Proliferation of cardiomyocytes and interstitial cells in the cardiac muscle of the mouse during pre- and postnatal development

Proliferation of cardiomyocytes and interstitial cells in the cardiac ventricle of the mouse during pre- and postnatal development was studied. Furthermore, the number of cardiomyocyte and interstitial cell nuclei per unit area was determined on histological sections. The labelling index of cardiomyocytes decreases from 23% on day 14 of gestation to about zero at 3 weeks after birth. The number of cardiomyocyte nuclei per unit area increases up to day 16 of gestation and then continuously declines. This coincides with the concept that the increase in size of the heart during early fetal life is mainly due to hyperplasia, while during late fetal life and after birth it is mainly, and during adult life exclusively, due to hypertrophy of cardiomyocytes. Proliferation of interstitial cells continues up to 5 days after birth and then decreases. The ratio of cardiomyocytes to interstitial cells decreases by a factor of about 10 between day 14 of gestation and 3 weeks after birth.     

Proliferation of Cardiomyocytes and Interstitial Cells In the Cardiac Muscle of the Mouse During Pre- and Postnatal Development

Proliferation of cardiomyocytes and interstitial cells in the cardiac ventricle of the mouse during pre- and postnatal development was studied. Furthermore, the number of cardiomyocyte and interstitial cell nuclei per unit area was determined on histological sections. The labelling index of cardiomyocytes decreases from 23% on day 14 of gestation to about zero at 3 weeks after birth. the number of cardiomyocyte nuclei per unit area increases up to day 16 of gestation and then continuously declines. This coincides with the concept that the increase in size of the heart during early fetal life is mainly due to hyperplasia, while during late fetal life and after birth it is mainly, and during adult life exclusively, due to hypertrophy of cardiomyocytes. Proliferation of interstitial cells continues up to 5 days after birth and then decreases. the ratio of cardiomyocytes to interstitial cells decreases by a factor of about 10 between day 14 of gestation and 3 weeks after birth.     

Cell cycle regulation in mouse heart during embryonic and postnatal stages

The regulation of cardiomyocyte proliferation is important for heart development and function. Proliferation levels of mouse cardiomyocytes are high during early embryogenesis and start to decrease at midgestation. Many cardiomyocytes undergo mitosis without cytokinesis, resulting in binucleated cardiomyocytes during early postnatal stages, following which the cell cycle arrests irreversibly. It remains unknown how the proliferation pattern is regulated, and how the irreversible cell cycle arrest occurs. To clarify the mechanisms, fundamental information about cell cycle regulators in cardiomyocytes and cell cycle patterns during embryonic and postnatal stages is necessary. Here, we show that the expression, complex formation, and activity of main cyclins and cyclin‐dependent kinases (CDKs) changed in a synchronous manner during embryonic and postnatal stages. These levels decreased from midgestation to birth, and then showed one wave in which the peak was around postnatal day 5. Detailed analysis of the complexes suggested that CDK activities were inhibited before the protein levels decreased. Analysis of DNA content distribution patterns in mono‐ and binucleated cardiomyocytes after birth revealed changes in cell cycle distribution patterns and the transition from mono‐ to binucleated cells. These analyses indicated that the wave of cell cycle regulator expression or activities during postnatal stages mainly produced binucleated cells from mononucleated cells. The data obtained should provide a basis for the analysis of cell cycle regulation in cardiomyocytes during embryonic and postnatal stages.     

