Contractile Cardiomyocytes Research Articles

Paracrine potential of adipose stromal vascular fraction cells to recover hypoxia-induced loss of cardiomyocyte function.

Cell-based therapies show promising results in cardiac function recovery mostly through paracrine-mediated processes (as angiogenesis) in chronic ischemia. In this study, we aim to develop a 2D (two-dimensional) in vitro cardiac hypoxia model mimicking severe cardiac ischemia to specifically investigate the prosurvival paracrine effects of adipose tissue-derived stromal vascular fraction (SVF) cell secretome released upon three-dimensional (3D) culture. For the 2D-cardiac hypoxia model, neonatal rat cardiomyocytes (CM) were cultured for 5 days at < 1% (approaching anoxia) oxygen (O2 ) tension. Typical cardiac differentiation hallmarks and contractile ability were used to assess both the cardiomyocyte loss of functionality upon anoxia exposure and its possible recovery following the 5-day-treatment with SVF-conditioned media (collected following 6-day-perfusion-based culture on collagen scaffolds in either normoxia or approaching anoxia). The culture at < 1% O 2 for 5 days mimicked the reversible condition of hibernating myocardium with still living and poorly contractile CM (reversible state). Only SVF-medium conditioned in normoxia expressing a high level of the prosurvival hepatocyte-growth factor (HGF) and insulin-like growth factor (IGF) allowed the partial recovery of the functionality of damaged CM. The secretome generated by SVF-engineered tissues showed a high paracrine potential to rescue the nonfunctional CM, therefore resulting in a promising patch-based treatment of specific low-perfused areas after myocardial infarction.

Human gingival fibroblast feeder cells promote maturation of induced pluripotent stem cells into cardiomyocytes

The use of human induced pluripotent stem (iPS) cells has been investigated in multiple regenerative medicine studies. However, although methods for efficient differentiation of iPS cells into heart tissues have been devised, it remains difficult to obtain cardiac tissue with high contractility. Herein, we established a method for differentiating iPS cells into highly contractile cardiomyocytes (CMs), and demonstrate that the use of human gingival fibroblasts (HGFs) as a feeder cells promotes maturation of iPS-derived CMs (iPS-CMs) in vitro. After CM differentiation of iPS cells, iPS-CMs showed increased mRNA expression of the CM specific maker cardiac troponin T (cTnT) in the absence and presence (on-feeder condition) of cocultured HGFs, and decreased expression of pluripotent markers was observed under both conditions. Protein expression of cTnT was also observed in immunocytochemical analyses, although on-feeder CMs showed comparatively robust sarcomere structure and significantly stronger contractility than feederless cardiomyocytes, suggesting that HGF feeder cells facilitate CM differentiation of iPS cells.

Heart regeneration after myocardial infarction: the role of microRNAs

The curative therapy after myocardial infarction (MI) is to regenerate new functional myocardial tissue: the formation of new contractile cardiomyocytes and the revascularization in the injured region. Although, it is admitted that adult mammal heart can regenerate during physiological ageing and after heart injuries. Its regenerative capacity is very limited and could not compensate the loss of an amount of functional tissue after MI. Therefore, stimulation of the regenerative capacity of the human heart could be the primary option to restore cardiac function after MI. MicroRNAs (miRNAs) are small noncoding RNAs that implicated in the gene expression. Several miRNAs have been shown to control important processes that contribute to the consequences of MI. MiRNAs play an important role during heart development and regulate cardiomyocytes proliferation and revascularization after MI. New data provide a proof of concept for identifying and activating conserved molecular programs to regenerate the damaged heart. In this review, we focus on the role of miRNAs in heart development and regeneration after MI.

Induction of cardiomyocyte proliferation, a new way forward for true myocardial regeneration?

Ischemic heart disease (IHD) and heart failure (HF) are major causes of morbidity and mortality worldwide and although acute treatment during myocardial infarction has improved significantly, chronic burden of the disease gained much attention recent years (1,2). The major cause of the failing hearts, however, the loss of contractile cardiomyocytes is currently still a problem to overcome.

