Expression and effects of TSP50 in mouse embryo and cardiac myocyte development.

TSP50, a testis-specific gene encoding a serine protease-like protein, was specifically expressed in the spermatocytes of testes but abnormally activated and expressed in many different kinds of cancers. Here, we aimed to analyze the expression of TSP50 in mouse embryo and its function in early embryonic development. Firstly, the distribution of TSP50 in oocytes and embryonic development was characterized by immunofluorescence, RT-PCR and western blotting, and the results showed that TSP50 was detected at all studied stages with a dynamic expression pattern. When overexpressed TSP50 in zygotes by microinjection, the zygotes development was highly accelerated. On the contrary, knocking down TSP50 expression by RNA interference greatly retarded the zygote development. Furthermore, TSP50 expression at embryonic day 6.5 (E6.5), day 8.5 (E8.5) and day 10.5 (E10.5) were increasingly enhanced, However, the expression of TSP50 decreased gradually in the development and differentiation of cardiac myocyte from E12.5 to postnatal (P0). Additionally, we found that TSP50 expression was decreased during cardiac myocyte differentiation of P19 cells. Overexpression of TSP50 could decrease the expression of GATA-4, and knockdown of TSP50 markedly increase the expression of GATA-4. Taken together, our data indicate that TSP50 may play an important role during the process of mouse embryonic development as well as myocardial cell differentiation.

Histone deacetylase 3 regulates smooth muscle differentiation in neural crest cells and development of the cardiac outflow tract.

RATIONALE: The development of the cardiac outflow tract (OFT) and great vessels is a complex process that involves coordinated regulation of multiple progenitor cell populations. Among these populations, neural crest cells make important contributions to OFT formation and aortic arch remodeling. Although numerous signaling pathways, including Notch, have been implicated in this process, the role of epigenetics in OFT development remains largely unexplored. OBJECTIVE: Because histone deacetylases (Hdacs) play important roles in the epigenetic regulation of mammalian development, we have investigated the function of Hdac3, a class I Hdac, during cardiac neural crest development in mouse. METHODS AND RESULTS: Using 2 neural crest drivers, Wnt1-Cre and Pax3(Cre), we show that loss of Hdac3 in neural crest results in perinatal lethality and cardiovascular abnormalities, including interrupted aortic arch type B, aortic arch hypoplasia, double-outlet right ventricle, and ventricular septal defect. Affected embryos are deficient in aortic arch artery smooth muscle during midgestation, despite intact neural crest cell migration and preserved development of other cardiac and truncal neural crest derivatives. The Hdac3-dependent block in smooth muscle differentiation is cell autonomous and is associated with downregulation of the Notch ligand Jagged1, a key driver of smooth muscle differentiation in the aortic arch arteries. CONCLUSIONS: These results indicate that Hdac3 plays a critical and specific regulatory role in the neural crest-derived smooth muscle lineage and in formation of the OFT.

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.

Carbonic Anhydrase III Attenuates Hypoxia-Induced Apoptosis and Activates PI3K/Akt/mTOR Pathway in H9c2 Cardiomyocyte Cell Line.

Myocardial ischemia can cause insufficient oxygen and functional damage to myocardial cells. Carbonic anhydrase III (CAIII) has been found to be closely related to the abnormality of cardiomyocytes. To investigate the role of CAIII in the apoptosis of myocytes under hypoxic conditions and facilitate the strategy for treating hypoxia-induced damage, in vitro experiments in H9c2 were employed. The protein expression of CAIII in H9c2 cells after hypoxia or normoxia treatment was determined by western blotting and immunohistochemistry. MTT assay was employed for cells viability measurement and LDH release was monitored. The apoptotic cells were observed using immunofluorescence assay, flow cytometric analysis, and TUNEL assay. CAIII-overexpression or -knockdown cells were constructed to determine the role of CAIII in regulating apoptosis-related proteins caspase-3, Bax, Bcl-2, and anti-apoptosis pathway PI3K/Akt/mTOR. The mRNA levels of CAIII and genes related to CAIII synthesis including REN, IGHM, APOBEC 3F, and SKOR2 were significantly upregulated in hypoxia fetal sheep. The expression of CAIII protein and content of apoptotic H9c2 cells were increased at 1, 3, 6, and 12 h after hypoxia treatment. Overexpression of CAIII significantly upregulated Bcl2 level and downregulated Bax and caspase-3 cleavage levels, while its knockdown led to the contrary results. Overexpressed CAIII promoted the HIF-1α level and activated the PI3K/Akt/mTOR pathway, thereby exerting an inhibitory effect on hypoxia-induced apoptosis. In conclusion, our findings revealed that CAIII could protect cell from hypoxia-apoptosis of H9c2 cells, in which, activated PI3K/Akt/mTOR signaling pathway may be involved.

