gene is classified as a 'tumour suppressor' and 'caretaker' on the basis of its proposed genome integrity maintenance functions32
. Germ-line mutations in BRCA1
predominantly predispose carriers to breast and ovarian cancer in addition to other cancer syndromes33
. BRCA1 is important for various cellular functions and exerts its effects on key cellular processes involved in DNA nucleotide excision and DSB repair, transcriptional regulation, ubiquitination and cell-cycle control4
. Mice harbouring complete ablation of BRCA1 die embryonically and display neuroepithelial, proliferative and morphogenetic abnormalities35
. The BRCA1
gene is composed of 24 exons. Exon 11 is the largest exon and covers ~60% of the entire BRCA1
. The exon 11 encoded domain interacts and colocalizes with RAD51 at the sites of DNA damage to activate RAD51-mediated repair of DSBs3
. Mice with systemic deletion of exon 11 in the BRCA1
gene show recessive embryonic lethality due to impaired DNA damage-induced activation of BRCA1, compromised RAD51-dependent repair pathway and increased apoptosis38
To understand the DNA damage repair function of BRCA1 in mature cardiac muscles, we utilized the Cre-loxP system to delete exon 11 of the BRCA1 gene specifically in cardiomyocytes and successfully generated CM-BRCA1−/− mice. Although the transcription and translation of the BRCA1Δ11 splice variant cannot be ruled out, it should be noted that if translated, it would still not encode the major part of the C-terminus domain and therefore would be non-functional with respect to the DNA damage repair properties of BRCA1. Baseline cardiac morphology as well as indices of cardiac functions along with apoptosis and DNA damage were indistinguishable between CM-BRCA1−/− and the WT control littermates. Strikingly, whereas almost 95% of the CM-BRCA1−/− mice and ~60% of the CM-BRCA1+/− mice were dead at the end of the study period (22 weeks), corresponding mortality in the WT mice was only ~20%. These mice died more frequently due to congestive heart failure and showed dilatation of the left ventricle. A closer analysis of the cardiac phenotype at 4 weeks post-MI revealed that CM-BRCA1−/− mice had increased infarct sizes, poor cardiac function, reduced contractility, dilated left ventricles and blunted compensatory hypertrophy compared with WT mice.
Ischaemia and accompanying hypoxic stress manifested with increased DNA breaks are the primary triggers for the induction of pro-apoptotic cascades leading to apoptotic cell death40
. Given the central role of BRCA1 in DSB repair, we postulated that an ischaemic insult may result in an increased burden of DSBs and that under conditions of BRCA1 deficiency there may be accumulation of unresolved DSBs thereby setting the stage for cardiomyocyte apoptosis and cardiac dysfunction4
. Congruent with our hypothesis, we observed ~2.5 fold greater DSBs in hearts from CM-BRCA1−/−
mice compared with those from the control littermates 48 h post-MI. Cardiac DSBs were resolved 72 h post-MI in WT control littermates but were persistently present in the hearts of CM-BRCA1−/−
mice. Peak cardiac BRCA1 expression in WT mice occurred 72 h post-MI providing a clear indication that BRCA1 is required for repairing ischaemia-induced DSBs. This finding is further strengthened by the degree of RAD51-foci formation, another marker of DSB repair, which temporally correlated with MI-induced BRCA1 expression in WT littermates. The observation that the extent of RAD51-foci formation was reduced in the hearts of CM-BRCA1−/−
mice post-MI suggests accumulation of unresolved DSBs, which would accordingly shift the balance towards cardiomyocyte death.
