Cardiac-specific Markers Research Articles

Abstract 345: Aggregation of Child Cardiac Progenitor Cells into Spheres Activates Notch Signaling and Improves Treatment of Right Ventricular Heart Failure

Congenital heart disease is rarely cured by surgery and can lead to life-threatening, intractable right ventricular heart failure (HF). Recent stem cell-based clinical trials have been limited by poor differentiation rates and low cell retention. We propose the aggregation of child cardiac progenitor cells (CPCs) into scaffold-free spheres to improve the differentiation of CPCs into mature cardiac phenotypes by enhancing intercellular Notch signaling, a key regulator of CPC fate decisions. Child CPC spheres were produced at a size of 1500 cells per sphere using a microwell array and cultured in suspension for up to 14 days with 70-80% cell viability. By 7 days in culture, aggregation of CPCs increased protein expression of Notch1 intracellular domain (3.8-fold) and transcript expression of Notch1 signaling pathway members, including ADAM10 (3.6-fold), DLL1 (25.0-fold), and JAG1 (3.2-fold), compared to parallel monolayer cultures. Increased Notch signaling activity was accompanied by high transcript levels of cardiac differentiation markers, including MEF2C, MYH7, and cTNNI. In particular, Nkx2.5, an important transcription factor for cardiomyocyte development, was robustly expressed within the nuclei of aggregated CPCs. Inhibition of Notch signaling activity using the small molecule γ-secretase inhibitor DAPT abrogated these effects, including the nuclear translocation of Nkx2.5. To evaluate the therapeutic effects of CPC spheres compared to single CPCs, right ventricular HF was induced in rats by pulmonary artery banding and cells were transplanted into the right ventricular free wall. By 28 days, echocardiographic measurements showed significantly improved right ventricular function with CPC sphere transplantation compared to single CPC transplantation (tricuspid annular plane systolic excursion: 2.03±0.2 vs. 1.61±0.22 mm, P<0.001). Additionally, CPC aggregation improved cell retention at 28 days compared to single CPCs as measured using DiR-labeled CPCs (29.2±4.0 vs. 11.1±7.3% of day 0 fluorescence, P<0.005). The results of our project will facilitate the development of autologous stem-cell based therapies for pediatric HF.

Isolation and Quantification of Nanovesicles in Isoproterenol-Induced Myocardial Infarcted Rats

Introduction and Aim: Myocardial infarction (MI) is one of the leading causes of death worldwide. The pathogenesis and aetiology of MI is still unclear and there is currently no cure for MI. Cardiac troponin is the only well-known cardiac-specific marker for the diagnosis of MI but due to the delayed release of troponin in the circulation, a novel cardiac biomarker is needed in the early stages of development of MI to reduce MI mortality. Recently, it has been reported that exosome concentration is highly regulated by stress and cardiovascular diseases (CVDs). We assessed the hypothesis that exosome secretion by the injured cardiomyocytes is increased during MI and thereby serve as biomarker for MI. The aim of this study was to quantify exosomes in an isoproterenol (ISO)-induced MI rats. Method: Twelve rats were divided into two groups (group A and B). Group-A (n=6) was the normal control rats and group-B (n=6) was the ISO-treated group. Group-B animals were injected with isoproterenol (85mg/kg/bw) for two consecutive days to induce MI. Blood pressure (BP), heart rate (HR) and body weight were monitored for 7 days in all animals prior the ISO injection and throughout the experiment. After second ISO-injection, all animals were sacrificed and blood, heart tissues were obtained. Histopathological analysis was performed in heart tissue samples and levels of cardiac markers (creatine kinase-MB, lactate dehydrogenase and troponin T) were measured from the serum. Exosomes were isolated from the plasma by differential ultracentrifugation. Exosomes were quantified and characterized using nanoparticle tracking analysis (NTA), transmission electron microscope (TEM) study and ELISA for the quantification of circulating exosomal protein (CD63). Result and Discussion: ISO injection caused the development of MI and was confirmed by the increase in BP, Cardiac markers (cardiac troponin T, lactate dehydrogenase and creatine kinase). NTA together with TEM analysis revealed particle sizes of the exosome and showed an elevated number of the exosome in ISO-treated animals. Levels of exosomal protein (CD63) were also increased in ISO-treated animals. Conclusion: The concentration of exosomes was increased in MI rats indicating that circulating exosomes may be used as a novel diagnostic marker for MI.

TGF-β1 combined with Sal-B promotes cardiomyocyte differentiation of rat mesenchymal stem cells.