Establishment of cardiac cytoarchitecture in the developing mouse heart

Cardiomyocytes are characterized by an extremely well-organized cytoarchitecture. We investigated its establishment in the developing mouse heart with particular reference to the myofibrils and the specialized types of cell-cell contacts, the intercalated discs (ICD). Early embryonic cardiomyocytes have a polygonal shape with cell-cell contacts distributed circumferentially at the peripheral membrane and myofibrils running in a random orientation in the sparse cytoplasm between the nucleus and the plasma membrane. During fetal development, the cardiomyocytes elongate, and the myofibrils become aligned. The restriction of the ICD components to the bipolar ends of the cells is a much slower process and is achieved for adherens junctions and desmosomes only after birth, for gap junctions even later. By quantifying the specific growth parameters of prenatal cardiomyocytes, we were able to identify a previously unknown fetal phase of physiological hypertrophy. Our results suggest (1) that myofibril alignment, bipolarization and ICD restriction happen sequentially in cardiomyocytes, and (2) that increase of heart mass in the embryo is not only achieved by hyperplasia alone but also by volume increase of the individual cardiomyocytes (hypertrophy). These observations help to understand the mechanisms that lead to the formation of a functional heart during development at a cellular level.     

MicroRNAs are dynamically regulated in hypertrophic hearts,and miR‐199a is essential for the maintenance of cell size in cardiomyocytes

Cardiac hypertrophy, which is characterized by an increase in cell size and reactivation of fetal genes, occurs as an adaptive response to diverse forms of stress and often results in heart failure and sudden death. Growing evidence indicates that microRNAs (miRNAs) are involved in cardiac hypertrophy, but the function of these miRNAs remains elusive. Here, using real time PCR analysis, we showed that several miRNAs were dynamically regulated in the rat hypertrophic hearts and miR‐199a was up‐regulated by 10‐fold in hypertrophic hearts after abdominal aorta constriction for 12 weeks. With tissue profiling analysis, we showed that miR‐199a was predominantly expressed in cardiomyocytes, but was also faintly detected in cardiac fibroblasts. To investigate whether miR‐199a was involved in cardiac hypertrophy, both over‐expression and knockdown of miR‐199a were performed in cultured cardiomyocytes. Over‐expression of miR‐199a in cardiomyocytes increased the cell size as measured by cell surface area, and also reduced the mRNA expression level of α‐myosin heavy chain. In accordance, knockdown of endogenous miR‐199a in cardiomyocytes reduced the cell size. Down‐regulation of miR‐199a also attenuated the phenylephrine‐induced increase of cell size. Furthermore, bioinformatic algorithms were used to predict the potential targets of miR‐199a in cardiac hypertrophy, and hypoxia‐inducible factor 1 alpha was confirmed by the luciferase reporter assay to be a potential target of miR‐199a. Taken together, our results demonstrated that miR‐199a, which was predominantly expressed in cardiomyocytes, was essential for the maintenance of cell size of cardiomyocytes and might play a role in the regulation of cardiac hypertrophy. J. Cell. Physiol. 225: 437–443, 2010. © 2010 Wiley‐Liss, Inc.     

The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro

Cardiac hypertrophy is a major risk factor for heart failure and associated patient morbidity and mortality. Research investigating the aberrant molecular processes that occur during cardiac hypertrophy uses primary cardiomyocytes from neonatal rat hearts as the standard experimental in vitro system. In addition, some studies make use of the H9C2 rat cardiomyoblast cell line, which has the advantage of being an animal-free alternative; however, the extent to which H9C2 cells can accurately mimic the hypertrophic responses of primary cardiac myocytes has not yet been fully established. To address this limitation, we have directly compared the hypertrophic responses of H9C2 cells with those of primary rat neonatal cardiomyocytes following stimulation with hypertrophic factors. Primary rat neonatal cardiomyocytes and H9C2 cells were cultured in vitro and treated with angiotensin II and endothelin-1 to promote hypertrophic responses. An increase in cellular footprint combined with rearrangement of cytoskeleton and induction of foetal heart genes were directly compared in both cell types using microscopy and real-time rtPCR. H9C2 cells showed almost identical hypertrophic responses to those observed in primary cardiomyocytes. This finding validates the importance of H9C2 cells as a model for in vitro studies of cardiac hypertrophy and supports current work with human cardiomyocyte cell lines for prospective molecular studies in heart development and disease.     

