小鼠胚胎心脏房室管心肌与心外膜的形态学变化

目的探讨小鼠胚胎心脏房室管分隔、重塑过程中房室管心肌与心外膜的变化规律。方法选用抗心肌肌球蛋白轻链Ⅱa(MLC2a)抗体、抗心肌肌球蛋白轻链Ⅱ(MLC-2)抗体、抗转录因子Tbx3(Tbx3)抗体、抗淋巴增强因子1(Lef1)抗体,对25只胚龄10~15 d小鼠胚胎切片进行免疫组织化学和免疫荧光染色。结果胚龄10~15 d,房室管心肌呈MLC2a阳性、MLC-2阴性,同时表达Tbx3。胚龄11~12 d,心外膜形成。胚龄12~13 d,两侧房室管心内膜垫彼此接近并融合形成房室瓣,心外膜来源间充质细胞数量增加,部分表达Lef1。胚龄13 d开始,部分心外膜来源间充质细胞穿过心肌延伸入壁侧房室瓣。胚龄15 d,房室瓣膜基部直接与MLC2a阳性的房室管心肌相连。结论小鼠胚胎房室管心肌发育为成体心脏房室环瓣膜基部的心肌;心外膜通过产生间充质细胞参与房室瓣的形成。     

Chondroitin sulfate expression is required for cardiac atrioventricular canal formation

Defects in cardiac valvulogenesis are a common cause of congenital heart disease, and the study of this process promises to provide mechanistic insights and lead to novel therapeutics. Normal valve development involves multiple signaling pathways, and recently roles have been identified for extracellular matrix components, including glycosaminoglycans. We, therefore, explored the role of the glycosaminoglycan chondroitin sulfate during zebrafish cardiac development. Beginning at 33 hr, there is a distinct zone of chondroitin sulfate expression in the atrioventricular (AV) boundary, in the cardiac jelly between the endocardium and myocardium. This expression is both spatially and temporally restricted, and is undetectable after 48 hr. Chemical as well as genetic inhibition of chondroitin synthesis results in AV canal (AVC) defects, including loss of the atrioventricular constriction, blood regurgitation, and failure of circulation. Lack of chondroitin disrupts a marker of cell migration, results in a loss of myocardial and endothelial markers of valvulogenesis, and misregulates bone morphogenetic protein expression, supporting an early role in AVC development. In summary, we have defined a requirement for chondroitin sulfate expression in the normal patterning of the AV boundary, suggesting that this component of the cardiac jelly provides a necessary signal in this critical transition in vertebrate cardiogenesis. Developmental Dynamics 238:3103–3110, 2009. © 2009 Wiley‐Liss, Inc.     

Apoptosis and epicardial contributions act as complementary factors in remodeling of the atrioventricular canal myocardium and atrioventricular conduction patterns in the embryonic chick heart

Background : During heart development, it has been hypothesized that apoptosis of atrioventricular canal myocardium and replacement by fibrous tissue derived from the epicardium are imperative to develop a mature atrioventricular conduction. To test this, apoptosis was blocked using an established caspase inhibitor and epicardial growth was delayed using the experimental epicardial inhibition model, both in chick embryonic hearts. Results : Chicken embryonic hearts were either treated with the peptide caspase inhibitor zVAD‐fmk by intrapericardial injection in ovo (ED4) or underwent epicardial inhibition (ED2.5). Spontaneously beating embryonic hearts isolated (ED7–ED8) were then stained with voltage‐sensitive dye Di‐4‐ANEPPS and imaged at 0.5–1 kHz. Apoptotic cells were quantified (ED5–ED7) by whole‐mount LysoTracker Red and anti‐active caspase 3 staining. zVAD‐treated hearts showed a significantly increased proportion of immature (base to apex) activation patterns at ED8, including ventricular activation originating from the right atrioventricular junction, a pattern never observed in control hearts. zVAD‐treated hearts showed decreased numbers of apoptotic cells in the atrioventricular canal myocardium at ED7. Hearts with delayed epicardial outgrowth showed also increased immature activation patterns at ED7.5 and ED8.5. However, the ventricular activation always originated from the left atrioventricular junction. Histological examination showed no changes in apoptosis rates, but a diminished presence of atrioventricular sulcus tissue compared with controls. Conclusions : Apoptosis in the atrioventricular canal myocardium and controlled replacement of this myocardium by epicardially derived HCN4‐/Trop1‐ sulcus tissue are essential determinants of mature ventricular activation pattern. Disruption can lead to persistence of accessory atrioventricular connections, forming a morphological substrate for ventricular pre‐excitation. Developmental Dynamics 247:1033‐1042, 2018. © 2018 Wiley Periodicals, Inc.     

