Maternal undernutrition induces differential cardiac gene expression in pulmonary hypertensive steers at high elevation

Pulmonary hypertension, characterized by elevated pulmonary arterial pressure (PAP) and right ventricular hypertrophy, is caused by decreased atmospheric oxygen at high altitude. We hypothesized that maternal undernutrition programs right ventricle gene expression and sensitivity to increasing PAP at high altitude (2,183 m). On day 30 of gestation, forty Angus x Gelbvieh cows received diets to induce either gain (Control) or loss of body weight (Restricted) until day 125 of gestation. On day 126 of gestation, Restricted cows were realimented to achieve the same body weight as Controls by day 250. Parturition occurred naturally. PAP, which ranged from 40 to 114 mmHg, was determined in 15-mo-old steers from Control or Restricted cows before necropsy. At necropsy, hearts were collected from steers, separated into right and left ventricles, atria, and septa and weighed. Ventricular thickness was recorded. Eight Affymetrix bovine microarrays were screened [four high PAP (two Control and two Restricted) and four low PAP (two Control and two Restricted)] with right ventricle mRNA. This analysis revealed that pentraxin-related protein, interferon-related developmental regulator, and peroxisome proliferator-activated receptor-gamma coactivator-1alpha were differentially expressed (P < 0.05) in steer right ventricle from high-PAP cows compared with low-PAP cows. Also, activation peptide and pancreas cationic trypsinogen, alpha-actin, similar to ubiquitin carboxylesterase, were differently expressed (P < 0.05) in steers from Restricted cows compared with those from Control cows. Upregulated genes in high-PAP right ventricle have been associated with pathological cardiac hypertrophy. It is concluded that right ventricle gene expression may be differentially programmed by maternal undernutrition in the fetus during early gestation and may be detrimental to health and longevity of offspring, particularly at high altitude.     

Dexamethasone Treatment of Newborn Rats Decreases Cardiomyocyte Endowment in the Developing Heart through Epigenetic Modifications

The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1), 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2''-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner.     

Cyclin A2 mediates cardiomyocyte mitosis in the postmitotic myocardium

Cell cycle withdrawal limits proliferation of adult mammalian cardiomyocytes. Therefore, the concept of stimulating myocyte mitotic divisions has dramatic implications for cardiomyocyte regeneration and hence, cardiovascular disease. Previous reports describing manipulation of cell cycle proteins have not shown induction of cardiomyocyte mitosis after birth. We now report that cyclin A2, normally silenced in the postnatal heart, induces cardiac enlargement because of cardiomyocyte hyperplasia when constitutively expressed from embryonic day 8 into adulthood. Cardiomyocyte hyperplasia during adulthood was coupled with an increase in cardiomyocyte mitosis, noted in transgenic hearts at all time points examined, particularly during postnatal development. Several stages of mitosis were observed within cardiomyocytes and correlated with the nuclear localization of cyclin A2. Magnetic resonance analysis confirmed cardiac enlargement. These results reveal a previously unrecognized critical role for cyclin A2 in mediating cardiomyocyte mitosis, a role that may significantly impact upon clinical treatment of damaged myocardium.     

A novel cardiomyocyte-enriched microRNA, miR-378, targets insulin-like growth factor 1 receptor: implications in postnatal cardiac remodeling and cell survival

