct samples.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_2": "Strengths\nThe paper described an exhaustive characterization of an overexpression model of Myofibrillar myopathy-6 (MFM6). This is\nthe first publication of such model. Producing a mice model of MFM6 has been extremely difficult since the KO are lethal and\ndouble KI for the P209L mutation do not show a phenotype and KI/KO only a very mild phenotype.\nThe paper makes all efforts to try to characterize clinically, histologically, biochemically and at the RNA expression level the\nimpact of the overexpression of a human hBAG3P209L. The authors are not clear early on that these mice are homozygous\nfor the insertion.\nThe sum of the work is impressive, and the results are well presented, and the paper is well written.\nWeaknesses\nThere should have been a better description of previous efforts to develop a mice model of MFM6.\nThe rational for generating a partially humanized, since the mice BAG3 are not inactivated, overexpression is not detailed in\nthe introduction. This is important, since the major reservation for this model is that all the findings may mostly be caused by\nthe overexpression of the hBAG3P209L. This is not well discussed in the paper and should be stated and discussed as one\nof the major limitations of the model.\nThe authors do not underline that very high levels of expression hBAG3P209L with an eGFP tag further suggests that it may\nhave a larger effect on aggregating of the targets of Bag3 and wildtype Bag3.\nMore discussion on the relative tissue expression of the wildtype and hBAG3P209L should have been included to insist on\nthe difference between heart and skeletal muscle.\nA model with at the most a hBAG3P209L expression level comparable to the wildtype would have been preferable.\nQuestions to be addressed\nPage 13: How significant was the “…increase of BAG3, αB-crystallin (Fig. 5c, d) and the small heat shock proteins HSPB7\nand 8 (Fig. 5e, f), of which HSPB8 binds BAG3” should be stated in this sentence.?\nPage 17: They provide strong evidence that overexpression of hBAG3P209L leads to the: “… 215 of proteins accumulating\nin the pellet fraction (log2(BAG3P209L/control)>0.25) showed reduced abundance in the soluble fraction (log2\n(BAG3P209L/control)<-0.25), indicating increased association to the cytoskeleton or aggregation (Fig. 6d). More data on\nrelative overexpression and phenotype should be included.\nPage 21: Over expression is likely the key driver to phenotype, which is not what is observed in the human pathology, as its\nlevel influence severity as stated: “This impairment correlated with the expression level of the BAG3P209L-eGFP transgene\nand explained the phenotypic variability we observed in this mouse line. The reason for the different expression levels of the\ntransgene are unclear as all mice analyzed were homozygous for the activated BAG3P209L-eGFP expression.”\nPage 22: Thie difference in expression between heart and skeletal muscle are important. Could we compare levels of\nexpression in the mice?\nMinor points\nA supplemental figure with the alignment of the hBAG3P209L compared to the mice Bags3 would be appreciated.\nPage 11 legend of figure 4: 20 most differentially expressed genes are shown. Not genes but pathways.\nPage 12: adherents rather than “adherens”\nPage 16: Figure d, the lines to dotes for hBAG3P209L and mice Bag3 are not clear.", "reviewer_3": "The authors present a mouse model for BAG3P209L myofibrillar myopathy that shows molecular and functional features of\nsevere muscle weakness as described in patients with MFM6. They demonstrate that accumulation of BAG3-containing\naggregates and loss of BAG3 function leads to sarcomere disintegration, mitochondrial dysfunction and subsequently\nmuscle weakness and this can be rescued by knockdown of hBAG3P209L.