Postnatal Myocardium Research Articles

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.

The post-natal heart contains a myocardial stem cell population

The recent identification of stem cell pools in a variety of unexpected tissue sources has raised the possibility that a pluripotent stem cell population may reside in the myocardium and contribute to the post-natal growth of this tissue. Here, we demonstrate that the post-natal myocardium contains a resident verapamil-sensitive side population (SP), with stem cell-like activity. When growth of the post-natal heart was attenuated through over-expression of a dominant negative cardiac transcription factor (MEF2C), the resident SP cell population was subject to activation, followed by a consequent depletion. In addition, cardiac SP cells are capable of fusion with other cell types, but do not adopt the corresponding gene expression profile. These observations suggest that a responsive stem cell pool resides in the adult myocardium, and may influence adaptation of the post-natal heart.

Metabolic adaptation of the fetal and postnatal ovine heart: regulatory role of hypoxia-inducible factors and nuclear respiratory factor-1.

Numerous metabolic adaptations occur in the heart after birth. Important transcription factors that regulate expression of the glycolytic and mitochondrial oxidative genes are hypoxia-inducible factors (HIF-1alpha and -2alpha) and nuclear respiratory factor-1 (NRF-1). The goal of this study was to examine expression of HIF-1alpha, HIF-2alpha, and NRF-1 and the genes they regulate in pre- and postnatal myocardium. Ovine right and left ventricular myocardium was obtained at four time points: 95 and 140 d gestation (term = 145 d) and 7 d and 8 wk postnatally. Steady-state mRNA and protein levels of HIF-1alpha and NRF-1 and protein levels of HIF-2alpha were measured along with mRNA of HIF-1alpha-regulated genes (aldolase A, alpha- and beta-enolase, lactate dehydrogenase A, liver and muscle phosphofructokinase) and NRF-1-regulated genes (cytochrome c, Va subunit of cytochrome oxidase, and carnitine palmitoyltransferase I ). HIF-1alpha protein was present in fetal myocardium but dropped below detectable levels at 7 d postnatally. HIF-2alpha protein levels were similar at the four time points. Steady-state mRNA levels of alpha-enolase, lactate dehydrogenase A, and liver phosphofructokinase declined significantly postnatally. Aldolase A, beta-enolase, and muscle phosphofructokinase mRNA levels increased postnatally. Steady-state mRNA and protein levels of NRF-1 decreased postnatally in contrast to the postnatal increases in cytochrome c, subunit Va of cytochrome oxidase, and carnitine palmitoyltransferase I mRNA levels. The in vivo postnatal regulation of enzymes encoding glycolytic and mitochondrial enzymes is complex. As transactivation response elements for the genes encoding metabolic enzymes continue to be characterized, studies using the fetal-to-postnatal metabolic transition of the heart will continue to help define the in vivo role of these transcription factors.

Metabolic Adaptation of the Fetal and Postnatal Ovine Heart: Regulatory Role of Hypoxia-Inducible Factors and Nuclear Respiratory Factor-1

Numerous metabolic adaptations occur in the heart after birth. Important transcription factors that regulate expression of the glycolytic and mitochondrial oxidative genes are hypoxia-inducible factors (HIF-1α and -2α) and nuclear respiratory factor-1 (NRF-1). The goal of this study was to examine expression of HIF-1α, HIF-2α, and NRF-1 and the genes they regulate in pre- and postnatal myocardium. Ovine right and left ventricular myocardium was obtained at four time points: 95 and 140 d gestation (term = 145 d) and 7 d and 8 wk postnatally. Steady-state mRNA and protein levels of HIF-1α and NRF-1 and protein levels of HIF-2α were measured along with mRNA of HIF-1α-regulated genes (aldolase A, α- and β-enolase, lactate dehydrogenase A, liver and muscle phosphofructokinase) and NRF-1-regulated genes (cytochrome c, Va subunit of cytochrome oxidase, and carnitine palmitoyltransferase I ). HIF-1α protein was present in fetal myocardium but dropped below detectable levels at 7 d postnatally. HIF-2α protein levels were similar at the four time points. Steady-state mRNA levels of α-enolase, lactate dehydrogenase A, and liver phosphofructokinase declined significantly postnatally. Aldolase A, β-enolase, and muscle phosphofructokinase mRNA levels increased postnatally. Steady-state mRNA and protein levels of NRF-1 decreased postnatally in contrast to the postnatal increases in cytochrome c, subunit Va of cytochrome oxidase, and carnitine palmitoyltransferase I mRNA levels. The in vivo postnatal regulation of enzymes encoding glycolytic and mitochondrial enzymes is complex. As transactivation response elements for the genes encoding metabolic enzymes continue to be characterized, studies using the fetal-to-postnatal metabolic transition of the heart will continue to help define the in vivo role of these transcription factors.

