Developing Rat Heart Research Articles

Nanopore sequencing reveals full‐length Tropomyosin 1 isoforms and their regulation by RNA‐binding proteins during rat heart development

Alternative splicing (AS) contributes to the diversity of the proteome by producing multiple isoforms from a single gene. Although short‐read RNA‐sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full‐length mRNA isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin‐binding protein required for cytoskeletal functions in non‐muscle cells and for contraction in muscle cells. Tpm1 undergoes AS regulation to generate muscle versus non‐muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full‐length Tpm1 isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long‐read cDNA sequencing without gene‐specific PCR amplification. In rat hearts, we identified full‐length Tpm1 isoforms composed of distinct exons with specific exon linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle‐specific exons are connected generating predominantly muscle‐specific Tpm1 isoforms in adult hearts. We found that the RNA‐binding protein RBFOX2 controls AS of rat Tpm1 exon 6a, which is important for cooperative actin binding. Furthermore, RBFOX2 regulates Tpm1 AS of exon 6a antagonistically to the RNA‐binding protein PTBP1. In sum, we defined full‐length Tpm1 isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into the regulation of Tpm1 AS by RNA‐binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine full‐length AS variants of muscle‐enriched genes.

Early L-T4 intervention improves fetal heart development in pregnant rats with subclinical hypothyroidism rats by activating BMP4/Smad4 signaling pathway

BackgroundIt is unclear whether the offspring of subclinical hypothyroidism (SCH) pregnant rats still have abnormal cardiac development, and whether early intervention with L-T4 can improve the abnormality of these offspring. Therefore, the aim of this study was to investigate the effect of early L-T4 intervention on the heart development of offspring of SCH pregnant rats and its possible molecular mechanism.MethodsEighty female Wistar rats were randomly divided into Sham group (placebo control), SCH group, LT4-E10 group (L-T4 treatment started on the 10th day of gestation), and LT4-E13 group (L-T4 treatment started on the 13th day of gestation). Each group was further divided into E16 (16th day of gestation), E18 (18th day of gestation), P5 (5th day postnatal day), and P10 (10th day postnatal day) subgroups. The levels of serum TT4 and TSH, the ratio of heart weight to body weight of offspring rats, the expression of metabolic enzymes, and the histopathology of cardiomyocytes were determined. To elucidate the effects of L-T4 on cardiac development of offspring of SCH pregnant rats, the expression levels of GATA4, Nkx2–5 and proteins involved in BMP4/Smad4 signaling pathway were detected by immunohistochemistry, real time quantitative polymerase chain reaction and Western blotting to elucidate the molecular mechanism of L-T4 regulating the heart development of the offspring of SCH pregnant rats.ResultsCompared with Sham group, serum TSH was significantly increased in SCH pregnant rats. Moreover, early L-T4 intervention significantly reduced the levels of serum TSH. Compared with the offspring in the SCH group, early L-T4 intervention significantly increased the heart weight, heart weight to body weight ratio, the activities of succinate dehydrogenase (SDH), Na+/K+-ATPase and Ca2+-ATPase, but reduced myocardial cell shrinkage and nuclear staining, hyperemia/congestion and vacuolar degeneration. In addition, early L-T4 intervention not only significantly increased the mRNA and protein expression of Gata4 and Nkx2–5, but also increased the protein expression involved in BMP4/Smad4 signal pathway in myocardium of the offspring of SCH pregnant rats.ConclusionsEarly L-T4 intervention can regulate the cardiac development of the offspring of SCH pregnant rats by activating BMP4/Smad4 signaling pathway and increasing the expression of Gata4 and Nkx2–5 proteins.