Modulation of Paraoxonase-1 and Apoptotic Gene Expression Involves in the Cardioprotective Role of Flaxseed Following Gestational Exposure to Diesel Exhaust Particles and/or Fenitrothion Insecticide.

The developmental exposure to a single chemical may elicit apoptosis in the different fetal organs, while the combined effects are restricted. We have examined the protective role of flaxseed (FS) against diesel exhaust particles (DEPs)- and/or fenitrothion (FNT)-induced fetal cardiac oxidative stress and apoptosis. A total of 48 timed pregnant rats were divided into eight groups (n = 6). The first group was saved as the control and the second fed on 20% FS diet. Animals in the third, fourth, and fifth groups were administered with DEPs (2.0 mg/kg), FNT (3.76 mg/kg), and their combination, respectively, while the sixth, seventh, and eighth groups were supplemented with 20% FS through intoxication with DEPs, FNT, and their combination, respectively. Our results revealed that DEPs and/or FNT significantly elevated the level of protein carbonyl and superoxide dismutase activity in the fetal cardiac tissues. However, the catalase activity and total thiol level were decreased; besides the histopathological alterations were remarked. Moreover, DEPs and/or FNT exhibited significant down-regulation in the anti-apoptotic (Bcl-2) and paraoxonase-1 gene expression, and up-regulation in the apoptotic (Bax and caspase-3) gene expression along with DNA fragmentation. Remarkably, FS supplementation significantly ameliorated the fetal cardiac oxidative injury, down-regulated the expression of the apoptotic genes, up-regulated the anti-apoptotic and paraoxonase-1 gene expression, reduced DNA fragmentation, and alleviated the myocardial cell architectures. These findings revealed that FS attenuates DEPs- and/or FNT-induced apoptotic cell death by repairing the disturbance in the anti-apoptotic/pro-apoptotic gene balance toward cell survival in the fetal myocardial cells.

Heart-specific ablation of Prkar1a causes failure of heart development and myxomagenesis.

BACKGROUND: Protein kinase A signaling has long been known to play an important role in cardiac function. Dysregulation of the protein kinase A system, caused by mutation of the protein kinase A regulatory subunit gene PRKAR1A, causes the inherited tumor syndrome Carney complex, which includes cardiac myxomas as one of its cardinal features. Mouse models of this genetic defect have been unsatisfactory because homozygote null animals die early in development and heterozygotes do not exhibit a cardiac phenotype. METHODS AND RESULTS: To study the cardiac-specific effects resulting from complete loss of Prkar1a, we used cre-lox technology to generate mice lacking this protein specifically in cardiomyocytes. Conditional knockout mice died at day 11.5 to 12.5 of embryogenesis with thin-walled, dilated hearts. These hearts showed elevated protein kinase A activity and decreased cardiomyocyte proliferation before demise. Analysis of the expression of transcription factors required for cardiogenesis revealed downregulation of key cardiac transcription factors such as the serum response factor, Gata4, and Nkx2-5. Although heart wall thickness was reduced overall, specific areas exhibited morphological changes consistent with myxomatous degeneration in the walls of knockout hearts. CONCLUSIONS: Loss of Prkar1a from the heart causes a failure of proper myocardial development with subsequent cardiac failure and embryonic demise. These changes appear to be due to suppression of cardiac-specific transcription by increased protein kinase A activity. These biochemical changes lead to myxoma-like changes, indicating that these mice may be a good model with which to study the formation of these tumors.

[Effect of hyperglycaemia on fetal heart in pregnant rats].