\nWhile potentially interesting, the study is essentially uncontrolled because they did not generate and characterize a mouse\nline that overexpresses WT BAG3 to the same level. Overexpression of many WT proteins leads to phenotypes so there is\nno way here of knowing if the observed phenotypes were due to the P209L mutation or gross overexpression of BAG3.", "reviewer_4": "In the manuscript the authors studied the role of BAG3 mutation in muscle physiopathology. They generated a humanized\ntransgenic mouse line that ubiquitously expressed the mutant BAG3P209L protein. These mice have been previously\ncharacterized from a cardiac point of view and indeed they showed a cardiomyopathy and died within 5-8 weeks. When the\nauthors checked the skeletal muscle function and structure, they found the typical features of Myofibrillar myopathies (MFM)\nsuch as myofibrillar disassembly, protein aggregates and weakness. Transcriptomics and proteomics analyses confirmed an\nalteration in proteostasis with impairment of autophagy/mitophagy and accumulation of dysfunctional mitochondria. Almost\nall these features were reverted by a genetic approach to knockdown BAG3 expression in skeletal muscles. The paper is\nprimarily descriptive of the muscle phenotype when BAG3P209L protein is expressed. The phenotype is very interesting but\nthe insights that explain the muscle features with BAG3P209L are superficially explored. The authors should consider the\nfollowing points\nPoint1. Figure 1f-g. The GFP pattern is not homogeneous among the different myofibers, especially in the soleus. Is there\nany fiber type specificity? From the pattern it looks like that it is higher expressed in the fast fibers. Authors should check the\nGFP expression in the different fiber types\nPoint2. Authors should perform morphometric analyses in different muscles such as Tibialis Anterior, Gastrocnemius and\nDiaphragm.\nPoint3. Fig2a. Check also Diaphragm, which is the most affected muscle in murine models of Muscle Dystrophy. Chek also\nfat infiltration with Sirius red staining\nPoint4 Fig3c-d are of poor quality. Show high resolution images\nPoint5. Fig3e-g. EM pictures suggest accumulation of vesicles/lysosomes. Show high magnification pictures to underline the\nfeatures. Authors should immunostain for Lamp1, p62 and LC3 and quantify the puncta.\nPoint6. Autophagy and mitophagy flux must be monitored to support the authors claims.\nPoint7, In vivo Bag3 knockdown results in phenotypic amelioration. Authors should also monitor whether mitochondrial\nfunction and autophagy was restored.\nPoint8. Author should put an effort in identifying which mechanisms downstream BAG3 dysfunction is the main trigger of the\nphenptype. Is autophagy reactivation sufficient to retore muscle function in BAG3P209L mutant? Or Is mitochondria\nimprovement via mitophagy? Or just mitochondrial function that when preserved also improves muscle function. These\nexperiments could be easily done (e.g. Beclin1 expression or rapamycin treatment for autophagy activation, BNIP3\nexpression for mitophagy, PGC1a expression for mitochondrial biogenesis and function) and would confirm/identify insights\nthat connect BAG3 mutation to MFM,"}, "author_response": "Point-by-point response to Reviewer Comments\nReviewer #2 (Remarks to the Author):\nStrengths\nThe paper described an exhaustive characterization of an overexpression model of Myofibrillar\nmyopathy-6 (MFM6). This is the first publication of such model. Producing a mice model of MFM6\nhas been extremely difficult since the KO are lethal and double KI for the P209L mutation do not\nshow a phenotype and KI/KO only a very mild phenotype.\nThe paper makes all efforts to try to characterize clinically, histologically, biochemically and at the\nRNA expression level the impact of the overexpression of a human hBAG3P209L. The authors are not\nclear early on that these mice are homozygous for the insertion.\nThe sum of the work is impressive, and the results are well presented, and the paper is well written.\nAnswer: We thank the reviewer for appreciating our work and his/her thoughtful and constructive\ncomments and suggestions.\nWeaknesses\nThere should have been a better description of previous efforts to develop a mice model of MFM6.