Myocardial Growth Research Articles

Abstract 314: Inactivation-Resistant GSK3αβ Dual Isoform Expression Attenuates Isoproterenol-Induced Myocardial Hypertrophy and Preserves Cardiac Function

Glycogen synthase kinase-3 (GSK-3) is implicated as an important signalling mediator in normal developmental myocardial growth and adverse hypertrophic remodelling. Upstream phosphorylation of isoform specific N-terminal serine residues - ser21(GSK-3α) and ser9(GSK-3β), respectively - inhibits kinase activity. Opposing effects of the two isoforms have recently been described in response to hypertrophic stimuli, but little is known of the overall effect of dual-isoform manipulation. We set out to characterise baseline and isoproterenol (ISO) stimulated phenotypes of mice with inactivation-resistant GSK-3αβ knockin (KI) alleles, in which ser21/9 are converted to non-phosphorylatable alanine. Between 5–15 weeks there was a non-significant trend towards higher heart weight (HW): body weight (BW) ratios in KI mice versus wild type (WT) mice. Mean left ventricular wall thickness (LVWT) at the level of the papillary muscle was comparable between genotypes (1.3±0.2mm vs 1.6±0.2mm, ns, n=8). 8–10 week old weight-matched mice received 2 weeks subcutaneous ISO (30mg/kg/d) or control saline (Con) infusions. WT hearts hypertrophied with ISO (LVWT 1.9±0.1mm vs 1.3±0.2mm Con, n=8; HW:BW ratio 6.7±0.6 vs 5.4±0.4 Con, n=10, both p< 0.05), but KI mice did not (1.4±0.1mm vs 1.6±0.2mm KI Con; HW:BW 6.2±1.0 vs 6.1±0.6 KI Con, respectively, both ns). Hypertrophy in the WT hearts was associated with reduced LV ejection fraction (41±9% vs 63±5% Con, n=6, p< 0.05) and greater interstitial fibrosis (2.1±0.5% vs 0.8±0.6%, n=6, p< 0.05). These were both attenuated in the KI mice subjected to ISO (70±13% vs 64±11% Con, and 1.3±0.7% vs 0.8±0.3% Con, respectively, both ns). GSK-3 inhibition with daily intraperitoneal 6-bromoindirubin-3′-oxime restored hypertrophic sensitivity to ISO in the KI mice (LVWT 1.8±0.2mm; HW:BW 7.3±0.5, p< 0.05 for both groups vs KI + ISO alone) but had no effect in the absence of hypertrophic stimulus (1.4±0.2mm and 5.4±0.7, respectively, for KI + Con + Bio, ns vs KI + Con alone). In conclusion, inactivation-resistant GSK-3αβ dual isoform expression does not affect age-related (eutrophic) myocardial growth in the mouse. However, it protects against pathological growth, maintaining cardiac function and attenuating interstitial fibrosis.

Angiotensin II type-1 receptor activation in the adult heart causes blood pressure-independent hypertrophy and cardiac dysfunction

Sustained hypertension leads to cardiac hypertrophy that can progress, through pathological remodelling, to heart failure. Abnormality of the renin-angiotensin system (RAS) has been strongly implicated in this process. Although hypertrophy in human is an established risk factor independent of blood pressure (BP), separation of remodelling in response to local cues within the differentiated myocardium from that related to pressure overload is unresolved. This study aimed to clarify the role of local RAS activity, specifically in the adult heart, in modulating cardiac hypertrophy and pathological remodelling. Transgenic mice with inducible cardiomyocyte-specific expression of a wild-type or N111G mutant form of the human angiotensin II (Ang II) type-1 receptor (hAT1R) were generated. The wild-type receptor is primarily stimulated by Ang II. In contrast, the N111G receptor can also be fully stimulated by the Ang II derivative, Ang IV, at levels that do not stimulate the wild-type receptor. The unique properties of these models were used to investigate the myocardial growth, remodelling and functional responses to hAT1R stimulation, specifically in adult cardiomyocytes, under normal conditions and following Ang IV infusion. Low-level expression of wild-type or N111G hAT1R at the cardiomyocyte membrane, from the onset of adolescence, induced enhanced myocyte growth and associated cardiac hypertrophy in the adult. This was not associated with change in resting BP or heart rate, measured by longitudinal telemetric analysis, and did not progress to pathological remodelling or heart failure. However, selective activation of cardiomyocyte-specific N111G receptors by Ang IV peptide infusion induced adverse ventricular remodelling within 4 weeks. This was characterized by increased interstitial fibrosis, dilatation of the left ventricle, and impaired cardiac function. Low-level local AT1R activity in differentiated myocardium causes compensated cardiac hypertrophy, that is, increased myocardial mass but with the retention of normal function, whereas short-term increased stimulation induces cardiac dysfunction with dilatation, reduced ejection fraction, and increased fibrosis in the absence of change in systemic BP.