The number and size of human ventricular cardiomyocytes and the number of nuclei in them

The study of isolated cardiomyocytes after alkaline dissociation allowed to reveal an individual variability of the absolute number and average volume of cells, as well as characteristics of these indices in the left and right ventricle myocardia of the dead adult patients' hearts. On the basis of the analysis of these indices and of other parameters of isolated cardiomyocytes, a possibility of hyperplasia of nuclei and of muscle cells themselves during their hypertrophy is discussed.     

Multiple influences of blood flow on cardiomyocyte hypertrophy in the embryonic zebrafish heart

Cardiomyocyte hypertrophy is a complex cellular behavior involving coordination of cell size expansion and myofibril content increase. Here, we investigate the contribution of cardiomyocyte hypertrophy to cardiac chamber emergence, the process during which the primitive heart tube transforms into morphologically distinct chambers and increases its contractile strength. Focusing on the emergence of the zebrafish ventricle, we observed trends toward increased cell surface area and myofibril content. To examine the extent to which these trends reflect coordinated hypertrophy of individual ventricular cardiomyocytes, we developed a method for tracking cell surface area changes and myofibril dynamics in live embryos. Our data reveal a previously unappreciated heterogeneity of ventricular cardiomyocyte behavior during chamber emergence: although cardiomyocyte hypertrophy was prevalent, many cells did not increase their surface area or myofibril content during the observed timeframe. Despite the heterogeneity of cell behavior, we often found hypertrophic cells neighboring each other. Next, we examined the impact of blood flow on the regulation of cardiomyocyte behavior during this phase of development. When blood flow through the ventricle was reduced, cell surface area expansion and myofibril content increase were both dampened, and the behavior of neighboring cells did not seem coordinated. Together, our studies suggest a model in which hemodynamic forces have multiple influences on cardiac chamber emergence: promoting both cardiomyocyte enlargement and myofibril maturation, enhancing the extent of cardiomyocyte hypertrophy, and facilitating the coordination of neighboring cell behaviors.     

Cardiomyocytes re-enter the cell cycle and contribute to heart development after differentiation from cardiac progenitors expressing Isl1 in chick embryo

Cardiomyocytes are generated from the precardiac mesoderm and the size of the heart increases dramatically during embryogenesis. However, it is unclear how differentiation and proliferation correlate in the cardiac cell line during development. Here, we show that cardiomyocytes re-entered into a proliferative state after differentiation with a concomitant cell cycle arrest in chick embryo. The cells in the course of differentiation from Isl1-positive cardiac precursors to cardiomyocytes did not proliferate, but differentiated cardiomyocytes proliferated even after the acquisition of contractile function. After differentiation, cardiomyocytes developed a proliferative potential to contribute to the increase in cell numbers during heart development. Almost all differentiated cardiomyocytes (82.8%) incorporated bromodeoxyuridine (BrdU) in vitro, indicating the ability of DNA replication. Furthermore, mitotic chromosomes were observed in the cardiomyocytes in which a sarcomeric structure was sustained in the cytoplasm. We conclude that the sequential events of the differentiation to contractile myocytes and the re-entry into the cell cycle are strictly regulated during cardiac cell maturation. These results provide an insight into the maturation mechanism of the cardiac cell line.     

Attenuation of microRNA-22 derepressed PTEN to effectively protect rat cardiomyocytes from hypertrophy