Transforming growth factor β1 (TGF-β1) and salvianolic acid B (Sal-B) are key signaling factors for stem cell differentiation into cardiomyocytes (CMs). The present study compared the biological effect of TGF-β1 and Sal-B, alone or in combination, on bone marrow mesenchymal stromal cells (BMSCs) that differentiate into myocardial-like cells in a simulated myocardial microenvironment in vitro. BMSCs were isolated from bones of limbs of 10 male Sprague Dawley rats and cultured. The 2nd-generation BMSCs were co-incubated with TGF-β1 and Sal-B, alone or in combination, for 72 h. The control group was BMSCs cultured without any inductive substance. The levels GATA binding protein 4 (GATA4) and homeobox protein NKx2.5 were determined by reverse-transcription quantitative polymerase chain reaction and immunofluorescence staining was used to evaluate α-sarcomeric actin and cardiac troponin I (cTNI) as cardiomyogenic differentiation markers. The ultrastructure of BMSCs in each group was also observed. BMSCs were initially spindle-shaped with irregular processes. The cells gradually increased in number 24 h post-inoculation and proliferated 7 days later. Compared with the control group, BMSCs in the treatment groups had fusiform shapes, orientating with one accord and were connected with adjoining cells forming myotube-like structures on day 28. The morphology and architecture/myotubes of BMSCs was similar among the treatment groups, but the amount of cells in the combined group was comparatively higher. The results of immunofluorescence staining revealed the expression of the CM-specific proteins α-sarcomeric actin and cTNI in these cells. The expression of these cardiac-specific markers in the combined group was significantly higher than that in the other groups (P<0.01 or P<0.05). In addition, the transcriptional expression of GATA4 and NKx2.5 in the treatment groups was stable and significantly higher than that in the control group on day 7. Transmission electron microscopy showed that BMSCs in the treatment groups all had myofilaments, rough endoplasmic reticulum and mitochondria in the cytoplasm when compared with the control group. Taken together, these results indicated that the combination of TGF-β1 and Sal-B effectively promotes cardiomyogenic differentiation of BMSCs in vitro and their application may represent a therapeutic strategy for the treatment of ischemic heart disease.

Strategic design of cardiac mimetic core-shell nanofibrous scaffold impregnated with Salvianolic acid B and Magnesium l-ascorbic acid 2 phosphate for myoblast differentiation

The major loss of myocardial tissue extracellular matrix after infarction is a serious complication that leads to heart failure. Regeneration and integration of damaged cardiac tissue is challenging since the functional restoration of the injured myocardium is an incredible task. The injured micro environment of myocardium fails to regenerate spontaneously. The emergence of nano-biomaterials would be a promising approach to regenerate such a damaged cardiomyocytes tissue. Here, we have fabricated a dual bioactive embedded nanofibrous cardiac patch via coaxial electrospinning technique, to mimic the topographical and chemical cues of the natural cardiac tissue. The proportion and the concentration of the polymers were optimized for tailored delivery of bioactives from a spatio-temporally designed scaffold. The functionalization of polymeric core shell nanofibrous scaffold with dual bioactives enhanced the physico-chemical and bio-mechanical properties of the scaffolds that has resulted in a 3-dimensional topography mimicking the natural cardiac like extracellular matrix. The sustained delivery of bioactive signals, improved cell adhesion, proliferation, migration and differentiation could be attributed to its highly interconnected nanofibrous matrix with good extended morphology. Further, the expression of cardiac specific markers were found to increase on investigation of mRNA by real time PCR studies and proteins by immunofluorescence and western blotting techniques, confirming cell - biomaterial interactions. Flow cytometry analysis authenticated a potent mitochondrial membrane potential of cells treated with nanocomposite. In addition, in ovo studies in chicken chorioallantoic membrane assay confirm the efficacy of the developed scaffold in inducing angiogenesis required for maintaining its viability after transplantation onto the infarcted zone. These promising results demonstrate the potential of the composite nanofibrous scaffold as an effective biomaterial substrate for cardiac regeneration providing cues for development of novel cardiac therapeutics.

P63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells

ObjectiveReprogramming of fibroblasts into induced cardiomyocytes represents a potential new therapy for heart failure. We hypothesized that inactivation of p63, a p53 gene family member, may help overcome human cell resistance to reprogramming. Methodsp63 Knockout (−/−) and knockdown murine embryonic fibroblasts (MEFs), p63−/− adult murine cardiac fibroblasts, and human cardiac fibroblasts were assessed for cardiomyocyte-specific feature changes, with or without treatment by the cardiac transcription factors Hand2–Myocardin (HM). ResultsFlow cytometry revealed that a significantly greater number of p63−/− MEFs expressed the cardiac-specific marker cardiac troponin T (cTnT) in culture compared with wild-type (WT) cells (38% ± 11% vs 0.9% ± 0.9%, P < .05). HM treatment of p63−/− MEFs increased cTnT expression to 74% ± 3% of cells but did not induce cTnT expression in wild-type murine embryonic fibroblasts. shRNA-mediated p63 knockdown likewise yielded a 20-fold increase in cTnT microRNA expression compared with untreated MEFs. Adult murine cardiac fibroblasts demonstrated a 200-fold increase in cTnT gene expression after inducible p63 knockout and expressed sarcomeric α-actinin as well as cTnT. These p63−/− adult cardiac fibroblasts exhibited calcium transients and electrically stimulated contractions when co-cultured with neonatal rat cardiomyocytes and treated with HM. Increased expression of cTnT and other marker genes was also observed in p63 knockdown human cardiac fibroblasts procured from patients undergoing procedures for heart failure. ConclusionsDownregulation of p63 facilitates direct cardiac cellular reprogramming and may help overcome the resistance of human cells to reprogramming.