Quantitative Control of Adaptive Cardiac Hypertrophy by Acetyltransferase p300

Acetyltransferase p300 is essential for cardiac development and is thought to be involved in cardiac myocyte growth through MEF2- and GATA4-dependent transcription. However, the importance of p300 in the modulation of cardiac growth in vivo is unknown. Pressure overload induced by transverse aortic coarctation, postnatal physiological growth, and human heart failure were associated with large increases in p300. Minimal transgenic overexpression of p300 (1.5- to 3.5-fold) induced striking myocyte and cardiac hypertrophy. Both mortality and cardiac mass were directly related to p300 protein dosage. Heterozygous loss of a single p300 allele reduced pressure overload-induced hypertrophy by approximately 50% and rescued the hypertrophic phenotype of p300 overexpressers. Increased p300 expression had no effect on total histone deacetylase activity but was associated with proportional increases in p300 acetyltransferase activity and acetylation of the p300 substrates histone 3 and GATA-4. Remarkably, a doubling of p300 levels was associated with the de novo acetylation of MEF2. Consistent with this, genes specifically upregulated in p300 transgenic hearts were highly enriched for MEF2 binding sites. Small increments in p300 are necessary and sufficient to drive myocardial hypertrophy, possibly through acetylation of MEF2 and upstream of signals promoting phosphorylation or nuclear export of histone deacetylases. We propose that induction of myocardial p300 content is a primary rate-limiting event in the response to hemodynamic loading in vivo and that p300 availability drives and constrains adaptive myocardial growth. Specific reduction of p300 content or activity may diminish stress-induced hypertrophy and forestall the development of heart failure.

