Preclinical Study of the Effect of Cardiotropic Agents on Isolated Heart

Direct effects of cardiotropic preparations on the hearts isolated from Wistar rats were examined. Deenergization of cardiomyocytes was modeled under conditions of hypoxic perfusion. Recovery of cardiac function during reperfusion was assessed by changes in the heart rate and contraction amplitude.     

Differential reparative phenotypes between zebrafish and medaka after cardiac injury

Results : Compared with zebrafish, the medaka heart responded differently to an injury: An excessive fibrotic response occurred in the medaka heart, and existing cardiomyocytes or cardiac progenitor cells remained dormant, resulting in no numerical difference between the uncut and injured heart with respect to the number of EdU‐incorporated cardiomyocytes. The results obtained from the analysis of the medaka raldh2‐GFP transgenic line showed a lack of raldh2 expression in the endocardium. Regarding periostin expression, the localization of medaka periostin‐b, a marker of fibrillogenesis, in the medaka heart remained at the wound site at 30 dpa; whereas zebrafish periostin‐b was no longer localized at the wound but was detected in the epicardium at that time. 相似文献   

Role of Phospholipase A2 in Activation of Isolated Cardiomyocyte Respiration in Postinfarction Cardiosclerosis

The rate of oxygen consumption by isolated cardiomyocytes was studied in rats with experimental postinfarction cardiosclerosis. The increase in oxygen consumption under these condition was comparable to that in melittin- and arachidonic acid-induced activation of phospholipase A₂ in cardiomyocytes of intact animals. Bromophenacyl bromide inhibition of phospholipase A₂ in cardiomyocytes of rats with postinfarction cardiosclerosis led to reduction of oxygen consumption rate to values characteristic of intact animal cardiomyocytes. The results confirm the hypothesis according to which high oxygen consumption in postinfarction cardiosclerosis is related to increased activity of phospholipase A₂. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 12, pp. 631–633, December, 2008     

Differentiation of mouse-induced pluripotent stem cells into cardiomyocytes in vitro

In order to investigate the effective method to induce mice-induced pluripotent stem (miPS) cells into cardiomyocytes in vitro and to investigate the effect of vitamin C on cardiomyocyte differentiation from miPS cells to find a highly efficient and clinically safe method. MiPS cells were isolated and expanded to form embryoid bodies (EBs) using the hanging drop way. EBs were induced using differentiation medium containing vitamin C (10^?4 mmol/ml). The control group did not receive any form of inducer. The time and frequency at which beating cardiomyocytes appeared and the percentage of beating colonies were determined to investigate the function of vitamin C on cardiac myocytes differentiation from miPS cells. Beating cell areas were found in (62.5 ± 1.7%) of EBs when using differentiation medium containing vitamin C, which was at a significantly greater frequency than in the control group (7.6 ± 2.6%). Beating cardiomyocytes within the two groups were positive for troponin (cTnT) staining. Vitamin C markedly increased the productivity of miPS cell differentiation into cardiomyocytes, as supported by expression of the unique cardiac protein cTnT. The vitamin C is suitable candidate for the induction of miPS cell differentiation into cardiomyocytes in vitro.     

Effect of Maternal Protein Restriction During Pregnancy and Lactation on the Number of Cardiomyocytes in the Postproliferative Weanling Rat Heart

Maternal protein restriction leads to a reduction in the number of cardiomyocytes in the rat heart at birth. However, in rats, cardiomyocytes continue to proliferate until about 2 weeks after birth. Hence, this study aimed to examine the effect of maternal protein restriction, on the number of cardiomyocytes in the young rat heart at a time point when the cardiomyocytes have ceased proliferating and are terminally differentiated. Female Wistar Kyoto rats were fed either a normal protein diet (NPD; 20% casein) or a low protein diet (LPD; 8.7% casein) during pregnancy and lactation. Offspring (seven males and seven females per group) were perfusion fixed at 4 weeks of age. Heart volume and total cardiomyocyte number were determined using stereological techniques. At 4 weeks of age, body weights in both male and female LPD offspring were significantly reduced compared with NPD controls whereas relative heart volumes were significantly increased in LPD offspring. Total number of cardiomyocytes was not significantly different between groups. In both groups, there was a significant linear correlation between cardiomyocyte number and heart volume. In conclusion, total cardiomyocyte number in the postproliferative rat heart does not appear to be affected by maternal protein restriction per se but is directly related to heart size. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Nodal signaling promotes the speed and directional movement of cardiomyocytes in zebrafish

