Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: Exerc Sport Sci Rev. 2021 Oct 1;49(4):267–273. doi: 10.1249/JES.0000000000000265

Skeletal Muscle Function Is Dependent Upon BRCA1 to Maintain Genomic Stability

Michael D Tarpey 1, Adam J Amorese 1, Elizabeth R LaFave 2, Everett C Minchew 1, Kelsey H Fisher-Wellman 1,3, Joseph M McClung 1,3, Eli G Hvastkovs 2, Espen E Spangenburg 1,3
PMCID: PMC8495729  NIHMSID: NIHMS1738980  PMID: 34091499

Abstract

Breast Cancer gene 1 (BRCA1) is a large, multi-functional protein that regulates a variety of mechanisms in multiple different tissues. Our work established that Brca1 is expressed in skeletal muscle and localizes to the mitochondria and nucleus. Here, we propose BRCA1 expression is critical for the maintenance of force production and mitochondrial respiration in skeletal muscle.

Keywords: muscle, DNA, mitochondria, nucleus, force, damage

Summary for Table of Contents:

Loss of Breast Cancer gene 1 (BRCA1) function leads to reductions in skeletal muscle specific force, altered mitochondrial energetics and accumulation of mitochondria DNA damage.

Introduction: What is BRCA1?

Breast Cancer 1, early onset gene (BRCA1) encodes for a tumor suppressor protein known as Breast Cancer Type Susceptibility Protein (Brca1). Efforts to identify the BRCA1 gene are largely credited to Dr. Mary Claire King, who used linkage analysis across numerous families with a strong family history of breast cancer to narrow the location of the gene to chromosome 17 (1). Ultimately the cloning of the BRCA1 gene occurred in 1994 by Dr. Mark Skolnick’s lab and independently by Myriad Genetics (2). Due to the ramifications of identifying the BRCA1 gene, the subsequent scientific investigations into the mechanisms regulated by Brca1 were and remain intense. However, to date much of our understandings concerning BRCA1 remain in their infancy as new discoveries are occurring frequently. Brca1 is most commonly described as a DNA repair protein, however a number of other roles and/or mechanisms have been prescribed to the protein. Most of the roles Brca1 plays appear to involve a form of genome surveillance with the primary goal of maintaining genetic health. In this review, we propose the hypothesis, that BRCA1 acts as a DNA repair protein in both the nucleus and mitochondria of skeletal muscle and the expression of BRCA1 is critical to maintenance of muscle function (Fig 1).

Figure 1.

Figure 1.

Open in a new tab

Hypothetical role of BRCA1 in skeletal muscle as a regulator of genomic health in the nucleus and mitochondria of skeletal muscle. Using recently published data, we propose that BRCA1 expression is necessary for the maintenance of skeletal muscle quality by ensuring activity of critical DNA repair mechanisms in the nucleus and mitochondria.

BRCA1 gene is susceptible to mutations.

BRCA1 is susceptible to mutations that can affect protein function, stability, and or folding with each mutation potentially altering cell function. The BRCA1 gene contains 22 exons with 110kb of DNA (Fig 2). Current estimates suggest there are over 1,700 described mutations, with nearly half being frameshift mutations and 323 being possible mis-sense mutations both of which are predicted to alter protein function (3). Approximately 1 in 400 individuals or 0.25% of the population carries a mutation in the BRCA1 gene. Data suggest that 12% of women will be diagnosed with breast cancer by the age 70, while 55–65% individuals with a BRCA1 mutation will be diagnosed with breast cancer by the age 70 (4). This increased risk of breast cancer initially drove much of the work on BRCA1 regulated mechanisms, however with 50% of the BRCA1 mutations inherited from fathers who are not affected by breast cancer it is unclear if the mutations affect other tissue functions (5), thus revealing a critical need to understand the impact of loss of BRCA1 function across all relevant tissues.

Figure 2.

Figure 2.

Open in a new tab

Graphical representation of the BRCA1 gene and protein. The gene contains 24 exons with exon 11 being over 3000 bp in length and the gene entire being approximately 110 kb in length. The BRCA1 protein is approximately 220 Kd in size with two critical regions; a RING domain in the N-terminus and tandem BRCT repeats in the C-terminus. The RING domain provides BRCA1 E3-ubquitin ligase activity, while the BRCT, a highly disordered region of the protein, allows protein-protein interaction through specific binding with phosphorylated proteins. The BRCT domain likely has a significant influence on cellular localization of BRCA1 based on the protein ‘piggybacking’ with a known binding partner. BRCA1 also contains a nuclear localization sequence (NLS) and nuclear exclusion sequence (NES), with the latter appearing critical to nuclear localization.

BRCA1 is a promiscuous protein with a number of splice variants making mechanisms hard to define.