Transcriptional activator DOT1L putatively regulates human embryonic stem cell differentiation into the cardiac lineage

BackgroundCommitment of pluripotent stem cells into differentiated cells and associated gene expression necessitate specific epigenetic mechanisms that modify the DNA and corresponding histone proteins to render the chromatin in an open or closed state. This in turn dictates the associated genetic machinery, including transcription factors, acknowledging the cellular signals provided. Activating histone methyltransferases represent crucial enzymes in the epigenetic machinery that cause transcription initiation by delivering the methyl mark on histone proteins. A number of studies have evidenced the vital role of one such histone modifier, DOT1L, in transcriptional regulation. Involvement of DOT1L in differentiating pluripotent human embryonic stem (hES) cells into the cardiac lineage has not yet been investigated.MethodsThe study was conducted on in-house derived (KIND1) and commercially available (HES3) human embryonic stem cell lines. Chromatin immunoprecipitation (ChIP) was performed followed by sequencing to uncover the cardiac genes harboring the DOT1L specific mark H3K79me2. Following this, dual immunofluorescence was employed to show the DOT1L co-occupancy along with the cardiac progenitor specific marker. DOT1L was knocked down by siRNA to further confirm its role during cardiac differentiation.ResultsChIP sequencing revealed a significant number of peaks characterizing H3K79me2 occupancy in the proximity of the transcription start site. This included genes like MYOF, NR2F2, NKX2.5, and HAND1 in cardiac progenitors and cardiomyocytes, and POU5F1 and NANOG in pluripotent hES cells. Consistent with this observation, we also show that DOT1L co-localizes with the master cardiac transcription factor NKX2.5, suggesting its direct involvement during gene activation. Knockdown of DOT1L did not alter the pluripotency of hES cells, but it led to the disruption of cardiac differentiation observed morphologically as well as at transcript and protein levels.ConclusionsCollectively, our data suggests the crucial role of H3K79me2 methyltransferase DOT1L for activation of NKX2.5 during the cardiac differentiation of hES cells.

Cyclic biaxial tensile strain promotes bone marrow-derived mesenchymal stem cells to differentiate into cardiomyocyte-like cells by miRNA-27a

A physical stimuli, it has been reported that cyclic tensile strain can promote bone marrow-derived mesenchymal stem cells (BMSCs) to differentiate into cardiomyocytes, but the underlying mechanisms have been poorly elucidated so far. Here, we used a mimicking loading strain, cyclic biaxial tensile strain (CBTS), and found it can promote BMSCs to differentiate into cardiomyocytes. When the CBTS were loaded, the cells expressed cardiac-specific markers GATA4, TNNT2, MEF-2c, and Cx43, meanwhile we found miR-27a decreased and stem cell factor (SCF) increased. When we overexpressed miR-27a, the cardiac-specific markers were down-regulated; we got the same results when SCF was knocked down by siRNA. Interestingly, we found SCF is a potential target of miR-27a by a bioinformatic analysis. So, we overexpressed miR-27a, and found SCF decreased both in mRNA and protein level. And, When miR-27a was co-transfected with SCF-3'UTR, it significantly reduced the luciferase activity, but not when co-transfected with SCF-3'UTR mutation plasmid. Furthermore, after transfected both miR-27a and SCF siRNA, and the protein expression of the markers were more down-regulated than that of single of them. Taken together, we found CBTS can promote BMSCs to differentiate into cardiomyocytes, and miR-27a functions as a mechano-sensitive miRNA in this process by targeting SCF.

Improving the Specificity of Cardiac Biomarkers-The Early Development of Cardiac Troponin I (cTnI) Assays.