\nThe rational for generating a partially humanized, since the mice BAG3 are not inactivated,\noverexpression is not detailed in the introduction. This is important, since the major reservation for\nthis model is that all the findings may mostly be caused by the overexpression of the hBAG3P209L.\nThis is not well discussed in the paper and should be stated and discussed as one of the major\nlimitations of the model.\nAnswer: As suggested by the reviewer, we have included a description of prior mouse models in the\nrevised version of the Introduction. Furthermore, we provide a more detailed explanation of the\nrationale for using an overexpression approach (page 4, lines 83-90).\nRegarding the limitations of the overexpression approach, we agree with the reviewer that\noverexpression of the BAG3 fusion protein will increase the probability of aggregate formation, which\nwill accelerate the manifestation of the phenotype. Nevertheless, we don’t think that the phenotype is\nnonspecific as our mouse model quite precisely mimics the human pathology, such as restrictive\ncardiomyopathy and skeletal muscle atrophy. Of note, restrictive cardiomyopathy in particular is\nspecific to MFM6 and is very rarely observed in mouse models for cardiac diseases, making its\ncoincidental occurrence highly unlikely. Furthermore, higher levels of BAG3P209L-eGFP lead to increased\nprotein accumulation and are therefore comparable to MFM6-patients in later stages of the disease.\nAlthough studies of MFM6-patients show no increase in the soluble fraction of BAG3 protein in heart\nor skeletal muscle, they clearly show the formation of BAG3-positive protein aggregates (Ruparelia et\nal. 2021 Autophagy and Schänzer et al., 2018 Molecular Genetics and Metabolism) as seen in our\nmouse model.\nThe authors do not underline that very high levels of expression hBAG3P209L with an eGFP tag\nfurther suggests that it may have a larger effect on aggregating of the targets of Bag3 and wildtype\nBag3.\nAnswer: We thank the reviewer for raising this important point. To address this, we have included new\ndata from a control mouse line overexpressing human BAG3WT-eGFP. This mouse line serves as an ideal\ncontrol, as it was generated in the same way as the CAG-BAG3P209L-eGFP mouse line (knockin in the\nRs26 locus), with the only difference being that the human BAG3 does not carry the P209L point\nmutation. We now show that there is a comparable induction of BAG3 mRNA in skeletal muscle of both\ntransgenic mouse lines, CAG-hBAG3P209L-eGFP and CAG-hBAG3WT-eGFP (control), while the total BAG3\nprotein (mouse + human) is 11.36-times increased in CAG-hBAG3P209L-eGFP-mice but only 4.46-times in\nCAG-hBAG3WT-eGFP-mice. This is due to the accumulation of hBAG3P209L-eGFP protein in aggregates,\nwhich is not seen in the control mouse line.\nRegarding the ratio of overexpressed to endogenous BAG3, there is 1.54 times more BAG3WT-eGFP\nprotein in skeletal muscle of the CAG-hBAG3WT-eGFP mouse line than mouse Bag3 protein. This is quite\nsimilar to the CAG-hBAG3P209L-eGFP mouse line, which has 1.92 times more BAG3WT-eGFP protein than\nmouse Bag3 protein. We have added a new paragraph discussing the differences in expression levels\nof protein and mRNA for mouse Bag3, human BAG3WT-EGFP, and BAG3P209L-eGFP in our transgenic\nmouse lines (page 6, lines 133-155).\nMore discussion on the relative tissue expression of the wildtype and hBAG3P209L should have been\nincluded to insist on the difference between heart and skeletal muscle.\nAnswer: We have included expression data comparing BAG3P209L protein levels in heart and skeletal\nmuscle (page 6, lines 145-150) and added a short discussion of the relative tissue expression to the\ndiscussion (page 28, lines 624-629). The total amount of BAG3 protein (human BAG3P209L and mouse\nBag3) in the hearts of these mice is 2.6 times higher than in skeletal muscle. This explains why the\nphenotype is more severe in hearts.\nA model with at the most a hBAG3P209L expression level comparable to the wildtype would have been\npreferable.