Brca1 is a 220 Kd protein that contains 3 distinct regions; a RING domain in the N terminus, nuclear localization signal (NLS), and a BRCT domain in the C terminus (Fig 2). All three of these domains have a higher frequency of mutation compared to the rest of the gene, with many of the mutations associated with an increased risk of breast cancer. The RING domain provides BRCA1 E3-ubiquitin ligase activity (6), although the role of the RING domain remains unclear because E3-ligase activity is not necessary for DNA repair, as a recent study elegantly discovered (7). In contrast, the same study found that the BRCA1 C-terminal domain (BRCT) is necessary for the DNA damage signaling response of the protein. BRCT domains are found in numerous proteins with most of these proteins having a described role in DNA repair and/or metabolism (8). BRCA1 has tandem BRCT domains that allow for protein-protein interaction specifically with phosphorylated proteins. BRCA1 recognizes the phosphorylated peptide motif consisting of four residues (pS/pT-X-X-X) (Fig 2) (8). The ability to interact with phosphorylated proteins has resulted in a constantly growing list of possible binding partners, expanding the regulatory role of BRCA1 to mechanisms beyond just DNA repair. For example, some of the identified binding partners for BRCA1 include XRCC1, p53 and PLK-1, all of which are thought to contribute to some aspect of skeletal muscle function (9–11). Known mutations in the tandem repeat BRCT region of BRCA1 is linked to both breast cancer and ovarian cancer. Interestingly, introduction of a mutation in the linker region between the two BRCT repeats also increases the cancer risk indicating that entire region and not just the BRCT sequence is critical for normal BRCA1 function (8). Current studies are underway using proteomic based approaches to identify potential BRCA1 binding partners in skeletal muscle.

Understanding of mechanisms regulated by BRCA1 is further complicated by alternative splicing that can occur during transcription or through post-transcriptional modifications of the transcript. Through alternative splicing, BRCA1 gene can produce alternative transcripts resulting in unique BRCA1 protein products that can mimic full-length BRCA1, induce unique mechanisms not activated by full-length BRCA1 or even inhibit mechanisms induced by full-length BRCA1 (12). Currently, over 100 alternative splicing variants have been identified, however only handful of the splice variants are known to be expressed at the protein level with some form of function in either fetal and/or adult tissues (12). At this time, we have documented that human skeletal muscle expresses the full-length isoform of BRCA1 and potentially the BRCA1b variant (12). We confirmed the same results in mouse muscle (unpublished data). It is unclear what role the variant protein products play in skeletal muscle and will require further investigation.

BRCA1 expression is transcriptionally regulated, and phosphorylation events affect function.

BRCA1 expression is regulated by transcriptional mediated mechanisms in non-muscle tissue, more specifically regulation at the level of the promoter can enhance or repress BRCA1 expression (13). At this time, the mechanisms that regulate BRCA1 expression in skeletal muscle are unknown. BRCA1 function is largely mediated by binding partners to the RING or BRCT domain, however phosphorylation of the BRCA1 protein can affect a number of cellular mechanisms. For example, phosphorylation of BRCA1 at residue Thr509 by Akt may influence cellular localization as the residue is found within the nuclear localization sequence (14). Phosphorylation of various serine residues also can influence various steps of the cell cycle particularly the G2 to M transition and also potentially affect BRCA1’s ability to complex with other binding partners (14). Finally, phosphorylation of BRCA1 has also been implicated to regulate the ability of BRCA to accumulate at sites of DNA damage (14). At this point, there a number of known post-translational modifications that can occur to BRCA1, but the effect on BRCA1 function is still largely undefined and remains unknown if any of these modifications occur in skeletal muscle.

BRCA1 function is necessary for tissues beyond just mammary tissue.

BRCA1 is commonly discussed as a breast cancer gene, which has led to substantial under estimation of the role BRCA1 plays in other cell types. Global genetic deletion of BRCA1 in the mouse leads to pre-natal lethality indicating that BRCA1 function is critical for development (15). BRCA1 is ubiquitously expressed, however the mRNA and protein content differ across a variety of tissues and the content does not appear to predict the importance of BRCA1 to tissue function. For example, BRCA1 mRNA is expressed at significantly lower levels in the mammary gland compared to thymus or testes (16,17), yet BRCA1 has clear documented roles in mammary gland development and is necessary for pregnancy-induced alterations in the mammary gland (17). Recent investigations have also provided clear evidence that BRCA1 expression is necessary for appropriate brain development (18). Furthermore, conditional deletion of the BRCA1 gene in heart tissue reduces lipid utilization and impairs survival in response to acute ischemic insults (19). In addition, deletion of BRCA1 within the vasculature increases the stress susceptibility in the vascular tissue pointing to important roles played by BRCA1 in the cardiovascular system (20). Thus, the necessity of BRCA1 in the brain and vasculature is clear even though early studies examining tissue expression of BRCA1 suggested the mRNA was expressed at very low abundance. Much of our recently gained knowledge concerning the importance of BRCA1 in individual tissues has instead relied on genetic deletion-based approaches. Collectively, the data suggest that reliance on mRNA expression or protein content to predict the importance of a gene to the function of a tissue may overlook evidence critical to understanding tissue function.