Members of the Nodal family regulate left‐right asymmetry during vertebrate organogenesis, but it is unclear how Nodal signaling controls asymmetric morphogenesis at the cellular level. We used high‐resolution time‐lapse imaging in zebrafish to compare the movements of cardiomyocytes in the presence or absence of Nodal signaling. Loss of Nodal signaling in late‐zygotic mutants for the Nodal co‐receptor one‐eyed pinhead (LZoep) abolished the leftward movement of cardiomyocytes. Global heart rotation was blocked but cardiomyocyte neighbor relationships were maintained as in wild type. Cardiomyocytes in LZoep mutants moved more slowly and less directionally than their wild‐type counterparts. The phenotypes observed in the absence of Nodal signaling strongly resemble abnormalities found in BMP signaling mutants. These results indicate that a Nodal‐BMP signaling cascade drives left‐right heart morphogenesis by regulating the speed and direction of cardiomyocyte movement. Developmental Dynamics 237:3624–3633, 2008. © 2008 Wiley‐Liss, Inc.     

Comparative pathomorphological study of contractile myocardium under conditions of increased left and right ventricular afterload

Structural changes in the myocardium under conditions of increased left and right ventricular afterload were studied using polarization microscopy and histological, histochemical, and stereological methods. Increased afterload not complicated by heart failure was characterized by low number of damaged cardiomyocytes (3.3–6.5%) and moderate structural changes in the ventricular myocardium (contractures of different severity). Increased afterload complicated by heart failure was characterized by high ratio of damaged cardiomyocytes (5.6–19.2%) and severe reversible (grade I and II contractures) and irreversible (grade III contractures and lump degradation of myofibrils) structural changes. Irreversible damage to most cardiomyocytes included plasmatic impregnation, which was most pronounced in the sub-endocardial layer of ventricles operating under conditions of increased afterload. Comparative study showed that increased left and right ventricular afterload induces similar pathomorphological changes in the contractile myocardium. Our results indicate that increased afterload to the right or left ventricle is accompanied by the development of stereotypical structural changes in the myocardium. Profound and severe disturbances can cause heart failure.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 12, pp. 693–697, December, 2004This revised version was published online in April 2005 with a corrected cover date.     

The homozygous variant c.245G > A/p.G82D in PNPLA2 is associated with arrhythmogenic cardiomyopathy phenotypic manifestations

Arrhythmogenic cardiomyopathy (ACM) is a familial cardiomyopathy featured by fibrofatty replacement of cardiomyocytes. Responsible genetic factors are not discernible in approximately one-third of ACM probands. To investigate this further, we performed whole genome sequencing in 14 mutation-negative ACM probands who underwent cardiac transplantation, and we identified one ACM proband with a rare homozygous missense variant in PNPLA2 (c.245G > A, p.G82D), a rate-limiting enzyme that hydrolyzes triglycerides into fatty acids and diacylglycerol. Bioinformatic analysis suggested that this missense variant may lead to loss of function and therefore impair lipid catabolism. Genetic screening in this proband's family also inferred that the homozygous variant cosegregated with disease. To validate the pathogenicity of this variant and confirm its association with ACM, we established a knockin mouse model carrying the orthologous human homozygous PNPLA2 variant. Interestingly, mice with the homozygous variant presented with arrhythmias and significant cardiac dysfunction at 12 weeks, whereas heterozygous mice were not affected. Moreover, those homozygous mice suffered sudden death and/or heart failure by the age of 14 weeks. Pathological examination showed that extensive lipogenesis in cardiomyocytes and cardiac fibrosis were prominent in the myocardium. Herein, our data demonstrated that the homozygous missense variant PNPLA2 (c.245G > A, p.G82D) associated with a recessive form of ACM.     