Roles of IL-6-gp130 Signaling in Vascular Inflammation

Interleukin-6 (IL-6) is a well-established, independent indicator of multiple distinct types of cardiovascular disease and all-cause mortality. In this review, we present current understanding of the multiple roles that IL-6 and its signaling pathways through glycoprotein 130 (gp130) play in cardiovascular homeostasis. IL-6 is highly inducible in vascular tissues through the actions of the angiotensin II (Ang II) peptide, where it acts in a paracrine manner to signal through two distinct mechanisms, the first being a classic membrane receptor initiated pathway and the second, a trans-signaling pathway, being able to induce responses even in tissues lacking the IL-6 receptor. Recent advances and new concepts in how its intracellular signaling pathways operate via the Janus kinase (JAK)-Signal Transducer and Activator of Transcription (STAT) are described. IL-6 has diverse actions in multiple cell types of cardiovascular importance, including endothelial cells, monocytes, platelets, hepatocytes and adipocytes. We discuss central roles of IL-6 in endothelial dysfunction, cellular inflammation by affecting monocyte activation/differentiation, cellular cytoprotective functions from reactive oxygen species (ROS) stress, modulation of pro-coagulant state, myocardial growth control, and its implications in metabolic control and insulin resistance. These multiple actions indicate that IL-6 is not merely a passive biomarker, but actively modulates adaptive and pathological responses to cardiovascular stress. Summary:IL-6 is a multifunctional cytokine whose presence in the circulation is linked with diverse types of cardiovascular disease and is an independent risk factor for atherosclerosis. In this review, we examine the mechanisms by which IL-6 signals and its myriad effects in cardiovascular tissues that modulate the manifestations of vascular inflammation.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section will spotlight significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Changing Tide (Dev Dyn 237:545–553) Like ridges in the sand carved by ocean waves receding toward low tide, the favored Clock and Wavefront model predicts that iterated somite boundary formation results from molecular juxtaposition of two inputs: a posterior moving determination wavefront, and a genetic oscillator, or somitogenesis clock. To enable quantitative study of the process, Schröter and colleagues document somitogenesis period in wild-type zebrafish (time between boundary formation) and somite length (length between boundaries) in wild-type zebrafish. Gauging these determinants is imprecise; therefore, groups of live embryos were imaged simultaneously to obtain mean population measurements. The authors find that somitogenesis period is linearly temperature-dependent, while somite length is temperature-compensated. In addition, both somite period and length varied from trunk to tail, but changes in the two measurements did not track together as expected, challenging the text-book version of the Clock and Wavefront model. The authors propose instead that wavefront velocity changes during somitogenesis, independent of clock periodicity. In other words, neither the rhythm of the lapping waves nor the rate at which the tide falls is constant. Theories ebb and flow, but the best adapt to accommodate new discoveries. The Great Uncoupler (Dev Dyn 237:713–724) To form a proportioned, mature embryo, growth and development must work hand-in-hand. But are these processes necessarily linked, or can they be genetically distinct? Supporting the former idea, Lavine and colleagues previously showed that development of coronary vasculature and myocardial growth were both outputs of fibroblast growth factor (FGF) signaling to the cardiomyoblast. Here, they test this concept further by teasing apart FGF outputs. Targeted ectopic expression of p27Kip1, a cyclin-dependent kinase inhibitor, enhances myocardial proliferation defects observed in mice lacking Fgfr1/2. By contrast, it has no effect on observed coronary vascular defects, including vascular density and expression of vascular genes, Vegf-A and Vegfr1. Lavine also previously demonstrated that Hedgehog (HH) functions downstream of FGF signaling to regulate vascular development. Here they find that HH has no affect on myocardial proliferation both in vitro and in vivo. The work unequivocally shows that outputs of FGF signaling to the cardiomyoblast are the sum of two parts. Stress management (Dev Dyn 237:725–735) How do you react to stress? Some act out while others become quiet. As it turns out, chick heart endothelial cells (CHECs) do both, depending on what kind of stress they are under. Cardiac cells are typically exposed to shear stress, drag force imposed by blood flow, and to stretch forces, caused by hydrostatic and blood pressures. Using QRT-PCR, Hierck et. al show that expression profiles of a handful of previously tested stress-response genes differ under shear stress and stretch forces, with an overall more robust response to the former conditions. CHECs harbor primary cilia-membrane extensions that are connected to the cytoskeleton. To test the hypothesis that these structures function as stress mechanosensors, the authors compare effects of shear stress on ciliated and nonciliated cells. They find that both chicken arterial ECs (CAECs) that do not bear cilia, and CHECs where cilia are disrupted by chloral hydrate, show a twofold reduced expression of KLF2, a shear responsive gene. Moreover CHECs exposed to colchicine and taxol, drugs that disrupt or stabilize cytoskeletal microtubules, respectively, have opposing expression profiles. These findings show that primary cilia rely on their microtubule-based cytoskeletal connections to transmit mechanosensory information. Together their results suggest that ECs have a self-regulatory mechanism for coping with stress. And you?

Inappropriate Left Ventricular Mass in Patients With Primary Aldosteronism

Assessment of appropriateness of left ventricular mass (LVM) for a given workload may better stratify hypertensive patients. Inappropriate LVM may reflect the interaction of genetic and neurohumoral factors other than blood pressure playing a significant role in myocardial growth. Primary aldosteronism (PA) represents a clinical model useful in assessing the effect of aldosterone increase on LVM. The aim of this study was to evaluate the inappropriateness of LVM in patients with PA. In 125 patients with PA (54 females; adrenal hyperplasia in 73 and adenoma in 52 patients) and in 125 age-, sex-, and blood pressure-matched, essential hypertensive patients, echocardiography was performed. The appropriateness of LVM was calculated by the ratio of observed LVM to the predicted value using a reference equation. In all of the subjects plasma renin activity and aldosterone, as well as clinic and 24-hour blood pressure, were measured. The prevalence of inappropriate LVM was greater in patients with traditionally defined left ventricular hypertrophy (70% and 44%, respectively; P=0.02) but also in patients without left ventricular hypertrophy (17% and 9%, respectively; P=0.085). In PA patients, a correlation was observed between the ratio of observed:predicted LVM and the ratio of aldosterone:plasma renin activity levels (r=0.29; P=0.003) or the postinfusion aldosterone concentration (r=0.44; P=0.004; n=42). In conclusion, in patients with PA, the prevalence of inappropriate LVM is increased, even in the absence of traditionally defined left ventricular hypertrophy. The increase in aldosterone levels could contribute to the increase of LV mass exceeding the amount needed to compensate hemodynamic load.