184 Establishment of Porcine Induced Pluripotent Stem Cell Lines by Adding LIN 28 Transcription Factor

The establishment of porcine induced pluripotent stem cells (piPSC) is important in the field of human biomedical regenerative medicine. The pig model is a more representative model than current rodent models because it better mimics human physiology in different organ systems. The piPSC can be traditionally generated by reprogramming somatic cells using 4 transcription factors (4TF: OCT4, SOX2, KLF4, and c-MYC), similarly in human. However, it is difficult to maintain the pluripotent state of reprogrammed cells and they exhibit poor differentiation capacity. Hence, the 4TF may be not enough to reprogram porcine somatic cells. This study aimed to establish piPSC by adding LIN28 (referred to as 5TF) to the traditional 4TF, via retroviral vector. Here, we report the successful establishment of 3 piPSC lines by using the 5TF. All 5TF-piPSC lines exhibited a normal karyotype (38, XY) and a typical mouse embryonic stem cell (ESC) morphology, including tightly packed and dome-like shape, even after they were propagated over 40 passages. All 5TF-piPSC lines were positive for alkaline phosphatase staining and expressed high levels of ESC-like markers (OCT4, SOX2, NANOG, and SSEA-1). Importantly, the 5TF-piPSC lines showed pluripotent capacity, as evidenced by differentiation into 3 germ layers in vitro following cystic embryoid body formation, as well as by efficiently forming teratomas containing all 3 embryonic germ layers in vivo. Moreover, the 5TF-piPSC lines showed spontaneously contractile cardiomyocytes and expressed cardiomyocyte markers (cardiac troponin T) during spontaneous cardiac differentiation using cell aggregation into spherical-like structures referred to as embryoid bodies. Thus, the addition of LIN28 TF promoted long-term pluripotency of piPSC and enhanced the ability to differentiate towards 3 embryonic germ layers and cardiac lineage. This research project is supported by grants from the Mahidol University, Thailand.

Telocytes in the human sinoatrial node.

The sinoatrial node (SAN) is composed mostly of pacemaker, transitional and Purkinje‐like cells. Pacemaker cells, especially in the centre of the SAN, are surrounded by dense fibrous tissue and do not have any contact with transitional cells. We hypothesize that the SAN contains telocytes that have contacts with pacemaker cells and contractile myocardium. Immunohistochemistry using antibodies against HCN4 and antibody combinations against CD34 and HCN4 was carried out on 12 specimens. Confocal laser scanning microscopy (CLSM) with two mixtures of primary antibodies, namely CD34/S100 and vimentin/S100, was performed in three cases. In two cases, CLSM was carried out with CD117 antibody. Specimens for electron microscopy and immunocytochemistry with HCN4 immunogold labelling were taken from another three patients. In our study, we found cells with the immunophenotype of telocytes in the SAN. There were twice as many of these cells in the centre of the SAN as in the periphery (20.3 ± 4.8 versus 10.8 ± 4.4 per high‐power field). They had close contact with pacemaker cells and contractile cardiomyocytes and expressed HCN4. The ultrastructural characteristics of these cells are identical to those of telocytes observed earlier in other organs. Our study provides evidence that telocytes are present in the SAN.

Translating Myocardial Remuscularization.

Myocardial remuscularization can be achieved by the implantation of cardiomyocyte-containing grafts. Critical steps toward clinical translation of the first generation cardiac remuscularization therapies include the choice of animal models with high predictive validity for therapeutic outcome in clinical trials. Clinically relevant remuscularization of the failing human heart remains unachieved to date. Proof-of-concept studies in small and large animal models provide compelling evidence as to the principle feasibility of myocardial remuscularization with human cardiomyocyte grafts.1,2 Clinical trials suggest myocardial protection and preservation by cell therapeutics.3,4 The mechanisms underlying the reported therapeutic effects remain under debate, but likely include the release of protective factors (growth factors, noncoding RNA) and modulation of the disease-related inflammatory response. Further studies are needed to define the paracrine milieu hypothesis and unleash its full therapeutic potential, aiming at delaying or preventing disease progression in patients with acute and subacute myocardial injury. The implantation of cardiomyocyte grafts follows a different strategy, namely remuscularization by integration of exogenously produced cardiomyocytes. Patients with chronically scarred myocardium presenting clinically with end-stage heart failure are the primary target. The suggested plug-and-play mechanism of direct cardiac remuscularization seems straight forward, but similar as for the paracrine milieu hypothesis there is a clear need for a better understanding of how, when, and where to integrate exogenous cardiomyocytes into the failing heart to achieve optimal results. Moreover, the path toward clinical application as off-the-shelf cardiomyocyte allograft therapeutics is less defined as for point-of-care autograft formulations and small chemical or biological compounds. A challenge to direct cardiac remuscularization was for many years the limited availability of bona fide cardiomyocytes. Cardiac biopsies can be used to harvest proliferative mesenchymal cells with progenitor cell properties, but without the capacity to spontaneously convert/transdifferentiate into terminally differentiated cardiomyocytes at a therapeutically relevant scale. Induced …