Featured Article: Adams JE III, Bodor GS, Davila-Roman VG, Delmez JA, Apple FS, Ladenson JH, Jaffe AS. Cardiac troponin I: a marker with high specificity for cardiac injury. Circulation 1993;88:101–6.2 By the late 1980s, it was clear that there were frequent increases of creatine kinase-muscle–brain (CK-MB)3 in patients with renal failure, hypothyroidism, pulmonary embolism, and a variety of skeletal muscle pathologies. Thus, there were often questions about whether such increases were due to the release from tissues other than the heart and especially from skeletal muscle where there is reexpression of B-chain CK protein in response to injury. These questions made the triage of patients with increased CK-MB values difficult, especially when conjoint skeletal muscle and cardiac muscle injury was present as is common postoperatively (1). Because of these difficulties, there was interest in developing more clinically specific cardiac markers. Initially, cardiac troponin T (cTnT) was introduced, but there were questions related to its clinical specificity. These questions subsequently were found to result from antibody cross-reactivity with skeletal muscle troponin, and that problem was …

Reciprocity of Action of Increasing Oct4 and Repressing p53 in Transdifferentiation of Mouse Embryonic Fibroblasts into Cardiac Myocytes.

p53 is a barrier to somatic cell reprogramming. Deletion or transient suppression of p53 increases the efficiency of reprogramming of somatic cells into induced pluripotent stem cells. Whether p53 represents an obstacle to a similar process transdifferentiation of somatic cells is unknown. However, it is predicted that inhibition of p53 would promote transdifferentiation of fibroblasts into cardiomyocytes. In this study, the effect of p53 on the capacity of cardiogenic transdifferentiation is evaluated using p53 wild-type (p53+/+), p53 heterozygous mutant (p53+/-), and p53 homozygous mutant (p53-/-) mouse embryonic fibroblasts (MEFs). Repression of p53 in MEFs increases the expression level of mesoderm transcription factors Brachyury (T) and MESP1. The cardiac-specific markers, Myh6 (Myosin, Heavy Chain 6), Myh7 (Myosin, Heavy Chain 7), and cTnI (cardiac muscle troponin I), show elevated expression in p53+/- and p53-/- MEFs compared with wild-type MEFs, but cardiac muscle troponin T (cTnT) showed a lower expression level when p53 was inhibited. After induction to cardiac differentiation, cTnT expression increased and markers of endoderm and ectoderm decreased in p53+/- and p53-/- MEFs. The effect of an important reprogramming factor Oct4 on cardiac transdifferentiation was also evaluated in the allelic series of p53 MEFs. We found that overexpression of Oct4 significantly enhanced Mesp1, Tbx5, and Isl1 expression in p53+/+ and p53+/- MEFs. Oct4 also enhanced cTnT expression in all three cell lines, especially in p53+/- MEFs. Thus, inhibition of p53 expression and viral expression of Oct4 both promote transdifferentiation of MEFs into cardiomyocytes, establishing reciprocity of action in the process.

Pivotal Role of Non-cardiomyocytes in Electromechanical and Therapeutic Potential of Induced Pluripotent Stem Cell-Derived Engineered Cardiac Tissue.

Although engineered cardiac tissues (ECTs) derived from induced pluripotent stem cells (iPSCs) are promising for myocardial regenerative therapy, the appropriate ratio of cardiomyocytes to non-cardiomyocytes is not fully understood. Here, we determined whether ECT-cell content is a key determinant of its structure/function, thereby affecting ECT therapeutic potential for advanced heart failure. Scaffold-free ECTs containing different ratios (25%, 50%, 70%, or 90%) of iPSC-derived cardiomyocytes were generated by magnetic-activated cell sorting by using cardiac-specific markers. Notably, ECTs showed synchronized spontaneous beating when cardiomyocytes constituted ≥50% of total cells, with the electrical-conduction velocity increasing depending on cardiomyocyte ratio; however, ECTs containing 90% cardiomyocytes failed to form stable structures. ECTs containing 25% or 50% cardiomyocytes predominantly expressed collagen and fibronectin, whereas ECTs containing 70% cardiomyocytes predominantly expressed laminin and exhibited the highest contractile/relaxation properties. Furthermore, transplantation of ECTs containing 50% or 70% cardiomyocytes into a rat chronic myocardial infarction model led to a more profound functional recovery as compared with controls. Notably, transplanted ECTs showed electrical synchronization with the native heart under Langendorff perfusion. Collectively, these results indicate that the quantity of non-cardiomyocytes is critical in generating functional iPSC-derived ECTs as grafts for cardiac-regeneration therapy, with ECTs containing 50–70% cardiomyocytes exhibiting stable structures and increased cardiotherapeutic potential.

Injectable OPF/graphene oxide hydrogels provide mechanical support and enhance cell electrical signaling after implantation into myocardial infarct.

