Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Dec 19;12(24):e029938.
doi: 10.1161/JAHA.123.029938. Epub 2023 Dec 18.

Dysregulated Autophagy and Sarcomere Dysfunction in Patients With Heart Failure With Co-Occurrence of P63A and P380S BAG3 Variants

Thomas G Martin  1 , Hana Pak  1 , Glenn S Gerhard  2 , Salim Merali  3 , Carmen Merali  3 , Bonnie Lemster  4 , Praveen Dubey  5 , Charles F McTiernan  4 , Michael R Bristow  6 , Arthur M Feldman  7 , Jonathan A Kirk  1
Affiliations

Dysregulated Autophagy and Sarcomere Dysfunction in Patients With Heart Failure With Co-Occurrence of P63A and P380S BAG3 Variants

Thomas G Martin et al. J Am Heart Assoc. .

Abstract

Background: Mutations to the co-chaperone protein BAG3 (B-cell lymphoma-2-associated athanogene-3) are a leading cause of dilated cardiomyopathy (DCM). These mutations often impact the C-terminal BAG domain (residues 420-499), which regulates heat shock protein 70-dependent protein turnover via autophagy. While mutations in other regions are less common, previous studies in patients with DCM found that co-occurrence of 2 BAG3 variants (P63A, P380S) led to worse prognosis. However, the underlying mechanism for dysfunction is not fully understood.

Methods and results: In this study, we used proteomics, Western blots, and myofilament functional assays on left ventricular tissue from patients with nonfailing, DCM, and DCM with BAG363/380 to determine how these mutations impact protein quality control and cardiomyocyte contractile function. We found dysregulated autophagy and increased protein ubiquitination in patients with BAG363/380 compared with nonfailing and DCM, suggesting impaired protein turnover. Expression and myofilament localization of BAG3-binding proteins were also uniquely altered in the BAG3,63/380 including abolished localization of the small heat shock protein CRYAB (alpha-crystallin B chain) to the sarcomere. To determine whether these variants impacted sarcomere function, we used cardiomyocyte force-calcium assays and found reduced maximal calcium-activated force in DCM and BAG363/380. Interestingly, myofilament calcium sensitivity was increased in DCM but not with BAG363/380, which was not explained by differences in troponin I phosphorylation.

Conclusions: Together, our data support that the disease-enhancing mechanism for BAG3 variants outside of the BAG domain is through disrupted protein turnover leading to compromised sarcomere function. These findings suggest a shared mechanism of disease among pathogenic BAG3 variants, regardless of location.

