Changes in cardiac troponins with gestational age explain changes in cardiac muscle contractility in the sheep fetus

The development of the adult cardiac troponin complex in conjunction with changes in cardiac function and cardiomyocyte binucleation has not been systematically characterized during fetal life in a species where maturation of the cardiomyocytes occurs prenatally as it does in the human. The aim of this study was to correlate the expression of each of the major adult troponin isoforms (T, I, and C) during late gestation (term of 150 days) to changes in both Ca(2+) sensitivity and maximum Ca(2+)-activated force of the contractile apparatus and the maturation of cardiomyocytes. The percentage of mononucleated cardiomyocytes in the right ventricle decreased with gestational age to 46% by 137-142 days of gestation. The length of binucleated cardiomyocytes did not change with gestational age, but the length of binucleated cardiomyocytes relative to heart weight decreased with gestational age. There was no change in the expression of adult cardiac troponin T with increasing gestation. The contractile apparatus was significantly more sensitive to Ca(2+) at 90 days compared with either 132 or 139 days of gestation, consistent with an ～30% increase in the expression of adult cardiac troponin I between 90 and 110 days of gestation. Maximum Ca(2+)-activated force significantly increased from 90 days compared with 130 days consistent with an increase of ～40% in cardiac troponin C protein expression. These data show that increased adult cardiac troponin I and C protein expression across late gestation is consistent with reduced Ca(2+) sensitivity and increased maximum Ca(2+)-activated force. Furthermore, changes in cardiac troponin C, not I, protein expression track with the timing of cardiomyocyte binucleation.     

Treatment of immature mice with gonadotrophins. Effects on some enzymic activities of unfractionated ovarian homogenates

Treatment of immature mice with both follicle-stimulating hormone and human chorionic gonadotrophin in vivo resulted in large increases in the specific activities of ovarian alkaline phosphatase and alkaline nucleotidase. The specific activities of other ovarian enzymes studied were not altered by gonadotrophin treatment. A simultaneous change in the Michaelis constant of ovarian alkaline phosphatase accompanied the increase in specific activity. These changes commenced 6-8h after injection of human chorionic gonadotrophin plus follicle-stimulating hormone. Injection of human chorionic gonadotrophin induced the change in Michaelis constant and increased ovarian alkaline phosphatase activity. Treatment with follicle-stimulating hormone had no effect on ovarian alkaline phosphatase. However, follicle-stimulating hormone synergistically augmented the response to human chorionic gonadotrophin. A latent period of about 24h elapsed before this augmentation was expressed. Augmentation of ovarian alkaline phosphatase was directly related to the dose of follicle-stimulating hormone at a fixed dose of chorionic gonadotrophin. No response of ovarian alkaline phosphatase was observed after treatment of immature mice in vivo with oestrogens, progesterone, growth hormone or prolactin. Unlike chorionic gonadotrophin, sheep luteinizing hormone over a wide dose range induced no response within 24h. However, a response in ovarian alkaline phosphatase was observed when sheep luteinizing hormone was administered in combination with follicle-stimulating hormone. The specific activity and K(m) of ovarian alkaline phosphatase increased during normal maturation. The Michaelis constant ceased to increase as sexual maturity was reached. The changes in alkaline phosphatase activity were of a similar magnitude to those induced by gonadotrophin treatment. It is concluded that the changes induced acutely by treatment in vivo with unphysiological doses of gonadotrophins occur in the maturing mouse under the influence of endogenous, homologous gonadotrophins at physiological concentrations.     

Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro

It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.     