BRCA1 is expressed at low levels in human and rodent skeletal muscle.

Brca1 was first identified in skeletal muscle at the protein level in differentiating C2C12 muscle cells (21). Since then a number of papers have confirmed BRCA1 expression in muscle, with a few suggestions that BRCA1 may not be expressed in skeletal muscle (22). The challenge in these equivocal results largely lay in the methodology used to assess BRCA1 content. When measuring BRCA1 mRNA with real time RT-PCR the ability to detect expression is fairly straightforward, however measuring protein content has proven to be more challenging in part due to antibodies that lack specificity. In human tissue, commercially available antibodies work with limited precision under very controlled conditions, while antibodies specific to murine BRCA1 have proven to be even less reliable with commonly used ones recently discontinued. To date it appears possible to assess BRCA1 using immunoblotting under very controlled conditions with appropriate positive/negative controls. Conversely, it is unclear if immunohistochemical procedures are possible due to the quality of available antibodies, with recent groups questioning the use of BRCA1 antibodies in immunohistochemistry (23). Due to the number of approaches that utilize antibodies for assessment of endogenous protein function, the lack of consistently reliable BRCA1 specific antibodies has hampered progress in the field. Undocumented, but often known is that developing antibodies specific to the murine form of BRCA1 have proven to be challenging for reasons that are elusive to the field. It is critical this limitation is overcome to continue progress in understanding BRCA1 function across all tissues.

BRCA1 localizes to multiple cellular compartments in the muscle cell.

As previously stated, assessing BRCA1 localization is difficult due to very poor functioning antibodies. To overcome this limitation, BRCA1 cDNA constructs were developed that were tagged with various cellular markers (e.g. FLAG) or fluorescent proteins (e.g. YFP) allowing investigators to assess localization in their tissue of interest. In non-muscles, BRCA1 has been found to localize to the nucleus, mitochondria and cytoplasm (24,25). The localization of BRCA1 to the nucleus is regulated by the nuclear localization signal (NLS) sequence with the nuclear exclusion sequence playing a critical role in the maintenance of BRCA1 in the nucleus. However, localization to the mitochondria is somewhat surprising because BRCA1 lacks a consensus mitochondrial localization sequence (MLS). It has been suggested that the BRCT domain likely allows BRCA1 to ‘piggy-back’ on other proteins that do contain a consensus MLS (25). In our hands, we have confirmed that in skeletal muscle BRCA1 localizes in the nucleus, mitochondria, and cytoplasm (26), when coupled with already published physiological and biochemical data it is very likely that BRCA1 plays a critical role in mitochondrial bioenergetics (27). Studies are ongoing to identify signals that regulate localization of BRCA1 within skeletal muscle.

BRCA1 is necessary for skeletal muscle function.

Since we have determined that BRCA1 is expressed in skeletal muscle and localizes to different areas of the cell, we sought to assess the importance of BRCA1 to the skeletal muscle cell. As it was unclear if genetic deletion of BRCA1 using a conditional approach would alter the development of the muscle, we employed an inducible deletion approach. In these experiments, the HSA-mER-Cre-mER mouse developed at the Univ of Kentucky (28) was crossed with BRCA1 fl/fl (29) resulting in a BRCA1fl/flHSA-mER-Cre-mER. The BRCA1 fl/fl has flox sites flanking exon 11, thus activation of Cre by delivery of tamoxifen to the BRCA1fl/flHSA-mER-Cre-mER results in excision of exon 11, the largest exon in the Brca1 gene, and loss of the full-length Brca1 protein in skeletal muscle. Exon 11 in the BRCA1 gene encodes for approximately 60% of the total protein and includes the nuclear localization signal. We found that we could detect recombination of the BRCA1 gene within days of the tamoxifen injection, however loss of the protein did not occur until weeks post tamoxifen delivery. In all of the data described, the animals were injected with tamoxifen when the animal reached 10–12 weeks of age and animals were aged for a further 8–20 weeks depending upon the study. Mice lacking Brca1 protein in their skeletal muscle are referred to as BRCAKO1smi (smi = skeletal muscle inducible) and the corresponding wild type animals were vehicle injected BRCA1fl/flHSA-mER-Cre-mER (WT), however control studies were run on tamoxifen injected HSA-mER-Cre-mER mice with no effects of the short-term tamoxifen delivery detected on any of our measured variables.