Direct effects of the angiotensin-converting enzyme inhibitor ramiprilat on adult rat ventricular cardiomyocytes

Aim: Angiotensin‐converting enzyme (ACE) inhibitors like ramiprilat bind to ACE expressed on the cell surface of endothelial cells and induce cell‐specific signalling including the activation of activator protein (AP)‐1. The present study addressed the question whether ramiprilat exerts a similar effect on adult ventricular cardiomyocytes, i.e. activates the AP‐1 or modifies contractile performance. It was further aimed to decide whether such effects depend on bradykinin receptors or whether they are directly mediated via ACE. Methods: Adult rat ventricular cardiomyocytes were isolated and cultured. mRNA expression of ACE was investigated by RT‐PCR, AP‐1 activation by gel mobility shift assays, and cardiac contractile performance by electrical pacing of isolated cells and analysis of cell shortening via a line‐camera. Results: Cardiomyocytes stably express ACE. Ramiprilat increased maximal contraction velocity and shortened the time‐to‐peak of contraction. In contrast to effects evoked by bradykinin, such effects caused by ramiprilat were not attenuated by HOE 140, a bradykinin‐receptor antagonist. These effects were also not attenuated in the presence of l ‐nitro‐arginine, used to mimic bradykinin‐dependent signalling. In cardiomyocytes, bradykinin but not ramiprilat activated AP‐1. Ramiprilat activates AP‐1 in endothelial cells that are known to respond to ramiprilat in this way. Conclusion: Ramiprilat exerts direct, bradykinin‐receptor independent effects on cardiomyocytes that improve cellular function without a corresponding effect on AP‐1 activation or induction of AP‐1 dependent effects. This newly described effect of ramiprilat may contribute to the protective effects seen by application of ACE inhibitors.     

The Influence of Naturally Occurring Differences in Birthweight on Ventricular Cardiomyocyte Number in Sheep

In most species including man, cardiomyocytes cease proliferating soon after birth when they become terminally differentiated. A reduced complement of cardiomyocytes in infancy may adversely impact on the function and adaptive capabilities of the heart in later life. Low birthweight is associated with an increased risk of heart disease in adults, but little is known about its effect on the number of cardiomyocytes. Using naturally occurring differences in birthweight, our aim was to determine the effect of birthweight on cardiomyocyte number in postnatal lambs. At 9 weeks after term birth, when the final number of cardiomyocytes is considered to be established, hearts were collected at necropsy from seven singleton and seven twin lambs. Hearts were perfusion‐fixed, and tissue samples were systematically taken from the left ventricle plus intraventricular septum (LV+S) and the right ventricle (RV). The number of cardiomyocyte nuclei was estimated using an unbiased optical disector–fractionator stereological technique, and the total number of cardiomyocytes was determined. Weights of the total heart, LV+S and RV were significantly related to both birthweight and necropsy weight. In the LV+S but not the RV, cardiomyocyte number was significantly and directly related to heart tissue weight, birthweight, and necropsy weight. We conclude that the final number of cardiomyocytes in the LV+S is related to prenatal and early postnatal growth, and is proportionate to the weight of heart tissue. A low cardiomyocyte number in the LV+S following restricted fetal growth may contribute to the increased incidence of heart disease in adults born with low birthweight. Anat Rec, 2009. © 2008 Wiley‐Liss, Inc.     