OBJECTIVE: To investigate the effect of hyperglycaemia on the cardiomyodial change of rat fetus. METHODS: Thirty clean SD pregnant rats were randomly dividing into group A, B and C, 10 in each group. Group A were injected intraperitoneally 50 mg/kg streptozotocin on the 6th day of pregnancy, Group B were injected the same dose on the 13th day of pregnancy, while Group C were injected intraperitoneally 0.1 mmol/L citrate buffer solution on the 6th day of pregnancy. All rats were killed on the 21st day of pregnancy, the total fetus, live fetus, weight, and length of fetus were recorded. The blood glucose in the fetal rats was measured, and the fetal hearts were collected. The fetal hearts were pathologically examined under light microscope and electron microscope. Immunohistochemical staining was applied to determine Caspase-3 in the heart of fetus. RESULTS: (1) The blood glucose of pregnant rats in the 3 groups showed no difference before intervening (P>0.05). There was significant difference between Group A and C, Group B and C after intervening (P<0.01), but no significant difference between Group A and B was found (P>0.05). (2 )The fetus in Group A and B was heavier and longer than in Group C, with significant difference (P<0.01), but not between Group A and B (P>0.05). The blood glucose of fetus in Group A and B was lower than that in Group C, with significant difference (P<0.01), but not between Group A and B (P>0.05). The rate of fetal death in Group A, B, and C were 31.96%,12.84%, and 3.88%, respectively. Significant deviation existed in the 3 groups (P<0.01). (3) Under light microscope, fetal hearts in Group A and B showed disorder, cardiac muscle cells swelled. There were vacuoles in cytolymph and necrosis in the myocardial tissue. Significant deviation in the integral of fetal necrosis existed in the 3 groups (P<0.01). (4) Caspase-3 was detected in the fetal hearts, the positive area ratio and mean OD value had significant deviation in the 3 groups (P<0.01).(5) Under the electron microscope, cardiomyocytes wrinkled, mitochondrion decreased, myofibril ruptured, while sarcomere blurred. The density of mitochondria in cardiamyocyte in Group A was lower than that in Group B and C (P<0.01), and the average volume of mitochondria of Group A and B was higher than that in Group C (P<0.01). CONCLUSION: There is apparent pathological change of fetal hearts in pregnant rats with hyperglycaemia. The longer the duration, the more obvious the change.

Expression patterns of protein kinase D 3 during mouse development.

BACKGROUND: The PKD family of serine/threonine kinases comprises a single member in Drosophila (dPKD), two isoforms in C. elegans (DKF-1 and 2) and three members, PKD1, PKD2 and PKD3 in mammals. PKD1 and PKD2 have been the focus of most studies up to date, which implicate these enzymes in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, immune responses, apoptosis and cell proliferation. Concerning PKD3, a role in the formation of vesicular transport carriers at the trans-Golgi network (TGN) and in basal glucose transport has been inferred from in vitro studies. So far, however, the physiological functions of the kinase during development remain unknown. RESULTS: We have examined the expression pattern of PKD3 during the development of mouse embryos by immunohistochemistry. Using a PKD3 specific antibody we demonstrate that the kinase is differentially expressed during organogenesis. In the developing heart a strong PKD3 expression is constantly detected from E10 to E16.5. From E12.5 on PKD3 is increasingly expressed in neuronal as well as in the supporting connective tissue and in skeletal muscles. CONCLUSION: The data presented support an important role for PKD3 during development of these tissues.

Mitogen-activated protein kinases in heart development and diseases.

Mitogen-activated protein (MAP) kinases belong to a highly conserved family of Ser-Thr protein kinases in the human kinome and have diverse roles in broad physiological functions. The 4 best-characterized MAP kinase pathways, ERK1/2, JNK, p38, and ERK5, have been implicated in different aspects of cardiac regulation, from development to pathological remodeling. Recent advancements in the development of kinase-specific inhibitors and genetically engineered animal models have revealed significant new insights about MAP kinase pathways in the heart. However, this explosive body of new information also has yielded many controversies about the functional role of specific MAP kinases as either detrimental promoters or critical protectors of the heart during cardiac pathological processes. These uncertainties have raised questions on whether/how MAP kinases can be targeted to develop effective therapies against heart diseases. In this review, recent studies examining the role of MAP kinase subfamilies in cardiac development, hypertrophy, and survival are summarized.

