Embryonic Cardiac Development Research Articles

Aceylated Cyclophilin D Regulates Mitochondrial Function in the Developing Mouse Heart

Mitochondria provide energy in form of ATP in mature eukaryotic cells. However, it is not clear, when and how during the embryonic cardiac development, mitochondria become able to regulate oxidative phosphorylation (OXPHOS) and prevent opening of the permeability transition pore (mPTP). Maturation of heart mitochondria is regulated by the activity of acetylated cyclophilin D (CypD). Cardiac tissue homogenates or isolated mitochondria ranging in age from embryonic (E) day 9.5 to adult wild-type mice were used to measure oxygen consumption, the expression of proteins of the electron transport chain and their assembly into supercomplexes was followed by denaturing and native electrophoresis. The expression of CypD and its acetylation status was assessed by western blotting and densitometry. n the heart of mouse embryos at E 9.5 mitochondrial electron transport chain (ETC) activity and OXPHOS are not coupled (respiratory control ratio (RCR) 1.48 ± 0.17, n=9). Addition of 1 µM cyclosporin A, an inhibitor of CypD D and the mPTP acutely increases the RCR to 3.69 ± 0.59 n=5). At E13.5, the end of the embryonic period in the mouse, OXPHOS is coupled and not significantly different from the adult heart. The enzymatic activity of the ETC complexes I, II, III and V increases significantly from E9.5 to the adult heart and the assembly of mitochondrial supercomplexes begins at about E13.5. Similarly, the expression of CypD increases as the heart matures. However, the detection of acetylated CypD decreases and the ratio of acetylated CypD to the total expressed CypD decreases from 1.1 ± 0.16 (n=3) at E9.5 to 0.54 ± 0.06 (n=3) in the adult heart. The activity of acetylated CypD regulates maturation of mitochondrial function in the developing heart.

The regulatory roles of p53 in cardiovascular health and disease.

Cardiovascular disease (CVD) remains the leading cause of mortality globally, so further investigation is required to identify its underlying mechanisms and potential targets for its prevention. The transcription factor p53 functions as a gatekeeper, regulating a myriad of genes to maintain normal cell functions. It has received a great deal of research attention as a tumor suppressor. In the past three decades, evidence has also shown a regulatory role for p53 in the heart. Basal p53 is essential for embryonic cardiac development; it is also necessary to maintain normal heart architecture and physiological function. In pathological cardiovascular circumstances, p53 expression is elevated in both patient samples and animal models. Elevated p53 plays a regulatory role via anti-angiogenesis, pro-programmed cell death, metabolism regulation, and cell cycle arrest regulation. This largely promotes the development of CVDs, particularly cardiac remodeling in the infarcted heart, hypertrophic cardiomyopathy, dilated cardiomyopathy, and diabetic cardiomyopathy. Roles for p53 have also been found in atherosclerosis and chemotherapy-induced cardiotoxicity. However, it has different roles in cardiomyocytes and non-myocytes, even in the same model. In this review, we describe the different effects of p53 in cardiovascular physiological and pathological conditions, in addition to potential CVD therapies targeting p53.

IP3R-Mediated Compensatory Mechanism for Calcium Handling in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Cardiac Ryanodine Receptor Deficiency.

In adult cardiomyocytes (CMs), the type 2 ryanodine receptor (RYR2) is an indispensable Ca2+ release channel that ensures the integrity of excitation-contraction coupling, which is fundamental for every heartbeat. However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood. Here, we generated two human induced pluripotent stem cell (iPSC)-based RYR2 knockout (RYR2–/–) lines using the CRISPR/Cas9 gene editing technology. We found that RYR2–/–-iPSCs could differentiate into CMs with the efficiency similar to control-iPSCs (Ctrl-iPSCs); however, the survival of iPSC-CMs was markedly affected by the lack of functional RYR2. While Ctrl-iPSC-CMs exhibited regular Ca2+ handling, we observed significantly reduced frequency and intense abnormalities of Ca2+ transients in RYR2–/–-iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular Ca2+ ([Ca2+ ]o) and caffeine in a concentration-dependent manner, while RYR2–/–-iPSC-CMs showed inconsistent reactions to [Ca2+ ]o and were insensitive to caffeine, indicating there is no RYR2-mediated Ca2+ release from the sarcoplasmic reticulum (SR). Instead, compensatory mechanism for calcium handling in RYR2–/–-iPSC-CMs is partially mediated by the inositol 1,4,5-trisphosphate receptor (IP3R). Similar to Ctrl-iPSC-CMs, SR Ca2+ refilling in RYR2–/–-iPSC-CMs is mediated by SERCA. Additionally, RYR2–/–-iPSC-CMs showed a decreased beating rate and a reduced peak amplitude of L-type Ca2+ current. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca2+ release is one of the major compensatory mechanisms for Ca2+ cycling in human CMs with the RYR2 deficiency.