AT 2 , Judgment Day

As reviewed recently in these pages,1 the vasoconstrictor angiotensin II (Ang II) is one of the circulating peptide growth factors, and clinically the most relevant, that fulfill many of Koch’s postulates for having additional effects locally in the myocardium. These criteria include local production of Ang II within the heart, its upregulation there by hypertrophic signals, its release from cardiac cells after mechanical load, the presence of its receptors on the surface of cardiac cells, and the responsiveness of both cardiac myocytes and cardiac fibroblasts to provocation by this peptide.1,2 This local circuit of Ang II production, release, binding, and transduction within the myocardium itself is the most cogent explanation for the effectiveness of ACE inhibitors in heart failure, even at doses that do not alter loading conditions.1,2 See p 346 Three related receptors exist for Ang II: AT1a, AT1b, and AT2, which communicate with a complex cell-wide web of signaling cascades through GTP-binding (G) proteins as the proximal transducer. The operation of a local circuit for Ang II within the myocardium poses two questions, which have attracted much recent experimental effort, and an even larger share of controversy. First, given that multiple receptors exist for Ang II, which type or types are responsible for pathobiology provoked by the local circuit? Second, given that cardiac muscle and nonmuscle cells are both potential targets for Ang II, what is the respective role of each? Genetic models that altogether lack the receptors individually provide an invaluable experimental tool beyond what is achievable by use of pharmacological inhibitors alone. Surprisingly, the Ang II receptor …

Integrins and the myocardium.

Extracellular matrix provides a structural, chemical, and mechanical substrate that is essential in cardiac development, growth, and responses to pathophysiological signals. Transmembrane receptors termed integrins provide a dynamic interaction of environmental cues and intracellular events. Integrins orchestrate multiple functions in the intact organism including organogenesis, regulation of gene expression, cell proliferation, differentiation, migration, and death. They are expressed in all cellular components of the cardiovascular system, including the vasculature, blood, cardiac myocytes and nonmuscle cardiac cells. The focus of this review will be on the role of integrins in the myocardium. We will provide background on integrin structure and function, discuss how the expression of integrins is critical to the form and function of the developing and postnatal myocardium, and review the known data on integrins as signaling molecules in the heart. Finally, we will offer insights to the future research directions into this important family of extracellular matrix receptors in the myocardium.

Inducible gene targeting in postnatal myocardium by cardiac-specific expression of a hormone-activated Cre fusion protein.

Cardiac-restricted expression of Cre recombinase can provoke lineage-specific gene excision in the myocardium. However, confounding early lethality may still preclude using loss-of-function models to study the postnatal heart. Here, we have tested whether inducible, heart-specific recombination can be triggered after birth by transgenic expression of a Cre fusion protein that incorporates a mutated progesterone receptor ligand binding domain (PR1) that is activated by the synthetic antiprogestin, RU486, but not by endogenous steroid hormones. CrePR1 driven by the alpha-myosin heavy chain (alphaMHC) promoter was expressed specifically in heart. Translocation of CrePR1 from cytoplasm to nuclei in ventricular myocytes was induced by RU486. To establish whether this approach can mediate cardiac-specific, drug-dependent excision between loxP sites in vivo, we mated alphaMHC-CrePR1 mice with a ubiquitously expressed (ROSA26) Cre reporter line. Offspring harboring alphaMHC-CrePR1 and/or the floxed allele were injected with RU486 versus vehicle, and the prevalence of beta-galactosidase (beta-gal)-positive cells was determined, indicative of Cre-mediated excision. Little or no baseline recombination was seen 1 week after birth. Cardiac-restricted, RU486-inducible recombination was demonstrated in bigenic mice at age 3 and 6 weeks, using each of 3 independent CrePR1 lines. Recombination in the absence of ligand paralleled the levels of CrePR1 protein expression and was more evident at 6 weeks. Thus, conditional, posttranslational activation of a Cre fusion protein can bypass potential embryonic and perinatal effects on the heart and permits inducible recombination in cardiac muscle. High levels of the chimeric Cre protein, in particular, were associated with progressive recombination in the absence of drug.