\nAnswer: We agree with the reviewer and present a control line in which hBAG3-eGFP protein is\noverexpressed to an extent comparable to the hBAG3P209L-eGFP protein (ratio of hBAG3WT-eGFP to\nmBag3 is 1.54 compared to a ratio of 1.92 for hBAG3P209L-eGFP to mBag3). Importantly, no obvious\nphenotype is observed in this control mouse line. Therefore, we conclude that our mouse model, despite\npotential drawbacks (overexpression of a non-physiological amount of the hBAG3-eGFP fusion protein),\nis still a valid model mimicking MFM6, as underscored by the observed phenotype.\nQuestions to be addressed\nPage 13: How significant was the “…increase of BAG3, αB-crystallin (Fig. 5c, d) and the small heat\nshock proteins HSPB7 and 8 (Fig. 5e, f), of which HSPB8 binds BAG3” should be stated in this\nsentence.?\nAnswer: We have added the word “statistically” to indicate a statistically significant increase of the\nmentioned proteins (page 16, line 361).\nPage 17: They provide strong evidence that overexpression of hBAG3P209L leads to the: “… 215 of\nproteins accumulating in the pellet fraction (log2(BAG3P209L/control)>0.25) showed reduced\nabundance in the soluble fraction (log2 (BAG3P209L/control)<-0.25), indicating increased association\nto the cytoskeleton or aggregation (Fig. 6d). More data on relative overexpression and phenotype\nshould be included.\nAnswer: We now include a correlation between the maximal force generation of the EDL and soleus\nmuscles and BAG3P209L-eGFP expression (Suppl. Fig. 4k,l). We show that the higher BAG3P209L-eGFP\nexpression, the lower the maximum force development. However, this does not apply to BAG3WT-EGFP\ncontrols, which indicates that mice with higher BAG3P209L-eGFP expression mimic patients in later\nstages of the disease with an increased build-up of protein aggregates.\nPage 21: Over expression is likely the key driver to phenotype, which is not what is observed in the\nhuman pathology, as its level influence severity as stated: “This impairment correlated with the\nexpression level of the BAG3P209L-eGFP transgene and explained the phenotypic variability we\nobserved in this mouse line. The reason for the different expression levels of the transgene are\nunclear as all mice analyzed were homozygous for the activated BAG3P209L-eGFP expression.”\nAnswer: We thank the reviewer for raising this point, and would like to explain this in more detail: We\nconsider the level of BAG3 protein expression, as measured by Western blot analysis, to be part of the\nphenotype. We now provide mRNA expression data for the CAG-BAG3P209L mouse line and demonstrate\nthat the mRNA-level of mouse Bag3 increases only 1.3-fold (a 30% increase, which is not statistically\nsignificant), while the amount of mBag3 protein increases 5.6-fold (460% increase). The mRNA level of\nhBAG3-eGFP is 1.8 times that of mBAG3 in non-transgenic controls. Total mRNA of BAG3 (mouse and\nhuman) is ~3-times higher than in non-transgenic CTRLs, but total BAG3 protein is ~11-times higher\nthan in CTRLs. This indicates the accumulation of BAG3P209L-protein, which is part of the phenotype and\nis only partly due to overexpression. This was further corroborated by analysis of our CAG-BAG3WT\nmouse line. In this line, we observed an approximately 5-fold increase in total BAG3 protein in skeletal\nmuscle without protein aggregate formation or functional impairment. mRNA-expression of mBAG3\nand hBAG3 in these mice was comparable to that of CAG-BAG3P209L-mice (mouse Bag3 mRNA\n0.038±0.018 in homozygous BAG3P209L-eGFP mice and 0.054±0.018 in homozygous BAG3WT-eGFP mice\nand human transgene mRNA was 0.061±0.033 for BAG3P209L-eGFP and 0.034±0.026 for BAG3WT-eGFP\n(Suppl. Fig. 3a)). From this, we conclude that expression BAG3WT-eGFP protein at levels up to at least 5\ntimes the basal level of endogenous Bag3 can be tolerated without causing protein accumulation or\ntriggering a typical MFM6 phenotype.\nPage 22: Thie difference in expression between heart and skeletal muscle are important. Could we\ncompare levels of expression in the mice?\nAnswer: As requested by the reviewer, data from Western Blot analysis comparing BAG3/Bag3\nexpression levels in hearts and skeletal muscle are now included in the revised version of the\nmanuscript new (Suppl. Fig. 3c,d). The total amount of human BAG3 and mouse Bag3 protein is\n6.36+-0.52 (a.u.) for heart and 2.45+-0.96 (a.u.) for skeletal muscle (new Suppl. Fig. 3d). Thus,\nexpression in the hearts of these mice is 2.6 times higher than in skeletal muscle.