Measurements of cardiac troponin I and creatine kinase myocardium isoform in dogs with diabetic ketoacidosis

Diabetic ketoacidosis (DKA) is a complication of diabetes mellitus (DM) which may cause cardiac injury. The study aimed to compare concentrations of cardiac troponin I (cTnI) and creatine kinase myocardium isoform (CK-MB) in DKA and normal dogs and determine the relationship between cTnI and CK-MB concentrations and blood profile values, complications, and the risk of death. Plasma samples from 20 normal and 24 DKA dogs were collected. The cTnI concentration of DKA dogs (0.053 [0.034–0.200] ng/ml) was higher than those of normal dogs (0.020 [0.015–0.043] ng/ml) (p < 0.0001). Dogs with higher cTnI concentration had a higher risk of death than those with lower cTnI concentrations (HR 12.34; 95 % CI 1.66, 91.97). DKA dogs (203.5 [142.8–290.0] U/l) had higher CK-MB concentrations than normal dogs (90 [78–134.5] ng/ml) (p = 0.008). In conclusion, cTnI and CK-MB concentrations increase in DKA dogs. With higher cTnI concentrations, dogs have a higher risk of death.     

Differences of creatine kinase MB and cardiac troponin I concentrations in normal and diseased human myocardium

The diagnosis of myocardial infarction (MI) is established in patients with chest pain and equivocal electrocardiogram changes by demonstrating a rise in blood levels of creatine kinase MB (CK-MB) and/or an increase in cardiac troponin I (cTnI) or cardiac troponin T (cTnT). Previous studies have shown that levels of CK-MB are increased in the left ventricle of individuals with heart disease; however, it has not been established whether there are differences in the ventricular myocardium concentrations of cTnI in diseased compared to healthy hearts. Using a simple extraction technique, concentrations of CK-MB and cTnI were measured in the left ventricle (LV) of six hearts obtained at autopsy from individuals ranging in age from 25 to 79 yr, with and without evidence of cardiac disease. The results show an 86-fold higher concentration of CK-MB and 7.7-fold lower concentration of cTnI in left ventricular myocardium of older men with and without cardiac disease, compared to that of younger men (< age 35 yr) without heart disease. These data suggest that age may need to be considered when setting cutoff limits for these markers for the diagnosis of myocardial infarction.     

Transient and stable transgene expression in human embryonic stem cells

Plasmid vectors remain a valuable yet capricious tool for the genetic manipulation of human embryonic stem (hES) cells. We have compared the efficacy of four promoters to mediate transient and stable transfection in hES and human embryonal carcinoma cell lines with the reporter enhanced green fluorescent protein (eGFP). In transient assays, the two mammalian promoters, UbiquitinC and Rosa26 (pUbiC and pR26), the human cytomegalovirus major immediate early promoter (HCMV-MIE; pCMV), and the HCMV-MIE/chicken beta-actin/rabbit beta-globin hybrid promoter (pCAGG) gave variable results that depended upon the cell line transfected but in an unpredictable way: each promoter supported strong transient expression in at least one cell line. The results for stable transfection were generally at variance with the transient assays. In each case, transgene silencing was quite marked, most notably with the pCMV, with which no eGFP-positive clones were obtained. An exception was the pCAG vector, in which the CAGG composite promoter is linked to the polyoma virus mutant enhancer PyF101; stable eGFP-positive transfectants were obtained, and these clones retained eGFP expression for over 120 passages, even in the absence of selection. However, if the PyF101 elements were removed, the resulting transfectants were also subjected to progressive gene silencing. Thus, the choice of promoter is critical for determining the desired effect of transgene expression in hES cells. Our data also demonstrate that pUbiC, pR26, pCAGG, and pCAG are more superior to the pCMV for generation of stable transfectants in hES cells. Disclosure of potential conflicts of interest is found at the end of this article.     

An inducible transgene expression system for regulated phenotypic modification of human embryonic stem cells