Geranylgeranyl pyrophosphate synthase facilitates the organization of cardiomyocytes during mid-gestation through modulating protein geranylgeranylation in mouse heart.

Aims: With the maturation of placenta, ventricular chamber maturation enhances cardiac contractile performance to adapt to the metabolic demand of growing embryo. The organization of cardiomyocytes is required for the morphological remodelling in ventricular chamber maturation. However, the mechanism governing the establishment of cardiac cytoarchitecture during ventricular chamber maturation is still poorly studied. Methods and results: Here, we found that the expression of geranylgeranyl pyrophosphate synthase (Ggpps), which mediates protein geranylgeranylation, increased in the mouse heart after the onset of placental function. By using different Cre lines, we found that the cardiac inactivation of Ggpps by the Nkx2.5Cre/+ line disrupted protein geranylgeranylation as early as E9.5, which affected ventricular chamber maturation and resulted in mid-gestational embryonic lethality. In contrast, α-SMA-Cre line mediated the disruption of protein geranylgeranylation from E13.5 did not affect embryonic heart development. Further analysis of Nkx2.5Cre/+; Ggppsfl/fl mutants showed that the loss of Ggpps caused disorganized cardiac cytoarchitecture as early as E11.5 by disturbing cell-cell junctions. Ggpps inactivation decreased Rho GTPase geranylgeranylation and their activity, which might account for the disruption of cell-cell junctions. Moreover, elevating the protein geranylgeranylation by supplement of geranylgeranyl pyrophosphate (GGPP) could recover the Ggpps deficient induced defects of cytoarchitecture and cell-cell junctions in vitro and in vivo. Conclusion: Our present study demonstrates that GGPPS-mediated protein geranylgeranylation plays an indispensable role in the ventricular chamber maturation and acts as a stage-specific signal to regulate the establishment of cardiac cytoarchitecture during mid-gestation.

Critical role of phosphodiesterase 2A in mouse congenital heart defects.

Aims: Phosphodiesterase 2 A (Pde2A), a cAMP-hydrolysing enzyme, is essential for mouse development; however, the cause of Pde2A knockout embryonic lethality is unknown. To understand whether Pde2A plays a role in cardiac development, hearts of Pde2A deficient embryos were analysed at different stage of development. Methods and results: At the stage of four chambers, Pde2A deficient hearts were enlarged compared to the hearts of Pde2A heterozygous and wild-type. Pde2A knockout embryos revealed cardiac defects such as absence of atrial trabeculation, interventricular septum (IVS) defects, hypertrabeculation and thinning of the myocardial wall and in rare cases they had overriding aorta and valves defects. E14.5 Pde2A knockouts showed reduced cardiomyocyte proliferation and increased apoptosis in the IVS and increased proliferation in the ventricular trabeculae. Analyses of E9.5 Pde2A knockout embryos revealed defects in cardiac progenitor and neural crest markers, increase of Islet1 positive and AP2 positive apoptotic cells. The expression of early cTnI and late Mef2c cardiomyocyte differentiation markers was strongly reduced in Pde2A knockout hearts. The master transcription factors of cardiac development, Tbx, were down-regulated in E14.5 Pde2A knockout hearts. Absence of Pde2A caused an increase of intracellular cAMP level, followed by an up-regulation of the inducible cAMP early repressor, Icer in fetal hearts. In vitro experiments on wild-type fetal cardiomyocytes showed that Tbx gene expression is down-regulated by cAMP inducers. Furthermore, Pde2A inhibition in vivo recapitulated the heart defects observed in Pde2A knockout embryos, affecting cardiac progenitor cells. Interestingly, the expression of Pde2A itself was dramatically affected by Pde2A inhibition, suggesting a potential autoregulatory loop. Conclusions: We demonstrated for the first time a direct relationship between Pde2A impairment and the onset of mouse congenital heart defects, highlighting a novel role for cAMP in cardiac development regulation.

Heterogeneity of beta-type myosin isozymes in the human heart and regulational mechanisms in their expression. Immunohistochemical study using monoclonal antibodies.