The loss of the Brca1 protein induced a number of measured phenotypic changes in the skeletal muscle of the adult animal. Specifically, we found a variable degree of kyphosis that developed in the BRCAKO1smi compared to the WT animals (30). This was accompanied by a loss in specific force production when assessed using ex vivo and in situ (stimulated via nerve) approaches suggesting that loss in muscle quality (defined as muscle force normalized to muscle mass) is not mediated at the level of the nerve, but instead within the muscle cell itself. We found that BRCAKO1ism exhibited slight, but significantly higher average individual fiber cross sectional area (CSA) as well as, surprisingly, lower IIb fiber percentage compared to the WT mice in the TA muscle. Collectively, the data suggest that induced deletion of BRCA1 specifically in the skeletal muscle leads to a reduction in muscle quality or more specifically reductions in force production that were not mediated by a loss in muscle mass. Collectively, the data suggest a mechanism within the muscle cell likely contributes to the lower muscle quality in the BRCAKO1smi. A logical target to consider is the sarcoplasmic reticulum, however, when the contractile kinetics (i.e. rates of contraction or rates of relaxation) were assessed no differences were detected between groups.

Loss of BRCA1 leads to altered mitochondrial respiration and morphology.

One consistent observation made was increased lactate production with repetitive stimulation in the BRCAKO1ism compared to the WT groups, which potentially suggested that deletion of BRCA1 results in an inability to meet an energetic demand via the endogenous mitochondria. Previous work had identified that deletion of BRCA1 alters various aspects of mitochondrial respiration across a number of different non-muscle cell types (31). In skeletal muscle, we found the loss of BRCA1 results in a reduced rate of maximal respiration in cultured human myotubes compared to myotubes that were treated with scrambled shRNA. Mitochondria isolated from BRCA1KOsmi exhibited reduced maximal ADP-respiration compared to mitochondria isolated from age-matched WT animals (26). Interestingly, we found that mitochondrial yield was reduced during isolation procedures in the BRCAKO1smi compared to the WT groups. Thus, it was possible that the isolation procedure was selectively isolating a specific population of mitochondria from the BRCAKO1smi group. To assess this concept, mitochondrial respiration was assessed using isolated fiber bundles from BRCA1KOsmi compared to the WT group. The advantage of using isolated fiber bundles is that it ensures that the native confirmation of the mitochondria remains intact during respiratory assessment. We found that the fiber bundles isolated from the BRCAKO1smi group exhibited reduced ADP-stimulated respiration kinetics across Complex I, II, III, and IV compared to the WT group (Fig 3) (30). Further, we found no difference in the protein content of the mitochondrial complexes in skeletal muscle from the WT and BRCAKO1smi group (30). Thus, regardless of approach employed, mitochondrial O2 consumption is reduced in skeletal muscle from BRCAKO1smi mice compared to the WT mice. Further, analysis using EM imaging found that various populations of mitochondria in the BRCAKO1smi exhibited significant hypertrophy of the mitochondria compared to the WT mice (Fig 4). More simply the BRCAKO1smi mice exhibited size heterogeneity in the mitochondria compared to the WT, which was associated with an increased susceptibility to Ca2+-induced swelling (30).

Figure 3.

Figure 3.

Open in a new tab

Mitochondrial respiration measures in fiber bundles isolated from red gastrocnemius muscles taken from age-matched WT and BRCAKO1smi littermates. (A) Representative tracing of high‐resolution respirometry measurement with skeletal muscle fiber bundles. Substrates and inhibitors were added sequentially to measure complex I leak and ADP‐stimulated complexes I, I+II, II and IV mitochondrial respiration. Data were corrected to muscle fiber dry weight. Complex I leak (state 3) respiration, complex I+II (state 3) respiration (C), complex II (state 3) respiration (D) and complex IV (state 3) respiration (E) were significantly lower in Brca1KOsmi fiber bundles compared to fiber bundles from the WT and Het mice. Black bars refer to WT mice, light grey bars refer to Het and dark grey bars refer to Brca1KOsmi mice. *Statistically different from Het (P < 0.05), #Statistically different from WT (P < 0.05). Data are the mean ± SEM. (Reprinted from (30). Copyright © 2018 John Wiley and Sons. Used with permission.)

Figure 4.

Figure 4.

Open in a new tab

Deletion of BRCA1 in the skeletal muscle of adult mice leads to the accumulation of abnormally large mitochondria compared to the WT mice. Transmission electron microscopy (TEM) images of soleus muscles taken from age-matched WT and BRCAKO1smi littermates. Scale Bar = 500nm.

Reductions in force production BRCAKO1smi are associated with alterations in mitochondrial respiration kinetics.