FGF-16 is required for embryonic heart development

Fibroblast growth factor 16 (FGF-16) expression has previously been detected in mouse heart at mid-gestation in the endocardium and epicardium, suggesting a role in embryonic heart development. More specifically, exogenously applied FGF-16 has been shown to stimulate growth of embryonic myocardial cells in tissue explants. We have generated mice lacking FGF-16 by targeting the Fgf16 locus on the X chromosome. Elimination of Fgf16 expression resulted in embryonic death as early as day 11.5 (E11.5). External abnormalities, including hemorrhage in the heart and ventral body region as well as facial defects, began to appear in null embryos from E11.5. Morphological analysis of FGF-16 null hearts revealed cardiac defects including chamber dilation, thinning of the atrial and ventricular walls, and poor trabeculation, which were visible at E10.5 and more pronounced at E11.5. These findings indicate FGF-16 is required for embryonic heart development in mid-gestation through its positive effect on myocardial growth.

Fibroblast growth factor regulation of neovascularization

Fibroblast growth factors are potent angiogenic inducers; however, their precise roles in angiogenesis have not been well understood. In this review, we will focus on specific roles played by fibroblast growth factors in neovascularization. Although fibroblast growth factors promote a strong angiogenic response, it has been suggested that FGF-induced angiogenesis requires activation of the vascular endothelial growth factor system. Recent findings have endorsed the view of indirect contribution of fibroblast growth factor signaling to vascular development. A study using embryoid bodies demonstrated a nonimmediate role played by fibrobalst growth factor receptor 1 in vasculogenesis as vascular endothelial growth factor supplementation was sufficient to promote vascular development in Fgfr1-/- embryoid bodies. Moreover, another line of evidence indicated that myocardial fibroblast growth factor signaling is essential for mouse coronary development. The key role of fibroblast growth factor signaling in this process is Hedgehog activation, which induces vascular endothelial growth factor expression and formation of the coronary vasculature. In addition to vascular endothelial growth factor interaction, fibroblast growth factors can control neovascularization by influencing other growth factors and chemokines such as platelet-derived growth factor, hepatocyte growth factor and monocyte chemoattractant protein-1, contributing to development of mature vessels and collateral arteries. Although fibroblast growth factors are potent angiogenic factors, they may indirectly control neovascularization in concert with other growth factors. Thus, the unique role played by fibroblast growth factors might be organization of various angiogenic pathways and coordination of cell-cell interactions in this process.

Disease-Modifying Mutations in Familial Hypertrophic Cardiomyopathy

Cardiomyopathies are primary disorders of cardiac muscle associated with abnormalities of cardiac wall thickness, chamber size, contraction, relaxation, conduction, and rhythm. They are a major cause of morbidity and mortality at all ages and, like acquired forms of cardiovascular disease, frequently progress into heart failure.1 In contrast to dilated cardiomyopathy (DCM), which is characterized by left or biventricular dilation in association with depressed myocardial contractility,1 familial hypertrophic cardiomyopathy (FHC) is characterized by increased cardiac mass with myocyte and myofibrillar disarray. The disease is associated with electrical instability (ie, atrial and ventricular arrhythmias), which makes it the leading cause of death in young athletes. There is marked diversity in the morphological features and clinical manifestations of both DCM and FHC.2 Article p 1820 Family studies have demonstrated that hypertrophic cardiomyopathy is a heritable disorder that is transmitted as an autosomal-dominant trait or as a sporadic disease. There is neither a racial nor an ethnic predisposition to this condition. Hypertrophic cardiomyopathy is not a rare condition; several noninvasive studies have demonstrated echocardiographic criteria for this disease in 0.2% of young adults (reviewed by Maron3). Genetic linkage studies in FHC have found disease loci on at least 14 different genes, most of them encoding sarcomeric genes; hence, FHC has been called a disease of the sarcomere. In addition, several mutations within the Ras pathway have been shown to cause at least syndromic forms of hypertrophic cardiomyopathy,4,5 pointing to the presence of additional but poorly understood disease-causing and/or modifier genes. Thus far, however, most of the clinical heterogeneity of FHC has been accounted for by the substantial diversity of gene defects that cause the condition (ie, up to 45% of disease-causing mutations have been observed in the β-myosin heavy chain [MHC] gene myh7 and up to 35% in …