Postnatal cardiac remodeling is characterized by a marked decrease in the insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R) expression. The underlying mechanism remains unexplored. This study examined the role of microRNAs in postnatal cardiac remodeling. By expression profiling, we observed a 10-fold increase in miR-378 expression in 1-week-old neonatal mouse hearts compared with 16-day-old fetal hearts. There was also a 4-6-fold induction in expression of miR-378 in older (10 months) compared with younger (1 month) hearts. Interestingly, tissue distribution analysis identified miR-378 to be highly abundant in heart and skeletal muscles. In the heart, specific expression was observed in cardiac myocytes, which was inducible by a variety of stressors. Overexpression of miR-378 enhanced apoptosis of cardiomyocytes by direct targeting of IGF1R and reduced signaling in Akt cascade. The inhibition of miR-378 by its anti-miR protected cardiomyocytes against H(2)O(2) and hypoxia reoxygenation-induced cell death by promoting IGF1R expression and downstream Akt signaling cascade. Additionally, our data show that miR-378 expression is inhibited by IGF1 in cardiomyocytes. In tissues such as fibroblasts and fetal hearts, where IGF1 levels are high, we found either absent or significantly low miR-378 levels, suggesting an inverse relationship between these two factors. Our study identifies miR-378 as a new cardioabundant microRNA that targets IGF1R. We also demonstrate the existence of a negative feedback loop between miR-378, IGF1R, and IGF1 that is associated with postnatal cardiac remodeling and with the regulation of cardiomyocyte survival during stress.     

Overexpression of microRNA-99a Attenuates Cardiac Hypertrophy

Pathological cardiomyocyte hypertrophy is associated with significantly increased risk of heart failure, one of the leading medical causes of mortality worldwide. MicroRNAs are known to be involved in pathological cardiac remodeling. However, whether miR-99a participates in the signaling cascade leading to cardiac hypertrophy is unknown. To evaluate the role of miR-99a in cardiac hypertrophy, we assessed the expression of miR-99a in hypertrophic cardiomyocytes induced by isoprenaline (ISO)/angiotensin-II (Ang II) and in mice model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Expression of miR-99a was evaluated in these hypertrophic cells and hearts. We also found that miR-99a expression was highly correlated with cardiac function of mice with heart failure (8 weeks after TAC surgery). Overexpression of miR-99a attenuated cardiac hypertrophy in TAC mice and cellular hypertrophy in stimuli treated cardiomyocytes through down-regulation of expression of mammalian target of rapamycin (mTOR). These results indicate that miR-99a negatively regulates physiological hypertrophy through mTOR signaling pathway, which may provide a new therapeutic approach for pressure-overload heart failure.     

Alcohol exposure during late gestation adversely affects myocardial development with implications for postnatal cardiac function

Prenatal exposure to high levels of ethanol is associated with cardiac malformations, but the effects of lower levels of exposure on the heart are unclear. Our aim was to investigate the effects of daily exposure to ethanol during late gestation, when cardiomyocytes are undergoing maturation, on the developing myocardium. Pregnant ewes were infused with either ethanol (0.75 g/kg) or saline for 1 h each day from gestational days 95 to 133 (term ～145 days); tissues were collected at 134 days. In sheep, cardiomyocytes mature during late gestation as in humans. Within the left ventricle (LV), cardiomyocyte number was determined using unbiased stereology and cardiomyocyte size and nuclearity determined using confocal microscopy. Collagen deposition was quantified using image analysis. Genes relating to cardiomyocyte proliferation and apoptosis were examined using quantitative real-time PCR. Fetal plasma ethanol concentration reached 0.11 g/dL after EtOH infusions. Ethanol exposure induced significant increases in relative heart weight, relative LV wall volume, and cardiomyocyte cross-sectional area. Ethanol exposure advanced LV maturation in that the proportion of binucleated cardiomyocytes increased by 12%, and the number of mononucleated cardiomyocytes was decreased by a similar amount. Apoptotic gene expression increased in the ethanol-exposed hearts, although there were no significant differences between groups in total cardiomyocyte number or interstitial collagen. Daily exposure to a moderate dose of ethanol in late gestation accelerates the maturation of cardiomyocytes and increases cardiomyocyte and LV tissue volume in the fetal heart. These effects on cardiomyocyte growth may program for long-term cardiac vulnerability.     