Central role of mitofusin 2 in autophagosome-lysosome fusion in cardiomyocytes

In the heart, autophagy has been implicated in cardioprotection and ischemia-reperfusion tolerance, and the dysregulation of autophagy is associated with the development of heart failure. Mitochondrial dynamic proteins are profoundly involved in autophagic processes, especially the initiation and formation of autophagosomes, but it is not clear whether they play any role in cardiac autophagy. We previously reported that mitofusin 2 (MFN2), a mitochondrial outer membrane protein, serves as a major determinant of cardiomyocyte apoptosis mediated by oxidative stress. Here, we reveal a novel and essential role of MFN2 in mediating cardiac autophagy. We found that specific deletion of MFN2 in cardiomyocytes caused extensive accumulation of autophagosomes. In particular, the fusion of autophagosomes with lysosomes, a critical step in autophagic degradation, was markedly retarded without altering the formation of autophagosomes and lysosomes in response to ischemia-reperfusion stress. Importantly, MFN2 co-immunoprecipitated with RAB7 in the heart, and starvation further increased it. Knockdown of MFN2 by shRNA prevented, whereas re-expression of MFN2 restored, the autophagosome-lysosome fusion in neonatal cardiomyocytes. Hearts from cardiac-specific MFN2 knock-out mice had abnormal mitochondrial and cellular metabolism and were vulnerable to ischemia-reperfusion challenge. Our study defined a novel and essential role of MFN2 in the cardiac autophagic process by mediating the maturation of autophagy at the phase of autophagosome-lysosome fusion; deficiency of MFN2 caused multiple molecular and functional defects that undermined cardiac reserve and gradually led to cardiac vulnerability and dysfunction.     

Regulation of Ncx1 expression. Identification of regulatory elements mediating cardiac-specific expression and up-regulation

The Na+-Ca2+ exchanger (NCX1) is up-regulated in hypertrophy and is often found up-regulated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. We have previously shown that the 1831-bp Ncx1 H1 (1831Ncx1) promoter directs cardiac-specific expression of the exchanger in both development and in the adult, and is sufficient for the up-regulation of Ncx1 in response to pressure overload. Here, we utilized adenoviral mediated gene transfer and transgenics to identify minimal regions and response elements that mediate Ncx1 expression in the heart. We demonstrate that the proximal 184 bp of the Ncx1 H1 (184Ncx1) promoter is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for up-regulation in response to pressure overload. Mutational analysis revealed that both the -80 CArG and the -50 GATA elements were required for expression in isolated adult cardiomyocytes. Chromatin immunoprecipitation assays in adult cardiocytes demonstrate that SRF and GATA4 are associated with the proximal region of the endogenous Ncx1 promoter. Transgenic lines were established for the 1831Ncx1 promoter-luciferase containing mutations in the -80 CArG or -50 GATA element. No luciferase activity was detected during development, in the adult, or after pressure overload in any of the -80 CArG transgenic lines. The Ncx1 -50 GATA mutant promoter was sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in development and for up-regulation in response to pressure overload but importantly, expression was no longer cardiac restricted. This work is the first in vivo study that demonstrates which cis elements are important for Ncx1 regulation.     

BMP-2 regulates cardiomyocyte contractility in a phosphatidylinositol 3 kinase-dependent manner

Bone morphogenetic protein-2 (BMP-2) regulates development of heart during vertebrate embryogenesis. In vitro BMP-2 induces differentiation of precardiac cells into mature cardiomyocytes by inducing the expression of cardiac-specific genes. However, the role of BMP-2 and its signaling in other cardiac functions have not been studied. We examined the action of phosphatidylinositol (PI) 3 kinase in isolated adult rat cardiomyocytes. Incubation of rat ventricular cardiomyocytes with BMP-2 increased the PI 3 kinase activity. Ly294002, a pharmacological inhibitor of PI 3 kinase, blocked BMP-2-induced PI 3 kinase activity completely. To investigate the contractility of isolated cardiomyocytes, fractional shortening was examined. BMP-2 significantly increased the percent fractional shortening of the cardiomyocytes. Inhibition of PI 3 kinase activity completely abolished this action of BMP-2. These data indicate that PI 3 kinase regulates BMP-2-induced myocyte contractility. To further confirm this observation, we used adenovirus-mediated gene transfer to express a constitutively active myristoylated catalytic subunit of PI 3 kinase in rat cardiomyocytes. Infection of cardiomyocytes with the adenovirus vector increased the expression of constitutively active PI 3 kinase within 24 h. Expression of constitutively active PI 3 kinase significantly increased cardiomyocyte contractility. Together, these data show for the first time that the growth and differentiation factor, BMP-2, stimulates cardiomyocyte contractility. Also we provide the first evidence that BMP-2-induced PI 3 kinase activity regulates this cardiomyocyte function.     