Self-renewing pluripotent human embryonic stem (hES) cells are capable of regenerating such non-dividing cells as neurons and cardiomyocytes for therapies and can serve as an excellent experimental model for studying early human development. Both the spatial and temporal relationships of gene expression play a crucial role in determining differentiation; to obtain a better understanding of hES cell differentiation, it will be necessary to establish an inducible system in hES cells that enables specific transgene(s) to reversibly and conditionally express (1) at specific levels and (2) at particular time points during development. Using lentivirus (LV)-mediated gene transfer and a tetracycline-controlled trans-repressor (TR), we first established in hES cells a doxycycline (DOX)-inducible expression system of green fluorescent protein (GFP) to probe its reversibility and kinetics. Upon the addition of DOX, the percentage of GFP(+) hES cells increased time dependently: The time at which 50% of all green cells appeared (T(50)(on)) was 119.5+/-3.2 h; upon DOX removal, GFP expression declined with a half-time (T(50)(off)) of 127.7+/-3.9 h and became completely silenced at day 8. Both the proportion and total mean fluorescence intensity (MFI) were dose-dependent (EC(50)=24.5+/-2.2 ng/ml). The same system when incorporated into murine (m) ES cells similarly exhibited reversible dose-dependent responses with a similar sensitivity (EC(50)=49.5+/-8.5 ng/ml), but the much faster kinetics (T(50)(on)=35.5+/-5.5 h, T(50)(off) = 71.5+/-2.4 hours). DOX-induced expression of the Kir2.1 channels in mES and hES cells led to robust expression of the inwardly rectifying potassium (K(+)) current and thereby hyperpolarized the resting membrane potential (RMP). We conclude that the LV-inducible system established presents a unique tool for probing differentiation.     

Efficient multicistronic expression of a transgene in human embryonic stem cells

The applicability of human embryonic stem cells (hESCs) will be greatly enhanced by techniques that permit efficient genetic modification with multiple transgenes. We report here on single-promoter-driven foot-and-mouth disease virus segment 2A-mediated multicistronic expression of a transgene in hESCs. Efficient multicistronic expression of the transgene was permitted by 2A-mediated separation with almost the same amounts of encoded proteins in hESC. In addition, the multicistronic protein expression was successful in hESC-derived differentiated cells in in vivo and in vitro differentiation assays. This technology may be a significant advance in the genetic engineering of hESCs and hESC-derived cells for purposes that require the reliable expression of multiple transgenes. Disclosure of potential conflicts of interest is found at the end of this article.     

Cardiomyopathy in dystrophin-deficient hearts is prevented by expression of a neuronal nitric oxide synthase transgene in the myocardium

Null mutation of dystrophin causes the lethal pathology of Duchenne muscular dystrophy (DMD) in which there is progressive pathology of skeletal and cardiac muscles. A large proportion of DMD patient deaths are attributable to cardiac dysfunction associated with ventricular fibrosis, arrhythmias and conduction abnormalities, although the relationships between the dystrophin mutation and the cardiac defects are unknown. Here, we tested whether cardiac pathology in dystrophin-deficient mdx mice can be corrected by the elevated production of nitric oxide (NO) by the myocardium. Dystrophin-deficient mdx mice were produced in which there was myocardial expression of a neuronal nitric oxide synthase (nNOS) transgene. Expression of the transgene prevented the progressive ventricular fibrosis of mdx mice and greatly reduced myocarditis. Electrocardiographs (ECG) attained by radiotelemetry of freely ambulatory mice showed that mdx mice displayed cardiac abnormalities that are characteristic of DMD patients, including deep Q-waves, diminished S:R ratios, polyphasic R-waves and frequent premature ventricular contractions. All of these ECG abnormalities in mdx mice were improved or corrected by nNOS transgene expression. In addition, defects in mdx cardiac autonomic function, which were reflected by decreased heart rate variability, were significantly reduced by nNOS transgene expression. These findings indicate that increasing NO production by dystrophic hearts may have therapeutic value.     

Cardiac troponin I and cardiac troponin T increases in pigs during ischemia-reperfusion damage.

In this study we addressed the question of whether the measurement of cardiac Troponin T (cTnT) and cardiac Troponin I (cTnI) is able to detect myocardial cell damage in an ischemia-reperfusion model in pigs. To answer the question 3 pigs were anaesthesized and a cardiac arrest was induced by electric fibrillation. After 5 minutes of global ischemia the cardiac arrest was reversed by electric defibrillation until normal perfusion was restored. We could clearly demonstrate an increase of cTnT and cTnI 30 minutes after reperfusion indicating myocardial injury during ischemia and subsequent reperfusion. The cTnT as well as the cTnI serum levels increased till 180 minutes after reperfusion. This ischemia-reperfusion injury is likely induced by oxygen radicals generated during hypoxia and subsequent reperfusion We conclude from our first results that troponin measurements with commercial available test kits may also reflect myocardial cell damage in pigs as it was recently demonstrated in rats. Further studies are needed for correlation of troponin serum levels and histopathological damage in this model especially if it is used to test beneficial or toxicological effects of radical neutralizing drugs.     