Research into the intervention effect of folic acid on arsenic-induced heart abnormalities in fetal rats during the periconception period

BackgroundThe incidence of CHD is the highest among birth defects and is increasing year to year. CHD seriously harms the health of infants and young children and presents a large economic burden to families and society. The pathogenesis of CHD and preventive measures are the focus of current research. Our research aimed to explore the intervention effect of folic acid on heart abnormalities resulting from sodium arsenic (NaAsO2) exposure during the periconception period.MethodsSixty 35-day-old female SD rats were randomly divided into 5 groups with 12 rats in each group. Group A was the control group. The rats were given distilled water and ordinary chow. The rats in group B were given distilled water containing 75 mg/L NaAsO2 and ordinary chow. The rats in groups C, D, and E were given distilled water containing 75 mg/L NaAsO2 and chow containing 0.53 mg/kg, 5.3 mg/kg, and 10.6 mg/kg folic acid, respectively. The general condition of the embryos and the histopathology of the embryonic hearts were examined. The acetylation levels of histone H3K9 in heart tissues and the expression levels of Mef2C (which is related to heart development) were observed.ResultsThe embryo weight and placental weight of groups B-E were significantly lower than those of group A (P < 0.05). The heart malformation rate of the fetal rats in groups B-E was significantly higher than that of the fetal rats in group A (P < 0.05). We found that the level of H3K9 acetylation in fetal rat cardiomyocytes in groups B-E was significantly higher than that in group A (P < 0.05) and that the level of H3K9 acetylation in groups C-E was lower than that in group B (P < 0.05). The mRNA level of Mef2C in fetal rat cardiomyocytes in group B-E was significantly higher than that in group A (P < 0.05), and the mRNA level of Mef2C in groups C-E was significantly lower than that in group B (P < 0.05).ConclusionSupplementation with folic acid during the periconception period can interfere with the toxic effects of arsenic on the heart. The mechanism may be that lowering the acetylation levels of histone H3K9 in heart tissues leads to decreased expression levels of Mef2C, which may play a protective role in heart development in fetal rats.

MiR-27a/b is a posttranscriptional regulator of Gpr126 (Adgrg6).

Gpr126 (Adgrg6), a member of the adhesion G protein-coupled receptor family, has been associated with a variety of human diseases. Yet, despite its clinical importance, the mechanisms regulating Gpr126 expression are poorly understood. Here, we aimed at identifying upstream regulatory mechanisms of Gpr126 expression utilizing the heart as model organ in which Gpr126 regulates trabeculation. Here, we focused on possible regulation of Gpr126 regulation by microRNAs, which have emerged as key players in regulating development, have a critical role in disease progression, and might serve as putative therapeutic targets. In silico analyses identified one conserved binding site in the 3' UTR of Gpr126 for microRNA 27a and 27b (miR-27a/b). In addition, miR-27a/b and Gpr126 expression were differentially expressed during rat heart development. A regulatory role of miR-27a/b in controlling Gpr126 expression was substantiated by reduced Gpr126 mRNA levels upon ectopic expression of miR-27a/b in HEK293T cells and miR-27b in zebrafish embryos. Regulation of Gpr126 expression by direct binding of miR-27a/b to the 3' UTR of Gpr126 was verified by luciferase reporter assays in HEK293T cells. Finally, the modulation of gpr126 expression in zebrafish by injection of either miR-27b or miR-27b inhibitor in single cell-stage embryos resulted in hypo- or hypertrabeculation, respectively. Collectively, the data indicate that Gpr126 expression is regulated by miR-27a/b.

Abstract 581: Determining the Nuclear Transcription Factor Network Controlling Expression of the Cardiac Inotropic Factor S100A1