The sodium channel NaV1.5 impacts on early murine embryonic cardiac development, structure and function in a non‐electrogenic manner

AimThe voltage‐gated sodium channel NaV1.5, encoded by SCN5A, is essential for cardiac excitability and ensures proper electrical conduction. Early embryonic death has been observed in several murine models carrying homozygous Scn5amutations. We investigated when sodium current (INa) becomes functionally relevant in the murine embryonic heart and how Scn5a/NaV1.5 dysfunction impacts on cardiac development.MethodsInvolvement of NaV1.5‐generated INa in murine cardiac electrical function was assessed by optical mapping in wild type (WT) embryos (embryonic day (E)9.5 and E10.5) in the absence and presence of the sodium channel blocker tetrodotoxin (30 µmol/L). INa was assessed by patch‐clamp analysis in cardiomyocytes isolated from WT embryos (E9.5‐17.5). In addition, cardiac morphology and electrical function was assessed in Scn5a‐1798insD−/− embryos (E9.5‐10.5) and their WT littermates.ResultsIn WT embryos, tetrodotoxin did not affect cardiac activation at E9.5, but slowed activation at E10.5. Accordingly, patch‐clamp measurements revealed that INa was virtually absent at E9.5 but robustly present at E10.5. Scn5a‐1798insD−/− embryos died in utero around E10.5, displaying severely affected cardiac activation and morphology. Strikingly, altered ventricular activation was observed in Scn5a‐1798insD−/− E9.5 embryos before the onset of INa, in addition to reduced cardiac tissue volume compared to WT littermates.ConclusionWe here demonstrate that NaV1.5 is involved in cardiac electrical function from E10.5 onwards. Scn5a‐1798insD−/− embryos displayed cardiac structural abnormalities at E9.5, indicating that NaV1.5 dysfunction impacts on embryonic cardiac development in a non‐electrogenic manner. These findings are potentially relevant for understanding structural defects observed in relation to NaV1.5 dysfunction.

The A-to-I RNA Editing Enzyme Adar1 Is Essential for Normal Embryonic Cardiac Growth and Development.

The A-to-I RNA Editing Enzyme Adar1 Is Essential for Normal Embryonic Cardiac Growth and Development.

Zebrafish Vestigial Like Family Member 4b Is Required for Valvulogenesis Through Sequestration of Transcription Factor Myocyte Enhancer Factor 2c.

A variety of cardiac transcription factors/cofactors, signaling pathways, and downstream structural genes integrate to form the regulatory hierarchies to ensure proper cardiogenesis in vertebrate. Major interaction proteins of the transcription cofactor vestigial like family member 4 (VGLL4) include myocyte enhancer factor 2 (MEF2) and TEA domain transcription factors (TEAD), both of which play important roles in embryonic cardiac development and in adulthood. In this study, we identified that the deficiency of zebrafish vgll4b paralog, a unique family member expressed in developing heart, led to an impaired valve development. Mechanistically, in vgll4b mutant embryos the disruption of Vgll4b-Mef2c complex, rather than that of Vgll4b-Tead complex, resulted in an aberrant expression of krüppel-like factor 2a (klf2a) in endocardium. Such misexpression of klf2a eventually evoked the valvulogenesis defects. Our findings suggest that zebrafish Vgll4b plays an important role in modulating the transcription activity of Mef2c on klf2a during valve development in a blood-flow-independent manner.