Cooperative interaction between GATA-4 and GATA-6 regulates myocardial gene expression.

Two members of the GATA family of transcription factors, GATA-4 and GATA-6, are expressed in the developing and postnatal myocardium and are equally potent transactivators of several cardiac promoters. However, several in vitro and in vivo lines of evidence suggest distinct roles for the two factors in the heart. Since identification of the endogenous downstream targets of GATA factors would greatly help to elucidate their exact functions, we have developed an adenovirus-mediated antisense strategy to specifically inhibit GATA-4 and GATA-6 protein production in postnatal cardiomyocytes. Expression of several endogenous cardiac genes was significantly down-regulated in cells lacking GATA-4 or GATA-6, indicating that these factors are required for the maintenance of the cardiac genetic program. Interestingly, transcription of some genes like the alpha- and beta-myosin heavy-chain (alpha- and beta-MHC) genes was preferentially regulated by GATA-4 due, in part, to higher affinity of GATA-4 for their promoter GATA element. However, transcription of several other genes, including the atrial natriuretic factor and B-type natriuretic peptide (ANF and BNP) genes, was similarly down-regulated in cardiomyocytes lacking one or both GATA factors, suggesting that GATA-4 and GATA-6 could act through the same transcriptional pathway. Consistent with this, GATA-4 and GATA-6 were found to colocalize in postnatal cardiomyocytes and to interact functionally and physically to provide cooperative activation of the ANF and BNP promoters. The results identify for the first time bona fide in vivo targets for GATA-4 and GATA-6 in the myocardium. The data also show that GATA factors act in concert to regulate distinct subsets of genes, suggesting that combinatorial interactions among GATA factors may differentially control various cellular processes.

No and neurogulins: Autacoid signaling in the heart

AS in other organs and tissues, the heart utilizes a number of autocrine, paracrine, and juxtacrine (autacoid) signaling pathways to respond both to moment-to-moment changes in demand and to longer term adaptation to physiologic stress, including (among others) nitric oxide (NO) and neuregulins. Aside from endothelium, NO within the heart is also produced by the NO synthase “eNOS” in atrial and ventricular myocytes, including SA and AV nodal cells, where it activates guanylyl cyclase in response to m2 muscarinic and purinergic agonists. Both coronary microvascular endothelial cells (CMEC) and cardiac myocytes also express inducible NOS (iNOS), a principal effector of “innate immunity”, in response to intracardiac inflammatory cytokines. CMEC, including capillary and venular endothelium, express a number of peptide autacoids, including angiotensins and endothelins, and also neuregulinI (NRGI), a family of autacoids essential for normal ventricular development. Cardiac myocytes, which express the neuregulin receptors erbB2, erbB3, and erbB4 during development, continue to express erbB4 (and, to a lesser extent erbB2) in the postnatal myocardium. An improved understanding of the complexity of autacoid signaling in the postnatal and adult myocardium will enable the development of new therapeutic strategies and targets for the stressed myocardium.

Neuregulins Promote Survival and Growth of Cardiac Myocytes: PERSISTENCE OF ErbB2 AND ErbB4 EXPRESSION IN NEONATAL AND ADULT VENTRICULAR MYOCYTES