Down-regulation of cardiac inotropic EF-hand calcium sensor protein S100A1 expression is a hallmark of human heart failure and drives progression and mortality. An AAV-S100A1 gene replacement therapy, which has been shown in preclinical models to rescue chronic heart failure, is in preparation for first clinical trials. However, the nuclear mechanisms that convey S100A1 gene locus silencing in the failing heart have not been determined yet. Given the steady increase of S100A1 expression in the developing heart until young adulthood and its decrease in the aged and failing heart, we developed a novel approach to decipher the mechanisms of S100a1 transcriptional regulation. We performed serial RNAseq analyses in an in vitro cardiac differentiation model of H9c2 cells that displays robust concurrent S100A1 and e.g. sarcoplasmic reticulum calcium ATPase 2 (SERCA2a) mRNA and protein during differentiation. Potential transcription factors (TF) were derived by computational motif analysis from S100A1’s gene locus regulatory elements that delivered more than 200 candidates. Subsequent computational TF footprint binding motive analysis from all differentially expressed transcripts of the serial RNAseq data provided a smaller group of less than 30 putatively TF families in the cH9C2 model that is actively during the in vitro biological process. Additional comparative TF expression analyses with developing rat hearts then enabled subtraction of a final TF group of 10 members for RNA interference screens in the cH9C2 model using S100A1 RNA expression as a read-out. Using a siRNA-mediated TF knock-down screen, we identified a small group of TFs such as zink finger and coiled-coil TFs (e.g. KLFs) and signal transducer and activator of transcription (STATs) TFs with an hitherto unknown ability to independently regulate S100A1 expression. Of note, other inotropic factors such as SERCA2a were found to be positively co-regulated by some of the TFs, such as KLFs, that enhance S100A1 expression. Further analyses were performed to confirm these results. In summary, we present a feasible computational filter and experimental strategy to identify relevant TF networks which we have successfully applied to identify cardiac regulatory networks.

Evidence of primary cilia in the developing rat heart

BackgroundA transient increase in cytosolic Ca2+ (the “Ca2+ transient”) determines the degree and duration of myocyte force development in the heart. However, we have previously observed that, under the same experimental conditions, the Ca2+ transients from isolated cardiac myocytes are reduced in amplitude in comparison to those from multicellular cardiac preparations. We therefore questioned whether the enzymatic cell isolation procedure might remove structures that modulate intracellular Ca2+ in some way. Primary cilia are found in a diverse range of cell types, and have an abundance of Ca2+-permeable membrane channels that result in Ca2+ influx when activated. Although primary cilia are reportedly ubiquitous, their presence and function in the heart remain controversial. If present, we hypothesized they might provide an additional Ca2+ entry pathway in multicellular cardiac tissue that was lost during cell isolation. The aim of our study was to look for evidence of primary cilia in isolated myocytes and ventricular tissue from rat hearts.MethodsImmunohistochemical techniques were used to identify primary cilia-specific proteins in isolated myocytes from adult rat hearts, and in tissue sections from embryonic, neonatal, young, and adult rat hearts. Either mouse anti-acetylated α-tubulin or rabbit polyclonal ARL13B antibodies were used, counterstained with Hoechst dye. Selected sections were also labelled with markers for other cell types found in the heart and for myocyte F-actin.ResultsNo evidence of primary cilia was found in either tissue sections or isolated myocytes from adult rat ventricles. However, primary cilia were present in tissue sections from embryonic, neonatal (P2) and young (P21 and P28) rat hearts.ConclusionThe lack of primary cilia in adult rat hearts rules out their contribution to myocyte Ca2+ homoeostasis by providing a Ca2+ entry pathway. However, evidence of primary cilia in tissue from embryonic and very young rat hearts suggests they have a role during development.

Titin isoforms are increasingly protected against oxidative modifications in developing rat cardiomyocytes

During the perinatal adaptation process N2BA titin isoforms are switched for N2B titin isoforms leading to an increase in cardiomyocyte passive tension (Fpassive). Here we attempted to reveal how titin isoform composition and oxidative insults (i.e. sulfhydryl (SH)-group oxidation or carbonylation) influence Fpassive of left ventricular (LV) cardiomyocytes during rat heart development. Moreover, we also examined a hypothetical protective role for titin associated small heat shock proteins (sHSPs), Hsp27 and αB-crystallin in the above processes. Single, permeabilized LV cardiomyocytes of the rat (at various ages following birth) were exposed either to 2,2′-dithiodipyridine (DTDP) to provoke SH-oxidation or Fenton reaction reagents (iron(II), hydrogen peroxide (H2O2), ascorbic acid) to induce protein carbonylation of cardiomyocytes in vitro. Thereafter, cardiomyocyte force measurements for Fpassive determinations and Western immunoblot assays were carried out for the semiquantitative determination of oxidized SH-groups or carbonyl-groups of titin isoforms and to monitor sHSPs’ expressions. DTDP or Fenton reagents increased Fpassive in 0- and 7-day-old rats to relatively higher extents than in 21-day-old and adult animals. The degrees of SH-group oxidation or carbonylation declined with cardiomyocyte age to similar extents for both titin isoforms. Moreover, the above characteristics were mirrored by increasing levels of HSP27 and αB-crystallin expressions during cardiomyocyte development. Our data implicate a gradual build-up of a protective mechanism against titin oxidation through the upregulation of HSP27 and αB-crystallin expressions during postnatal cardiomyocyte development.