Our data demonstrate an association of muscle weakness with reductions in ADP-stimulated mitochondrial respiration, increased mitochondrial swelling, and abnormal mitochondrial morphology in the BRCAKO1smi compared to age-matched WT littermates. However, these findings do not demonstrate a clear mechanistic linkage between altered mitochondrial energetics and muscle weakness when BRCA1 function is lost. We and others have speculated that lost BRCA1 function compromises the ability of the mitochondria to meet the increased energetic demand of a cell leading to altered physiological function (27, 30, 31). Specifically, free energy of ATP hydrolysis (ΔGATP) is a non- linear function of the ratio of [ATP]/[ADP] (i.e., energy charge) that must be maintained to support critical cellular reactions. For example, a 10 kJ/mol drop in ΔGATP induces a near complete loss of contractile function during ischemia (32). A less extreme example is the sacro/endoplasmic reticulum Ca2+-ATPase pump (SERCA) which is particularly sensitive to small changes in ΔGATP as shown both through modeling and experimental approaches (33,34) SERCA is a key regulator of skeletal muscle relaxation rates, which are slowed by 10–20% depending upon the contraction protocol employed in the BRCAKO1smi compared to the WT mice (unpublished data, Spangenburg et al). Thus, collectively our data suggest a potential reduction in ΔGATP due to altered mitochondrial energetics in the BRCAKO1smi compared to the WT mice, which appears to manifest itself in measurable alterations in skeletal muscle contractile kinetics. Thus, it is critical to pursue more in-depth experiments that would directly link altered contractile kinetics to changes in mitochondrial bioenergetic regulation when BRCA1 function is lost.

How does deletion of BRCA1 in skeletal muscle lead to altered mitochondrial respiration dynamics?

As previously stated BRCA1 is largely known as a DNA repair protein and the role BRCA1 plays in the mitochondria remains largely unknown. Interestingly, Coene et al. identified that the intramitochondrial distribution of BRCA1 was predominantly localized to the mitochondrial matrix in rat hepatic cells, implying a relationship between Brca1 and DNA repair because mtDNA is housed within the mitochondrial matrix (36). To test this hypothesis, we utilized a random mutation capture assay where we identified an accumulation of mutations in the mitochondrial DNA isolated from the BRCAKO1smi compared to the WT groups (30). Using two additional analytical approaches to assess DNA structural changes in order to seek chemically-based answers, we have since confirmed that mitochondrial DNA from BRCAKO1smi accumulates mutations compared to mitochondrial DNA from the WT groups (Eli Hvastkovs, data unpublished). Our data specifically demonstrate that BRCAKO1smi mtDNA exhibits losses in guanine content, consistent with mutations stemming from initial damage. To date, our data suggest that induced deletion of BRCA1 in skeletal muscle leads to visible heterogeneity of mitochondrial size and reduced respiratory kinetics both of which are associated with an accumulation of mtDNA mutations. It should be noted that we have also identified the accumulation of damaged DNA, specifically the increased presence of oxidatively damaged guanine, in the nucleus of skeletal muscle from BRCAKO1ism compared to the WT groups (Eli Hvastkovs, data unpublished). Thus, the data collectively indicate a strong association between the accumulation of DNA damage and mutations both in the mitochondria and nucleus which appear to contribute to altered mitochondrial respiration and reduced muscle quality. Critical next steps will be to perform experiments that prevent or attenuate the DNA damage in the face of lost BRCA1 function to determine if the loss of function remains or not.

Does post-mitotic tissue need the ability to repair DNA?

The average mutation rate per nucleotide site is approximately 2.5 × 10−8, which is approximately 175 new mutations per generation (37). Estimates suggest over 70,000 DNA lesions develop per cell/day due to oxidative damage from metabolic activity (38). Skeletal muscle requires mitochondrial ATP production to meet energetic demand of the muscle both at rest and during repetitive contraction. Mitochondria require oxygen (O2) to act as a terminal electron acceptor in the electron transport chain (ETC) to drive ATP generation. In all cells, including skeletal muscle, mitochondria are not perfectly coupled (i.e. O2 consumed per ATP formed) resulting in a perpetual electron leak from the ETC that leads to reactive oxygen species (ROS) formation inducing potential oxidative damage to the nuclear and mitochondrial DNA (39). Like other cells, muscle cells require a DNA repair program to reverse harmful mutations or prevent genomic instability, unfortunately the identification of DNA repair mechanisms in skeletal muscle remains elusive. Most of our understanding about DNA damage/repair in muscle was elucidated using exogenous non-physiological genotoxic agents, thus our understanding of DNA repair under physiological conditions is not elucidated yet making it unclear if DNA repair in skeletal muscle utilizes similar mechanisms as other cells. We believe we have identified that Brca1 is a key regulator of endogenous DNA repair in skeletal muscle. BRCA1 has been implicated in numerous forms of DNA repair including homologous recombination (HR), non-homologous end-joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER), etc…(for detailed review see (35)).