Forced expression of the cell cycle inhibitor p57Kip2 in cardiomyocytes attenuates ischemia-reperfusion injury in the mouse heart

BackgroundMyocardial hypoxic-ischemic injury is the cause of significant morbidity and mortality worldwide. The cardiomyocyte response to hypoxic-ischemic injury is known to include changes in cell cycle regulators. The cyclin-dependent kinase inhibitor p57Kip2 is involved in cell cycle control, differentiation, stress signaling and apoptosis. In contrast to other cyclin-dependent kinase inhibitors, p57Kip2 expression diminishes during postnatal life and is reactivated in the adult heart under conditions of cardiac stress. Overexpression of p57Kip2 has been previously shown to prevent apoptotic cell death in vitro by inhibiting stress-activated kinases. Therefore, we hypothesized that p57Kip2 has a protective role in cardiomyocytes under hypoxic conditions. To investigate this hypothesis, we created a transgenic mouse (R26loxpTA-p57k/+) that expresses p57Kip2 specifically in cardiac tissue under the ventricular cardiomyocyte promoter Mlc2v.ResultsTransgenic mice with cardiac specific overexpression of p57Kip2 are viable, fertile and normally active and their hearts are morphologically indistinguishable from the control hearts and have similar heart weight/body weight ratio. The baseline functional parameters, including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), LVdp/dtmax, heart rate (HR) and rate pressure product (RPR) were not significantly different between the different groups as assessed by the Langendorff perfused heart preparation. However, after subjecting the heart ex vivo to 30 minutes of ischemia-reperfusion injury, the p57Kip2 overexpressing hearts demonstrated preserved cardiac function compared to control mice with higher left ventricular developed pressure (63 ± 15 vs 30 ± 6 mmHg, p = 0.05), rate pressure product (22.8 ± 4.86 vs 10.4 ± 2.1 × 103bpm × mmHg, p < 0.05) and coronary flow (3.5 ± 0.5 vs 2.38 ± 0.24 ml/min, p <0.05).ConclusionThese data suggest that forced cardiac expression of p57Kip2 does not affect myocardial growth, differentiation and baseline function but attenuates injury from ischemia-reperfusion in the adult mouse heart.

Heart failure – are there gender aspects?

Gender differences in the syndrome of heart failure (HF) occur in etiology and pathophysiology, in the clinical presentation and course of the syndrome. In addition, gender specific treatment responses and gender associated differences in the behavior of treating physicians are found. Hypertension and diabetes play a major role as causes of HF in women and both interact in their pathophysiology with the renin angiotensin system (RAS). Modulation of the RAS by estrogens explains specific differences between pre- and postmenopausal women and men. Myocardial growth processes and myocardial calcium handling are differentially regulated in female and male myocytes. Myocardial remodeling with age and as a consequence of mechanical load differs in women and men. For yet unknown reasons, HF with preserved systolic function seems to be more frequent in women than in men and the clinical course of systolic failure is different in both genders.

Novel tool to suppress cell proliferation in vivo demonstrates that myocardial and coronary vascular growth represent distinct developmental programs

Cell proliferation, differentiation, and vascular growth are coordinated processes that are essential for embryonic development, tissue repair, and disease pathogenesis. Of interest, whether these critical processes are dependent upon each other has not been thoroughly explored. We have generated mice that conditionally express the cell cycle inhibitor p27(Kip1), following Cre-mediated recombination, as a tool to separate tissue proliferation from other cellular processes. Using the embryonic heart as a model, we show that myocardial proliferation and coronary development are genetically separable processes. Forced expression of p27, in both a wild-type and in a genetically sensitized background, resulted in ventricular hypoplasia without having any substantial effects on coronary development. We further demonstrate that Hedgehog signaling, which is essential for coronary vascular growth, does not control myocardial proliferation. Together, these studies strongly suggest that myocardial cell proliferation and coronary development are genetically separable programs exemplifying one of the many potential uses of this genetic tool.