Multiple Nav1.5 isoforms are functionally expressed in the brain and present distinct expression patterns compared with cardiac Nav1.5.

It has previously been demonstrated that there are various voltage gated sodium channel (Nav) 1.5 splice variants expressed in brain tissue. A total of nine Nav1.5 isoforms have been identified, however, the potential presence of further Nav1.5 variants expressed in brain neurons remains to be elucidated. The present study systematically investigated the expression of various Nav1.5 splice variants and their associated electrophysiological properties in the rat brain tissue, via biochemical analyses and whole-cell patch clamp recording. The results demonstrated that adult Nav1.5 was expressed in the rat, in addition to the neonatal Nav1.5, Nav1.5a and Nav1.5f isoforms. Further studies indicated that the expression level ratio of neonatal Nav1.5 compared with adult Nav1.5 decreased from 1:1 to 1:3 with age development from postnatal (P) day 0 to 90. This differed from the ratios observed in the developing rat hearts, in which the expression level ratio decreased from 1:4 to 1:19 from P0 to 90. The immunohistochemistry results revealed that Nav1.5 immunoreactivity was predominantly observed in neuronal cell bodies and processes, whereas decreased immunoreactivity was detected in the glial components. Electrophysiological analysis of Nav1.5 in the rat brain slices revealed that an Na current was detected in the presence of 300 nM tetrodotoxin (TTX), however this was inhibited by ~1 µM TTX. The TTX-resistant Na current was activated at −40 mV and reached the maximum amplitude at 0 mV. The results of the present study demonstrated that neonatal and adult Nav1.5 were expressed in the rat brain and electrophysiological analysis further confirmed the functional expression of Nav1.5 in brain neurons.

Changes in the composition of fatty acids and lipofuscin-like pigments during development of rat heart.

Postnatal heart development is characterized by critical periods of heart remodeling. In order to characterize the changes in the lipophilic fraction induced by free radicals, fatty acids and their oxidized products, lipofuscin-like pigments (LFP), were investigated. Fatty acids were analyzed by gas chromatography and LFP were studied by fluorescence techniques. A fluorophore characterized by spectral methods was further resolved by HPLC. Major changes in the composition of fatty acids occurred immediately after birth and then during maturation. Fluorescence of LFP changed markedly on postnatal days 1, 4, 8, and 14, and differed from the adult animals. LFP comprise several fluorophores that were present since fetal state till adulthood. No new major fluorophores were formed during development, just the abundances of individual fluorophores have been modulated which produced changes in the shape of the spectral arrays. HPLC resolved the fluorophore with excitation maximum at 360 nm and emission maximum at 410 nm. New chromatographically distinct species appeared immediately on postnatal day 1, and then on days 30 and 60. Consumption of polyunsaturated fatty acids immediately after birth and subsequent formation of LFP suggests that oxidative stress is involved in normal heart development.

Changes in short-chain acyl-coA dehydrogenase during rat cardiac development and stress.