To investigate the existence of heterogeneity of beta-type myosin isozymes (HC beta) in human hearts, immunohistochemical studies using monoclonal antibodies (MoAbs) raised against human ventricular myosin heavy chains were performed. Two types of MoAbs recognized some muscle fibers in the atrium, whereas both reacted with all ventricular muscle fibers. Since atrial muscle fibers reactive with each MoAb were found to be clearly different, the existence of two immunologically distinct HC beta (beta 1, and beta 2) was suggested in the atrium. By using affinity chromatography, two molecular variants of HC beta were isolated from the bovine atrium, and differences in the primary structure of beta 1 and beta 2 were confirmed by analysis of peptides produced by chymotryptic digestion. In pressure-overloaded human atria, myofibers containing beta 1 and/or beta 2 increased in accordance with decrement of myofibers containing alpha-type myosin isozyme (P less than 0.01). But they differed in expression during the developmental stage, since beta 2 did not exist in the early embryonic bovine heart, but beta 1 did. Thus, there are two distinct HC beta whose expression is regulated by at least two factors: pressure overload and developmental stage.

Disruption of ECE-1 and ECE-2 reveals a role for endothelin-converting enzyme-2 in murine cardiac development.

Endothelin-converting enzyme-1 and -2 (ECE-1 and -2) are membrane-bound metalloproteases that can cleave biologically the inactive endothelin-1 (ET-1) precursor to form active ET-1 in vitro. We previously reported developmental defects in specific subsets of neural crest-derived tissues, including branchial arch-derived craniofacial structures, aortic arch arteries, and the cardiac outflow tract in ECE-1 knockout mice. To examine the role of ECE-2 in cardiovascular development, we have now generated a null mutation in ECE-2 by homologous recombination. ECE-2 null mice develop normally, are healthy into adulthood, are fertile in both sexes, and live a normal life span. However, when they are bred into an ECE-1-null background, defects in cardiac outflow structures become more severe than those in ECE-1 single knockout embryos. In addition, ECE-1(-/-); ECE-2(-/-) double null embryos exhibited abnormal atrioventricular valve formation, a phenotype never seen in ECE-1 single knockout embryos. In the developing mouse heart, ECE-2 mRNA is expressed in the endocardial cushion mesenchyme from embyronic day (E) 12.5, in contrast to the endocardial expression of ECE-1. Levels of mature ET-1 and ET-2 in whole ECE-1(-/-); ECE-2(-/-) embryos at E12.5 do not differ appreciably from those of ECE-1(-/-) embryos. The significant residual ET-1/ET-2 in the ECE-1(-/-); ECE-2(-/-) embryos indicates that proteases distinct from ECE-1 and ECE-2 can carry out ET-1 activation in vivo.

Responsiveness to glucagon in fetal hearts. Species variability and apparent disparities between changes in beating, adenylate cyclase activation, and cyclic AMP concentration.

Previous studies of the ability of the immature heart to respond to glucagon have yielded conflicting results. To test the possibility that the apparent discrepancies might be explained in part by species variability, isolated hearts of fetal mice and rats (13-22 days' gestational age) were studied under identical conditions in vitro. Changes in atrial rate and ventricular contractility were measured in spontaneously beating hearts exposed to glucagon, and activation of adenylate cyclase was assayed in cardiac homogenates. In mice of 16 days' gestational age or less, there was no change in heart rate in response to glucagon; at 17-18 days, minimal responsiveness was present; and after 19 days, 10muM glucagon caused an increase in spontaneous atrial rate of 30 +/- 4% (SEM) (P less than 0.001). Measurement of the extent and speed of volume displacement of the isotonically contracting hearts with a specially constructed capacitance transducer revealed that ventricular inotropic responsiveness also appeared after 17-19 days. Cardiac stores of glycogen were reduced in older hearts exposed to glucagon, but not in those aged less than 16 days. In contrast, glucagon failed to activate adenylate cyclase in homogenates of hearts of fetal mice at any age. Furthermore, glucagon failed to elicit an increase in the concentration of cyclic AMP in spontaneously beating hearts that developed tachycardia. Responses in hearts of fetal rats were distinctly different from those in mouse hearts: at no age was there any change in heart rate, strength of contraction, glycogen content, or adenylate cyclase activation. Thus, there are major species differences in cardiac pharmacological maturation. Although the mouse heart develops the ability to increase its rate and strength of contraction and to undergo glycogenolysis in response to glucagon well before birth, the rat heart does not. In addition, there is an apparent disparity in late fetal mouse hearts between the ability of glucagon to induce functional responses and its ability to stimulate adenylate cyclase and increase cyclic AMP levels. It is impossible, of course, to rule out absolutely the possibility that localized increases in a critical cyclic AMP pool were present but too small to measure in the entire tissue. Nevertheless, the most obvious interpretation of our results is that they are compatible with the hypothesis that glucagon may exert some of its hemodynamic effects independently from the adenylate cyclase-cyclic AMP system in the late-fetal mouse heart.