In non-muscle cells Brca1 is a regulator of multiple types of DNA repair and may play a similar role in skeletal muscle.

Brca1 was implicated as a key component in the DNA repair process when cells with a genetic deletion of BRCA1 exhibited a hyper-sensitivity to DNA damaging insults (40). Brca1 is also a key component of a protein complex that localizes to sites of damaged DNA (40). Brca1 contains two unique regions, a RING domain in the N-terminus and a tandem BRCT repeat domain in the C-terminus. It appears that the BRCT domain, a highly disordered region that encourages binding of phosphorylated proteins, is necessary for the DNA repair properties of BRCA1 (41). In non-muscle cells, Brca1 contributes non-homologous end-joining (NHEJ) which appears to be one mechanism for DNA repair in skeletal muscle (42). In addition, nucleotide excision repair (NER) and/or base excision repair (BER) both of which are affected by Brca1 activity, appear to occur in the mitochondria of skeletal muscle (35,43). Current dogma indicates that Brca1 regulates DNA repair through translocation to the site of damage as a part of a larger complex of proteins, however the members of the complex differ based on the tissue of interest (35). At this time, no one has identified what proteins interact with Brca1 in skeletal muscle. However, known Brca1 modulators and binding partners are present in skeletal muscle. For example, in response to DNA damage Brca1 is phosphorylated (Ser1164) by Polo-like Kinase 1 (PLK1) and Jia et al. elegantly demonstrated that PLK1 is critical for myogenesis (9). Also, brain and reproductive organ-expressed protein (BRE) responds to DNA damage, facilitates satellite cell differentiation and is a known Brca1 binding partner (44) as is XRCC1 which also appears necessary for myogenesis (11). When considered collectively, the data would suggest that BRCA1 could regulate DNA repair in skeletal muscle and the loss of BRCA1 function would be detrimental to the long-term health of the muscle.

Do these findings translate to BRCA1 mutations in patients?

A logical extension of our work and that of others is to characterize the phenotype of skeletal muscle in individuals with known mutations in BRCA1. The question is challenging to address with defined precision for a number of reasons. Specifically, many of the known mutations that associate with risk of cancer are based on genetic ancestry work or on large population studies, thus the studies are correlative and often lack cause/effect understanding. More importantly, the effect of a given single mutation on BRCA1 function is not always known, as with over 1700 known mutations it is unclear how each unique mutation effects BRCA1 function, in other words it is unlikely that they all result in a complete loss of function. In addition, current estimates indicate the 0.25% of the population has one defined mutation in the BRCA1 gene, thus identifying a cohort of individuals with the same mutation has proven to be challenging. The data collected within our lab is specific to cases of loss of function, thus our focus in future experiments will be on documented mutations that lead to loss of BRCA1 function to determine if patients with these mutations exhibit altered skeletal muscle function.

Conclusions: Future directions for BRCA1 in skeletal muscle biology.

Key next steps include identifying if BRCA1 can play a protective role allowing skeletal muscle to recover from acute or chronic insults. Critical to this concept, will be determining if an inability to repair damaged DNA is directly responsible for the development of skeletal muscle dysfunction. This is a challenging distinction to separate and will require the identification of the mechanism by which BRCA1 is contributing to skeletal muscle function. We are currently assessing the necessity of specific regions within the Brca1 protein to determine which are critical to function within skeletal muscle. We expect these data will help elucidate which known BRCA1 mutations should be focused on in human patients. Overall, our work has demonstrated that BRCA1 should be considered a critical regulator of skeletal muscle function.

Key Points:

  • Breast Cancer gene 1 (BRCA1) is a novel regulator of skeletal muscle.

  • Loss of BRCA1 leads to reduced muscle quality and lower mitochondrial respiratory kinetics.

  • Loss of BRCA1 leads to the accumulation of DNA damage in the nucleus and mitochondria.

Acknowledgements

The authors have no conflicts to declare.