Regulation of Cardiomyocyte Proliferation and Myocardial Growth During Development by FOXO Transcription Factors

Cardiomyocytes actively proliferate during embryogenesis and withdraw from the cell cycle during neonatal stages. FOXO (Forkhead O) transcription factors are a direct target of phosphatidylinositol-3 kinase/AKT signaling in skeletal and smooth muscle and regulate expression of the Cip/Kip family of cyclin kinase inhibitors in other cell types; however, the interaction of phosphatidylinositol-3 kinase/AKT signaling, FOXO transcription factors, and cyclin kinase inhibitor expression has not been reported for the developing heart. Here, we show that FOXO1 and FOXO3 are expressed in the developing myocardium concomitant with increased cyclin kinase inhibitor expression from embryonic to neonatal stages. Cell culture studies show that embryonic cardiomyocytes are responsive to insulin-like growth factor 1 stimulation, which results in the induction of the phosphatidylinositol-3 kinase/AKT pathway, cytoplasmic localization of FOXO proteins, and increased myocyte proliferation. Likewise, adenoviral-mediated expression of AKT promotes cardiomyocyte proliferation and cytoplasmic localization of FOXO. In contrast, increased expression of FOXO1 negatively affects myocyte proliferation. In vivo myocyte-specific transgenic expression of FOXO1 during heart development causes embryonic lethality at embryonic day 10.5 because of severe myocardial defects that coincide with premature activation of p21(cip1), p27(kip1), and p57(kip2) and decreased myocyte proliferation. Transgenic expression of dominant negative FOXO1 in cardiomyocytes does not obviously affect heart development at embryonic day 10.5, but results in abnormal morphology of the myocardium by embryonic day 18.5 along with decreased cyclin kinase inhibitor expression and increased myocyte proliferation. These data support FOXO transcription factors as negative regulators of cardiomyocyte proliferation and promoters of neonatal cell cycle withdrawal during heart development.

Mutation of the HEXIM1 Gene Results in Defects During Heart and Vascular Development Partly Through Downregulation of Vascular Endothelial Growth Factor

Our previous studies and those of others indicated that the transcription factor Hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) is a tumor suppressor and cyclin-dependent kinase inhibitor, and that these HEXIM1 functions are mainly dependent on its C-terminal region. We provide evidence here that the HEXIM1 C-terminal region is critical for cardiovascular development. HEXIM1 protein was detected in the heart during critical time periods in cardiac growth and chamber maturation. We created mice carrying an insertional mutation in the HEXIM1 gene that disrupted its C-terminal region and found that this resulted in prenatal lethality. Heart defects in HEXIM1(1 to 312) mice included abnormal coronary patterning and thin ventricular walls. The thin myocardium can be partly attributed to increased apoptosis. Platelet endothelial cell adhesion molecular precursor-1 staining of HEXIM1(1 to 312) heart sections revealed decreased vascularization of the myocardium despite the presence of coronary vasculature in the epicardium. The expression of vascular endothelial growth factor (VEGF), known to affect angioblast invasion and myocardial proliferation and survival, was decreased in HEXIM1(1 to 312) mice compared with control littermates. We also observed decreased fibroblast growth factor 9 (FGF9) expression, suggesting that effects of HEXIM1 in the myocardium are partly mediated through epicardial FGF9 signaling. Together our results suggest that HEXIM1 plays critical roles in coronary vessel development and myocardial growth. The basis for this role of HEXIM1 is that VEGF is a direct transcriptional target of HEXIM1, and involves attenuation a repressive effects of C/EBPalpha on VEGF gene transcription.

δ‐OPIOID RECEPTOR STIMULATION ENHANCES THE GROWTH OF NEONATAL RAT VENTRICULAR MYOCYTES VIA THE EXTRACELLULAR SIGNAL‐REGULATED KINASE PATHWAY

1. The aims of the present study were to determine whether delta-opioid receptor stimulation enhanced proliferation of and to investigate the role of the extracellular signal-regulated kinase (ERK) pathway in ventricular myocytes from neonatal rats. 2. At concentratins ranging from 10 nmol/L to 10 micromol/L, [D-Ala2,D-Leu5]enkephalin (DADLE) concentration-dependently promoted myocardial growth and DNA synthesis and altered the cytoskeleton. 3. At 1 micromol/L, DADLE also increased the expression and phosphorylation of ERK. 4. These effects of 1 micromol/L DADLE were abolished by 10 micromol/L naltrindole, a selective delta-opioid receptor antagonist, 10 nmol/L U0126, a selective ERK antagonist, 1 micromol/L staurosporine, an inhibitor of protein kinase (PK) C, and 100 micromol/L Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt hydrate (Rp-cAMPS), an inhibitor of PKA. 5. In conclusion, delta-opioid receptor stimulation enhances the proliferation and development of the ventricular myocytes of neonatal rats. The ERK pathway and related signalling mechanisms, namely PKC and PKA, are involved.