Maternal exercise upregulates mitochondrial gene expression and increases enzyme activity of fetal mouse hearts.

Maternal exercise during pregnancy has been shown to improve the long-term health of offspring in later life. Mitochondria are important organelles for maintaining adequate heart function, and mitochondrial dysfunction is linked to cardiovascular disease. However, the effects of maternal exercise during pregnancy on mitochondrial biogenesis in hearts are not well understood. Thus, the purpose of this study was to test the hypothesis that mitochondrial gene expression in fetal myocardium would be upregulated by maternal exercise. Twelve-week-old female C57BL/6 mice were divided into sedentary and exercise groups. Mice in the exercise group were exposed to a voluntary cage-wheel from gestational day 1 through 17. Litter size and individual fetal weights were taken when pregnant dams were sacrificed at 17 days of gestation. Three to four hearts from the same group were pooled to study gene expression, protein expression, and enzyme activity. There were no significant differences in litter size, sex distribution, and average fetal body weight per litter between sedentary and exercised dams. Genes encoding mitochondrial biogenesis and dynamics, including nuclear respiratory factor-1 (Nrf1), Nrf2, and dynamin-related GTPase termed mitofusin-2 (Mfn2) were significantly upregulated in the fetal hearts from exercised dams. Cytochrome c oxidase activity and ATP production were significantly increased, while the hydrogen peroxide level was significantly decreased in the fetal hearts by maternal exercise. Our results demonstrate that maternal exercise initiated at day 1 of gestation could transfer the positive mitochondrial phenotype to fetal hearts.

Embryonic epinephrine synthesis in the rat heart before innervation: association with pacemaking and conduction tissue development.

Epinephrine is a potent neurotransmitter and hormone that can influence cardiac performance beginning shortly after the first myocardial contractions occur in developing vertebrate embryos. In the present study, we provide evidence that the heart itself may produce epinephrine during embryonic development. Using antibodies that selectively recognize the catecholamine biosynthetic enzymes, tyrosine hydroxylase, dopamine ss-hydroxylase, and phenylethanolamine N-methyltransferase, we used coimmunofluorescent staining techniques to identify cardiac cells that have the capability of producing catecholamines. Initially, cells expressing catecholamine biosynthetic enzymes were found interspersed throughout the myocardium, but by embryonic day 11.5 (E11.5), they became preferentially localized to the dorsal venous valve and atrioventricular canal regions. As development proceeded, catecholamine biosynthetic enzyme expression decreased in these regions but became quite strong along the crest of the interventricular septum by E16.5. This expression pattern was also transient, decreasing in the ventricular septum by E19.5. These data are consistent with a transient and progressive association of catecholamine-producing cells within regions of the heart that become the sinoatrial node, atrioventricular node, and bundle of His. This is the first evidence demonstrating that intrinsic cardiac adrenergic cells may be preferentially associated with early pacemaking and conduction tissue development.

Apoptotic versus autophagic cell death in heart failure.