After myocardial infarction (MI), the scar tissue contributes to ventricular dysfunction by electrically uncoupling viable cardiomyocytes in the infarct region. Injection of a conductive hydrogel could not only provide mechanical support to the infarcted region, but also synchronize contraction and restore ventricular function by electrically connecting isolated cardiomyocytes to intact tissue.Methods: We created a conductive hydrogel by introducing graphene oxide (GO) nanoparticles into oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels. The hydrogels were characterized by AFM and electrochemistry workstation. A rat model of myocardial infarction was used to investigate the ability of OPF/GO to improve cardiac electrical propagation in the injured heart in vivo. Echocardiography (ECHO) was used to evaluate heart function 4 weeks after MI. Ca2+ imaging was used to visualize beating cardiomyocytes (CMs). Immunofluorescence staining was used to visualize the expression of cardiac-specific markers.Results: OPF/GO hydrogels had semiconductive properties that were lacking in pure OPF. In addition, the incorporation of GO into OPF hydrogels could improve cell attachment in vitro. Injection of OPF/GO 4 weeks after myocardial infarction in rats enhanced the Ca2+ signal conduction of cardiomyocytes in the infarcted region in comparison with PBS or OPF alone. Moreover, the injection of OPF/GO hydrogel into the infarct region enhanced the generation of cytoskeletal structure and intercalated disc assembly. Echocardiography analysis showed improvement in load-dependent ejection fraction/fractional shortening of heart function 4 weeks after injection.Conclusions: We prepared a conductive hydrogel (OPF/GO) that provide mechanical support and biological conduction in vitro and in vivo. We found that injected OPF/GO hydrogels can provide mechanical support and electric connection between healthy myocardium and the cardiomyocytes in the scar via activating the canonical Wnt signal pathway, thus upregulating the generation of Cx43 and gap junction associated proteins. Injection of OPF/GO hydrogel maintained better heart function after myocardial infarction than the injection of a nonconductive polymer.

Isolation, Characterization and Differentiation of Mouse Cardiac Progenitor Cells.

Despite several strategies developed for replenishing the dead myocardium after myocardial infarction (MI), stem cell therapy remains the leading method to regenerate new myocardium. Although induced pluripotent stem cells (iPS) and transdifferentiation of the differentiated cells have been used as novel approaches for myocardial regeneration, these approaches did not yield very successful results for myocardial regeneration in in vivo studies. Asynchronous contractility of newly formed cardiomyocytes with the existing cardiomyocytes is the most important issue with iPS approach, while very low yield of transdifferentiated cardiomyocytes and theirless chances to beat in the same rhythm as existing cardiomyocytes in the MI heart are important caveats with transdifferentiation approach. CSCs are present in the heart and they have the potential to differentiate into myocardial cells. However, the number of resident CSCs is very low. Therefore, it is important to get maximum yield of CSCs during isolation process from the heart. Increasing the number of CSCs and initiating their differentiation ex vivo are crucial for CSC-based stem cell therapy. Here, we present a better method for isolation, characterization and differentiation of CSCs from the mouse heart. We also demonstrated morphological changes in the CSCs after 2days, 3days, and 7days in maintenance medium and a separate group of CSCs cultured for 12days in differentiation medium using Phase-Contrast microscopy. We have used different markers for identification of CSCs isolated from the mouse heart such as marker for mouse CSC, Sca-1, cardiac-specific markers NKX2-5, MEF2C, GATA4, and stemness markers OCT4 and SOX2. To characterize the differentiated CSCs, we used CSCs maintained in differentiation medium for 12days. To evaluate differentiation of CSCs, we determined the expression of cardiomyocyte-specific markers actinin and troponin I. Overall; we described an elegant method for isolation, identification, differentiation and characterization of CSCs from the mouse heart.

Altered Functional Expression of β-Adrenergic Receptors in Rhesus Monkey Embryonic Stem Cell-Derived Cardiomyocytes.

Pluripotent stem cells have demonstrated the potential to generate large numbers of functional cardiomyocytes (CMs) from different cell sources. Besides Wnt signaling, additional pathways are involved in early cardiac development and function. To date however, no study exists showing the effects of perturbing the canonical Wnt pathway using nonhuman primate embryonic stem (ES) cells. In this study, we investigated the effect of canonical Wnt inhibition during differentiation of nonhuman primate ES cell-derived CMs under defined, growth factor conditions. Rhesus monkey ES (rES) cells were differentiated into spontaneously beating CMs in the absence (control) or presence (treated) of Wnt inhibitor Dickkopf1 (DKK1), vascular endothelial growth factor, and basic fibroblast growth factor combined or added in a sequential manner during differentiation. Quantification and functional characterization of CMs were assessed by molecular and electrophysiological techniques. Analysis revealed no difference in average ratio of spontaneously beating clusters in both control and treated groups. However, the percentage of CMs was significantly reduced and the expressions of specific cardiac markers tested were also decreased in the treated group. Interestingly, we found that in CMs obtained from treated group, β-adrenergic receptors (β-ARs) were less expressed, their function was altered and electrophysiological studies revealed differences in action potential responsiveness to β-AR stimulation. We demonstrated that the Wnt/β-catenin pathway inhibitor, DKK1 associated with other growth factors repressed functional expression of β-ARs in rES cell-derived CMs. Thus, control of this pathway in each cell line and source is important for proper basic research and further cell therapy applications.