Cardiac hypertrophy, which is characterized by the enlargement of cell size, reactivation of fetal genes, remains one of the most important triggers to heart failure. Increasing evidence shows that microRNA (miRNA) is extensively involved in the pathogenesis of cardiac hypertrophy. But the effects of miRNAs on cardiomyocyte hypertrophy have not been completely solved yet. Here, we showed that a collection of miRNAs was aberrantly expressed in hypertrophic cardiomyocytes induced by phenylephrine (PE) or angiotensin II (Ang II). Among them, miR-22 was the most strikingly up-regulated miRNA. To investigate the role of miR-22 in hypertrophy, both over-expression and knock-down assays were performed on cardiomyocytes. The results showed that up-regulation of miR-22 significantly increased the cell size and markedly influenced the expression of hypertrophic markers, including induction of nppa and reduction of myh6. In contrast, reduction of miR-22 level attenuated either PE- or Ang II-induced hypertrophic reaction. Furthermore, several genes, including PTEN, were identified as potential targets of miR-22 by bioinformatic algorithms. Using luciferase analysis, miR-22 could significantly suppress the luciferase activity of reporter fused with 3' untranslated region of PTEN mRNA. Furthermore, up-regulation of miR-22 could suppress the protein level of PTEN and reduction of miR-22 level markedly increased the protein level of PTEN in cardiomyocytes by Western blot analysis, suggesting that the contribution of miR-22 to cardiomyocyte hypertrophy may be partially through targeting PTEN. Taken together, miRNAs were dynamically regulated in cardiomyocyte hypertrophy and attenuation of miR-22 in rat cardiomyocytes efficiently protected from hypertrophic effects through derepressing PTEN.     

MiR-221 promotes cardiac hypertrophy in vitro through the modulation of p27 expression

Cardiac hypertrophy has been known as an independent predictor for cardiovascular morbidity and mortality. Molecular mechanisms underlying the development of heart failure remain elusive. Recently, microRNAs (miRs) have been established as important regulators in cardiac hypertrophy. Here, we reported miR-221 was up-regulated in both transverse aortic constricted mice and patients with hypertrophic cardiomyopathy (HCM). Forced expression of miR-221 by transfection of miR-221 mimics increased myocyte cell size and induced the re-expression of fetal genes, which were inhibited by the knockdown of endogenous miR-221 in cardiomyocytes. The TargetScan algorithm-based prediction identified that p27, a cardiac hypertrophic suppressor, is the putative target of miR-221, which was confirmed by luciferase assay and Western blotting. In conclusion, our results demonstrated that miR-221 regulated cardiomyocyte hypertrophy probably through down-regulation of p27, suggesting that miR-221 may be a new intervention target for cardiac hypertrophy.     

In vitro effects of exercise on the heart

Pathologic and physiologic factors acting on the heart can produce consistent pressure changes, volume overload, or increased cardiac output. These changes may then lead to cardiac remodeling, ultimately resulting in cardiac hypertrophy. Exercise can also induce hypertrophy, primarily physiologic in nature. To determine the mechanisms responsible for each type of remodeling, it is important to examine the heart at the functional unit, the cardiomyocyte. Tests of individual cardiomyocyte function in vitro provide a deeper understanding of the changes occurring within the heart during hypertrophy. Examination of cardiomyocyte function during exercise primarily follows one of two pathways: the addition of hypertrophic inducing agents in vitro to normal cardiomyocytes, or the use of trained animal models and isolating cells following the development of hypertrophy in vivo. Due to the short lifespan of adult cardiomyocytes, a proportionately scant amount of research exists involving the direct stimulation of cells in vitro to induce hypertrophy. These attempts provide the only current evidence, as it is difficult to gather extensive data demonstrating cell growth as a result of in vitro physical stimulation. Researchers have created ways to combine skeletal myocytes with cardiomyocytes to produce functional muscle cells used to repair pathologic heart tissue, but continue to struggle with the short lifespan of these cells. While there have been promising findings regarding the mechanisms that surround cardiac hypertrophy in vitro, the translation of in vitro findings to in vivo function is not consistent. Therefore, the focus of this review is to highlight recent studies that have investigated the effect of exercise on the heart, both in vitro and in vivo.     