Nitric oxide depresses connexin 43 after myocardial infarction in mice

Aims: Heart failure (HF) is a major cause of death and morbidity. Connexin 43 (Cx43) content is reduced in the failing myocardium, but regulating factors have not been identified. In HF, inducible nitric oxide synthase (iNOS)‐induced high levels of nitric oxide (NO) cause apoptosis and cardiac dysfunction. However, a direct iNOS–Cx43 link has not been demonstrated. We investigated this relationship in mice after myocardial infarction. Methods: Effects of myocardial infarction were evaluated 2 weeks after coronary artery ligation in wild‐type C57BL/6 (WT) and iNOS^?/? knockout mice. Myocardial Cx43 and Cx45 content were assessed by immunofluorescence confocal imaging and western blotting. Cardiac function was evaluated in anaesthetized mice using a micro pressure‐tipped catheter inserted into the left ventricle. Results: Despite similar infarct size, deficiency in iNOS resulted in significantly lower plasma nitrate/nitrite levels, better haemodynamic performance and lower mortality 2 weeks after coronary ligation. Myocardial Cx43, but not Cx45, content was lower in WT mice following ligation. The reduction in Cx43 was less in iNOS^?/? compared with WT mice. To assess the direct effect of NO on Cx43 expression, cultured neonatal mouse cardiomyocytes were employed. Incubation with the NO donor, S‐nitroso‐N‐acetylpenicillamine, elicited a dose‐dependent decrease in Cx43 content in cultured neonatal cardiomyocytes. Conclusions: Increased NO production from iNOS depressed cardiac performance and contributed to the decreased myocardial Cx43 content 2 weeks after myocardial infarction.     

Electrical restitution in rat ventricular muscle

The mechanism of electrical restitution was studied in isolated rat ventricular muscle using drugs that inhibit specific ion currents. The effect of transient changes in cytosolic Ca concentration and Na/Ca exchange in relation to the restitution process was also studied in single ventricular cardiomyocytes. Conventional microelectrode techniques were applied to record action potentials having gradually increasing coupling intervals, each evoked following a train of stimuli with a frequency of 1 Hz. Ion currents were recorded from enzymatically isolated cells using the whole cell patch clamp technique. Ca transients were monitored in myocytes loaded with the fluorescent dye, indo-1. The electrical restitution process in multicellular rat ventricular preparations at 37 °C was described as a sum of three exponential components: an early positive component, a subsequent fast negative component and a late negative component, having time constants of 21.9±1.9, 73.1±6.0 and 1053±61 ms, respectively (n=9). Inhibition of the transient outward K current, the delayed rectifier K current, or the chloride current did not substantially alter these time constants. The early positive and fast negative components were fully abolished by nifedipine or MnCl₂. In the presence of caffeine, the fast negative component was absent, while the time constant of the early positive component increased to 39.5±5.8 ms (n=5). In single myocytes loaded with indo-1, the Ca transients decayed with a time constant of 151±12 ms at room temperature (n=5). These Ca transients were accompanied by inward current tails, identified as a Na/Ca exchange current, having a decay time constant of 140±4.5 ms. It is concluded that electrical restitution in rat ventricular muscle is relatively little affected by recovery from voltage-dependent inactivation of ion channels, it is rather governed by transient changes in cytosolic Ca concentration possible via Ca-dependent inactivation of the L-type Ca current and activation of the Na/Ca exchange current.     

Embryonic cardiomyocyte expression of endothelial genes.

Vertebrate precardiac mesoderm contains cells destined to become cardiomyocyte or endothelial cells. To determine the stability of these phenotypes freshly isolated embryonic day (E) 2.5-E6 chicken hearts were immunostained for myosin heavy chain (MyHC) to identify cardiomyocytes, and von Willebrand factor (vWF) and Flk-1 to identify endothelial cells. At E2.5-E3, 90% of cells express only MyHC and 6% express only vWF/Flk-1. However, 2% MyHC+ cells in E2.5-E3 hearts and 0.3% in E4-E6 hearts, also express vWF/Flk-1; and when cultured 3 days, >40% of the MyHC+ cells express vWF/Flk-1, but they do not express Vezf1, vascular endothelial cadherin, or Tie2. Thus, only a subset of endothelial genes are induced in cultured cardiomyocytes. While the subsequent developmental fate of embryonic heart cells exhibiting a vWF+/MyHC+ phenotype is unknown, analysis of this phenotype may provide information pertinent to mechanisms of cell phenotype stability, cellular transdifferentiation, and induction of stable cell types from embryonic stem cells.     