This study was designed to investigate the expression of short-chain acyl-CoA dehydrogenase (SCAD), a key enzyme of fatty acid β-oxidation, during rat heart development and the difference of SCAD between pathological and physiological cardiac hypertrophy. The expression of SCAD was lowest in the foetal and neonatal heart, which had time-dependent increase during normal heart development. In contrast, a significant decrease in SCAD expression was observed in different ages of spontaneously hypertensive rats (SHR). On the other hand, swim-trained rats developed physiological cardiac hypertrophy, whereas SHR developed pathological cardiac hypertrophy. The two kinds of cardiac hypertrophy exhibited divergent SCAD changes in myocardial fatty acids utilization. In addition, the expression of SCAD was significantly decreased in pathological cardiomyocyte hypertrophy, however, increased in physiological cardiomyocyte hypertrophy. SCAD siRNA treatment triggered the pathological cardiomyocyte hypertrophy, which showed that the down-regulation of SCAD expression may play an important role in pathological cardiac hypertrophy. The changes in peroxisome proliferator-activated receptor α (PPARα) was accordant with that of SCAD. Moreover, the specific PPARα ligand fenofibrate treatment increased the expression of SCAD and inhibited pathological cardiac hypertrophy. Therefore, we speculate that the down-regulated expression of SCAD in pathological cardiac hypertrophy may be responsible for ‘the recapitulation of foetal energy metabolism’. The deactivation of PPARα may result in the decrease in SCAD expression in pathological cardiac hypertrophy. Changes in SCAD are different in pathological and physiological cardiac hypertrophy, which may be used as the molecular markers of pathological and physiological cardiac hypertrophy.

Functional Study of TREK-1 Potassium Channels During Rat Heart Development and Cardiac Ischemia Using RNAi Techniques

To explore the physiological and pathological significance of the 2-pore domain potassium channel TWIK-related K(+) (TREK)-1 in rat heart, its expression and role during heart development and cardiac ischemia were investigated. In the former study, the ventricles of Sprague Dawley rats were collected from embryo day 19 to postnatal 18 months and examined for mRNA and protein expression of TREK-1. It was found that both increased during development, reached a maximum at postnatal day 28, and remained higher at postnatal day 3 through to postnatal 18 months. In the latter study, protein expression of TREK-1 was examined after initiation of acute heart ischemia by ligation of the left anterior descending coronary artery. TREK-1 expression was found to be increased in the endocardium but unchanged in the epicardium. In primary cultured rat neonatal ventricular myocytes subjected to hypoxia (oxygen-glucose deprivation), TREK-1 expression was increased. In cultured neonatal cardiomyocytes, silencing of the TREK-1 gene by lentivirus delivery of the short-hairpin RNAs, L-sh-492 and L-sh-605, was found to promote their viability and number. In addition, both short-hairpin RNA provided protection against hypoxia-induced injury to cardiomyocytes in vitro. These results suggest that TREK-1 plays an important role in neonatal rat heart development and downregulation of TREK-1 may provide protection against ischemic injury. It seems that TREK-1 is a potential drug target for treatment of acute heart ischemia.

Anti-hypertrophic effects of oxytocin in rat ventricular myocytes

BackgroundOxytocin (OT) and functional OT receptor (OTR) are expressed in the heart and are involved in blood pressure regulation and cardioprotection. Cardiac OTR signaling is associated with atrial natriuretic peptide (ANP) and nitric oxide (NO) release. During the synthesis of OT, its precursor, termed OT-Gly-Lys-Arg (OT-GKR), is accumulated in the developing rat heart. Consequently, we hypothesized that an OT-related mechanism of ANP controls cardiomyocyte (CM) hypertrophy. MethodsThe experiments were carried out in newborn and adult rat CM cultures. The enhanced protein synthesis and increased CM volume were mediated by a 24-h treatment with endothelin-1 or angiotensin II. ResultsThe treatment of CM with OT or its abundant cardiac precursor, OT-GKR, revealed ANP accumulation in the cell peri-nuclear region and increased intracellular cGMP. Consequently, the CM hypertrophy was abolished by the treatment of 10nM OT or 10nM OT-GKR. The ANP receptor antagonist (anantin) and NO synthases inhibitor (l-NAME) inhibited cGMP production in CMs exposed to OT. STO-609 and compound C inhibition of anti-hypertrophic OT effects in CMs indicated the contribution of calcium–calmodulin kinase kinase and AMP-activated protein kinase pathways. Moreover, in ET-1 stimulated cells, OT treatment normalized the reduced Akt phosphorylation, prevented abundant accumulation of ANP and blocked ET-1-mediated translocation of nuclear factor of activated T-cells (NFAT) into the cell nuclei. ConclusioncGMP/protein kinase G mediates OT-induced anti-hypertrophic response with the contribution of ANP and NO. OT treatment represents a novel approach in attenuation of cardiac hypertrophy during development and cardiac pathology.