Keywords: BAG3; autophagy; dilated cardiomyopathy; sarcomere.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Unbiased quantitative proteomics assessment suggests altered autophagy with BAG3 63/380 variants.
A, BAG3 protein sequence with domains and known BAG3‐binding partners; the BAG3 P63A mutation lies just outside of the WW domain, and the P380S mutation is located within the PXXP domain. B, Volcano plot of all proteins identified by bottom‐up mass spectrometry and analyzed by label‐free quantification; proteins with a +/− 0.3 log2 fold change in expression relative to nonfailing controls and a P value <0.05 were considered as having significantly different expression; upregulated proteins compared with nonfailing=red, downregulated proteins compared with nonfailing=blue; n=3 nonfailing, 2 BAG3 63/380; samples were not pooled for this analysis. C, DAVID GO analysis of the top 5 groups of differentially expressed proteins organized by biological process. AMOTL1 indicates angiomotin like 1; BCL‐2, B‐cell lymphoma 2; GO, gene ontology; HSC70, heat shock cognate protein 70; HSF‐1, heat shock transcription factor 1; HSPB/70, heat shock protein B/70; LATS1/2, large tumor suppressor kinase 1/2; LPS, lipopolysaccharide; PLCγ, phospholipase C γ; PXXP, proline‐rich; TSC1, tuberous sclerosis 1; and WW, tryptophan–tryptophan.
Figure 2
Figure 2. Dysregulated autophagy in left ventricle of patients with BAG3 63/380.
A, Western blots for LC3, P62, and ubiquitin in the triton‐soluble protein fraction of left ventricles from patients with NF, DCM, and BAG3 63/380 mutation, with corresponding Revert total protein stain. B through E, Quantitative densitometry analysis of the LC3‐I (B), LC3‐II (C), P62 (D), and ubiquitin (E) signals normalized to total protein; n=4 NF, 5 DCM, 2 BAG3 63/380; data are presented as the mean±standard error and were analyzed by 1‐way permutational ANOVA. DCM indicates dilated cardiomyopathy; NF, nonfailing; and LC3, microtubule‐associated proteins 1A/1B light chain 3B.
Figure 3
Figure 3. Assessment of BAG3‐binding partners in the soluble protein fraction.
A, Western blot for BAG3 and HSPB8 in the triton‐soluble protein fraction with corresponding Revert total protein stain. B, Western blot for SYNPO2, HSP70, and CRYAB in the triton‐soluble protein fraction with corresponding Revert total protein stain. C through G, Quantitative densitometry analysis of BAG3 (C), HSPB8 (D), SYNPO2 (E), HSP70 (F), and CRYAB (G) signals normalized to total protein signal. For all, n=4 NF, 5 DCM, 2 BAG3 63/380; data are presented as the mean±standard error and were analyzed by 1‐way permutational ANOVA. CRYAB indicates crystallin alpha B; DCM, dilated cardiomyopathy; HSP70/B8, heat shock protein 70/β‐8; NF, nonfailing; and SYNPO2, synaptopodin 2.
Figure 4
Figure 4. Assessment of BAG3‐binding partners in the myofilament protein fraction.
A, Western blot for BAG3 and HSPB8 in the myofilament protein fraction with corresponding Revert total protein stain. B, Western blot for SYNPO2, HSP70, and CRYAB in the myofilament protein fraction with corresponding Revert total protein stain. C through G, Quantitative densitometry analysis of BAG3 (C), HSPB8 (D), SYNPO2 (E), HSP70 (F), and CRYAB (G) signals normalized to total protein signal. For all, n=4 NF, 5 DCM, 2 BAG3 63/380; data are presented as the mean±standard error. CRYAB indicates crystallin alpha B; DCM, dilated cardiomyopathy; HSP70/B8, heat shock protein 70/β‐8; NF, nonfailing; and SYNPO2, synaptopodin 2.
Figure 5
Figure 5. Cardiomyocytes from BAG3 63/380 patients exhibit reduced sarcomere function and increased myofilament protein ubiquitination.
A. Skinned cardiomyocyte force‐calcium curves for the NF, DCM, and BAG3 63/380 samples. B and C, Summary data for cardiomyocyte maximal calcium‐activated force (Fmax) (B) and EC50 (calcium concentration required to elicit half‐maximal force) (C) from the 3 groups; n=9 NF cardiomyocytes from 3 samples (1 sample not used due to lack of remaining tissue), 15 DCM from 5 samples, 6 BAG3 63/380 from 2 samples. D, Western blot for phosphorylated (S23/S24) cardiac troponin I (cTnI), total TnI, and sarcomeric α‐Actin. E, Phosphorylated cTnI signal normalized to total cTnI signal. F, Slow skeletal TnI (ssTnI) signal normalized to α‐Actin. G. Western blot for ubiquitin in the myofilament protein fraction with corresponding total protein stain. H, Ubiquitin signal normalized to total protein. All data are presented as the mean±standard error and were analyzed by 1‐way ANOVA. (p‐)cTnI indicates (phosphorylated) cardiac troponin I; DCM, dilated cardiomyopathy; NF, nonfailing; and ssTnI, slow skeletal troponin I.
Figure 6
Figure 6. Secondary structure prediction suggests increased β‐sheet propensity and reduced disorder with P63A variants.
A, PepFold predicted secondary peptide structure with the P63A variant compared with the wild‐type BAG3 peptide sequence. B, Pepfold predicted secondary peptide structure with the P380S variant compared with the wild‐type BAG3 peptide sequence.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

  • Cardiac fibroblasts: answering the call.
    Kleinbongard P, Senyo SE, Lindsey ML, Garvin AM, Simpson JA, de Castro Braz LE. Kleinbongard P, et al. Am J Physiol Heart Circ Physiol. 2024 Sep 1;327(3):H681-H686. doi: 10.1152/ajpheart.00478.2024. Epub 2024 Aug 2. Am J Physiol Heart Circ Physiol. 2024. PMID: 39093000 Free PMC article. Review.

References

    1. Kirk JA, Cheung JY, Feldman AM. Therapeutic targeting of BAG3: considering its complexity in cancer and heart disease. J Clin Invest. 2021;131:e149415. doi: 10.1172/JCI149415 - DOI - PMC - PubMed
    1. Martin TG, Myers VD, Dubey P, Dubey S, Perez E, Moravec CS, Willis MS, Feldman AM, Kirk JA. Cardiomyocyte contractile impairment in heart failure results from reduced BAG3‐mediated sarcomeric protein turnover. Nat Commun. 2021;12:2942. doi: 10.1038/s41467-021-23272-z - DOI - PMC - PubMed
    1. Hishiya A, Kitazawa T, Takayama S. BAG3 And Hsc70 interact with Actin capping protein CapZ to maintain myofibrillar integrity under mechanical stress. Circ Res. 2010;107:1220–1231. doi: 10.1161/CIRCRESAHA.110.225649 - DOI - PMC - PubMed
    1. Ulbricht A, Gehlert S, Leciejewski B, Schiffer T, Bloch W, Höhfeld J. Induction and adaptation of chaperone‐assisted selective autophagy CASA in response to resistance exercise in human skeletal muscle. Autophagy. 2015;11:538–546. doi: 10.1080/15548627.2015.1017186 - DOI - PMC - PubMed
    1. Arndt V, Dick N, Tawo R, Dreiseidler M, Wenzel D, Hesse M, Fürst DO, Saftig P, Saint R, Fleischmann BK, et al. Chaperone‐assisted selective autophagy is essential for muscle maintenance. Curr Biol. 2010;20:143–148. doi: 10.1016/j.cub.2009.11.022 - DOI - PubMed

Publication types

MeSH terms