References

  • 1.Hall JM, Lee MK, Newman B, Morrow JE, Anderson LA, Huey B, et al. Linkage of early-onset familial breast cancer to chromosome 17q21. Science. 1990; 250(4988) :1684–9. [DOI] [PubMed] [Google Scholar]
  • 2.Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 1994; 266(5182):66–71. [DOI] [PubMed] [Google Scholar]
  • 3.Fleming MA, Potter JD, Ramirez CJ, Ostrander GK, Ostrander EA. Understanding missense mutations in the BRCA1 gene: An evolutionary approach. Proc Natl Acad Sci U S A. 2003. February 4;100(3):1151–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Kuchenbaecker KB, Hopper JL, Barnes DR, Phillips KA, Mooij TM, Roos-Blom MJ, et al. Risks of breast, ovarian, and contralateral breast cancer for BRCA1 and BRCA2 mutation carriers. JAMA. 2017. June 20; 317(23):2402–16. [DOI] [PubMed] [Google Scholar]
  • 5.King MC. “The race” to clone BRCA1. Vol. 343, Science; 2014. p. 1462–5. [DOI] [PubMed] [Google Scholar]
  • 6.Ruffner H, Joazeiro CAP, Hemmati D, Hunter T, Verma IM. Cancer-predisposing mutations within the RING domain of BRCA1: Loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity. Proc Natl Acad Sci U S A. 2001; 98(9):5134–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Shakya R, Reid LJ, Reczek CR, Cole F, Egli D, Lin CS, et al. BRCA1 tumor suppression depends on BRCT phosphoprotein binding, but not its E3 ligase activity. Science 2011;334 (6055):525–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Leung CCY, Glover JNM. BRCT domains: Easy as one, two, three. Vol. 10, Cell Cycle. Taylor and Francis Inc.; 2011. p. 2461–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Jia Z, Nie Y, Yue F, Kong Y, Gu L, Gavin TP, et al. A requirement of polo-like Kinase 1 in murine embryonic myogenesis and adult muscle regeneration. Elife. 2019. August 1; 8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Park JY, Wang PY, Matsumoto T, Sung HJ, Ma W, Choi JW, et al. P53 improves aerobic exercise capacity and augments skeletal muscle mitochondrial DNA content. Circ Res. 2009;105(7):705–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Al-Khalaf MH, Blake LE, Larsen BD, Bell RA, Brunette S, Parks RJ, et al. Temporal activation of XRCC1-mediated DNA repair is essential for muscle differentiation. Cell Discov. 2016; 2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Li D, Harlan-Williams LM, Kumaraswamy E, Jensen RA. BRCA1 - No matter how you splice it. Vol. 79, Cancer Research; 2019. p. 2091–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Bindra RS, Gibson SL, Meng A, Westermark U, Jasin M, Pierce AJ, et al. Hypoxia-induced down-regulation of BRCA1 expression by E2Fs. Cancer Res. 2005; 65(24): 11597–604. [DOI] [PubMed] [Google Scholar]
  • 14.Ouchi T BRCA1 phosphorylation: Biological consequences. Vol. 5, Cancer Biology and Therapy. Landes Bioscience; 2006. p. 470–5. [DOI] [PubMed] [Google Scholar]
  • 15.Xu X, Wagner K-U, Larson D, Weaver Z, Li C, Ried T, et al. Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation. Nat Genet. 1999;22(1):37–43. [DOI] [PubMed] [Google Scholar]
  • 16.Marquis ST, Rajan JV., Wynshaw-Boris A, Xu J, Yin G-Y, Abel KJ, et al. The developmental pattern of Brca1 expression implies a role in differentiation of the breast and other tissues. Nat Genet. 1995;11(1):17–26. [DOI] [PubMed] [Google Scholar]
  • 17.Lane TF, Deng C, Elson A, Lyu MS, Kozak CA, Leder P. Expression of Brca1 is associated with terminal differentiation of ectodermally and mesodermally derived tissues in mice. Genes Dev. 1995;9(21):2712–22. [DOI] [PubMed] [Google Scholar]
  • 18.Pao GM, Zhu Q, Perez-Garcia CG, Chou S-J, Suh H, Gage FH, et al. Role of BRCA1 in brain development. Proc Natl Acad Sci U S A. 2014;111(13):E1240–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Shukla PC, Singh KK, Quan A, Al-Omran M, Teoh H, Lovren F, et al. BRCA1 is an essential regulator of heart function and survival following myocardial infarction. Nat Commun. 2011;2(1). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Lovren F, Pan Y, Quan A, Singh KK, Khan R, Gupta N, et al. BRCA1 shields vascular smooth muscle cells from oxidative stress. J Thorac Cardiovasc Surg. 2014;147(6):1946–1955 e1. [DOI] [PubMed] [Google Scholar]
  • 21.Kubista M, Rosner M, Kubista E, Bernaschek G, Hengstschlager M. Brca1 regulates in vitro differentiation of mammary epithelial cells. Oncogene. 2002;21(31):4747–56. [DOI] [PubMed] [Google Scholar]
  • 22.Blackshear PE, Goldsworthy SM, Foley JF, McAllister KA, Bennett LM, Collins NK, et al. Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice. Oncogene. 1998. January 8;16(1):61–8. [DOI] [PubMed] [Google Scholar]