Formation of highly organized intracellular structure and energy metabolism in cardiac muscle cells during postnatal development of rat heart

Adult cardiomyocytes have highly organized intracellular structure and energy metabolism whose formation during postnatal development is still largely unclear. Our previous results together with the data from the literature suggest that cytoskeletal proteins, particularly βII-tubulin, are involved in the formation of complexes between mitochondria and energy consumption sites. The aim of this study was to examine the arrangement of intracellular architecture parallel to the alterations in regulation of mitochondrial respiration in rat cardiomyocytes during postnatal development, from 1day to 6months.Respirometric measurements were performed to study the developmental alterations of mitochondrial function. Changes in the mitochondrial arrangement and cytoarchitecture of βII- and αIV-tubulin were examined by confocal microscopy.Our results show that functional maturation of oxidative phosphorylation in mitochondria is completed much earlier than efficient feedback regulation is established between mitochondria and ATPases via creatine kinase system. These changes are accompanied by significant remodeling of regular intermyofibrillar mitochondrial arrays aligned along the bundles of βII-tubulin. Additionally, we demonstrate that formation of regular arrangement of mitochondria is not sufficient per se to provide adult-like efficiency in metabolic feed-back regulation, but organized tubulin networks and reduction in mitochondrial outer membrane permeability for ADP are necessary as well. In conclusion, cardiomyocytes in rat heart become mature on the level of intracellular architecture and energy metabolism at the age of 3months.

Chronic Intermittent Hypobaric Hypoxia Ameliorates Ischemia/Reperfusion-Induced Calcium Overload in Heart via Na+/Ca2+ Exchanger in Developing Rats

Background/Aims: Chronic intermittent hypobaric hypoxia (CIHH) protects the heart against ischemia/reperfusion (I/R) injury. This study investigated the calcium homeostasis mechanism and the role of Na⁺/Ca²⁺ exchanger (NCX) in the cardiac protective effect of CIHH in developing rats. Methods: Neonatal male rats received CIHH treatment or no treatment (control) in a hypobaric chamber simulating 3000-meter altitude for 42 days. The left ventricular function of isolated hearts was evaluated after 30 minutes of ischemia and 60 minutes of reperfusion. Myocardial infarct size, intracellular Ca²⁺ concentration ([Ca²⁺]_i), Na⁺-Ca²⁺ exchanger currents (I_Na/Ca) in ventricular myocytes, and NCX1 protein level in the sarcolemmal membrane were determined. Results: The recovery of cardiac function after I/R was improved, with the myocardial infarct size reduced, in CIHH rats compared with control rats (p<0.05). These effects were attenuated by Bay K8644, an L-type Ca²⁺ channel agonist, or ryanodine, a sarcoplasmic reticulum Ca²⁺ channel receptor activator. Furthermore, the increases in [Ca²⁺]_i during I/R were blunted in CIHH rats, but this effect was abolished by Bay K8644 or chelerythrine, a protein kinase C (PKC) inhibitor. The I_Na/Ca was decreased and the reversal potential of I_Na/Ca was shifted toward negative potential during simulated ischemia in the control cardiomyocytes (p<0.05). The inhibition of NCX1 protein expression during I/R was smaller in the CIHH rats than in the control rats (p<0.05). Conclusion: These data suggest that CIHH protects developing rat hearts during I/R by enhancing the resistance against calcium overload and by preserving normal I_Na/Ca and NCX1 protein. PKC activation might be involved in this protective process of CIHH.