The Immature Heart: The Roles of Bone Marrow Stromal Stem Cells in Growth and Myocardial Repair

Studies have shown that adult bone marrow derived stem cells (MSCs) can participate in repair of myocardial injury in adult hearts, as well as in cardiac growth during fetal development in utero. Yet, no studies have evaluated the role of MSCs with respect to normal growth or tissue repair in immature hearts after birth. The present study examines whether MSCs may participate in the myocardial growth and injury in the post-natal immature hearts. MSCs were isolated from adult Lewis rats and labeled with Lac-Z gene using retroviral vectors. These MSCs were injected systemically into groups of neonatal (NB=2days-old), immature (B=30days-old) and adult (A=>3months-old) isogeneic Lewis rats. Additionally, left coronary artery ligation was carried out in subgroups of immature (BL) and adult (AL) rats one week after MSCs injection. The hearts were harvested serially from 2-days to 6-weeks, stained with X-Gal for labeled MSCs. Cardiomyocyte phenotypic expression was evaluated by immunohistological staining for Troponin I-C and Connexin-43. Labeled MSCs were found to home into the bone marrow in all rats of different developmental stages. They could be recruited from bone marrow into the infarcted site of myocardium only in groups AL and BL. They were also capable of differentiating into cardiomyocyte phenotype after myocardial injury. In contrast to that reported in the developing fetus, MSCs did not appear to contribute to the growth of non-injured hearts after birth. However, they can be recruited from the bone marrow and regenerate damaged myocardium both in the adult and in the immature hearts.

Specific LPA receptor subtype mediation of LPA‐induced hypertrophy of cardiac myocytes and involvement of Akt and NFκB signal pathways

Lysophosphatidic acid (LPA) is a bioactive phospholipid with diverse functions mediated via G-protein-coupled receptors (GPCRs). In view of the elevated levels of LPA in acute myocardial infarction (MI) patients we have conducted studies aimed at identifying specific LPA receptor subtypes and signaling events that may mediate its actions in hypertrophic remodeling. Experiments were carried out in cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and in a rat MI model. In NRCMs, LPA-induced hypertrophic growth was completely abrogated by DGPP, an LPA1/LPA3 antagonist. The LPA3 agonist OMPT, but not the LPA2 agonist dodecylphosphate, promoted hypertrophy as examined by 3[H]-Leucine incorporation, ANF-luciferase expression and cell area. In in vivo experiments, LPA1, LPA2 and LPA3 mRNA levels as well as LPA1 and LPA3 protein levels increased together with left ventricular remodeling (LVRM) after MI. In addition, LPA stimulated the phosphorylation of Akt and p65 protein and activated NF-kappaB-luciferase expression. Inhibitors of PI3K (wortmannin), mTOR (rapamycin), and NF-kappaB (PDTC or SN50) effectively prevented LPA-induced 3[H]-Leucine incorporation and ANF-luciferase expression. Furthermore, ERK inhibitors (U0126 and PD98059) suppressed LPA-stimulated activation of NF-kappaB and p65 phosphorylation whereas wortmannin showed no effect on NF-kappaB activation. Our findings indicate that LPA3 and/or LPA1 mediate LPA-induced hypertrophy of NRCMs and that LPA1 and LPA3 may be involved in LVRM of MI rats. Moreover, Akt and NF-kappaB signaling pathways independently implicate in LPA-stimulated myocardial hypertrophic growth.