P5388N-cadherin promotes cardiac regeneration by stabilizing beta-catenin

Abstract Background Although the adult mammalian heart fails to regenerate after injury, it is known that newborn mice within a week have full cardiac regenerative capacity. The molecular determinants underlying the disparate regenerative capacity between neonatal and adult mice, however, remain incompletely understood. Exploiting RNA sequencing in isolated cardiomyocytes from neonatal and adult mouse heart, we identified Cdh2, which encodes the adherence junction protein N-cadherin, as a potential novel mediator of cardiac regeneration. Cdh2 expression levels were much higher in neonatal, compared with adult, cardiomyocytes and showed a strong positive correlation with that of multiple cell cycle genes. N-cadherin has been reported to be essential for embryonic cardiac development; its role in cardiac regeneration, however, remains unknown. Purpose To determine the role of Cdh2 (N-cadherin) in cardiac regeneration and to investigate the underlying molecular mechanisms. Methods Apical resection in postnatal day 1 mice was used as a cardiac regenerative model. The in vitro gain/loss-of function studies of Cdh2/N-cadherin was performed in postnatal day 1 neonatal mouse cardiomyocytes (P1CM) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM). N-cadherin inhibitor exherin was used to study the effects of N-cadherin in vivo. Results Comparing to sham-operated control, Cdh2 was significantly upregulated in mouse cardiac apex and border zone following apical resection, which was accompanied with increased cardiomyocyte proliferation activity. In vitro, knocking down Cdh2 or inhibition of N-cadherin activity with exherin in P1CM significantly reduced the proliferative activity of cardiomyocytes, whereas overexpression of Cdh2 markedly increased the proliferation of P1CM. In addition, forced expression of Cdh2 resulted in significant upregulation of multiple cell cycle genes, including Ccnd1 (Cyclin D1) and Pcna (proliferating cell nuclear antigen), in P1CM. In vivo inhibition of N-cadherin in P1 neonatal mice with exherin following apical resection impaired cardiac regeneration and increased scar formation (Figure). Knocking down CDH2 in human iPSC-CMs significantly reduced the proliferative activity and the expression levels of cell cycle gene CCND1 in iPSC-CMs. Mechanistically, we demonstrated that the pro-mitotic effects of N-cadherin in cardiomyocytes were mediated, at least partially, by stabilizing β-catenin, a pro-mitotic transcription factor, through direct interaction with its cytoplasmic domain and/or inactivation of GSK3β, a critical component of β-catenin destruction complex. N-Cad blocker impairs heart regeneration Conclusion Our study uncovered a previously unrecognized role of Cdh2 (N-cadherin) in cardiomyocyte proliferation and cardiac regeneration. Enhancing cardiac expression or activity of N-cadherin, therefore, could be a potential novel therapeutic approach to promote cardiac regeneration and restore cardiac function in adult heart following injury.

236: Micro RNA expression profile in pregnant women complicated fetuses with muscular ventricular septal defects

Ventricular septal defects (VSD) account for 20% of congenital heart defects (CHDs) and the pathogenesis and molecular mechanism have not been elucidated. MicroRNAs (miRs) are small single-stranded, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Recent research demonstrated that miRs help regulate embryonic cardiac development, cardiac mophogenesis, and cardiomyocyte growth and differentiation and are closely related to the occurrence of CHDs. This study aimed to measure differences in miR expression in maternal serum in patients complicated with fetal muscular VSD. This was a prospective study including fetuses at risk for CHD (pregestational diabetes, anticonvulsant use, or history of cardiac defects) during 2nd trimester echocardiographic examination. Fetuses without cardiac abnormalities were marked as control. Fetuses with isolated mVSD were marked as abnormal. Maternal venous serum was collected, RNA extracted, and an expression profile of 797 human miRs was measured using NanoString nCounter technology as counts (molecules/100ng RNA). Expression levels of >2fold increase or <50% decrease compared to control were considered up- or downregulated, respectively. Placental-derived miRs within the entire expression profile were also evaluated separately. miRs profiling was obtained in 6 controls and 7 mVSDs cases. Expression levels ranged from 3-198 counts, indicating relatively low expression. The median count did not vary between normal (20.7) and mVSD (21.4). Of the 797, 12 miRs were significantly upregulated (Fig. 1) and 36 miRs downregulated (P<0.01 for both). There were no differences in expression among the 114 known placentally-derived miRs between groups. This study identifies a select serum miR expression profile in women with mVSD fetuses in the 2nd trimester that differs from women with normal fetuses. This approach may provide a quantitative screening tool for confirming fetal CHDs diagnosed by echocardiography. This will be valuable as a biomarker approach for early detection of fetal CHDs in the first trimester to aid diagnosis by fetal echocardiography.