CARDIOPROTECTIVE PROPERTIES OF PROGESTINES: INFLUENCE OF DROSPIRENONE ON MYOCARD IN EXPERIMENTAL HEART FAILURE

Aim. To assess the influence of synthetic progesterone — drospirenone, on remodeling of the left ventricle myocardium (LV) in ovariectomized (OE) female rats under circumstances of experimental heart failure (EHF). Material and methods. The study was done on 25 female Wistar rats, mass 260-300 g. Twenty animals (in 2 months after bilateral ovariectomy) underwent modeling of EHF by subcutaneous load of 0,1% mesaton solution for 14 days with following swimming to profound exhaustion. Four rats were removed on 14th day of EHF. Sixteen experimental animals with EHF were selected to 3 groups: 10 underwent 14 days subcutaneous load of drospirenon (Sigma-Aldrich, USA), dose 2 mg/kg (n=5) and 0,5 mg/kg (n=5), 6 were being loaded saline 0,2 mL daily. On 28th day the animals were removed from the experiment. With light microscopy method, immune cytochemistry, morphometry the LV myocardium was investigated. Results . In OE rodents on the 14th day of EHF there was clear and significant heteromorphism of contractile cardiomyocytes (CMC) with the signs of hypertrophy and dystrophy; increase of the volume density (VD) of stroma, reorganized extracellular matrix (EM), expression of metalloproteases-2,9 (MMP-2,9) and tissue inhibitor of metalloproteases-1 (TIMP-1), CMC with apoptosis. After remodeling of EHF at 28th day in myocardium there was further increase of CMC number with significantly changed morphology and tinctorial properties, degradation of EM components. In rats receiving 14 days drospirenon, regardless of dosage, there was regression of pathological changes, decrease of CMC hypertrophy grade, as the nuclei, decrease of CMC with the signs of apoptosis, and stabilized EM components. Conclusion. The cardioprotective properties of drospirenon are discussed, that even in minimal therapeutic concentrations facilitates structural and functional rehabilitation of myocardium under conditions of gonade hormones deficiency and EHF.

Distinct and Shared Determinants of Cardiomyocyte Contractility in Multi-Lineage Competent Ethnically Diverse Human iPSCs.

The realization of personalized medicine through human induced pluripotent stem cell (iPSC) technology can be advanced by transcriptomics, epigenomics, and bioinformatics that inform on genetic pathways directing tissue development and function. When possible, population diversity should be included in new studies as resources become available. Previously we derived replicate iPSC lines of African American, Hispanic-Latino and Asian self-designated ethnically diverse (ED) origins with normal karyotype, verified teratoma formation, pluripotency biomarkers, and tri-lineage in vitro commitment. Here we perform bioinformatics of RNA-Seq and ChIP-seq pluripotency data sets for two replicate Asian and Hispanic-Latino ED-iPSC lines that reveal differences in generation of contractile cardiomyocytes but similar and robust differentiation to multiple neural, pancreatic, and smooth muscle cell types. We identify shared and distinct genes and contributing pathways in the replicate ED-iPSC lines to enhance our ability to understand how reprogramming to iPSC impacts genes and pathways contributing to cardiomyocyte contractility potential.

Abstract 203: A Shh Coreceptor Cdo is Required for Efficient Cardiomyogenesis of Pluripotent Stem Cells

Sonic hedgehog (Shh) signaling plays an important role for early heart development, such as heart looping and cardiomyogenesis of pluripotent stem cells. A multifunctional receptor Cdo functions as a Shh coreceptor together with Boc and Gas1 to activate Shh signaling and these coreceptors seem to play compensatory roles in early heart development. Thus in this study, we examined the role of Cdo in cardiomyogenesis by utilizing an in vitro differentiation of pluripotent stem cells. Here we show that Cdo is required for efficient cardiomyogenesis of pluripotent stem cells by activation of Shh signaling. Cdo is induced concurrently with Shh signaling activation upon induction of cardiomyogenesis of P19 embryonal carcinoma (EC) cells. Cdo -depleted P19 EC and Cdo –/– mouse embryonic stem (ES) cells display decreased expression of key cardiac regulators, including Gata4, Nkx2.5 and Mef2c and this decrease coincides with reduced Shh signaling activities. Furthermore Cdo deficiency causes a stark reduction in formation of mature contractile cardiomyocytes. This defect in cardiomyogenesis is overcome by reactivation of Shh signaling at the early specification stage of cardiomyogenesis. The Shh agonist treatment restores differentiation capacities of Cdo -deficient ES cells into contractile cardiomyocytes by recovering both the expression of early cardiac regulators and structural genes such as cardiac troponin T and Connexin 43. Therefore Cdo is required for efficient cardiomyogenesis of pluripotent stem cells and an excellent target to improve the differentiation potential of stem cells for generation of transplantable cells to treat cardiomyopathies.