\nMinor points\nA supplemental figure with the alignment of the hBAG3P209L compared to the mice Bags3 would be\nappreciated.\nAnswer: A new supplemental figure depicting the alignment of the amino acid sequences was added\nto the supplement (new Suppl. Fig. 14) and mentioned in the revised Discussion (page 27, lines 620-\n622). The identities at the amino acid level are 484/581, which corresponds to 83%.\nPage 11 legend of figure 4: 20 most differentially expressed genes are shown. Not genes but\npathways.\nAnswer: In the Volcano plot of Fig. 4a, the 20 most differentially expressed genes are shown. We see\nno need to change the legend.\nPage 12: adherents rather than “adherens”\nAnswer: We thank the reviewer for this suggestion. However, after studying various publications, we\ncome to the conclusion that “adherens” rather than “adherents” junctions is the correct term for this\nkind of cell-cell contact.\nPage 16: Figure d, the lines to dotes for hBAG3P209L and mice Bag3 are not clear.\nAnswer: We have changed Fig. 6d accordingly: The lines pointing to the dots have been made thicker.\nThe “Bag3” labeling has been moved so that it can no longer be confused with the labeling of the\nhBAGP209L-eGFP.\nReviewer #3 (Remarks to the Author):\nThe authors present a mouse model for BAG3P209L myofibrillar myopathy that shows molecular and\nfunctional features of severe muscle weakness as described in patients with MFM6. They\ndemonstrate that accumulation of BAG3-containing aggregates and loss of BAG3 function leads to\nsarcomere disintegration, mitochondrial dysfunction and subsequently muscle weakness and this can\nbe rescued by knockdown of hBAG3P209L.\nWhile potentially interesting, the study is essentially uncontrolled because they did not generate and\ncharacterize a mouse line that overexpresses WT BAG3 to the same level. Overexpression of many\nWT proteins leads to phenotypes so there is no way here of knowing if the observed phenotypes\nwere due to the P209L mutation or gross overexpression of BAG3.\nAnswer: We appreciate Reviewer #3’s critical observation regarding the need for a control mouse line\nthat overexpresses a fusion protein of human BAG3WT and eGFP. We have generated a transgenic\nmouse line that overexpresses human BAG3WT-eGFP under the control of the ubiquitous CAG-promoter.\nSince the transgenic construct was knocked into the Rs26 locus, is inducible, and was crossed with a\nPGK-Cre mouse line and bred to homozygosity, it provides the ideal control, as requested, to\ndemonstrate the specificity of BAG3P209L-eGFP overexpression in mimicking MFM6. To strengthen our\nconclusions, we have conducted key experiments with this hBAG3WT-eGFP mouse line and did not\nobserve any obvious phenotype (new Suppl. Figs. 1, 3, 7). From these experiments we can conclude that\nthe phenotype observed in CAG-BAG3P209L mice is not an artifact caused by uncontrolled overexpression\nof a human BAG3WT-eGFP fusion protein, but rather a specific manifestation of the MFM6 phenotype.\nReviewer #4 (Remarks to the Author):\nIn the manuscript the authors studied the role of BAG3 mutation in muscle physiopathology.\nThey generated a humanized transgenic mouse line that ubiquitously expressed the mutant\nBAG3P209L protein. These mice have been previously characterized from a cardiac point of\nview and indeed they showed a cardiomyopathy and died within 5-8 weeks. When the authors\nchecked the skeletal muscle function and structure, they found the typical features of\nMyofibrillar myopathies (MFM) such as myofibrillar disassembly, protein aggregates and\nweakness. Transcriptomics and proteomics analyses confirmed an alteration in proteostasis\nwith impairment of autophagy/mitophagy and accumulation of dysfunctional mitochondria.