Intracellular calcium and electrical restitution in mammalian cardiac cells

The role of calcium current and changes in intracellular calcium concentration ([Ca²⁺]_i) in regulation of action potential duration (APD) during electrical restitution process was studied in mammalian ventricular preparations. Properly timed action potentials were recorded from multicellular preparations and isolated cardiomyocytes using conventional microelectrodes and EGTA-containing patch pipettes. APD increased monotonically in canine and guinea pig ventricular preparations with increasing diastolic interval (DI), while in rabbit papillary muscles the restitution process was biphasic: APD first lengthened, then shortened as the DI increased. When the restitution process was studied in single cardiomyocytes using EGTA-containing patch pipettes, the restitution pattern was similar in the three species studied. Similarly, no difference was observed in the recovery time constant of calcium current (I_Ca-L) measured under these conditions in voltage clamped myocytes. Loading the myocytes with the [Ca²⁺]_i-chelator BAPTA-AM had adverse effects in rabbit and canine cells. In rabbit myocytes steady-state APD lengthened and the late shortening component of restitution was abolished in BAPTA-loaded cells. In canine myocytes BAPTA-load shortened steady-state APD markedly, and during restitution, APD decreased with increasing DI. The late shortening component of restitution, observed in untreated rabbit preparations, was greatly reduced after nifedipine treatment, but remained preserved in the presence of 4-aminopyridine or nicorandil. Beat to beat changes in APD, peak I_Ca-L and [Ca²⁺]_i, measured using the fluorescent dye, Fura-2, were monitored in rabbit ventricular myocytes after a 1-min period of rest. In these cells, the shortening of APD was accompanied by a gradual reduction of the peak I_Ca-L and elevation of diastolic [Ca²⁺]_i during the initial eight post-rest action potentials. It is concluded that elevation of [Ca²⁺]_i shortens, while reduction of [Ca²⁺]_i lengthens APD in rabbit, but not in canine ventricular myocytes. These differences may probably be related to different distributions of [Ca²⁺]_i-dependent ion currents and/or to differences in calcium handling between the two species.     

CUG‐BP,Elav‐like family member 1 (CELF1) is required for normal myofibrillogenesis,morphogenesis, and contractile function in the embryonic heart

Background: CUG‐BP, Elav‐like family member 1 (CELF1) is a multifunctional RNA binding protein found in a variety of adult and embryonic tissues. In the heart, CELF1 is found exclusively in the myocardium. However, the roles of CELF1 during cardiac development have not been completely elucidated. Results: Myofibrillar organization is disrupted and proliferation is reduced following knockdown of CELF1 in cultured chicken primary embryonic cardiomyocytes. In vivo knockdown of Celf1 in developing Xenopus laevis embryos resulted in myofibrillar disorganization and a trend toward reduced proliferation in heart muscle, indicating conserved roles for CELF1 orthologs in embryonic cardiomyocytes. Loss of Celf1 also resulted in morphogenetic abnormalities in the developing heart and gut. Using optical coherence tomography, we showed that cardiac contraction was impaired following depletion of Celf1, while heart rhythm remained unperturbed. In contrast to cardiac muscle, loss of Celf1 did not disrupt myofibril organization in skeletal muscle cells, although it did lead to fragmentation of skeletal muscle bundles. Conclusions: CELF1 is required for normal myofibril organization, proliferation, morphogenesis, and contractile performance in the developing myocardium. Developmental Dynamics 245:854–873, 2016. © 2016 Wiley Periodicals, Inc.     

Activation of μ-opiate receptors as a factor of regulation of heart resistance to ischemia-reperfusion and oxidative stress

Intravenous injection of the selective μ-opiate receptor agonist DAMGO (0.1 mg/kg, 15 min before isolation of the heart) improved resistance of isolated perfused rat heart to ischemia (45 min) and reperfusion (60 min) damages.In vivo administration of DAMGO prevented reperfusion-induced damages to cardiomyocytes and decreased the content of conjugated dienes in the myocardium during ischemia-reperfusionin vitro. Furthermore, stimulation of μ-opiate receptors promoted recovery of myocardial contractility during reoxygenation, but had no effect on heart resistance to free radical-induced damages during perfusion of isolated heart with a solution containing Fe²⁺ and ascorbic acid.