Морфологические изменения миокарда крыс в условиях гемической гипоксии на фоне коррекции цитофлавином

Целью исследования явилось изучение влияния цитофлавина на морфологические особенности сократительных кардиомиоцитов самок крыс, перенесших беременность, а также их потомства в условиях гемической гипоксии, индуцированной нитритом натрия (NaNO2). Эксперимент выполнен на 28 трехмесячных самках белых крыс линии Вистар и их 24 семидневных крысятах. На протяжении всей беременности и в течение 7 дней периода молочного вскармливания всем самкам ежедневно внутрибрюшинно вводили нитрит натрия (NaNO2) в дозе 5 мг/100 г массы тела. Самкам второй опытной группы дополнительно внутрибрюшинно вводили цитофлавин из расчета 0,5 мл/100 г массы тела животного. Выявлено, что гемическая гипоксия оказывает выраженное патогенное действие на сократительные кардиомиоциты и сосуды гемомикроциркуляторного русла, которое проявляется в виде гипоксических и ишемических повреждений, сопровождающихся нарушением целостности клеточных и митохондриальных мембран. Гистологическое и морфометрическое изучение миокарда животных первой опытной группы выявило достоверное уменьшение длины и площади кардиомиоцитов соответственно на 16,9 и 36,04 % (p ≤ 0,05), в то время как во второй группе животных, получавших цитофлавин, эти изменения носили менее выраженный характер, уменьшение длины и площади, в среднем, происходило на 8,2 и 18,6 %, соответственно (p ≤ 0,05). Применение цитофлавина позволяет снизить степень повреждения кардиомиоцитов у самок после гестации и новорожденных крысят, по-видимому, за счет его антиоксидантных и мембранопротекторных свойств.

A Shh coreceptor Cdo is required for efficient cardiomyogenesis of pluripotent stem cells

Sonic hedgehog (Shh) signaling plays an important role for early heart development, such as heart looping and cardiomyogenesis of pluripotent stem cells. A multifunctional receptor Cdo functions as a Shh coreceptor together with Boc and Gas1 to activate Shh signaling and these coreceptors seem to play compensatory roles in early heart development. Thus in this study, we examined the role of Cdo in cardiomyogenesis by utilizing an in vitro differentiation of pluripotent stem cells. Here we show that Cdo is required for efficient cardiomyogenesis of pluripotent stem cells by activation of Shh signaling. Cdo is induced concurrently with Shh signaling activation upon induction of cardiomyogenesis of P19 embryonal carcinoma (EC) cells. Cdo-depleted P19 EC and Cdo−/− mouse embryonic stem (ES) cells display decreased expression of key cardiac regulators, including Gata4, Nkx2.5 and Mef2c and this decrease coincides with reduced Shh signaling activities. Furthermore Cdo deficiency causes a stark reduction in formation of mature contractile cardiomyocytes. This defect in cardiomyogenesis is overcome by reactivation of Shh signaling at the early specification stage of cardiomyogenesis. The Shh agonist treatment restores differentiation capacities of Cdo-deficient ES cells into contractile cardiomyocytes by recovering both the expression of early cardiac regulators and structural genes such as cardiac troponin T and Connexin 43. Therefore Cdo is required for efficient cardiomyogenesis of pluripotent stem cells and an excellent target to improve the differentiation potential of stem cells for generation of transplantable cells to treat cardiomyopathies.

SPARC-Dependent Cardiomyopathy in Drosophila.