\nAlmost all these features were reverted by a genetic approach to knockdown BAG3 expression\nin skeletal muscles. The paper is primarily descriptive of the muscle phenotype when\nBAG3P209L protein is expressed. The phenotype is very interesting but the insights that\nexplain the muscle features with BAG3P209L are superficially explored. The authors should\nconsider the following points\nWe thank the reviewer for his/her thoughtful criticisms and suggestions. We have performed, as\nrequested, new experiments and these helped to further clarify the disease mechanisms underlying the\nMFM in our transgenic mouse model.\nPoint1. Figure 1f-g. The GFP pattern is not homogeneous among the different myofibers,\nespecially in the soleus. Is there any fiber type specificity? From the pattern it looks like that it\nis higher expressed in the fast fibers. Authors should check the GFP expression in the different\nfiber types\nAnswer: We thank the reviewer for raising this point. We have included new data on fiber type in the\nrevised version of the manuscript (Suppl. Fig. 4) and found a switch from fast to slow fibers in soleus\nand EDL muscles from 4 week old CAG-BAG3P209L-mice. Specifically, the percentage of MHC-I type fibers\nincreased, while the percentage of MHC-IIb fibers decreased. This has been observed in several\nneuromuscular diseases, such as Duchenne muscular dystrophy (Talbot and Maves, Wiley Interdiscip\nRev Dev Biol. 2016, 5: 518–534.).\nInterestingly, there was no preference for BAG3P209L-eGFP expression in the different fiber types in the\nsoleus and EDL muscles, as the expression levels were not statistically significantly different.\nNevertheless, we observed a very high variability when comparing individual values.\nPoint2. Authors should perform morphometric analyses in different muscles such as Tibialis Anterior,\nGastrocnemius and Diaphragm.\nAnswer: We thank the reviewer for this helpful suggestion. We do include an analysis of the diaphragm,\nas the failure of this muscle is one of the main causes of MFM6 fatality (new Fig. 1f,k,l,p; new Fig.2a,d;\nnew Fig. 3d; new Suppl. Fig. 1b). We found BAG3P209L-eGFP positive protein aggregates, increased\nfibrosis and a higher number of centralized nuclei.\nIn addition, our extensive analysis of different skeletal muscle types focused on the soleus, quadriceps,\ndiaphragm, and EDL muscles, because quadriceps and EDL mainly consist of fast twitch type II fibers,\nand soleus and diaphragm mainly of slow twitch type I fibers, type II fibers, and the quadriceps mostly\nof a mixed type (quadriceps). With this selection, we believe we have effectively characterized the\nvarious skeletal muscle types.\nPoint3. Fig2a. Check also Diaphragm, which is the most affected muscle in murine models of Muscle\nDystrophy. Chek also fat infiltration with Sirius red staining\nAnswer: We examined the diaphragm muscle and now present histological data (see also above). We\nshow homogeneous BAG3P209L-eGFP expression (new Fig. 1f) and the formation of eGFP-positive protein\naggregates (Fig. 3d). Histology revealed an increase in centralized nuclei (new Fig. 1k,l,p) in Soleus,\nQuadriceps, and EDL muscles, indicating muscle pathology. We have also performed Sirius red staining,\nas requested by the reviewer, on the diaphragm of CAG-BAG3P209L-mice (new Fig. 2a) and report a\nstatistically significant increase in fibrosis (new Fig. 2d). We have not observed fat infiltration with\neither Sirius red or H & E stainings in the diaphragm, EDL muscles, soleus muscle, or quadriceps muscle.\nPoint4 Fig3c-d are of poor quality. Show high resolution images\nAnswer: The images in Fig. 3c-d have been replaced with high-resolution images.\nPoint5. Fig3e-g. EM pictures suggest accumulation of vesicles/lysosomes. Show high magnification\npictures to underline the features. Authors should immunostain for Lamp1, p62 and LC3 and quantify\nthe puncta.\nAnswer: We have added SQSTM1 and LC3 staining of skeletal muscle from CAG-BAG3P209L and control\nmice and quantified, as requested by the reviewer, the puncta (new Fig. 3e,f). We observed a\nstatistically significant increase in SQSTM1 and LC3B positive puncta.\nPoint6. Autophagy and mitophagy flux must be monitored to support the authors claims.