BRCA1 Reflects Myocardial Adverse Remodeling in Idiopathic Dilated Cardiomyopathy

BRCA1 Reflects Myocardial Adverse Remodeling in Idiopathic Dilated Cardiomyopathy J.K. Nozynskia,*, D. Konecka-Mrowkaa, M. Zakliczynskib, E. Zembala-Nozynskac, D. Langec, and M. Zembalab a Department of Histopathology, Silesian Centre for Heart Diseases, Zabrze, Upper Silesia, Poland; bDepartment of Cardiac, Vascular and Endovascular Surgery and Transplantology, Silesian Medical University, Zabrze, Upper Silesia, Poland; cDepartment of Tumor Pathology, Center of Oncology - Maria Sklodowska-Curie Memorial Institute, Gliwice, Upper Silesia, Poland

ABSTRACT Background. The role of BRCA1 in chronic ischemic episodes seems to be pivotal for adverse remodeling and development of ischemic cardiomyopathy, because of its role in DNA repair and apoptosis. The aim of this study was to investigate the role of BRCA-1 in idiopathic dilated cardiomyopathy (IDCM). Material and Methods. The study group (IDCM) comprised myocardial samples from hearts explanted before transplantation owing to IDCM in 10 males (age 44
5.3 years) without clinical symptoms of ischemic heart disease. The control group consisted myocardial fragments taken from 10 male heart valve and pulmonary artery donors with diagnosed cerebral death (age 40
2.3 years). BRCA1 was detected immunohistochemically with rabbit anti-BRCA1 polyclonal antibody. The intensity of BRCA1 expression was semiquantitatively assessed for cardiocytes, small vessels including capillaries, and interstitial cells. The significances between groups were estimated using the Mann-Whitney U test. Results. All IDCM cases were positive and presented nonuniform BRCA1 expression: hypertrophied cardiocytes showed very intense staining and typical cardiomyopathic cardiocytes were stained weakly forming mosaic. Control cases showed weak-to-moderate uniform staining. Intensity of staining was significantly higher in IDCM cardiocytes, whereas small vessels and interstitial were stained similarly in both groups. Conclusions. IDCM adverse remodeling results in more intense but nonuniform BRCA1 expression. This phenomenon seems to reflect IDCM biology: lower BRCA1 expression in myocytes with lower anti-apoptotic and DNA damage repair activity, with the opposite in hypertrophied myofibers.

B

RCA1 BELONGS TO multifunctional proteins acting in transcriptional regulation, DNA damage repair, and cell cycle checkpoint [1]. The gene encoding BRCA1 was found on chromosome 17q12-21 in humans [2]. The crucial role of BRCA1 in DNA repair depends on the interaction with multiple proteins resulting in creation of BRCA1genome surveillance complex, which plays a role in the recognition of abnormal or damaged DNA [3,4]. It should be emphasized that in some studies hypoxia induced downregulation of BRCA1 [5]. BRCA1 overexpression, as an anti-apoptotic factor, was documented in endothelial

cells subjected to doxorubicin; moreover, BRCA1 diminished the inflammatory response and improved capillary density in ischemic limb muscle, playing a suppressive role in ischemic episodes [6]. Studies of myocardial tissue

Supported by Silesian Medical University Grant No: KNW-1169/N/4/0. *Address correspondence to Jerzy K. Nozynski, Department of Histopathology, Silesian Centre for Heart Diseases, SklodowskaCurie 9, Zabrze, Upper Silesia 41-800, Poland. E-mail: [email protected]

0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2015.12.141

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Transplantation Proceedings, 48, 1746e1750 (2016)

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Fig 1. BRCA1 expression in control cases. (A) Longitudinal section revealed regular diffuse pale brown stained expression of BRCA1 along the sarcoplasm. Interstitial cells (fibroblasts), small vessel and perivascular cells (at the top) shows no typical staining. Magnification 100-fold. (B) Longitudinal section at higher magnification showed regular diffuse expression of BRCA1 along the sarcoplasm. Capillaries also exhibits weak positive reaction. Magnification 400-fold. (C) Transverse section revealed generally regular weak to intense BRCA1 staining inside cardiocytes sarcoplasm. Negatively stained cardiocytes were never seen. A small vessel (center) showed a weak reaction in endothelial and perivascular cells. Magnification 200-fold. (D) Small arteriole (at center) with moderate BRCA1 expression. Around the arteriole transversely sectioned cardiocytes stained intensely. Interstitial cells could be considered as negatively or weakly stained. Magnification 400-fold.

revealed similar results: the increase of myocardial BRCA1 expression after an acute ischemic episode was protective from adverse myocardial remodeling [7]. The role of BRCA1 in chronic ischemic episodes seems to be pivotal for adverse remodeling of the left ventricle in heart failure caused by coronary artery disease; however, its role in development of nonischemic cardiomyopathy is poorly described. Thus, we conducted a pilot study, checking the role of this protein in idiopathic dilated cardiomyopathy (IDCM). MATERIAL AND METHODS The IDCM group was composed of the archived tissue samples from 10 male hearts (44
5.3 years old) explanted before transplantation in whom ischemic heart disease was excluded by

coronary angiography. The heart was excised after administration of a cardioplegic solution, rinsed in sterile cold Ringer solution, and immediately fixed. The control group was composed of 10 heart specimens taken from heart valve and pulmonary artery donors with diagnosed cerebral death. All hearts were harvested with the intention to perform orthotropic heart transplantation; however, the procedure was canceled owing to nonmedical reasons. The group included 10 males with normal cardiac morphology, blood pressure, and blood glucose levels and a mean glomerular filtration rate of 60 mL/min/1.73 m2 (40
2.3 years old). These hearts were harvested from multiorgan donors. After excision, the organ was rinsed with cold Ringer solution (4 C) and transported to a specialized laboratory under cold sterile conditions. Before undergoing histologic evaluation, each organ was inspected grossly to confirm its normal morphology. Additional evaluations concerning microbiological and serologic tests for exclusion of bacterial and/or viral infections or contamination were done routinely. In cases of

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NOZYNSKI, KONECKA-MROWKA, ZAKLICZYNSKI ET AL

Fig 2. BRCA1 expression in dilated cardiomyopathy cases. (A) Longitudinal section reveals irregular diffuse and granular brown stained expression of BRCA1 along the sarcoplasm. Interstitial cells (fibroblasts), small vessel and perivascular cells (at the top) shows no typical staining. Magnification 200-fold. (B) Another longitudinal section trough fibrotic area (bottom) and hypertrophied cardiocytes (right). Hypertrophied cardiocyte with strong but irregular diffuse and granular positive reaction along the sarcoplasm. Fibrotic area with no BRCA1 expression. Magnification 200-fold. (C) Transverse section reveals irregular staining of single cardiocytes. A predominant part of cardiocytes with strong BRCA1 expression surrounds few cells with trace or negative expression. Magnification 400-fold. (D) Another transverse section trough area with predominant weak or negative BRCA1 expression, especially in typical cardiomyopathic cardiocytes, with enlarged nuclei. Magnification 400-fold. (E) Typical cardiomyopathic cardiocyte with bizarre nucleus, perinuclear vacuole (halo) and focal weak expression of BRCA1 in sarcoplasm (longitudinal section). Adjacent cardiocytes also exhibits focal BRCA1 staining and areas of sarcoplasm with negative reaction. Magnification 600-fold. (F) Small vessel with absent BRCA1 staining. Magnification 600-fold.

MYOCARDIAL ADVERSE REMODELING IN IDCM

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the lack of gross and microscopic abnormalities, absent atherosclerotic lesions, and with negative result in bacteriology and serology, the organ was classified as healthy. The cold ischemia time was <3 hours. One full-thickness transmural fragment from grossly unchanged left ventricular anterior wall from each case was excised 2 cm below the aortic root (1.5  1.5 cm). Then, tissues were fixed in 6% buffered solution of depolymerized paraformaldehyde and subjected to routine histologic examination using a typical paraffin procedure. The immunohistochemical examination consisted of tissue rehydration in diminished ethanol concentrations, then washing and antigen retrieval procedure using 0.1 mmol citrate buffer pH 6.0 in pressure cooker for 1 hour. After this step the slides were cooled, washed 3 times, and endogenous peroxidase was blocked (3% aqueous solution of H2O2). Incubation with rabbit anti-BRCA1 polyclonal antibody (Bio-Genex Clone PU345-UP) was conducted in humid chamber (at room temperature, 1 hour). Coupled antibody was developed with DAB-based Ultravision Quanto Detection System (Thermo Fisher Scientific). Cell nuclei were counterstained with hematoxylin. Finally, slides were dehydrated and mounted with DPX. The intensity of BRCA1 expression was assessed semiquantitatively for cardiocytes, small vessels including capillaries and interstitial cells as 0 (no staining), 1 (weak staining; pale brown), or 2 (intense staining; dark brown) for minimal and maximal expression (including focal changes) and average expression suggestive for all analyzed elements, independently by 2 observers. The significances between groups were estimated using the Mann-Whitney U test.

RESULTS

In the control group, weak, diffuse, regular, and focally granular BRCA1 cytoplasmic expression was seen. In these “granular” cardiocytes the subtle staining was distributed along the cytoplasmic myofibrils. Moreover, small vessels shown also weak reaction, similarly stained interstitial cells (Fig 1). IDCM cases shown nonuniform BRCA1 expression,

but generally the staining was stronger than in control cases. At lower magnification, in almost all IDCM cases, the areas of weakly stained cardiocytes were located between myofibers with stronger BRCA1 staining. The groups of hypertrophied cardiocytes shown very intense staining, whereas typical cardiopathic myocytes were stained weakly with perinuclear halo around bizarre nucleus. All scars and fibrotic areas were stained negatively, whereas surrounding cardiocytes showed negative or diminished BRCA1 expression. Vessels and interstitial cells also showed diversities of staining intensity, from negative to strong (Fig 2). Statistical results and semiquantitative assessments are presented in Table 1. The average grade of BRCA1 expression was significantly higher in IDCM group, which was also characterized by nonuniformities of cardiocyte BRCA1 staining in cardiomyopathy, whereas the control group showed lower but uniform BRCA1 expression.

DISCUSSION

The role of BRCA1 was studied extensively from the time of identifying the BRCA1 and 2 as a clue factors in the development of breast, prostate, ovarian, and colorectal cancers [8e11]. The mutations of BRCA genes resulting in the lack of functional BRCA1/2 proteins were proved to worsen prognosis in patients suffering from these types of neoplasm. From a cytophysiologic point of view, the loss of functional BRCA1/2 could activate p53, causing cell cycle arrest or apoptosis [12]. Singh et al. [6] demonstrated greater mortality in BRCA1-knockout mice after myocardial infarction; moreover, increased apoptosis after doxorubicin administration was seen in the BRCA1/ group. It should be pointed out that baseline BRCA1 expression in our control group could be described as similar to the one

Table 1. BRCA1 Expression in Studied Hearts Intensity

MinimalCONTR

Cardiocytes Median 1 Upper quartile 1 Lower quartile 1 Mean
SD 1
0 P* Small vessels (including capillaries) Median 0.5 Upper quartile 1 Lower quartile 0 Mean
SD 0.50
0.53 P* Interstitial cells Median 0.5 Upper quartile 1 Lower quartile 0 Mean
SD 0.50
0.53 P*

MinimalIDCM

MaximalCONTR

MaximalIDCM

AverageCONTR

AverageIDCM

0 1 0 0.30
0.48

2 2 1 1.60
0.52

2 2 2 1.90
0.32

1 1 1 1
0

2 1 2 1.60
0.52

.003

.3 1 1 0 0.0
0.52

1 2 1 1.40
0.52

.69

2 2 1 1.60
0.52

1 1 1 1
0

.65 0 1 0 0.40
0.52

1

.01

1 1 1 1
0

.47 1 1.25 1 1.20
0.42

.47

1 1.25 1 1.20
0.42

1 1 1 1
0

1 1 1 0.90
0.31 1

Abbreviations: Minimal, minimal BRCA1 expression, represents focal intensity; Maximal, maximal BRCA1 expression represents focal intensity; Average, representative intensity of BRCA1 expression in whole slide; CONTR (lower index), control group; IDCM (lower index), idiopathic dilated cardiomyopathy group. *ManneWhitney U test.

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observed in control mice as slight staining of cardiocyte sarcoplasm and small vessels. Further studies of BRCA1 expression on human myocardium in patients undergoing valvular surgery, presented in the same paper, showed a lower baseline BRCA1 expression than in control mice and our control group [6]. This difference could be explained by the possible influence of cold ischemia on healthy myocardium. The irregular BRCA1 expression in IDCM cases shown in our study suggests that in part cardiocytes synthesis of BRCA1 was diminished or absent making them, more susceptible for detrimental factors, resulting in apoptosis and the progression of IDCM [13e16]. It should be mentioned that autophagy, another type of programmed cell death, also plays an important or even essential role in the progression of IDCM [17,18]. Experimental studies with BRCA1 and tumor cells demonstrated the increased formation of autophagic vacuoles correlated with the decrease of BRCA1 levels and promotion of autophagy in BRCA1negative cells [19,20]. Thus, activation of both programmed cell death pathways could reflect the loss of BRCA1 expression in cardiocytes. Results of our study are not sufficient to elucidate the origin of BRCA1 lack in some cardiocytes. Further studies to explain the mechanisms of BRCA1 downregulation in IDCM are warranted. REFERENCES [1] Hu Y. BRCA1, hormone, and tissue-specific tumor suppression. Int J Biol Sci 2009;5:20e7. [2] Miki Y, Swensen J, Shattuck-Eidens D, et al. A strong candidate for breast and ovarian susceptibility gene BRCA1. Science 1994;266:66e71. [3] Wang Y, Cortez D, Yazdi P, et al. BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev 2000;14:927e39. [4] Moynahan ME, Cui TY, Jasin M. Homology-directed DNA repair, mitomycin-C resistance, and chromosome stability is restored with correction of a Brca1 mutation. Cancer Res 2001;61: 4842e50. [5] Glazer PM, Hegan DC, Lu Y, et al. Hypoxia and DNA repair. Yale J Biol Med 2013;86:443e51.

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