The Drosophila heart is an important model for studying the genetics underpinning mammalian cardiac function. The system comprises contractile cardiomyocytes, adjacent to which are pairs of highly endocytic pericardial nephrocytes that modulate cardiac function by uncharacterized mechanisms. Identifying these mechanisms and the molecules involved is important because they may be relevant to human cardiac physiology. This work aimed to identify circulating cardiomodulatory factors of potential relevance to humans using the Drosophila nephrocyte-cardiomyocyte system. A Kruppel-like factor 15 (dKlf15) loss-of-function strategy was used to ablate nephrocytes and then heart function and the hemolymph proteome were analyzed. Ablation of nephrocytes led to a severe cardiomyopathy characterized by a lengthening of diastolic interval. Rendering adult nephrocytes dysfunctional by disrupting their endocytic function or temporally conditional knockdown of dKlf15 led to a similar cardiomyopathy. Proteomics revealed that nephrocytes regulate the circulating levels of many secreted proteins, the most notable of which was the evolutionarily conserved matricellular protein Secreted Protein Acidic and Rich in Cysteine (SPARC), a protein involved in mammalian cardiac function. Finally, reducing SPARC gene dosage ameliorated the cardiomyopathy that developed in the absence of nephrocytes. The data implicate SPARC in the noncell autonomous control of cardiac function in Drosophila and suggest that modulation of SPARC gene expression may ameliorate cardiac dysfunction in humans.

The importance of neuregulin in the development of heart and diseases of the cardiovascular system

Neuregulin-1 belongs to the epidermal growth factors (EGF) which have a role in cell proliferation, differentiation and function maintenance of epithelial breast cells, glial cells, neurons and myocytes. It acts in a paracrine and jucstacrine manner through family ErbB tyrosine kinases receptors (ErbB2, ErbB3, and ErbB4) which contain extracellular places for ligands, transmembrane domains and tyrosin kinase domains. NRG-ErbB signaling pathway that is important in embryonic development of the heart also has its role in adult heart. Neuregulin is mainly synthesized in the endocardial endothelial cells and endothelial cells of the myocardial capillaries. ErbB3 receptors are distributed on the endothelial cells and the ErbB2/ErbB4 complex in cardiomyocytes. Soluble NRG-1 induces proliferation of cardiomyocytes in the embryo, increases survival, affects the apoptosis of the culture of cardiomyocytes and induces hypertrophic growth in neonatal and adult ventricular cardiomyocytes. In addition to the effect on the proliferation and differentiation of cardiomyocytes, NRG-1 also promotes the proper differentiation and recruitment of cells of the vascular system of the heart from contractile cardiomyocytes. Endogenous neuregulin may also affect cardiac function by acting on the nekardiomiocite promoting angiogenesis. The cardio-protective effects of neuregulin-1 through nekardiomiocita can be achieved by increasing the activity of cardiac fibroblasts, where neuregilina administration results in reduction of the size of the scar. The ischemic damage of cardiomyocytes and physical strain lead to the activation of the tissue neuregulin 1, which is a potent inducer of endothelial progenitor cell mobilization. Since neuregulin is necessary in the development of the cardiovascular system, regulation of the cardio-protective mechanism of signaling, it is possible that the application of recombinant neuregulin isoforms may have potential application in the treatment of certain cardiac diseases. However, the use of a recombinant NRG-1 can stimulate the proliferation non-cardiac cells as well. The question is whether the use of neuregulin in the cardioprotection will trigger only the receptors intended for that because if NRG-1 is given systemically it can activate the receptors in other tissues and promote unwanted reactions.

CHANGES IN THE CONTRACTILE CARDIOMYOCYTES AND HEMOMICROCIRCULATORY BED IN PREGNANT RATS AND THEIR PUPS EXPOSED TO HEMIC HYPOXIA

The performed analysis of the morphology of cardiomyocytes and myocardial vascular bed in 12 pregnant rats and 16 newborn pups exposed to the hemic hypoxia induced by NaNO 2 revealed some similar degenerative and destructive changes which were more pronounced in pups. The main morphological characteristics of hypoxic myocardial damage in pregnant and newborn rats were presented in the form of phenomena of mixed dystrophy, swelling and destruction of endothelial cells and contractile cardiomyocytes along with the lysis of myofibrils, perivascular edema, capillary hyperemia, emptiness and spasm of the arterioles and contracture changes. Revealed pathological changes in the myocardium reflect the development of the myocardiodystrophy processes in pregnant rats and newborn pups.

Endocrine and other physiologic modulators of perinatal cardiomyocyte endowment.