\nAnswer: We have addressed this interesting point with new experiments using primary skeletal\nmyocytes from CAG-BAG3P209L mice. We isolated satellite cells from 4-week-old mice and treated them\nwith Bafilomycin A1 (BAF A1) to inhibit autophagy and mitophagy. As a readout for autophagy, we\nused SQSTM1, and as a readout for mitophagy, PINK1 stainings. We demonstrate that autophagy and\nmitophagy are stalled (blocked) in skeletal myocytes from CAG-BAG3P209L-mice, as there was no\ndifference in total amount after BAF A1 treatment (new Fig. 7b,c). Additionally, there is no change in\nBAG3P209L-eGFP fluorescence intensity, which suggests no degradation of BAG3-protein aggregates\n(new Fig. 7d). As a positive control for autophagic and mitophagy flux, we overexpressed Beclin1 and\nBNIP3 in skeletal myocytes and observed a significant increase of SQSTM1 and PINK1, respectively (new\nFig.7b,c).\nPoint7, In vivo Bag3 knockdown results in phenotypic amelioration. Authors should also monitor\nwhether mitochondrial function and autophagy was restored.\nAnswer: This is addressed in our Western Blot analysis results and functional data: Both mitochondrial\nfunction and autophagy are restored, as we see a decrease in mitophagy, as indicated by PINK1\nexpression (new Suppl. Fig. 7a,m) and a decrease in blockage of autophagy, as measured by LC3B and\nSQSTM1 expression (Suppl. Fig. 7a,f,l). In addition, muscle force increased significantly (Fig. 8h,i), which\nis an indicator (although indirect) of improved mitochondrial function.\nPoint8. Author should put an effort in identifying which mechanisms downstream BAG3 dysfunction\nis the main trigger of the phenptype. Is autophagy reactivation sufficient to retore muscle function in\nBAG3P209L mutant? Or Is mitochondria improvement via mitophagy? Or just mitochondrial function\nthat when preserved also improves muscle function. These experiments could be easily done (e.g.\nBeclin1 expression or rapamycin treatment for autophagy activation, BNIP3 expression for\nmitophagy, PGC1a expression for mitochondrial biogenesis and function) and would confirm/identify\ninsights that connect BAG3 mutation to MFM,\nAnswer: We thank the reviewer for raising the criticism, whether autophagy or mitophagy primarily\nunderlie the pathological phenotype. Therefore, we have performed experiments with primary skeletal\nmyoblasts from the skeletal muscles of CAG-BAG3P209L mice, which we differentiated into skeletal\nmyocytes. As suggested by the reviewer, we transfected primary differentiated myocytes with\nexpression vectors for Beclin1, which activates autophagy, BNIP3, which induces mitophagy, and PGC-\n1α, which induces mitochondrial biogenesis. Interestingly, only the induction of autophagy had a\nsignificant impact on the phenotype, as there were fewer SQSTM1-positive and BAG3-positive protein\naggregates in the cells (new Fig. 7a,b,d). The cells also displayed improved cross-striation and were\nlarger. We measured autophagic flux in these cells by treating them with Bafilomycin A1. Again, an\nincrease in SQSTM1 was observed only in Beclin1- treated cells consistent with the induction of\nautophagic flux (new Fig. 7b). Mitophagy flux, measured based on PINK1 levels, was increased after\nBNIP3 overexpression (Fig. 7c), but did not reduce SQSTM1 or BAG3P209L-eGFP levels (new Fig. 7b,d). In\naddition, sarcomere morphology was assessed using the SarcAsM algorithm (Haertter et al., 2024 eLife)\nand revealed a statistically significant increased Z-band length in BECN1 transfected cells compared to\nCTRL and PGC-1α transfected cells (new Suppl. Fig. 11a).\nFurthermore, we have explored the downstream effects of BAG3P209L by inducing autophagy with\nrapamycin in CAG-BAG3P209L mice. This led to an amelioration of the phenotype, as shown by increased\ngrip strength, better motor coordination (rota-rod), and more voluntary movement (new Suppl. Fig.\n11e-g). Measuring LC3B-II content in the quadriceps muscle of these mice showed an increase in this\nautophagy marker (new Suppl. Fig. 11h).\nWe conclude from these experiments that impaired autophagy, not impaired mitophagy is responsible\nfor the skeletal muscle phenotype."