HMGA1 is a new target of miR-195 involving isoprenaline-induced cardiomyocyte hypertrophy

Emerging data have shown that microRNAs (miRNAs) have important functions in the processes of cardiac hypertrophy and heart failure that occur during the postnatal period. Cardiac overexpression of miR-195 results in pathological cardiac growth and heart failure in transgenic mice. In the present study, we analyzed the roles of miR-195 in cardiomyocyte hypertrophy and found that miR-195 was greatly upregulated during isoprenaline-induced cardiomyocyte hypertrophy. By using mRNA microarray and molecular approach, we identified a novel putative target of miR-195 called high-mobility group A1 (HMGA1). Total mRNA microarray showed that HMGA1 was downregulated in primary cardiomyocytes that overexpressed miR-195. Using luciferase activity assay, we demonstrated that miR-195 interacts with the 3′-untranslated region of HMGA1 mRNA. Moreover, we showed that miR-195 in primary cardiomyocytes downregulates the expression of HMGA1 at the protein level. Taken together, our data demonstrated that miR-195 can negatively regulate a new target, HMGA1, which is involved in cardiomyocyte hypertrophy.     

Deficiency of Cardiomyocyte-specific MicroRNA-378 Contributes to the Development of Cardiac Fibrosis Involving a Transforming Growth Factor β (TGFβ1)-dependent Paracrine Mechanism

Understanding the regulation of cardiac fibrosis is critical for controlling adverse cardiac remodeling during heart failure. Previously we identified miR-378 as a cardiomyocyte-abundant miRNA down-regulated in several experimental models of cardiac hypertrophy and in patients with heart failure. To understand the consequence of miR-378 down-regulation during cardiac remodeling, our current study employed a locked nucleic acid-modified antimiR to target miR-378 in vivo. Results showed development of cardiomyocyte hypertrophy and fibrosis in mouse hearts. Mechanistically, miR-378 depletion was found to induce TGFβ1 expression in mouse hearts and in cultured cardiomyocytes. Among various secreted cytokines in the conditioned-media of miR-378-depleted cardiomyocytes, only TGFβ1 levels were found to be increased. The increase was prevented by miR-378 expression. Treatment of cardiac fibroblasts with the conditioned media of miR-378-depleted myocytes activated pSMAD2/3 and induced fibrotic gene expression. This effect was counteracted by including a TGFβ1-neutralizing antibody in the conditioned-medium. In cardiomyocytes, adenoviruses expressing dominant negative N-Ras or c-Jun prevented antimiR-mediated induction of TGFβ1 mRNA, documenting the importance of Ras and AP-1 signaling in this response. Our study demonstrates that reduction of miR-378 during pathological conditions contributes to cardiac remodeling by promoting paracrine release of profibrotic cytokine, TGFβ1 from cardiomyocytes. Our data imply that the presence in cardiomyocyte of miR-378 plays a critical role in the protection of neighboring fibroblasts from activation by pro-fibrotic stimuli.     

MicroRNA-375-3p inhibitor suppresses angiotensin II-induced cardiomyocyte hypertrophy by promoting lactate dehydrogenase B expression

Cardiac hypertrophy is a myocardial enlargement due to overload pressure, and the primary cause of heart failure. We investigated the function of miR-375-3p in cardiac hypertrophy and its regulating mechanisms. miR-375-3p was upregulated in hearts of the transverse aortic constriction rat model and angiotensin II (Ang II)-induced primary cardiomyocyte hypertrophy model; the opposite was observed for lactate dehydrogenase B (LDHB) protein expression. miR-375-3p knockdown reduced the surface area of primary cardiomyocytes increased by Ang II treatment and decreased the B-natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) messenger RNA (mRNA) and protein levels. miR-375-3p was also observed to directly target LDHB. LDHB knockdown increased the surface area of Ang II-treated primary cardiomyocytes and increased the BNP and β-MHC mRNA and protein levels. LDHB knockdown attenuated the effects of miR-375-3p on the surface area of primary cardiomyocytes and BNP and β-MHC levels. Therefore, miR-375-3p inhibitor suppresses Ang II-induced cardiomyocyte hypertrophy by promoting LDHB expression.     