Immature contractile cardiomyocytes proliferate to rapidly increase cell number, establishing cardiomyocyte endowment in the perinatal period. Developmental changes in cellular maturation, size and attrition further contribute to cardiac anatomy. These physiological processes occur concomitant with a changing hormonal environment as the fetus prepares itself for the transition to extrauterine life. There are complex interactions between endocrine, hemodynamic and nutritional regulators of cardiac development. Birth has been long assumed to be the trigger for major differences between the fetal and postnatal cardiomyocyte growth patterns, but investigations in normally growing sheep and rodents suggest this may not be entirely true; in sheep, these differences are initiated before birth, while in rodents they occur after birth. The aim of this review is to draw together our understanding of the temporal regulation of these signals and cardiomyocyte responses relative to birth. Further, we consider how these dynamics are altered in stressed and suboptimal intrauterine environments.

Cardiac protein kinases: the cardiomyocyte kinome and differential kinase expression in human failing hearts.

Protein kinases are potential therapeutic targets for heart failure, but most studies of cardiac protein kinases derive from other systems, an approach that fails to account for specific kinases expressed in the heart and the contractile cardiomyocytes. We aimed to define the cardiomyocyte kinome (i.e. the protein kinases expressed in cardiomyocytes) and identify kinases with altered expression in human failing hearts. Expression profiling (Affymetrix microarrays) detected >400 protein kinase mRNAs in rat neonatal ventricular myocytes (NVMs) and/or adult ventricular myocytes (AVMs), 32 and 93 of which were significantly up-regulated or down-regulated (greater than two-fold), respectively, in AVMs. Data for AGC family members were validated by qPCR. Proteomics analysis identified >180 cardiomyocyte protein kinases, with high relative expression of mitogen-activated protein kinase cascades and other known cardiomyocyte kinases (e.g. CAMKs, cAMP-dependent protein kinase). Other kinases are poorly investigated (e.g. Slk, Stk24, Oxsr1). Expression of Akt1/2/3, BRaf, ERK1/2, Map2k1, Map3k8, Map4k4, MST1/3, p38-MAPK, PKCδ, Pkn2, Ripk1/2, Tnni3k, and Zak was confirmed by immunoblotting. Relative to total protein, Map3k8 and Tnni3k were up-regulated in AVMs vs. NVMs. Microarray data for human hearts demonstrated variation in kinome expression that may influence responses to kinase inhibitor therapies. Furthermore, some kinases were up-regulated (e.g. NRK, JAK2, STK38L) or down-regulated (e.g. MAP2K1, IRAK1, STK40) in human failing hearts. This characterization of the spectrum of kinases expressed in cardiomyocytes and the heart (cardiomyocyte and cardiac kinomes) identified novel kinases, some of which are differentially expressed in failing human hearts and could serve as potential therapeutic targets.

Microgrooved ultra-thin films as building blocks of future bio-hybrid actuators.

This paper aims to demonstrate the possibility of exploiting poly-L-lactic acid (PLLA) ultra-thin films as platforms for bio-hybrid actuation. Firstly, flat PLLA nanofilms at different concentrations (15 and 25 mg/ml in dichloromethane) were tested with contractile cardiomyocytes. The results obtained using motion vector analysis, a non-invasive method capable of estimating flow velocities on recorded videos, demonstrated that PLLA nanofilms were able to move under the contraction of muscle cells. Immunofluorescence images reflected good cell spreading, thus confirming that these films are promising matrices for bio-hybrid actuation. Subsequently, microgrooved PLLA nanofilms were fabricated, in order to drive muscle cell distribution on an anisotropic surface, thus optimizing the system's efficiency. After matrix characterization, in terms of AFM and SEM imaging, we investigated the viability and morphology of C2C12 skeletal muscle cells (a more controllable muscle cell type), 24 h after cell seeding as well as at the 7-day differentiation state.