Ischaemia-induced increased mortality and cardiac dysfunction were further associated with significantly higher numbers of TUNEL-positive nuclei and cleaved caspase-3 expression demonstrating increased cardiomyocyte death in the hearts of CM-BRCA1−/−
mice compared with corresponding WT control littermates post-MI. Systemic loss of BRCA1-associated embryonic lethality in mice can be partially rescued by the heterozygous deletion of the tumour suppressor gene p5318
. Intriguingly, loss of p53 protects the heart from rupture and associated death, whereas overexpression of p53 induces cardiomyocyte apoptosis24
. Therefore, we sought to determine if p53 is a modulator of cardiomyocyte apoptosis post-MI in CM-BRCA1−/−
mice. We found significantly higher p53 protein levels along with increased p53-regulated pro-apoptotic downstream target Bax post-MI in the hearts of CM-BRCA1−/−
mice. The most important factor in the p53-dependent survival and/or apoptotic signalling pathway is the shift in the ratio between the pro-apoptotic Bax and pro-survival molecule Bcl-2 (ref. 45
), both of which are recognized as major regulators of cardiomyocyte apoptosis and/or survival decisions during heart failure25
. We observed that the MI-induced increase in cardiomyocyte apoptosis in CM-BRCA1−/−
mice was associated with an ~10 fold increase in the Bax to Bcl-2 ratio, shifting the balance from a survival to an apoptotic milieu. As the systemic loss of BRCA1-associated embryonic lethality in mice can be partially rescued by the heterozygous deletion of p53
), we investigated how deletion of one allele of p53
would affect the MI-induced cardiac phenotype associated with cardiomyocyte-specific loss of BRCA1. To this aim, we generated double heterozygous knockout mice (CM-BRCA1+/−
). We report that the post-MI phenotype associated with loss of full-length BRCA1 could be prevented with systemic deletion of one allele of p53
, such that the heart from the these mice show reduced apoptosis as well as improved cardiac function and contractility post-MI. We further confirm the direct role of p53 in BRCA1 deficiency-associated post-MI cardiac phenotype by systemically deleting one allele of both BRCA1
, which mimics more of a human BRCA1-haploinsufficient condition and demonstrate that deletion of one allele of p53 was able to rescue the BRCA1-deficiency-associated MI-induced cardiac phenotype.
Chemotherapeutic agents function by directly or indirectly damaging DNA through various mechanisms47
. Doxorubicin is a potent chemotherapeutic agent used for a wide variety of malignancies, but the clinical utility has been limited by its dose-dependent cardiotoxicity, which eventually results in refractory cardiac dysfunction48
. Doxorubicin induces DSBs49
and BRCA1 has been previously implicated in the repair of DSBs4
. Thus the role of BRCA1 in response to doxorubicin-induced cardiotoxicity is an important subject for investigation. In our hands, CM-BRCA1−/−
mice showed reduced cardiac performance, increased DSBs, reduced DSB repair and enhanced cardiomyocyte death in comparison to the WT mice following treatment with doxorubcin.
Of note, we provide herein evidence of clinical relevance to our in vivo
observations. We report that ischaemia significantly upregulated BRCA1 expression in adult human atrial and ventricular tissues, and also in human fetal cardiomyocytes. Importantly, we also show that ischaemia significantly induced DSBs in human atrial tissues. BRCA1 physically interacts with RAD51 to regulate RAD51 nuclear transport, which is required for DSB repair29
. We propose that ischaemia induced-BRCA1 upregulation is associated with increased DSB repair and this is confirmed by greater RAD51-foci formation in ischaemic atrial tissues. We demonstrate for the first time that BRCA1 protects atrial and ventricular tissues against DSBs by promoting DSB repair through RAD51 interaction, and thus loss of function or BRCA1 haplo-insufficiency may lead to impaired DSB repair with subsequent accumulation of DNA damage, apoptosis and/or cardiac dysfunction.
Taken together this study provides evidence that in the heart BRCA1 mediates a survival pathway that acts to block the onset of apoptosis resulting in cardiac dysfunction following ischaemic or genotoxic stress. Our data identifying BRCA1 as an essential and novel regulator of heart function affords important translational implications. From a therapeutic standpoint, our data suggest that BRCA1 may represent a novel therapeutic target to limit cardiac failure, a leading cause of death worldwide50
. From an epidemiological standpoint, these observations provide important clues regarding a potential susceptibility of BRCA1 mutation carriers to cardiovascular disease, in addition to the well-documented predisposition towards cancer syndromes. Recent data suggest that BRCA1/2 mutation carriers exhibit an increase in non-neoplastic death particularly at older ages51
. Although the causes of non-neoplastic death in the reported population remain unknown, our data raise the possibility that the excess mortality may be due to increased rates of death from ischaemic heart disease. Finally, from a pharmacogenomic point of view, this data may indicate a heightened susceptibility of BRCA1
mutation carriers to anthracycline-induced cardiac failure53
, a cornerstone of chemotherapy for breast and ovarian cancer.