Cyclin I and p53 are differentially expressed during the terminal differentiation of the postnatal mouse heart

In this study, we have used Ki-67 and MF20 mAb to determine how extensively cardiomyocytes proliferate in the postnatal mouse heart. It was established that the cardiomyocytes divided rapidly in 2-day-old hearts. However, at 13 days, the majority of cardiomyocytes had entered into terminal growth arrest and differentiation. We exploited this finding in order to identify proteins that were associated with cardiomyocyte growth and differentiation. The protein profiles of 2- and 13-day-old hearts were established by two-dimensional electrophoresis and compared. Seventeen protein spots were found to be differentially expressed at day 13. Eight of them were up-regulated while the remaining nine protein spots were down-regulated. We focused our attention on 2 of the proteins identified by MALDI-TOF MS, cyclin I and p53, because they are both believed to be involved in cell cycle regulation. Western blot analysis confirmed that both proteins were positively up-regulated in the 13-day-old postnatal heart. To determine directly whether these proteins were associated with cell proliferation, we examined their expression patterns in H9c2 cardiomyocytes maintained in vitro. We established that cyclin I expression was low during the growing phase of H9c2 culture and high during the growth arrest/differentiation phases. In contrast, p53 expression was unchanged during both phases. The various growth phases were confirmed by the presence of cyclin A and growth arrest-specific 1 proteins. We investigated whether silencing cyclin I expression using cyclin I-siRNA could promote an increase in H9c2 cell proliferation. It was determined that silencing cyclin I could enhance a small, but significant, increase in H9c2 cell division. Similar results were obtained for cardiomyocytes extracted from 13-day-old hearts. These results imply that the reason why cardiomyocytes in 13-day-old hearts increased cyclin I expression was probably associated with terminal growth arrest. However, the increase in p53 expression was probably associated with cardiomyocyte differentiation, rather than growth arrest.     

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.     

Maternal hypoxia alters matrix metalloproteinase expression patterns and causes cardiac remodeling in fetal and neonatal rats

Fetal hypoxia leads to progressive cardiac remodeling in rat offspring. The present study tested the hypothesis that maternal hypoxia results in reprogramming of matrix metalloproteinase (MMP) expression patterns and fibrillar collagen matrix in the developing heart. Pregnant rats were treated with normoxia or hypoxia (10.5% O(2)) from day 15 to 21 of gestation. Hearts were isolated from 21-day fetuses (E21) and postnatal day 7 pups (PD7). Maternal hypoxia caused a decrease in the body weight of both E21 and PD7. The heart-to-body weight ratio was increased in E21 but not in PD7. Left ventricular myocardium wall thickness and cardiomyocyte proliferation were significantly decreased in both fetal and neonatal hearts. Hypoxia had no effect on fibrillar collagen content in the fetal heart, but significantly increased the collagen content in the neonatal heart. Western blotting revealed that maternal hypoxia significantly increased collagen I, but not collagen III, levels in the neonatal heart. Maternal hypoxia decreased MMP-1 but increased MMP-13 and membrane type (MT)1-MMP in the fetal heart. In the neonatal heart, MMP-1 and MMP-13 were significantly increased. Active MMP-2 and MMP-9 levels and activities were not altered in either fetal or neonatal hearts. Hypoxia significantly increased tissue inhibitors of metalloproteinase (TIMP)-3 and TIMP-4 in both fetal and neonatal hearts. In contrast, TIMP-1 and TIMP-2 were not affected. The results demonstrate that in utero hypoxia reprograms the expression patterns of MMPs and TIMPs and causes cardiac tissue remodeling with the increased collagen deposition in the developing heart.     

Newborn Hypoxia/Anoxia Inhibits Cardiomyocyte Proliferation and Decreases Cardiomyocyte Endowment in the Developing Heart: Role of Endothelin-1