Advances in the mechanism of Tbx2 regulation in embryonic cardiac development

Embryonic cardiogenesis requires precise expression of various genes.This process involves a complex model of gene regulation, in which any deviation will lead to the occurrence of heart defects.Expression of Tbx2 in different species is ultimately confined to the atrioventricular canal in the non-ventricular myocardium, suggesting that the temporal and spatial expression of Tbx2 gene during cardiac development is highly consistent and evolutionarily conserved.As a member of the T-box transcription factor family, Tbx2 plays an important role in the differentiation of outflow tract and atrioventricular canal, leading to a series of changes in the regulatory pathway by regulating the transcription levels of the downstream target genes.At present, more and more studies have indicated that aberrant expressions or regulations of Tbx2 result in cardiac malformations in different model animals.In clinical studies, microdeletions/duplications in the fragments involving TBX2 and genetic variants in the non-coding region of this gene have also been reported in human congenital heart defects.Thus, this article is to review the advances in the effect and possible mechanisms for Tbx2 gene in cardiac development, and to discuss the relationship between this gene and congenital heart defects. Key words: Tbx2; Cardiac development; Congenital heart defects

Abstract 349: Sall4 Blocks Cardiac Trans-differentiation but Stimulates Cardiac Stem-like Cell (iPSC) Generation and Improve Post MI Function In Vivo

Background: Sall4 is an essential component in maintaining embryonic stem cell self-renewal and pluripotency. Given its functional and protein level interaction with cardiac trans-differentiation factors Tbx5/Gata4, as well as its critical role in modulating embryonic cardiac development, we hypothesized that Sall4 may play an important role in cardiac reprogramming. Material/Method: We conducted gain- and loss- of function studies on neonatal and adult cardiac fibroblast from both mouse and rat. We also evaluated Sall4 function on infarcted rat heart. Results: Gata4/Mef2c/Tbx5 (GMT) administration markedly upregulated mRNA expression levels of stem factors including Sall4 , Bmi1 , and p63 , while inhibition of Sall4 largely enhanced GMT- mediated Cardiac troponin T ( cTNT ) induction. Conversely, overexpression of Sall4 plus Gata4/Mef2C in cardiac fibroblast for 7 days induces over 30-fold increase of Oct4 and Nanog by real-time PCR; longer culture induced rapidly-dividing iPSC-like clusters in both neonatal mouse and adult rat cardiac fibroblasts. Further tests showed that these iPSC-like cells were partially positive in alkaline phosphatases staining, capable of forming cardiosphere and differentiating into various cell types. Sall4 delivery also improved post-infarct cardiac function to a level that is comparable with Gata4 in rat. Conclusion: These results suggested that Sall4 is a promising target for cardiac reprogramming even though further characterization in vitro as well as in vivo is needed.

Tead1 is required for maintaining adult cardiomyocyte function, and its loss results in lethal dilated cardiomyopathy.