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have made very significant modifications to the manuscript, including adding data on a new mice line that\nfurther supports that the original line is a good model for myofibrillar myopathy 6 and not due to overexpression of the tagged\ntransgene. They have made very constructive modifications in response to the different reviewers comments that have\nimproved clarity. This is an important contribution to the field.", "reviewer_3": "Whilst the authors have made a valiant attempt to address my concern that the phenotypes associated with transgenic\noverexpression of mutant BAG3P209L might simply be due to overexpression and not the mutation, their control transgenic\nline only expresses WT BAG3 protein to 39% of the level acheived with BAG3P209L so unfortunately I do not think they can\nrule out simple overexpression as a source of any or all phenotypes.", "reviewer_4": "The authos addressed my requests. The paper has been improved"}, "author_response": "Point-by-point response to Reviewer Comments\nReviewer #2 (Remarks to the Author):\nThe authors have made very significant modifications to the manuscript, including adding data on a\nnew mice line that further supports that the original line is a good model for myofibrillar myopathy 6\nand not due to overexpression of the tagged transgene. They have made very constructive\nmodifications in response to the different reviewers comments that have improved clarity. This is an\nimportant contribution to the field.\nAnswer:\nWe thank the reviewer for his/her positive comments and his/her appreciation of our work.\nResponse to Reviewer #3:\nWhilst the authors have made a valiant attempt to address my concern that the phenotypes\nassociated with transgenic overexpression of mutant BAG3P209L might simply be due to\noverexpression and not the mutation, their control transgenic line only expresses WT BAG3 protein\nto 39% of the level acheived with BAG3P209L so unfortunately I do not think they can rule out simple\noverexpression as a source of any or all phenotypes.\nAnswer:\nWe have a different view from the reviewer on this specific point for the following reasons:\n1. mRNA-expression of mBAG3 and hBAG3 in the CAG-BAG3WT-eGFP mice was similar to that in\nthe CAG-BAG3P209L-mice (total BAG3 mRNA induction 3.37±1.77 in homozygous BAG3P209L-\neGFP mice and 3.01±1.41 in homozygous BAG3WT-eGFP mice compared to CTRL (Suppl. Fig.\n3a)). As previously explained, the high level of hBAG3P209L-eGFP protein results from the\npathomechanism, not its cause, as evidenced by the similar levels of mRNA expression and the\ndefective autophagy with subsequent protein accumulation in skeletal muscles, as shown in\nFigs. 3a-e, 5b, 6g. As expected, we observed the formation of hBAG3P209L-eGFP protein\naggregates in the striated muscle of CAG-BAG3P209L-mice over time, whereas this was not seen\nin our control mouse line, where hBAG3WT-eGFP protein is overexpressed.\n2. Additional evidence regarding protein accumulation is shown when comparing the amount of\nmouse Bag3 protein in the two mouse lines: Although the mRNA level of mBag3 is higher in\nmice expressing hBAGWT-eGFP (mouse Bag3 mRNA 0.038±0.018 in homozygous CAG-\nBAG3P209L-mice and 0.054±0.018 in homozygous CAG-BAG3WT mice (Suppl. Fig. 3a)), the\nprotein level is only increased by a factor of 1.76, whereas in CAG-BAG3P209L mice it is increased\nby a factor of 5.2.\n3. Overexpression of the hBAG3P209L-eGFP fusion protein mimics MFM6 in the hearts of our\nmouse model, specifically restrictive cardiomyopathy (Kimura et al., 2021, Nat Commun.,\n12:3575.), which is extremely rare in transgenic mouse models (Liu et al., 2016, Front Physiol.,\n7:629.) as well as in skeletal muscle exhibiting the pathognomonic phenotype.\nThus, all these findings collectively show, as also acknowledged by Reviewer 2, that the phenotype and\ncell biological results are caused by BAG3 dysfunction and not merely by overexpression of the protein."}]

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