Abstract 170: Protein Kinase C regulates alternative splicing transitions during heart development

Introduction: Highly conserved genetic and molecular pathways coordinate gene expression during heart development. Alternative splicing (AS) regulates gene expression by generating distinct spliced isoforms of proteins such that embryonic variants are expressed only in fetus but not in adult. Importantly, mutations that affect splicing of embryonic genes such as Nkx2.5 and GATA4 can lead to congenital heart defect. Although AS is critical for proper gene expression in developing heart, the signals that regulate AS are not well known. We have previously shown that Protein Kinase C α/β (PKCα/β) phosphorylates splicing regulator CELF1 that controls AS in heart, suggesting that PKC signaling could be responsible for AS regulation during heart development. Hypothesis: PKCα/β contributes to embryonic gene expression by regulating AS. Methods: We used H9c2 rat embryonic cell differentiation into cardiomyocyte-like cells and rat heart tissues at different stages of development to determine whether PKC is responsible for AS transitions. The localization and levels of proteins were assessed by western blot and immunohistochemistry. Quantitative RT-PCR was performed to determine the splicing pattern of nine developmentally regulated genes using specific primers. Results: The results indicate that AS of developmentally important genes transitioned during rat heart development and H9c2 differentiation. PKCα/β regulated embryonic to neonatal AS transitions by phosphorylating splicing regulators CELF1 and Rbfox2. Blocking PKCα/β activity by bisindolylmaleimide IX reduced both the phosphorylation and protein levels of CELF1 and Rbfox2 with concurrent changes in AS transitions. Depletion of PKCα was sufficient to alter a subset of AS events by down-regulating CELF1 and Rbfox2 proteins. Finally, our data suggest that nuclear localization of PKCα/β at embryonic stages could be the mechanism for AS transitions and increased steady state levels of splicing regulators. Conclusions: Our results show that PKCα/β regulate splicing regulators and AS during heart development. Changes in PKCα/β subcellular localization is one of the determinants for embryonic to neonatal AS transitions in developing rat heart and during cardiomyocyte differentiation.

The Ambiguous Role of NKX2-5 Mutations in Thyroid Dysgenesis

NKX2-5 is a homeodomain-containing transcription factor implied in both heart and thyroid development. Numerous mutations in NKX2-5 have been reported in individuals with congenital heart disease (CHD), but recently a select few have been associated with thyroid dysgenesis, among which the p.A119S variation. We sequenced NKX2-5 in 303 sporadic CHD patients and 38 families with at least two individuals with CHD. The p.A119S variation was identified in two unrelated patients: one was found in the proband of a family with four affected individuals with CHD and the other in a sporadic CHD patient. Clinical evaluation of heart and thyroid showed that the mutation did not segregate with CHD in the familial case, nor did any of the seven mutation carriers have thyroid abnormalities. We tested the functional consequences of the p.A119S variation in a cellular context by performing transactivation assays with promoters relevant for both heart and thyroid development in rat heart derived H10 cells and HELA cells. There was no difference between wildtype NKX2-5 and p.A119S NKX2-5 in activation of the investigated promoters in both cell lines. Additionally, we reviewed the current literature on the topic, showing that there is no clear evidence for a major pathogenic role of NKX2-5 mutations in thyroid dysgenesis. In conclusion, our study demonstrates that p.A119S does not cause CHD or TD and that it is a rare variation that behaves equal to wildtype NKX2-5. Furthermore, given the wealth of published evidence, we suggest that NKX2-5 mutations do not play a major pathogenic role in thyroid dysgenesis, and that genetic testing of NKX2-5 in TD is not warranted.

Abstract 150: Determining Changes in Contractility, Myofilament Expression and Myofilament Sensitivity in the Developing Human Ventricle