Heart disease remains the leading cause of death worldwide, highlighting a pressing need to identify novel regulators of cardiomyocyte (CM) function that could be therapeutically targeted. The mammalian Hippo/Tead pathway is critical in embryonic cardiac development and perinatal CM proliferation. However, the requirement of Tead1, the transcriptional effector of this pathway, in the adult heart is unknown. Here, we show that tamoxifen-inducible adult CM-specific Tead1 ablation led to lethal acute-onset dilated cardiomyopathy, associated with impairment in excitation-contraction coupling. Mechanistically, we demonstrate Tead1 is a cell-autonomous, direct transcriptional activator of SERCA2a and SR-associated protein phosphatase 1 regulatory subunit, Inhibitor-1 (I-1). Thus, Tead1 deletion led to a decrease in SERCA2a and I-1 transcripts and protein, with a consequent increase in PP1-activity, resulting in accumulation of dephosphorylated phospholamban (Pln) and decreased SERCA2a activity. Global transcriptomal analysis in Tead1-deleted hearts revealed significant changes in mitochondrial and sarcomere-related pathways. Additional studies demonstrated there was a trend for correlation between protein levels of TEAD1 and I-1, and phosphorylation of PLN, in human nonfailing and failing hearts. Furthermore, TEAD1 activity was required to maintain PLN phosphorylation and expression of SERCA2a and I-1 in human induced pluripotent stem cell-derived (iPS-derived) CMs. To our knowledge, taken together, this demonstrates a nonredundant, novel role of Tead1 in maintaining normal adult heart function.

Backbone and side-chain resonance assignments of (Ca2+)4-calmodulin bound to beta calcineurin A CaMBD peptide.

Calcineurin (CaN) is a heterodimeric and highly conserved serine/threonine phosphatase (PP2B) that plays a critical role in coupling calcium signals to physiological processes including embryonic cardiac development, NF-AT-regulated gene expression in immune responses, and apoptosis. The catalytic subunit (CaNA) has three isoforms (α, β, and γ,) in humans and seven isoforms in Paramecium. In all eukaryotes, the EF-hand protein calmodulin (CaM) regulates CaN activity in a calcium-dependent manner. The N- and C-domains of CaM (CaMN and CaMC) recognize a CaM-binding domain (CaMBD) within an intrinsically disordered region of CaNA that precedes the auto-inhibitory domain (AID) of CaNA. Here we present nearly complete 1H, 13C, and 15N resonance assignments of (Ca2+)4-CaM bound to a peptide containing the CaMBD sequence in the beta isoform of CaNA (βCaNA-CaMBDp). Its secondary structure elements predicted from the assigned chemical shifts were in good agreement with those observed in the high-resolution structures of (Ca2+)4-CaM bound to CaMBDs of multiple enzymes. Based on the reported literature, the CaMBD of the α isoform of CaNA can bind to CaM in two opposing orientations which may influence the regulatory function of CaM. Because a high resolution structure of (Ca2+)4-CaM bound to βCaNA-CaMBDp has not been reported, our studies serve as a starting point for determining the solution structure of this complex. This will demonstrate the preferred orientation of (Ca2+)4-CaM on the CaMBD as well as the orientations of CaMN and CaMC relative to each other and to the AID of βCaNA.

Functional Analyses of a Novel CITED2 Nonsynonymous Mutation in Chinese Tibetan Patients with Congenital Heart Disease.

CITED2 gene is an important cardiac transcription factor that plays a fundamental role in the formation and development of embryonic cardiovascular. Previous studies have showed that knock-out of CITED2 in mice might result in various cardiac malformations. However, the mechanisms of CITED2 mutation on congenital heart disease (CHD) in Chinese Tibetan population are still poorly understood. In the present study, 187 unrelated Tibetan patients with CHD and 200 unrelated Tibetan healthy controls were screened for variants in the CITED2 gene; we subsequently identified one potential disease-causing mutation p.G143A in a 6-year-old girl with PDA and functional analyses of the mutation were carried out. Our study showed that the novel mutation of CITED2 significantly enhanced the expression activity of vascular endothelial growth factor (VEGF) under the role of co-receptor hypoxia inducible factor 1-aipha (HIF-1A), which is closely related with embryonic cardiac development. As a result, CITED2 gene mutation may play a significant role in the development of pediatric congenital heart disease.

CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

Epicardium-derived cells (EPDCs) play a fundamental role in embryonic cardiac development and are reactivated in the adult heart in response to myocardial infarction (MI). In this study, EPDCs from post-MI rat hearts highly expressed the ectoenzyme CD73 and secreted the profibrotic matricellular protein tenascin-C (TNC). CD73 on EPDCs extensively generated adenosine from both extracellular ATP and NAD. This in turn stimulated the release of additional nucleotides from a Brefeldin A-sensitive intracellular pool via adenosine-A2BR signaling, forming a positive-feedback loop. A2BR activation, in addition, strongly promoted the release of major regulatory cytokines, such as IL-6, IL-11, and VEGF. TNC was found to stimulate EPDC migration and, together with ATP-P2X7R signaling, to activate inflammasomes in EPDCs via TLR4. Our results demonstrate that EPDCs are an important source of various proinflammatory factors in the post-MI heart controlled by purinergic and TNC signaling.-Hesse, J., Leberling, S., Boden, E., Friebe, D., Schmidt, T., Ding, Z., Dieterich, P., Deussen, A., Roderigo, C., Rose, C. R., Floss, D. M., Scheller, J., Schrader, J. CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

Cyclophilin D Acetylation Regulates Cardiac Myocyte Differentiation

INTRODUCTION: Protein acetylation is an important regulator of cellular function. Acetylation of cyclophilin D (CyPD) at lysine 166 increases its ability to open the mitochondria permeability transition pore (PTP), which is an important regulator of mitochondrial metabolism and myocyte differentiation. HYPOTHESIS: Acetylation of CyPD controls PTP activity, mitochondrial structure and function, and myocyte differentiation in the developing heart. METHODS: We use immunoblotting and immunoprecipitation to determine the global protein acetylation state and that of CyPD during embryonic cardiac development in wild type (WT) and CyPD-null mice. We also re-expressed WT and mutant CyPD in CyPD-null embryonic and neonatal cardiac myocyte cultures to determine effects on mitochondrial structure and function and myocyte differentiation. RESULTS: Immunoblotting revealed that global protein acetylation was high in the embryo but fell during later development. In contrast, CyP-D was highly acetylated in the early embryonic heart, but this decreased dramatically during subsequent development. When re-expressed in cultures of CyPD-null myocytes, WT CyPD recapitulated the WT cell phenotype of fragmented mitochondrial structure, low mitochondrial membrane potential (Δψm), and decreased differentiation. Re-expression of a CyPD acetylation mimic mutant (K166Q) had similar or greater effects on these parameters than WT CyPD, while an inactivating (R96G) and de-acetylation mimic (K166R) CyPD mutant did not. CONCLUSION: These data suggest that myocyte differentiation during cardiac development is associated with a decrease in overall protein acetylation, perhaps because of the changes that occur in metabolism as the heart develops. However, specific acetylation of CyPD appears to be particularly important for maturation of mitochondrial function and regulation of cardiac myocyte differentiation.

Cardiomyopathy-Associated Gene 1-Sensitive PKC-Dependent Connexin 43 Expression and Phosphorylation in Left Ventricular Noncompaction Cardiomyopathy

Background/Aims: Cardiomyopathy-associated gene 1 (CMYA1) plays an important role in embryonic cardiac development, postnatal cardiac remodeling and myocardial injury repair. Abnormal CMYA1 expression may be involved in cardiac dysplasia and primary cardiomyopathy. Our study aims to establish the relationship between CMYA1 and Left ventricular noncompaction cardiomyopathy (LVNC) pathogenesis. Methods: We explored the effects of CMYA1 on connexins (Cx), which contribute to gap junction intercellular communication (GJIC), and the underlying signaling pathway in human normal tissues, LVNC myocardial tissues and HL1 cells by means of western blotting, RT-qPCR, immunohistochemistry, immunofluorescence, co-immunoprecipitation and scrape loading-dye transfer. Results: CMYA1 expression was inversely associated with Cx43 and Cx40 expression, as determined by gap junction PCR array analysis. An increased expression and disordered distribution of CMYA1 at the intercalated discs in LVNC myocardial tissue was also observed. CMYA1 and Cx43 are co-expressed and interact in myocardial cells. CMYA1 expression was positively correlated with p-Cx43 (S368) via the Protein kinase C (PKC) signaling pathway in myocardial tissue and HL1 cells. The diffusion distance of Lucifer Yellow in the HL1 cells in which CMYA1 was over-expressed or knocked down was significantly less or more than that of the control group, respectively. Conclusion: Abnormal CMYA1 expression affects the expression and phosphorylation of Cx43 through the PKC signaling pathway, which is involved in the regulation of GJIC. CMYA1 participates in the molecular mechanism of LVNC pathogenesis.

Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy

Phospholamban (PLN) plays a central role in Ca2+ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase 2A (SERCA2A) Ca2+ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function.

MicroRNAs in congenital heart disease.

Congenital heart disease (CHD) is a broad term which encompasses a spectrum of pathology, the most common phenotypes include atrial septal defects (ASDs), ventricular septal defects (VSDs), patent ductus arteriosus (PAD) and tetralogy of Fallot (TOF). The impact of CHD is profound and it is estimated to be responsible for over 40% of prenatal deaths. MicroRNAs (miRs) are small, highly conserved, non-coding RNAs which have complex roles in a variety of pathophysiological states. miRs are post-transcriptional negative regulators of gene expression. Individual miRs are known to exert effects in multiple target genes, therefore the altered expression of a single miR could influence an entire gene network resulting in complex pathological states. Recent evidences suggest a role in the dysregulation of miRs in CHD. Mouse knock out models have contributed to our knowledge base revealing specific patterns of miR expression in cardiovascular physiology and pathological states. Specific miRs necessary for embryonic cardiac development have been revealed. Dysregulation of these miRs has been shown to cause structural abnormalities in the heart and vasculature, thus furthering our understanding of the processes which result in CHD. These advances have provided new insight into the signalling pathways responsible for CHD. Furthermore, this new appreciation for miRs in the development of CHD has uncovered their potential for new therapeutic targets where modulated miR activity may reduce the burden of disease. Here, we summarize current knowledge of the cause-effect relationships of miRs in CHD and consider their potential as a therapeutic targets and biomarkers in this clinical setting.

Blood flow through the embryonic heart outflow tract during cardiac looping in HH13-HH18 chicken embryos.

Blood flow is inherently linked to embryonic cardiac development, as haemodynamic forces exerted by flow stimulate mechanotransduction mechanisms that modulate cardiac growth and remodelling. This study evaluated blood flow in the embryonic heart outflow tract (OFT) during normal development at each stage between HH13 and HH18 in chicken embryos, in order to characterize changes in haemodynamic conditions during critical cardiac looping transformations. Two-dimensional optical coherence tomography was used to simultaneously acquire both structural and Doppler flow images, in order to extract blood flow velocity and structural information and estimate haemodynamic measures. From HH13 to HH18, peak blood flow rate increased by 2.4-fold and stroke volume increased by 2.1-fold. Wall shear rate (WSR) and lumen diameter data suggest that changes in blood flow during HH13-HH18 may induce a shear-mediated vasodilation response in the OFT. Embryo-specific four-dimensional computational fluid dynamics modelling at HH13 and HH18 complemented experimental observations and indicated heterogeneous WSR distributions over the OFT. Characterizing changes in haemodynamics during cardiac looping will help us better understand the way normal blood flow impacts proper cardiac development.

4D subject-specific inverse modeling of the chick embryonic heart outflow tract hemodynamics.

Blood flow plays a critical role in regulating embryonic cardiac growth and development, with altered flow leading to congenital heart disease. Progress in the field, however, is hindered by a lack of quantification of hemodynamic conditions in the developing heart. In this study, we present a methodology to quantify blood flow dynamics in the embryonic heart using subject-specific computational fluid dynamics (CFD) models. While the methodology is general, we focused on a model of the chick embryonic heart outflow tract (OFT), which distally connects the heart to the arterial system, and is the region of origin of many congenital cardiac defects. Using structural and Doppler velocity data collected from optical coherence tomography, we generated 4D ([Formula: see text]) embryo-specific CFD models of the heart OFT. To replicate the blood flow dynamics over time during the cardiac cycle, we developed an iterative inverse-method optimization algorithm, which determines the CFD model boundary conditions such that differences between computed velocities and measured velocities at one point within the OFT lumen are minimized. Results from our developed CFD model agree with previously measured hemodynamics in the OFT. Further, computed velocities and measured velocities differ by [Formula: see text]15% at locations that were not used in the optimization, validating the model. The presented methodology can be used in quantifications of embryonic cardiac hemodynamics under normal and altered blood flow conditions, enabling an in-depth quantitative study of how blood flow influences cardiac development.