Multiple immunoassay systems are negatively interfered by circulating cardiac troponin I autoantibodies

Circulating cardiac troponin I (cTnI) autoantibodies have recently been detected in more and more patients with myocardial injury. In the present study, a total of 121 patients with acute myocardial infarction (AMI) were screened for cTnI autoantibodies using an indirect ELISA. Positive results were further confirmed by Western blot analysis. As a result, 13 autoantibody-positive sera were identified, in which cTnI values detected by different immunoassay systems are very different. Further evaluation revealed low recovery in one of the 13 samples with the Access 2 system (Beckman Coulter, 2^ generation), one low and one moderate recovery sample with Architect i2000 (Abbott), one low and two moderate with AxSYM (Abbott), two low and three moderate with Dimension Xpand (Dade Behring, 2^ generation), and four low and one moderate with Vidas (bioMérieux). Our work demonstrates that circulating cTnI autoantibodies occur in part of patients with AMI and, for the first time to our knowledge, shows that these autoantibodies can result in considerable negative interference in all the five commonly used cTnI immunoassay systems, which may lead to incorrect diagnoses and following treatments. The indirect ELISA established in our laboratory is suitable for a rapid preliminary screening for cTnI autoantibody in clinical work.     

High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors

Here we describe the sustained expression of transgenes introduced into human embryonic stem (ES) cells using self-inactivating lentiviral vectors. At low multiplicity of infection, vesicular stomatitis virus-pseudotyped vectors containing a green fluorescent protein (GFP) transgene under the control of a human elongation factor 1alpha promoter transduced human ES cells at high efficiency. The majority of the transduced ES cells, which harbored low numbers of integrated vectors, continued to express GFP after 60 days of culture. Incorporation of a scaffold attachment region (SAR) from the human interferon-beta gene into the lentiviral vector backbone increased the average level of GFP expression, and inclusion of the SAR together with a chromatin insulator from the 5' end of the chicken beta-globin locus reduced the variability in GFP expression. When the transduced ES cells were induced to differentiate into CD34(+) hematopoietic precursors in vitro, GFP expression was maintained with minimal silencing. The ability to efficiently introduce active transgenes into human ES cells will facilitate gain-of-function studies of early developmental processes in the human system. These results also have important implications for the possible future use of gene-modified human ES cells in transplantation and tissue regeneration applications.     

A very high level in a cardiac troponin I assay

Cardiac troponin I (TnIc) is a very sensitive and also a specific marker of myocardial injuries. We report here, the clinical case of a patient with a particularly important and brutal increase of the troponin during a myocardial infarction. A 64-year-old man was admitted to hospital. He had a myocardial infarction associated to a cardiac necrosis and important cytolysis. The troponin assay was normal when the patient was hospitalized. The angioplasty coronary reperfusion brought about a massive troponin Ic release in systemic circulation: the assay made 10 hours after the appearance of the symptoms shows us an exceptional TnIc concentration that is greater than 4000 microg/L (baseline: 1,5 microg/L). This might be the highest value ever reported.     

Release of cardiac troponin I from viable cardiomyocytes is mediated by integrin stimulation

Elevated cardiac troponin-I (cTnI) levels have been demonstrated in serum of patients without acute coronary syndromes, potentially via a stretch-related process. We hypothesize that this cTnI release from viable cardiomyocytes is mediated by stimulation of stretch-responsive integrins. Cultured cardiomyocytes were treated with (1) Gly–Arg–Gly–Asp–Ser (GRGDS, n = 22) to stimulate integrins, (2) Ser–Asp–Gly–Arg–Gly (SDGRG, n = 8) that does not stimulate integrins, or (3) phosphate-buffered saline (control, n = 38). Cells and media were analyzed for intact cTnI, cTnI degradation products, and matrix metalloproteinase (MMP)-2. Cell viability was examined by assay of lactate dehydrogenase (LDH) activity and by nuclear staining with propidium iodide. GRGDS-induced integrin stimulation caused increased release of intact cTnI (9.6 ± 3.0%) as compared to SDGRG-treated cardiomyocytes (4.5 ± 0.8%, p < 0.001) and control (3.0 ± 3.4%, p < 0.001). LDH release from GRGDS-treated cardiomyocytes (15.9 ± 3.8%) equalled that from controls (15.2 ± 2.3%, p = n.s.), indicating that the GRGDS-induced release of cTnI is not due to cell necrosis. This result was confirmed by nuclear staining with propidium iodide. Integrin stimulation increased the intracellular and extracellular MMP2 activity as compared to controls (both p < 0.05). However, despite the ability of active MMP2 to degrade cTnI in vitro, integrin stimulation in cardiomyocytes was not associated with cTnI degradation. The present study demonstrates that intact cTnI can be released from viable cardiomyocytes by stimulation of stretch-responsive integrins.