Synergistic role of 5-azacytidine and ascorbic acid in directing cardiosphere derived cells to cardiomyocytes in vitro by downregulating Wnt signaling pathway via phosphorylation of β-catenin.

BackgroundCardiosphere derived cells (CDCs) represent a valuable source in stem cell based therapy for cardiovascular diseases, yet poor differentiation rate hinders the transplantation efficiency. The aim of this study is to check the ability of 5-Azacytidine (Aza) alone and in combination with ascorbic acid (Aza+AA) in delineating CDCs to cardiomyogenesis and the underlying Wnt signaling mechanism in induced differentiation.MethodsCDCs were treated with Aza and Aza+AA for a period of 14 days to examine the expression of cardiac specific markers and Wnt downstream regulators by immunofluorescence, real time PCR and western blot.ResultsResults revealed that Aza+AA induced efficient commitment of CDCs to cardiomyogenic lineage. Immunofluorescence analysis showed significant augment for Nkx 2.5, GATA 4 and α-Sarcomeric actinin markers in Aza+AA group than control group (p = 0.0118, p = 0.009 and p = 0.0091, respectively). Relative upregulation of cardiac markers, Nkx 2.5 (p = 0.0156), GATA 4 (p = 0.0087) and down regulation of Wnt markers, β-catenin (p = 0.0107) and Cyclin D1 (p = 0. 0116) in Aza+AA group was revealed by RNA expression analysis. Moreover, the Aza+AA induced prominent expression of GATA 4, α-Sarcomeric actinin and phospho β-catenin while non phospho β-catenin and Cyclin D1 expression was significantly suppressed as displayed in protein expression analysis. Generation of spontaneous beating in Aza+AA treated CDCs further reinforced that Aza+AA accelerates the cardiomyogenic potential of CDCs.ConclusionCombined treatment of Aza along with AA implicit in inducing cardiomyogenic potential of CDCs and is associated with down regulating Wnt signaling pathway. Altogether, CDCs represent a valuable tool for the treatment of cardiovascular disorders.

Inhibitory effect of oxidative damage on cardiomyocyte differentiation from Wharton's jelly-derived mesenchymal stem cells.

Ischemic heart diseases are a serious health problem worldwide. The transplantation of mesenchymal stem cells (MSCs) has been investigated in numerous clinical trials on various other diseases due to the self-renewal capacity of these cells and their potential to differentiate into a variety of cell types. The presence of excess reactive oxygen species in injured myocardium causes cardiac dysfunction and leads to inefficient repair of the heart. The poor outcomes of stem cell transplantation have been suggested to result from residual oxidative damage affecting the transplanted cells. The aim of the present study was to compare the effects of hydrogen peroxide (H2O2) on Wharton's jelly-derived MSCs (WJ-MSCs) and bone marrow-derived MSCs (BM-MSCs) in vitro, in order to provide information useful for the future selection of MSC types for cardiac differentiation and transplantation. H2O2 at concentrations of 200, 500 and 1,000 µM was applied to WJ-MSCs and BM-MSCs under cardiogenic differentiation conditions. The morphology of MSCs treated with H2O2 was similar to that of untreated cells, whereas the cell density decreased in direct association with the dose of H2O2. Cardiac differentiation markers were then evaluated by immunofluorescence analysis of GATA4 and cardiac troponin T (cTnT). The fluorescence intensity levels of the two markers were identified to be diminished by increasing doses of H2O2 from 500 to 1,000 µM. The expression levels of homeobox protein Nkx2.5, cTnT and cardiac α-actin were also examined, and were identified to be low in the WJ-MSCs treated with 1,000 µM H2O2, which was similar to the findings observed in BM-MSCs. These results suggested that oxidative stress affects cardiomyocyte differentiation via the downregulation of cardiac genes and cardiac proteins. Furthermore, it should be noted that there was a marked difference in the effect depending on the source of MSCs. This evidence provided supportive information for the use of stem cells in transplantation.

Oxidized low-density lipoprotein (ox-LDL) promotes cardiac differentiation of bone marrow mesenchymal stem cells via activating ERK1/2 signaling.