As pediatric cardiac surgery is increasingly performed in the first year of life, the need for understanding contractility regulation becomes increasingly important. We examined contractility and myofilament changes in ventricular biopsies removed as part of the surgical correction of congenital heart defects from newborns (NB) (hypoplastic left heart syndrome, &lt; 1 wk old) and infants (IF) (tetralogy of Fallot, 3-12 mo old). Sarcomere shortening and calcium (Ca) transients were measured in isolated ventricular cells stimulated at 0.5 and 1 Hz. Increasing pacing frequency caused a significant increase in sarcomere shortening (p&lt; 0.05)in only the IF, but the Ca transient amplitude was relatively unchanged in both groups. Consistent with work in the developing rat heart, we found an isoform switch with increasing developmental age in myofilament proteins, troponin T (TnT) and troponin I (TnI). Western blot analysis revealed that total TnI (the sum of cardiac TnI (cTnI) and slow skeletal TnI (ssTnI)) was not significantly different between the two age groups. However, when comparing only the cTnI isoforms, we found that the NB had a significantly lower levels compared to the IF (11556±789 a.u. vs. 21770±1700 a.u., p&lt;0.001). Analysis of the RT-PCR revealed nearly significant lower levels of TnT isoform 1 in the IF group than the NB group (p=0.078), no significant difference in the levels of TnT isoform 2 (p= 0.536), and significantly higher levels of TnT isoform 3 in the IF group than the NB group (p= 0.039). We also observed developmental changes in myofilament sensitivity to calcium as assessed by measuring the gradient of the fura-2 cell length trajectory on phase-plane diagrams during the late relaxation of the twitch contraction. These results collectively suggest that contractility and myofilament changes, or the lack of changes, may serve as targets for clarifying elements associated with cardiac dysfunction in the developing human ventricle.

Chronological and Morphological Study of Heart Development in the Rat

Adult and embryonic laboratory rats have been used as a mammalian model organism in biomedical research, descriptive and experimental cardiac embryology, and experimental teratology. There have been, however, considerable variations and discrepancies concerning the developmental staging of the rat embryo in the reported literature, which have resulted in several controversies and inconsistencies. Therefore, we carried out a careful anatomical and histological study of rat cardiac morphogenesis from the premorphogenetic period to the mature heart in a newborn pup. A correlation between the chronology and morphological features of the heart and embryo or newborn was made. We provide a simple and comprehensive guide relating the developmental timing and fate of the embryonic components of the heart and their morphological changes in the rat based on in vivo labeling studies in the chick. We also compare the timing of heart development in rats, humans, and mice.

Generation of hydrogen peroxide in the developing rat heart: the role of elastin metabolism

Reports describing production of reactive oxygen species in neonatal heart are missing. As lysyl oxidase is potentially important source of H(2)O(2), we studied its role during ontogenic development of rat heart. H(2)O(2) was detected in thin sections of developing rat heart by fluorescence microscopy with the use of fluorescence probe 2'-7'-dichlorofluorescin. The experimental design comprised foetuses 21 days after conception, and then the animals sampled on the 1st, 4th, 7th, 10th, 15th, 30th and 60th day after birth. We also used 7-month-old animals as an example of ageing effects. Since the day 4 on, H(2)O(2) was produced only extracellularly up to the day 15, between days 30 and 60 intracellular production was detected as well, and in 7-month-old animals only extracellular production was observed. The specific inhibitors of lysyl oxidase almost completely quenched the H(2)O(2)-dependent fluorescence. Starting from day 7, blue autofluorescence specific to oxidized proteins developed in the vessel wall. Intracellular blue autofluorescence specific to autoxidation products developed after day 30. Chloroform extraction diminished the intracellular blue fluorescence, leaving the extracellular fluorescence intact. This confirmed the protein nature of the fluorophores. Lysyl oxidase is significant source of H(2)O(2) in the heart vessel wall during development and H(2)O(2) oxidatively modifies elastin producing protein blue autofluorescence.

Stereological and Histological Analysis of the Developing Rat Heart

We studied with quantitative and microscopical methods the heart of rats divided into five age groups: embryos at the age of 11 days, fetuses at the age of 16 days and 20 days and also heart samples of 3-day-old pups and young adults (5 weeks of age) were used (n = 10 samples in each group). At the end of the study; heart samples were obtained from all animals. Stereological estimations were performed on heart volume, volume of heart lumen (ventricles and atria), volume of myocardium, numerical density of the myocyte nuclei and mean nuclear diameter of myocytes. Volumetric values and numerical data were estimated via Cavalieri method and physical dissector, respectively. In this study, histological examination was performed at light and electron microscopic levels. The numerical density of the myocyte nuclei increased from fetuses to young adults. Differences between embryos and fetuses, between fetuses and 3-day-old pups, and between 3-day-old pups and young adults were statistically significant. These results indicate that myogenesis continued in the rat myocardium during prenatal life and after birth.