Ghrelin signaling in heart remodeling of adult obese mice

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), has been suggested to be associated to obesity, insulin secretion, cardiovascular growth and homeostasis. GHS-R has been found in most of the tissues, and among the hormone action it is included the regulation of heart energy metabolism. Therefore, hypernutrition during early life leads to obesity, induces cardiac hypertrophy, compromises myocardial function, inducing heart failure in adulthood. We examined ghrelin signaling process in cardiac remodeling in these obese adult mice. The cardiomyocytes (cmy) of left ventricle were analyzed by light microscopy and stereology, content and phosphorilation of cardiac proteins: ghrelin receptor (growth hormone secretagogue receptor 1a, GHSR-1a), protein kinase B (AKT and pAKT), phosphatidil inositol 3 kinase (PI3K), AMP-activated protein kinase (AMPK and pAMPK) and actin were achieved by Western blotting. GHSR-1a gene expression was analyzed by Real Time-PCR. We observed hyperglycemia and higher liver and visceral fat weight in obese when compared to control group. Obese mice presented a marked increase in heart weight/tibia length, indicating an enlarged heart size or a remodeling process. Obese mice had increased GHSR-1a content and expression in the heart associated to PI3K content and increased AKT content and phosphorylation. In contrast, AMPK content and phosphorylation in heart was not different between experimental groups. Ghrelin plasma levels in obese group were decreased when compared to control group. Our data suggest that remodeled myocardial in adult obese mice overnourished in early life are associated with higher phosphorylation of GHSR-1a, PI3K and AKT but not with AMPK.     

Effects of raloxifene and estradiol on bone turnover parameters in intact and ovariectomized rats

This study was designed to investigate effects of raloxifene (RLX) and estradiol on bone formation and resorption in intact and ovariectomized (ovx) rat models. In the intact model, a total of 24 adult female rats were divided into three groups: Controls subcutaneously received saline alone. RLX (2 mg/kg) and estradiol (30 μg/kg) were injected to two groups of animals for a period of 6 weeks at two daily intervals. In the second model, rats (n = 24) were ovx and allowed to recover for a period of at least 3 weeks. Control group received vehicle alone. Remaining rats were divided into two groups and injected with RLX (2 mg/kg) and estradiol (30 μg/kg) for 6 weeks. Urine samples were collected from all animals 24 h after the last drug administration. Urinary deoxypyridinoline (DPD) was measured by ELISA. Serum parathyroid hormone (PTH), calcitonin, and osteocalcin levels were measured by immunoradiometric method. Serum concentrations of alkaline phosphatase (ALP), Ca, and inorganic phosphate were determined by enzymatic–colorimetric method. Lumbar vertebrae (L2) of all animals were dissected out and processed for histopathological evaluation. Removal of ovaries significantly elevated urinary DPD levels (p < 0.01) compared with intact controls. Treatment of both intact and ovx rats with estradiol resulted in significant decreases (p < 0.01) in DPD values. RLX administration had no significant effect in the intact rats, but it remarkably reduced bone turnover in the ovx animals (p < 0.001). Both estradiol and RLX produced conflicting effects on serum ALP, osteocalcin, and PTH levels in both animal models. These findings suggest that RLX exerts its protective effects by reducing bone resorption, similar to that of estradiol, in ovx rats.     