The differentiation efficiency of bone marrow mesenchymal stem cells (BM-MSCs) is low in vivo after transplantation. Therefore, it is necessary to look for effective reagents for enhancing cardiac differentiation of BM-MSCs. It has been reported that cardiac differentiation of stem cells depends on the activation of extracellular signal-regulated protein 1/2 (ERK1/2) signaling. Oxidized low-density lipoprotein (ox-LDL) is a potent reagent for ERK1/2 activation. This indicates that ox-LDL may be a potential reagent to stimulate cardiac differentiation of stem cells. In this study, we investigated the effect of ox-LDL on cardiac differentiation of BM-MSCs and its relationship with ERK1/2 signaling. BM-MSCs were isolated from mouse bone marrow, cultured in DMEM supplemented with 15% FBS, and passaged up to the 3rd passage. Following culture with 5μg/mL ox-LDL for 3weeks, the cardiac differentiation of the 3rd passage BM-MSCs was identified by immunostaining, Western blotting, and RT-PCR assays for measuring the expression of cardiac-specific markers. To further explore the role of ERK1/2 signaling in cardiac differentiation of BM-MSCs, we simultaneously exposed BM-MSCs to ERK1/2 inhibitor (U0126) and ox-LDL, and identified the cardiac differentiation again. The expressions of cardiac-specific markers including α-cardiac actin, α-MHC, β-MHC, ANP, and BNP were markedly increased in BM-MSCs following treatment with ox-LDL (P<.05), which indicates a directional differentiation of BM-MSCs to cardiac cells. Further, ox-LDL could also activate ERK1/2 in BM-MSCs, and application of U0126 markedly inhibited ox-LDL-induced cardiac transformation of BM-MSCs. Ox-LDL induces cardiac differentiation of BM-MSCs via activation of ERK1/2 signaling.

Involment of RAS/ERK1/2 signaling and MEF2C in miR-155-3p inhibition-triggered cardiomyocyte differentiation of embryonic stem cell.

MicroRNAs (miRNAs) are short, noncoding RNAs that regulate post-transcriptional gene expression by targeting messenger RNAs (mRNAs) for cleavage or translational repression. Growing evidence indicates that miR-155 expression changes with the development of heart and plays an important role in heart physiopathology. However, the role of miR-155 in cardiac cells differentiation is unclear. Using the well-established embryonic stem cell (ESC), we demonstrated that miR-155-3p expression was down-regulated during cardiogenesis from mouse ESC. By contrast, the myogenic enhance factor 2C (MEF2C), a predicted target gene of miR-155-3p, was up-regulated. We further demonstrated that miR-155-3p inhibition increased the percentage of embryoid bodies (EB) beating and up-regulated the expression of cardiac specific markers, GATA4, Nkx2.5, and cTnT mRNA and protein. Notably, miR-155-3p inhibition caused upregulation of MEF2C, KRAS and ERK1/2. ERK1/2 inhibitor, PD98059 significantly decreased the expression of MEF2C protein. These findings indicate that miR-155-3p inhibition promotes cardiogenesis, and its mechanisms are involved in the RAS-ERK1/2 signaling and MEF2C.

MODELING THE DILATED CARDIOMYOPATHY WITH ATAXIA SYNDROME (DCMA), A PEDIATRIC MITOCHONDRIAL CARDIOMYOPATHY, USING CARDIOMYOCYTES DERIVED FROM INDUCED PLURIPOTENT STEM CELLS

Cardiomyopathy is a predominant and lethal feature of many inborn metabolic disorders but results from derangements in cellular energetics rather than ischemic myocardial injury. This fundamental difference is likely why standard heart failure medications appear to be ineffective. This problem is exemplified by the dilated cardiomyopathy with ataxia syndrome (DCMA). DCMA is related to Barth syndrome with abnormal cardiolipin and mitochondria causing heart failure, conduction defects and other systemic features. Isolated cases of DCMA have been identified worldwide but the largest known group of patients is found in the Hutterites of southern Alberta. No effective therapy exists and in our cohort there is 39% mortality in early childhood related to cardiac dysfunction despite recommended optimal medical therapy. The lack of useful drugs for mitochondrial cardiomyopathies means that there are few therapies available outside of mechanical circulatory support or heart transplantation. Since the rarity and heterogeneity of the pediatric cardiomyopathies preclude randomized drug trials, the use of patient cells and in vitro drug modeling represents the best possible option to identify effective therapeutics. Here, we describe the creation and characterization of cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) (as an in vitro model of DCMA) from 4 children with DCMA and varying severity of cardiac dysfunction. Purified peripheral blood mononuclear cells derived from 4 DCMA patients (Table 1) were reprogrammed into induced pluripotent stem cells (iPSCs) using Sendai virus and the 4 Yamanaka factors, and characterized using standard pluripotency tests. Our iPSCs expressed markers of pluripotency including SSEA4 and OCT4. The DCMA-derived iPSCs were sequenced to detect the DCMA related mutation in their genomic DNA. Results showed that the iPSCs carried the DNAJC19 mutation (rs137854888) responsible for the abnormal splicing causing the disease phenotype. These iPSCs were successfully differentiated into beating cardiomyocytes using a modified protocol and lactate as a sole carbon source to enrich purified iPSC-CMs. The resulting iPSC-CMs expressed the cardiac-specific markers TNNT2 and NKX2.5. We describe the development of iPSC-CMs for DCMA which represent a novel in vitro model for mitochondrial cardiomyopathy and provide a platform to study the efficacy of potential therapeutics and genome editing approaches.