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5

Embryonic organs attain their final dimensions through the generation of proper cell number and size, but the control mechanisms remain obscure. Here, we establish Gridlock (Grl), a Hairy-related basic helix-loop-helix (bHLH) transcription factor, as a negative regulator of cardiomyocyte proliferative growth in zebrafish embryos. Mutations in grl cause an increase in expression of a group of immediate-early growth genes, myocardial genes, and development of hyperplastic hearts. Conversely, cardiomyocytes with augmented Grl activity have diminished cell volume and fail to divide, resulting in a marked reduction in heart size. Both bHLH domain and carboxyl region are required for Grl negative control of myocardial proliferative growth. These Grl-induced cardiac effects are counterbalanced by the transcriptional activator Gata5 but not Gata4, which promotes cardiomyocyte expansion in the embryo. Biochemical analyses show that Grl forms a complex with Gata5 through the carboxyl region and can repress Gata5-mediated transcription via the bHLH domain. Hence, our studies suggest that Grl regulates embryonic heart growth via opposing Gata5, at least in part through their protein interactions in modulating gene expression.

111Indium-trastuzumab visualises myocardial human epidermal growth factor receptor 2 expression shortly after anthracycline treatment but not during heart failure: A clue to uncover the mechanisms of trastuzumab-related cardiotoxicity

Aim Trastuzumab can induce cardiotoxicity, particularly when combined with anthracyclines. Myocardial human epidermal growth factor receptor 2 (HER2) expression may be transiently upregulated by a compensatory mechanism following cardiac stress. 111In-DTPA-trastuzumab, scintigraphy can detect HER2 positive tumour lesions, however previously, we found myocardial uptake in only 1 of the 15 anthracycline-pre-treated patients with a median of 11 months after the last anthracycline administration. To evaluate whether myocardial HER2 expression is upregulated by anthracycline-induced cardiac stress or in case of heart failure by chronic pressure or volume overload, we performed 111In-DTPA-trastuzumab scans in patients shortly after anthracyclines and with non-anthracycline-related heart failure. Methods Patients within 3 weeks after undergoing 4–6 cycles first-line anthracycline-based chemotherapy and patients with heart failure due to cardiac disease underwent gammacamera imaging 48 and 96 h after 111In-DTPA-trastuzumab intravenously. Results Myocardial 111In-DTPA-trastuzumab uptake was observed in 5 out of 10 anthracycline-treated patients, who all were without symptomatic cardiac dysfunction. None of the 10 heart failure patients showed myocardial uptake. Conclusion Shortly after completion of anthracycline treatment, myocardial HER2 over-expression was detectable in 50% of the patients. 111In-DTPA-trastuzumab scintigraphy after anthracyclines prior to adjuvant trastuzumab potentially identifies patients susceptible for trastuzumab-related cardiotoxicity and thus may facilitate the optimal timing of trastuzumab therapy.

Forensic pathological investigation of myocardial hypoxia-inducible factor-1α, erythropoietin and vascular endothelial growth factor in cardiac death

The present study investigated the immunohistochemical distributions and mRNA expressions of myocardial hypoxia-inducible factor (HIF)-1α and its downstream factors, erythropoietin (Epo) and vascular endothelial growth factor (VEGF), in cardiac deaths. Medico-legal autopsy cases ( n = 114, within 48-h postmortem) of cardiac deaths ( n = 58) and control cases ( n = 56) were examined. Immunohistochemical positivities of HIF-1α, Epo and VEGF were patchily observed in cardiomyocytes in the acute ischemic lesions of myocardial infarction ( n = 37), showing a relationship to morphological cardiomyocyte damage: the staining was intense in the regions with early ischemic changes and weak in the necrotic regions. Immunopositivities were sporadically detected in cardiomyocytes in some cases of sudden cardiac death without infarction (SCD, n = 13). In chronic congestive heart disease (CHD, n = 8), weak positivities were diffusely observed in the cardiomyocytes. However, there were no such findings in cases of mechanical asphyxiation ( n = 16) or drowning ( n = 18). HIF-1α, Epo and VEGF mRNA expressions, as measured by real-time reverse transcription-polymerase chain reaction (RT-PCR), showed localized elevations related to acute myocardial infarction (AMI) lesions, whereas such findings were mild in recurrent myocardial infarction (RMI) and SCD cases. CHD showed significant elevations of these mRNAs irrespective of the sampling site. The mRNA expressions were significantly lower in cases of drowning. These findings suggest that focal immunopositivities and increased mRNAs of these factors are indicative of short and substantial duration of myocardial ischemia, respectively. The combined analyses may not only be useful for investigating the site, phase and severity of acute myocardial ischemia and the severity of chronic ischemic stress, but also contribute to differentiating cardiac deaths from asphyxiation and drowning or interpreting the possible contribution of cardiac disease in traumatic death.