In the developing heart, cardiomyocytes undergo terminal differentiation during a critical window around birth. Hypoxia is a major stress to preterm infants, yet its effect on the development and maturation of the heart remains unknown. We tested the hypothesis in a rat model that newborn anoxia accelerates cardiomyocyte terminal differentiation and results in reduced cardiomyocyte endowment in the developing heart via an endothelin-1-dependent mechanism. Newborn rats were exposed to anoxia twice daily from postnatal day 1 to 3, and hearts were isolated and studied at postnatal day 4 (P4), 7 (P7), and 14 (P14). Anoxia significantly increased HIF-1α protein expression and pre-proET-1 mRNA abundance in P4 neonatal hearts. Cardiomyocyte proliferation was significantly decreased by anoxia in P4 and P7, resulting in a significant reduction of cardiomyocyte number per heart weight in the P14 neonates. Furthermore, the expression of cyclin D2 was significantly decreased due to anoxia, while p27 expression was increased. Anoxia has no significant effect on cardiomyocyte binucleation or myocyte size. Consistently, prenatal hypoxia significantly decreased cardiomyocyte proliferation but had no effect on binucleation in the fetal heart. Newborn administration of PD156707, an ET_A-receptor antagonist, significantly increased cardiomyocyte proliferation at P4 and cell size at P7, resulting in an increase in the heart to body weight ratio in P7 neonates. In addition, PD156707 abrogated the anoxia-mediated effects. The results suggest that hypoxia and anoxia via activation of endothelin-1 at the critical window of heart development inhibits cardiomyocyte proliferation and decreases myocyte endowment in the developing heart, which may negatively impact cardiac function later in life.     

Tom70 serves as a molecular switch to determine pathological cardiac hypertrophy

Pathological cardiac hypertrophy is an inevitable forerunner of heart failure. Regardless of the etiology of cardiac hypertrophy, cardiomyocyte mitochondrial alterations are always observed in this context. The translocases of mitochondrial outer membrane (Tom) complex governs the import of mitochondrial precursor proteins to maintain mitochondrial function under pathophysiological conditions; however, its role in the development of pathological cardiac hypertrophy remains unclear. Here, we showed that Tom70 was downregulated in pathological hypertrophic hearts from humans and experimental animals. The reduction in Tom70 expression produced distinct pathological cardiomyocyte hypertrophy both in vivo and in vitro. The defective mitochondrial import of Tom70-targeted optic atrophy-1 triggered intracellular oxidative stress, which led to a pathological cellular response. Importantly, increased Tom70 levels provided cardiomyocytes with full resistance to diverse pro-hypertrophic insults. Together, these results reveal that Tom70 acts as a molecular switch that orchestrates hypertrophic stresses and mitochondrial responses to determine pathological cardiac hypertrophy.     

Overexpression of bone morphogenetic protein 10 in myocardium disrupts cardiac postnatal hypertrophic growth

Postnatal cardiac hypertrophies have traditionally been classified into physiological or pathological hypertrophies. Both of them are induced by hemodynamic load. Cardiac postnatal hypertrophic growth is regarded as a part of the cardiac maturation process that is independent of the cardiac working load. However, the functional significance of this biological event has not been determined, mainly because of the difficulty in creating an experimental condition for testing the growth potential of functioning heart in the absence of hemodynamic load. Recently, we generated a novel transgenic mouse model (alphaMHC-BMP10) in which the cardiac-specific growth factor bone morphogenetic protein 10 (BMP10) is overexpressed in postnatal myocardium. These alphaMHC-BMP10 mice appear to have normal cardiogenesis throughout embryogenesis, but develop to smaller hearts within 6 weeks after birth. alphaMHC-BMP10 hearts are about half the normal size with 100% penetrance. Detailed morphometric analysis of cardiomyocytes clearly indicated that the compromised cardiac growth in alphaMHC-BMP10 mice was solely because of defect in cardiomyocyte postnatal hypertrophic growth. Physiological analysis further demonstrated that the responses of these hearts to both physiological (e.g. exercise-induced hypertrophy) and pathological hypertrophic stimuli remain normal. In addition, the alphaMHC-BMP10 mice develop subaortic narrowing and concentric myocardial thickening without obstruction by four weeks of age. Systematic analysis of potential intracellular pathways further suggested a novel genetic pathway regulating this previously undefined cardiac postnatal hypertrophic growth event. This is the first demonstration that cardiac postnatal hypertrophic growth can be specifically modified genetically and dissected out from physiological and pathological hypertrophies.