ANALYSIS OF CHANGE IN NT-proBNP AFTER ANGIOTENSIN RECEPTOR BLOCKER (ARB) THERAPY IN PATIENT WITH HEART FAILURE

NT-proBNP is an inactive fragment of BNP secreted by stretched ventricle as response to wall stress in patients with heart failure. As a specific cardiac marker, elevated NT-proBNP correlates well with heart failure severity. The principle of heart failure therapy is modulation on neurohormonal activation. ARB can modulate neurohormon on RAA system, that result in decreasing NT-proBNP level and favorable outcomes. Reduction in NT-proBNP more than biologic variability (&gt; 25%) shows a therapy response.This study was to analyze change of NT-proBNP after ARB therapy in ambulatory HF patients. This observational prospective study was carried from September to December 2015. Blood sampling was performed on patients who meet the inclusion criteria of the study at first visit and after 2 months therapy. NT-proBNP was measured by IMMULITE® as primary parameter and creatinin as secondary parameter. There are 14 patients met the inclusion criteria of the study (11 males and 3 females). ARB therapy used in patients were Valsartan (64%), Telmisartan (22%) and Candesartan (14%). After 2 months ARB therapy, a decrease in level of NT-proBNP with initial median 3092.5 (216 – 32112) pg/ml to 2135.5 (350 – 16172) pg/ml respectively were statistically significant (p=0.003). And the secondary parameter creatinin serum convert to eGFR shows a change in eGFR with initial median 73.33 (37.05 – 266.68) ml/minute to 81.04 (39.31 – 167.02) ml/minute respectively were statistically not significant (p=0.657). There were 7 patients (50%) have a decrease &gt; 25%. In this study, we found that ARB therapy can change NT-proBNP level significantly after 2 months therapy.

Epigenetic reprogramming converts human Wharton\u2019s jelly mesenchymal stem cells into functional cardiomyocytes by differential regulation of Wnt mediators

BackgroundLineage commitment of mesenchymal stem cells (MSCs) to cardiac differentiation is controlled by transcription factors that are regulated by epigenetic events, mainly histone deacetylation and promoter DNA methylation. Here, we studied the differentiation of human Wharton’s jelly MSCs (WJMSCs) into the cardiomyocyte lineage via epigenetic manipulations.MethodsWe introduced these changes using inhibitors of DNA methyl transferase and histone deacetylase, DC301, DC302, and DC303, in various combinations. We characterized for cardiogenic differentiation by assessing the expression of cardiac-specific markers by immunolocalization, quantitative RT-PCR, and flow cytometry. Cardiac functional studies were performed by FURA2AM staining and Greiss assay. The role of Wnt signaling during cardiac differentiation was analyzed by quantitative RT-PCR. In-vivo studies were performed in a doxorubicin-induced cardiotoxic mouse model by injecting cardiac progenitor cells. Promoter methylation status of the cardiac transcription factor Nkx2.5 and the Wnt antagonist, secreted frizzled-related protein 4 (sFRP4), after cardiac differentiation was studied by bisulfite sequencing.ResultsBy induction with DC301 and DC302, WJMSCs differentiated into cardiomyocyte-like structures with an upregulation of Wnt antagonists, sFRP3 and sFRP4, and Dickkopf (Dkk)1 and Dkk3. The cardiac function enhancer, vinculin, and DDX20, a DEAD-box RNA helicase, were also upregulated in differentiated cardiomyocytes. Additionally, bisulfite sequencing revealed, for the first time in cardiogenesis, that sFRP4 is activated by promoter CpG island demethylation. In vivo, these MSC-derived cardiac progenitors could not only successfully engraft to the site of cardiac injury in mice with doxorubicin-induced cardiac injury, but also form functional cardiomyocytes and restore cardiac function.ConclusionThe present study unveils a link between Wnt inhibition and epigenetic modification to initiate cardiac differentiation, which could enhance the efficacy of stem cell therapy for ischemic heart disorders.