Neuregulins (i.e. neuregulin-1 (NRG1), also called neu differentiation factor, heregulin, glial growth factor, and acetylcholine receptor-inducing activity) are known to induce growth and differentiation of epithelial, glial, neuronal, and skeletal muscle cells. Unexpectedly, mice with loss of function mutations of NRG1 or of either of two of their cognate receptors, ErbB2 and ErbB4, die during midembryogenesis due to the aborted development of myocardial trabeculae in ventricular muscle. To examine the role of NRG and their receptors in developing and postnatal myocardium, we studied the ability of a soluble NRG1 (recombinant human glial growth factor 2) to promote proliferation, survival, and growth of isolated neonatal and adult rat cardiac myocytes. Both ErbB2 and ErbB4 receptors were found to be expressed by neonatal and adult ventricular myocytes and activated by rhGGF2. rhGGF2 (30 ng/ml) provoked an approximate 2-fold increase in embryonic cardiac myocyte proliferation. rhGGF2 also promoted survival and inhibited apoptosis of subconfluent, serum-deprived myocyte primary cultures and also induced hypertrophic growth in both neonatal and adult ventricular myocytes, which was accompanied by enhanced expression of prepro-atrial natriuretic factor and skeletal alpha-actin. Moreover, NRG1 mRNA could be detected in coronary microvascular endothelial cell primary cultures prepared from adult rat ventricular muscle. NRG1 expression in these cells was increased by endothelin-1, another locally acting cardiotropic peptide within the heart. The persistent expression of both a neuregulin and its cognate receptors in the postnatal and adult heart suggests a continuing role for neuregulins in the myocardial adaption to physiologic stress or injury.

Transforming growth factor-beta signal transduction.

The TGF-β superfamily of cytokines comprises an array of more than two dozen secreted peptides that have been implicated as multifunctional regulators of cell growth, differentiation, and function.1 2 Members of this family are pivotal for normal development in a variety of species, providing diffusible signals that influence pattern formation, body axes, cell fate, and other aspects of morphogenesis. In the embryonic heart, developmental functions for TGF-β inferred from the genes’ program of expression and from model systems for cardiac organogenesis include the specification of cardiogenic precursor cells in lateral mesoderm as well as epithelial-mesenchymal transitions involved in valve formation.2 In postnatal myocardium, TGF-β controls the mixed histocompatibility genes, whose deregulation may explain the myocarditis found in mice lacking TGF-β1. In isolated cardiac muscle cells, TGF-β counters the suppression of myocyte contraction by interleukin-1β, at least in part through a block to induction of nitric oxide synthase. Conversely, TGF-β evokes a “fetal” program of myocardial gene expression, which, together with the upregulation of TGF-β by mechanical load and other trophic signals, suggests the operation of an autocrine or paracrine loop in certain forms of cardiac hypertrophy.2 The TGF-β superfamily consists of at least 25 different peptides, classified into three subgroups based on sequence similarities: (1) TGF-βs themselves, of which three isoforms are found in mammals, (2) activins, and (3) a complex third subfamily of proteins (BMPs, nodal, Xenopus Vg-1, Drosophila dpp, and screw) with prominent effects on mesoderm induction and formation of axial structures.1 2 All known members of the superfamily are produced as large precursor molecules that are cleaved at a conserved RXXR motif into the mature C-terminal dimer and the N-terminal proregion; binding of the ligand by the N-terminal remnant results in a biologically inactive latent complex. This requirement for activation of …

Troponin I gene expression during human cardiac development and in end-stage heart failure.

Recent reports have demonstrated the presence of two isoforms of troponin I in the human fetal heart, namely, cardiac troponin I and slow skeletal muscle troponin I. Structural and physiological considerations indicate that these isoforms would confer differing contractile properties on the myocardium, particularly on the phosphorylation-mediated regulation of contractility by adrenergic agonists. We have investigated the developmental expression of these isoforms in the human heart from 9 weeks of gestation to 9 months of postnatal life, using Western blots revealed with troponin I antibodies to detect troponin protein isoforms and Northern blots to detect the corresponding mRNAs. The results show the following: 1) Slow skeletal muscle troponin I is the predominant isoform throughout fetal life. 2) After birth, the slow skeletal isoform is lost, with cardiac troponin I being the only isoform detectable by 9 months of postnatal development. 3) The protein isoforms and their corresponding mRNAs follow the same pattern of accumulation, suggesting that the transition in troponin expression is regulated at the level of gene transcription. The developmental transition in troponin I isoform content has implications for contractility of the fetal and postnatal myocardium. We further analyzed right and left ventricular muscle samples from 17 hearts in end-stage heart failure resulting from pulmonary hypertension, ischemic heart disease, or dilated cardiomyopathy. Cardiac troponin I mRNA remained abundant in each case, and slow skeletal muscle troponin I mRNA was not detectable in any of sample. We conclude that alterations in troponin I isoform content do not therefore contribute to the altered contractile characteristics of the adult failing ventricle.