OBJECTIVE: Progressive loss of cardiomyocytes is one of the most important pathogenic characteristics of heart failure. Apoptosis may be an important mode of cell death in heart failure but it must be demonstrated by multiple criteria and not just TUNEL staining alone. Previously, we and others have demonstrated that besides apoptosis other phenomena like active gene transcription can result in TUNEL positivity. Moreover, other types of cell death that are caspase-independent could be important in heart failure. This study examined the hypothesis whether TUNEL labeling parallels caspase activation. METHODS: Cardiac tissue of patients in the terminal stage of heart failure as a consequence of ischaemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM) were studied. Embryonic mice hearts were used for positive control for detection of the classical apoptosis. RESULTS: In mice embryonic hearts we could clearly find apoptotic cell death detected by TUNEL labeling and immunohistochemistry for activated caspase-3. In heart failure, TUNEL-positive cardiomyocytes were negative for active caspase-3 but showed signs of active gene transcription (SC-35). However, autophagic cell death could be found in 0.3% of the cardiomyocytes. Autophagic cell death was demonstrated by granular cytoplasmic ubiquitin inclusions, an established marker of autophagocytosis in neurons. Interestingly, these autophagic cardiomyocytes were TUNEL and activated caspase-3 negative but were also negative for C9, a marker for necrosis. Western blot analysis confirmed that in cardiomyopathies no cleavage of caspase-3 and caspase-7 occurred. CONCLUSION: The present study demonstrates two fundamentally different situations of cell death in cardiac tissue. In embryonic mice, cardiomyocytes undergo caspase-dependent cell death. However, cardiomyocytes in heart failure show caspase-independent autophagic cell death rather than apoptotic cell death.

Thyroxine and propylthiouracil-induced changes in the activity of monoamine oxidase in the fetal rat.

The activity of monoamine oxidase (MAO) towards tryptamine has been determined in heart, brain and liver of the 21.5 day old rat fetus. The activity was compared between normal, thyroxine and propylthiouracil (PTU) treated animals. Hypothyroidism induced by PTU leads to a decrease in the activity of the three organs, while hyperthyroidism causes an increase in the enzymatic activity. These differences seem to be rather independent of the protein content of the organs. It appears thar rat fetal MAO is under a strong thyroid control.

Hypoglycemia and embryonic heart development.

Abnormal embryonic development is a complication of the diabetic pregnancy, and heart defects are among the most common and detrimental congenital malformations of the diabetic embryopathy. Hypoglycemia is a common side effect of diabetes therapy and is a potential teratogen. An association between hypoglycemia and congenital defects has been difficult to demonstrate in humans, but in vivo and in vitro animal studies have illustrated the importance of glucose as a substrate for normal development. Hypoglycemia alters embryonic heart morphology, producing abnormal looping and chamber expansion, decreased myocardial thickness, disorganized layers, and decreased overall size. Hypoglycemia decreases embryonic heart rate and vascularity, and it alters embryonic heart metabolism by increasing glucose uptake and glycolysis. Hypoglycemia also affects protein expression in the embryonic heart, increasing the expression of glucose regulated proteins, hexokinase, and glucose transport protein. Thus, hypoglycemia interferes with normal cardiogenesis and alters morphology, function, metabolism, and expression of certain proteins in the developing heart. It is likely that these factors contribute to heart defects observed in the diabetic embryopathy, but the definitive link has yet to be made. Future studies are expected to further elucidate mechanisms mediating hypoglycemia-induced cardiac dysmorphogenesis.

beta 2-Adrenoceptors regulate induction of myocardial ornithine decarboxylase in mice in vivo.

The pharmacological characteristics of the myocardial adrenoceptor of the mouse have been examined during embryogenesis by measuring ornithine decarboxylase (ODC, EC 4.1.1.17) induction. 2 A four fold elevation of ODC activity was observed after isoprenaline (10 mg/kg, s.c.), and enzyme activity was increased two to three fold following adrenaline (1 mg/kg, s.c.) or terbutaline given by direct injection to the foetus (10 microgram/500 mg). 3 Pretreatment with the beta-adrenoceptor antagonist, propranolol (10 mg/kg), totally blocked the increase in ODC activity. 4 Elevation of myocardial ODC activity was not inhibited by metoprolol, a relatively specific beta-adrenoceptor antagonist, at a dose of 10 mg/kg. 5 Since the increase in ODC activity was blocked by a beta-adrenoceptor antagonist (propranolol) and enzyme activity was stimulated by terbutaline, a beta 2-agonist, we conclude that beta 2-adrenoceptors are selectively coupled to the regulation of murine cardiac ODC activity following catecholamine stimulation.