  • 23.Pérez-Vallés A, Martorell-Cebollada M, Nogueira-Vázquez E, García-García JA, Fuster-Diana E. The usefulness of antibodies to the BRCA1 protein in detecting the mutated BRCA1 gene. An immunohistochemical study. J Clin Pathol. 2001;54(6):476–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Maniccia AW, Lewis C, Begum N, Xu J, Cui J, Chipitsyna G, et al. Mitochondrial localization, ELK-1 transcriptional regulation and growth inhibitory functions of BRCA1, BRCA1a, and BRCA1b proteins. J Cell Physiol. 2009;219(3):634–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Henderson BR. Regulation of BRCA1, BRCA2 and BARD1 intracellular trafficking. Bioessays. 2005;27(9):884–93. [DOI] [PubMed] [Google Scholar]
  • 26.Jackson KC, Tarpey MD, Valencia AP, Iñigo MR, Pratt SJ, Patteson DJ, et al. Induced Cre-mediated knockdown of Brca1 in skeletal muscle reduces mitochondrial respiration and prevents glucose intolerance in adult mice on a high-fat diet. FASEB J. 2018; 32(6): 3070–3084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Singh KK, Shukla PC, Yanagawa B, Quan A, Lovren F, Pan Y, et al. Regulating cardiac energy metabolism and bioenergetics by targeting the DNA damage repair protein BRCA1. J Thorac Cardiovasc Surg. 2013;146(3):702–9. [DOI] [PubMed] [Google Scholar]
  • 28.McCarthy JJ, Srikuea R, Kirby TJ, Peterson CA, Esser KA. Inducible Cre transgenic mouse strain for skeletal muscle-specific gene targeting. Skelet Muscle. 2012;2(1):8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Xu X, Wagner KU, Larson D, Weaver Z, Li C, Ried T, et al. Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation. Nat Genet. 1999;22(1):37–43. [DOI] [PubMed] [Google Scholar]
  • 30.Tarpey MD, Valencia AP, Jackson KC, Amorese AJ, Balestrieri NP, Renegar RH, et al. Induced in vivo knockdown of the Brca1 gene in skeletal muscle results in skeletal muscle weakness. J Physiol. 2019; 597(3): 869–887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Privat M, Radosevic-Robin N, Aubel C, Cayre A, Penault-Llorca F, Marceau G, et al. BRCA1 induces major energetic metabolism reprogramming in breast cancer cells. PLoS One. 2014;9(7). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Kammermeier H, Roeb E. Regulation of systolic force and control of free energy of ATP-hydrolysis in hypoxic hearts. J Mol Cell Cardiol. 1990;22(6):707–13. [DOI] [PubMed] [Google Scholar]
  • 33.Tarpey MD, Amorese AJ, Balestrieri NP, Fisher-Wellman KH, Spangenburg EE. Doxorubicin causes lesions in the electron transport system of skeletal muscle mitochondria that are associated with a loss of contractile function. J Biol Chem. 2019. December 20;294(51):19709–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Barclay CJ. Energetics of contraction. Compr Physiol. 2015;5(2):961–95. [DOI] [PubMed] [Google Scholar]
  • 35.Wu J, Lu LY, Yu X. The role of BRCA1 in DNA damage response. Vol. 1, Protein and Cell. Higher Education Press; 2010. p. 117–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Coene ED, Hollinshead MS, Waeytens AA, Schelfhout VR, Eechaute WP, Shaw MK, et al. Phosphorylated BRCA1 is predominantly located in the nucleus and mitochondria. Mol Biol Cell. 2005;16(2):997–1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Nachman MW, Crowell SL. Estimate of the mutation rate per nucleotide in humans. Genetics. 2000;156(1):297–304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Tubbs A, Nussenzweig A. Endogenous DNA Damage as a Source of Genomic Instability in Cancer. Vol. 168, Cell. Cell Press; 2017. p. 644–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Fisher-Wellman KH, Neufer PD. Linking mitochondrial bioenergetics to insulin resistance via redox biology. Vol. 23, Trends in Endocrinology and Metabolism. Trends Endocrinol Metab; 2012. p. 142–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Rosen EM. BRCA1 in the DNA damage response and at telomeres. Front Genet. 2013;4:85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Rodriguez MC, Zhou S. BRCT domains: Phosphopeptide binding and signaling modules. Vol. 13, Frontiers in Bioscience. Front Biosci; 2008. p. 5905–15. [DOI] [PubMed] [Google Scholar]
  • 42.Vahidi Ferdousi L, Rocheteau P, Chayot R, Montagne B, Chaker Z, Flamant P, et al. More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny. Stem Cell Res. 2014. November 1;13(3):492–507. [DOI] [PubMed] [Google Scholar]
  • 43.Wagatsuma A, Sakuma K. Molecular mechanisms for age-associated mitochondrial deficiency in skeletal muscle. Vol. 2012, Journal of Aging Research. J Aging Res; 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Xiao L, Lee KKH. BRE facilitates skeletal muscle regeneration by promoting satellite cell motility and differentiation. Biol Open. 2016. February 15;5(2):100–11. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES