Immunohistochemical expression of cell differentiation and growth in neonate cardiomyocytes

Abstracts

FUNDAMENTO: As alterações cardíacas na fase de transição do coração fetal para a vida extrauterina vêm sendo exploradas por inúmeras pesquisas em animais, e os mecanismos celulares responsáveis por essas modificações ainda não estão bem documentado em seres humanos. OBJETIVO: Avaliar o mecanismo de diferenciação celular em cardiomiócitos ocorridas nos primeiros dias de vida, por meio da análise imunoistoquímica de proteínas envolvidas com processos de proliferação e contração muscular, em amostras de miocárdio de recém-natos humanos. MÉTODO: Estudo transversal de amostras parafinadas de miocárdio provenientes de banco de necropsias de recémnascidos humanos, divididos em dois grupos amostrais: recém-nascidos a termo que foram a óbito com no máximo dois dias de vida (NEO1) com 10 casos, e recém- nascidos a termo que foram a óbito entre três e 10 dias de vida (NEO2) com 14 casos, a fim de seguir uma linha de tempo que contemplasse a fase de transição da circulação fetal a vida extrauterina. As amostras foram estudadas em tissue microarray e os anticorpos utilizados foram o Ki67, PCNA, PTEN, Bcl2 (proliferação) e HHF35 e actina sarcomérica (proteínas contráteis). RESULTADOS: Foi encontrada diferença com o Ki67 p = 0,02, HHF35 p < 0,01 e actina sarcomérica p = 0,02, e a expressão do Ki67 foi mais alta no grupo NEO1 e a expressão do HHF35 e da actina sarcomérica foi mais alta no grupo NEO2. CONCLUSÃO: Os resultados sugerem que os cardiomiócitos apresentam uma característica proliferativa (Ki67) nos NEO1 e que essa vai, seguindo uma linha temporal, sendo substituída por um caráter de diferenciação (HHF35 e actina sarcomérica) nos NEO2.

Recém-nascido; miócitos cardíacos; imunoistoquímica; transição de fase; circulação placentária

^IPontifícia Universidade Católica do Paraná - PUC-PR

^IIUniversidade Federal do Paraná - UFPR, PR - Brazil

Mailing Address

ABSTRACT

BACKGROUND: The cardiac alterations during the fetal heart transition to extrauterine life have been explored by several animal studies and the cell mechanisms responsible for these modifications are not well documented in humans.

OBJECTIVE: To evaluate the mechanism of cell differentiation into cardiomyocytes that occur in the first days of life, through immunohistochemical analysis of proteins involved in proliferation and muscle contraction processes, in samples of human neonate myocardium.

METHODS: Cross-sectional study of paraffin-sample sections of myocardium from an autopsy database of human neonates, divided into two sample groups: full-term neonates who died after a maximum of two days of life (NEO1) with 10 cases, and full-term infants who died between 3 and 10 days of life (NEO2) with 14 cases, in order to follow a temporal line that would contemplate the transition from fetal circulation to extrauterine life. The samples were studied in tissue microarray and the antibodies used were Ki67, PCNA, PTEN, Bcl2 (proliferation), HHF35 and sarcomeric actin (contractile proteins).

RESULTS: Difference was observed regarding Ki67, p = 0.02; HHF35, p <0.01 and sarcomeric actin, p = 0.02, with Ki67 expression being higher in NEO1 group, whereas HHF35 and sarcomeric actin expression was higher in the NEO2 group.

CONCLUSION: The results suggest that cardiomyocytes have a proliferation characteristic (Ki67) in NEO1 which, following a temporal line, will be replaced by a differentiation characteristic (HHF35 and sarcomeric actin) in NEO2.

Keywords: Infant, newborn; myocytes, cardiac / classification; immunohistochemistry; phase transition; placental circulation.

Introduction

During cardiac development, in animal studies, cardiomyocytes undergo numerous phenotypic alterations, going from an intrauterine proliferative phenotype (hyperplastic) to a cell growth and differentiation pattern in extrauterine life (hypertrophic), according to MacLellan and Schneider and Cortius et al.^1,2.

Anversa and Nadal-Ginard reported that the transition of cardiomyocytes from a hyperplastic to a hypertrophic phenotype would provide a significant increase in cardiac contractile force³.

At the hypertrophic stage of heart development, ventricular cardiomyocytes increase in size due to increased synthesis of contractile proteins and also to decrease in DNA synthesis with a consequent decrease in cell proliferation.

The cell mechanisms that are responsible for triggering and regulating this transition are not fully understood⁴.

Porrello et al. performed partial resection of the heart in rats aged one and seven days of life. They observed regeneration only in one-day-old rats, thus demonstrating that, for a brief period after birth, the mammalian heart seems to have capacity for regeneration⁵.

The design of these mechanisms is particularly important to understand cardiomyocyte regeneration processes, through undifferentiated primitive cells, after myocardial lesions in adults. The knowledge of intrinsic proliferation and cardiomyogenic potential of newborns can lead to a better understanding of these processes in adults, resulting in future clinical benefits in patients with severe myocardial lesions⁵.

The hypothesis of the present study is based on the fact that cardiomyocytes can undergo a gradual transition from the hyperplastic to the hypertrophic phenotype, following the temporal evolution of neonates, involving alterations in the immunohistochemical expression of some proteins that act on cell growth and differentiation. Therefore, the objective of this study is to evaluate, by immunohistochemical analysis of some proteins involved in the processes of muscle proliferation and contraction, the mechanism of cell differentiation of cardiomyocytes in humans that can demonstrate the transition from fetal circulation to extrauterine life.

Methods

This study analyzed samples of left ventricular myocardium fixed in formalin and embedded in paraffin, from autopsies of full-term newborns (gestational age between 38 and 42 weeks), who died within 10 days of post-natal life (n = 106 cases) at the Pediatric and Perinatal Pathology Unit, Department of Pathology, Hospital de Clinicas, Universidade Federal do Parana (UFPR), between the years 1985 and 2005. This study was approved by the UFPR Ethics Committee under protocol # 1319.167/2006-11.

The cases were divided into two groups: NEO1, full-term newborns that died no more than two days after birth, and NEO2, who died between three and 10 days of post-natal life.

The study consisted initially of 56 cases in NEO1 and 50 cases in NEO2. After exclusion criteria were applied, 10 cases were selected for the NEO1 group and 14 cases for the NEO2 group.

The exclusion criteria included all cases of malformed, syndromic fetuses, fetal erythroblastosis cases, placental alterations or those with a gestational history of chronic arterial or specific gestational hypertension, pre-pregnancy or gestational diabetes, maternal infections, intrauterine growth retardation, oligohydramnios and polyhydramnios.

All cases were classified according to sex, gestational age, age and cause of death. Samples of myocardium were fixed in formalin and preserved in paraffin blocks, which led us to adopt the immunohistochemistry technique as the method of analysis of protein expression.

Immunohistochemistry seems to be the most appropriate technique for materials fixed in formalin and embedded in paraffin. It has been described over the years, as an important tool in the study of protein expression, in addition to being an inexpensive and easy-to-perform technique.

The results, however, are influenced by factors such as fixation time and processing of the material, the quality of the fixation agent and the chosen antibodies, the reactions themselves and the subjectivity when interpreting the slides. Nevertheless, immunohistochemistry has been widely used in material fixed in formalin and processed in paraffin due to its expression capacity even when using poor-quality tissues⁶.

Myocardium samples from each group were then mounted in tissue microarrays (TMA), using three fragments of LV myocardium per case, and each fragment measured 3 mm in diameter.

The immunohistochemical technique was used to demonstrate the specific antigens in myocardial samples fixed in formalin and embedded in paraffin and all antibodies were diluted with antibody diluent (Dakocytomation ^®). The EnVision ^® + Dual Link / Peroxidase (Dakocytomation ^®) was used as the secondary antibody. As chromogen, used to reveal the reaction, 3,3'-diaminobenzidine or DAB, chromogen-substrate system (Dakocytomation ^®) was used. The following primary antibodies and dilution ratios were used: anti-PTEN (1:400; ^® Novocastra), anti-Ki67 (1:150, Dako ^®), anti-PCNA (1:400, Dako ^®) and anti-Bcl2 (1:200, Dako ^®) to assess cell proliferation, and HHF35 (1:400, Dako ^®) and sarcomeric anti-actin (1:400, Dako ^®) to assess contractile proteins. The reactions were controlled reactions with positive and negative controls.

The reactions of antibodies Ki67, PCNA, Bcl2 and PTEN for cell proliferation were analyzed by simple quantitative method by counting the nuclei of positive cardiomyocytes in the entire sample, under 400 X magnification.

The reactions of HHF35 and sarcomeric actin antibodies for contractile proteins were analyzed by the morphometric quantitative method, and this method reads the color intensity of the region with positive immunohistochemistry reaction and converts it into an area measurement, using the micrometer as unit. For that purpose an optical microscope was used with a 200 X magnification, in a binocular microscope (Olympus BX50 ^®) coupled to a computer in which the Image Pro Plus ^® software had been installed to perform the analysis. To compare the groups in terms of quantitative variables, the ANOVA model was considered, with a significance level of 0.05.

Results

A total of 24 newborn hearts were analyzed, showing, regarding gender, mean gestational age or days of life and cause of death, the following profile shown in Table 1⁷. Among the immunohistochemical markers that suggest cell proliferation, Ki67 with p = 0.02 showed significant difference between NEO1 and NEO2 groups. Table 2 shows the descriptive statistics and p values for antibodies PCNA, Ki67, Bcl2 and PTEN that evaluate cell proliferation. The sarcomeric actin and HHF35 antibodies that evaluate contractile proteins showed significant differences between the two groups, with higher values in the NEO2 group, with their results shown in Table 3.

Discussion

PCNA and Ki67 have been used to study the cell cycle as important markers of proliferation and have been used in studies on cardiomyocyte cell growth^1,2,8.

In the mammalian embryo, proliferation and differentiation of cardiomyocytes occur concomitantly during embryonic and fetal periods and continue to occur until a few days after birth².

Studies in rats have shown that cardiomyocytes often stop dividing at approximately 17 days of post-natal development. The expression of PCNA in this phase shows that 6% of cardiomyocytes were in synthesis activity at birth, but only 1.5% of these cells would be in synthesis activity at 11 days of life, and only 0.5% of adult cardiomyocytes could enter the synthesis phase. This inability of adult cardiomyocytes to regenerate through the increase of cell divisions has been broadly studied and is as one of the major problems for the clinical treatment of heart disease^1,2,8.

Recent studies of the immunohistochemical expression of Ki67 in infarcted human hearts have shown, however, that 4% of the cardiomyocytes showed positivity for this proliferative protein in infarcted regions. Furthermore, the phenotypic alterations found in the transition from fetal circulation to extrauterine life could include a gradual decrease in PCNA and Ki67 expression^1,2,8-12.

In the present study, Ki67 showed a decrease in expression when groups NEO1 and NEO2 were compared (p = 0.02). This is in agreement with the idea of a decrease in the proliferative characteristics of cardiomyocytes in this period.

Another antibody that has an important role in cell cycle regulation is PTEN, which is involved in cell cycle progression, cell migration and growth, as well as apoptosis¹³. Samples in this study showed a difference in PTEN expression between the two groups; however, the differences were not statistically significant. PTEN, when activated, could be involved in blocking the cell capacity to move from G1 to S phase through cyclin inhibition, consequently blocking the proliferative capacity of cardiomyocytes in the extrauterine pathway¹³.

Increased expression of Bcl2 was observed, which followed the temporal evolution of the sample groups, although with no statistically significant difference. This fact might indicate greater resistance to apoptosis in the group of newborns who died after two days of life and therefore, a greater tendency to cell growth and differentiation^2,9.

The HHF35 antibodies and sarcomeric actin, which are used for the study of contractile proteins, showed statistically significant difference between the two sample groups, and the Neo2 group showed higher expression of both proteins (p < 0.01 and p = 0.02, respectively). This fact possibly demonstrates that the differentiation of cardiomyocytes occurs by increasing the volume of cardiac cells, through changes in the expression of sarcomeric proteins, which would result in changes in the functional properties of cardiomyocytes with an increase in capacity of power generation by the heart muscles during the postnatal development¹⁴.

Conclusions

We observed a decrease in Ki67 expression in relation to sample temporal evolution, showing a reduction in cardiomyocyte proliferative phenotype. The antibodies that marked contractile proteins (sarcomeric anti-actin and HHF35) showed increased expression following the temporal evolution of the sample groups. These data are consistent with the substitution of the hyperplasic proliferative characteristic of this tissue during intrauterine life by a cell differentiation characteristic in extrauterine life.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Sources of Funding

There were no external funding sources for this study.

Study Association

This article is part of the thesis of doctoral submitted by Tarcísio Fulgêncio Alves da Silva, from Pontífica Universidade Católica do Paraná.

References

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Immunohistochemical expression of cell differentiation and growth in neonate cardiomyocytes

Tarcísio Fulgêncio Alves da Silva^I; Greyce Kelly de Souza^I; Mona Adalgisa Simões^I; Francisco Cesar Pabis^I; Lucia de Noronha^I,II

Publication Dates

• Publication in this collection
  09 Aug 2012
• Date of issue
  Sept 2012

History

• Received
  21 Feb 2012
• Accepted
  16 Apr 2012
• Reviewed
  21 Feb 2012