Morphology of contractile cardiomyocytes and vessels in pregnant rats and their pups exposed to hemic hypoxia

Zadnipryany I. V., Tret'yakova O. S., Sataieva T. P., Zukow W. Morphology of contractile cardiomyocytes and vessels in pregnant rats and their pups exposed to hemic hypoxia. Journal of Education, Health and Sport. 2015;5(4):72-80. ISSN 2391-8306. DOI: 10.5281/zenodo.16595 http://ojs.ukw.edu.pl/index.php/johs/article/view/2015%3B5%284%29%3A72-80 https://pbn.nauka.gov.pl/works/553142 http://dx.doi.org/10.5281/zenodo.16595 Formerly Journal of Health Sciences. ISSN 1429-9623 / 2300-665X. Archives 2011 – 2014 http://journal.rsw.edu.pl/index.php/JHS/issue/archive Deklaracja. Specyfika i zawartośc merytoryczna czasopisma nie ulega zmianie. Zgodnie z informacją MNiSW z dnia 2 czerwca 2014 r., ze w roku 2014 nie bedzie przeprowadzana ocena czasopism naukowych; czasopismo o zmienionym tytule otrzymuje tyle samo punktow co na wykazie czasopism naukowych z dnia 31 grudnia 2014 r. The journal has had 5 points in Ministry of Science and Higher Education of Poland parametric evaluation. Part B item 1089. (31.12.2014). © The Author (s) 2015; This article is published with open access at Licensee Open Journal Systems of Kazimierz Wielki in Bydgoszcz, Poland and Radom in Radom, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. This is an open access article licensed under the terms of the Creative Commons Attribution Non Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 20.01.2014. Revised 27.02.2015. Accepted: 15.03.2015. UDC 611.08: 611: 611.12 + 611.16 morphology of contractile cardiomyocytes and vessels IN PREGNANT rats AND their pups exposed to HEMIC HYPOXIA I. V. Zadnipryany, O. S. Tret'yakova, T. P. Sataieva , W. Zukow Medical Academy named after S.I. Georgievskiy Federal State Autonomous Educational Institution of H igher Education Crimean Federal University Kazimierz Wielki University, Bydgoszcz Summary The performed morphological analysis of cardiomyocytes and myocardial vessels in 12 pregnant rats and 16 newborn pups exposed to the hemic hypoxia induced by NaNO 2 revealed some similar degenerative and destructive changes which were more pronounced in pups. The main morphological characteristics of hypoxic myocardial damage in pregnant and newborn rats were presented in the form of phenomena of mixed dystrophy, swelling and destruction of endothelial cells and contractile cardiomyocytes along with the lysis of myofibrils, perivascular edema, capillary hyperemia, emptiness and spasm of the arterioles and contracture changes. Revealed pathological changes in the myocardium reflect the onset of myocardiodystrophy processes in pregnant rats and their newborn pups. Keywords: hemic hypoxia, cardiomyocytes, vessels, myocardium, pregnant rats, pups.

Isolation of contractile cardiomyocytes from human pluripotent stem-cell-derived cardiomyogenic cultures using a human NCX1-EGFP reporter.

The prospective isolation of defined contractile human pluripotent stem cell (hPSC)-derived cardiomyocytes is advantageous for regenerative medicine and drug screening applications. Currently, enrichment of cardiomyocyte populations from such cultures can be achieved by combinations of cell surface markers or the labor-intensive genetic modification of cardiac developmental genes, such as NKX2.5 or MYH6, with fluorescent reporters. To create a facile, portable method for the isolation of contractile cardiomyocytes from cardiomyogenic hPSC cultures, we employed a highly conserved cardiac enhancer sequence in the SLC8A1 (NCX1) gene to generate a lentivirally deliverable, antibiotic-selectable NCX1cp-EGFP reporter. We show that human embryonic stem cells (and induced pluripotent stem cells) transduced with the NCX1cp-EGFP reporter cassette exhibit enhanced green fluorescent protein (EGFP) expression in cardiac progenitors from 5 days into the directed cardiac hPSC differentiation protocol, with all reporter-positive cells transitioning to spontaneously contracting foci 3 days later. In subsequent stages of cardiomyocyte maturation, NCX1cp-EGFP expression was exclusively limited to contractile cells expressing high levels of cardiac troponin T (CTNT), MLC2a/v, and α-actinin proteins, and was not present in CD90/THY1(+) cardiac stromal cells or CD31/PECAM(+) endothelial cells. Flow-assisted cytometrically sorted EGFP(+) fractions of differentiated cultures were highly enriched in both early (NKX2.5 and TBX5) and late (CTNT/TNNI2, MYH6, MYH7, NPPA, and MYL2) cardiomyocyte markers, with a significant proportion of cells displaying a ventricular-like action potential pattern in patch-clamp recordings. We conclude that the use of the cardiac-specific promoter of the human SLC8A1(NCX1) gene is an effective strategy to isolate contractile cardiac cells and their progenitors from hPSC-derived cardiomyogenic cultures.