Progenitor cell therapy for heart disease

Many cell types are currently being studied as potential sources of cardiomyocytes for cell transplantation therapy to repair and regenerate damaged myocardium. The question remains as to which progenitor cell represents the best candidate. Bone marrow-derived cells and endothelial progenitor cells have been tested in clinical studies. These cells are safe, but their cardiogenic potential is controversial. The functional benefits observed are probably due to enhanced angiogenesis, reduced ventricular remodeling, or to cytokine-mediated effects that promote the survival of endogenous cells. Human embryonic stem cells represent an unlimited source of cardiomyocytes due to their great differentiation potential, but each step of differentiation must be tightly controlled due to the high risk of teratoma formation. These cells, however, confront ethical barriers and there is a risk of graft rejection. These last two problems can be avoided by using induced pluripotent stem cells (iPS), which can be autologously derived, but the high risk of teratoma formation remains. Cardiac progenitor cells have the advantage of being cardiac committed, but important questions remain unanswered, such as what is the best marker to identify and isolate these cells? To date the different markers used to identify adult cardiac progenitor cells also recognize progenitor cells that are outside the heart. Thus, it cannot be determined whether the cardiac progenitor cells identified in the adult heart represent resident cells present since fetal life or extracardiac cells that colonized the heart after cardiac injury. Developmental studies have identified markers of multipotent progenitors, but it is unknown whether these markers are specific for adult progenitors when expressed in the adult myocardium. Cardiac regeneration is dependent on the stability of the cells transplanted into the host myocardium and on the electromechanical coupling with the endogenous cells. Finally, the promotion of endogenous regenerative processes by mobilizing endogenous progenitors represents a complementary approach to cell transplantation therapy.     

A novel type of self-beating cardiomyocytes in adult mouse ventricles

This study was designed to investigate the presence of resident heart cells that are distinct from terminally-differentiated cardiomyocytes. Adult mouse heart was coronary perfused with collagenase, and ventricles were excised and further digested. After spinning cardiomyocyte-containing fractions down, the supernatant fraction was collected and cultured without adding any chemicals. Two to five days after plating, some of rounded cells adhered to the culture dish, gradually changed their shape and then started self-beating. These self-beating cells did not appreciably proliferate but underwent a further morphological maturation process to form highly branched shapes with many projections. These cells were mostly multinucleated, well sarcomeric-organized and expressed cardiac marker proteins, defined as atypically-shaped cardiomyocytes (ACMs). Patch-clamp experiments revealed that ACMs exhibited spontaneous action potentials arising from the preceding slow diastolic depolarization. We thus found a novel type of resident heart cells in adult cardiac ventricles that spontaneously develop into self-beating cardiomyocytes.     

The pro-angiogenic cytokine pleiotrophin potentiates cardiomyocyte apoptosis through inhibition of endogenous AKT/PKB activity

Pleiotrophin is a development-regulated cytokine and growth factor that can promote angiogenesis, cell proliferation, or differentiation, and it has been reported to have neovasculogenic effects in damaged heart. Developmentally, it is prominently expressed in fetal and neonatal hearts, but it is minimally expressed in normal adult heart. Conversely, we show in a rat model of myocardial infarction and in human dilated cardiomyopathy that pleiotrophin is markedly up-regulated. To elucidate the effects of pleiotrophin on cardiac contractile cells, we employed primary cultures of rat neonatal and adult cardiomyocytes. We show that pleiotrophin is released from cardiomyocytes in vitro in response to hypoxia and that the addition of recombinant pleiotrophin promotes caspase-mediated genomic DNA fragmentation in a dose- and time-dependent manner. Functionally, it potentiates the apoptotic response of neonatal cardiomyocytes to hypoxic stress and to ultraviolet irradiation and of adult cardiomyocytes to hypoxia-reoxygenation. Moreover, UV-induced apoptosis in neonatal cardiomyocytes can be partially inhibited by small interfering RNA-mediated knockdown of endogenous pleiotrophin. Mechanistically, pleiotrophin antagonizes IGF-1 associated Ser-473 phosphorylation of AKT/PKB, and it concomitantly decreases both BAD and GSK3beta phosphorylation. Adenoviral expression of constitutively active AKT and lithium chloride-mediated inhibition of GSK3beta reduce the potentiated programmed cell death elicited by pleiotrophin. These latter data indicate that pleiotrophin potentiates cardiomyocyte cell death, at least partially, through inhibition of AKT signaling. In conclusion, we have uncovered a novel function for pleiotrophin on heart cells following injury. It fosters cardiomyocyte programmed cell death in response to pro-apoptotic stress, which may be critical to myocardial injury repair.