Light differentially regulates cell division and the mRNA abundance of pea nucleolin during de-etiolation

The abundance of plant nucleolin mRNA is regulated during de-etiolation by phytochrome. A close correlation between the mRNA abundance of nucleolin and mitosis has also been previously reported. These results raised the question of whether the effects of light on nucleolin mRNA expression were a consequence of light effects on mitosis. To test this we compared the kinetics of light-mediated increases in cell proliferation with that of light-mediated changes in the abundance of nucleolin mRNA using plumules of dark-grown pea (Pisum sativum) seedlings. These experiments show that S-phase increases 9 h after a red light pulse, followed by M-phase increases in the plumule leaves at 12 h post-irradiation, a time course consistent with separately measured kinetics of red light-induced increases in the expression of cell cycle-regulated genes. These increases in cell cycle-regulated genes are photoreversible, implying that the light-induced increases in cell proliferation are, like nucleolin mRNA expression, regulated via phytochrome. Red light stimulates increases in the mRNA for nucleolin at 6 h post-irradiation, prior to any cell proliferation changes and concurrent with the reported timing of phytochrome-mediated increases of rRNA abundance. After a green light pulse, nucleolin mRNA levels increase without increasing S-phase or M-phase. Studies in animals and yeast indicate that nucleolin plays a significant role in ribosome biosynthesis. Consistent with this function, pea nucleolin can rescue nucleolin deletion mutants of yeast that are defective in rRNA synthesis. Our data show that during de-etiolation, the increased expression of nucleolin mRNA is more directly regulated by light than by mitosis.     

Phosphorylation of nucleolin is indispensable to upregulate miR-21 and inhibit apoptosis in cardiomyocytes

Nucleolin is a multifunctional phosphoprotein and is involved in protecting from myocardial ischemia/reperfusion (I/R) injury. The function of nucleolin is regulated by posttranslational modifications, including phosphorylation and glycosylation. To study whether phosphorylation of nucleolin (P-nucleolin) was involved in the protection from myocardial I/R injury. We investigated the expression pattern of P-nucleolin (Thr-76 and 84) in hearts subjected to I/R injury, or rat cardiac myoblast cells (H9C2) subjected to hydrogen peroxide (H ₂O ₂). The results showed that the expression of P-nucleolin and the ratio of P-nucleolin/nucleolin were significantly increased both in vivo and in vitro. Mutant nucleolin was obtained by site directed mutagenesis in vitro: threonine at 76 and 84 was replaced by alanine, and we found that the protective effect of nucleolin on apoptosis induced by oxidative stress was dependent on its phosphorylation at 76 and 84 in H9C2 cells. Furthermore, the cardio-protective roles of P-nucleolin (Thr-76 and 84) in H9C2 cardiomyocytes, were attributable to the upregulation of microRNA (miR)-21. Further analysis found that P-nucleolin (Thr-76 and 84) could bind to miR-21, and P-nucleolin colocalized with argonaute 2 (Ago2) in cytoplasm and could interact with Ago2 in a RNA-independent manner under cell oxidative stress. The current study revealed that P-nucleolin (Thr-76 and 84) increased in I/R injury myocardium, P-nucleolin was indispensable to upregulate miR-21 and inhibited apoptosis induced by H ₂O ₂ in H9C2 cardiomyocytes. These findings provided new insight into the molecular mechanisms of nucleolin in myocardial I/R injury and oxidative stress cells.     

Structural and functional heterogeneity of cardiomyocytes during hemodynamic loading of the rat heart

Ultrastructural studies of cardiomyocytes during experimental aorta coarctation enabled one to divide them into 6 types: with mitochondrial swelling and enlargement of the sarcoplasmic reticulum; with primary damage to myofibrils; with disintegration of ultrastructure because of edema; with hypertrophy and hyperplasia of ultrastructures; without essential changes in organelles; and with concomitant changes in mitochondria and myofibrils. Such different reactions of cardiomyocytes are regarded as an adaptation mechanism that ensures the maintenance of heart function under extreme conditions.