The art of cobbling a running pump—Will human embryonic stem cells mend broken hearts?

The heart is one of the least regenerative organs in the body, and highly vulnerable to the increasing incidence of cardiovascular diseases in an aging world population. Cell-based approaches aimed at cardiac repair have recently caused great public excitement. But clinical trials of patients’ own skeletal myoblasts or bone marrow cells for transplantation have been disappointing. Human embryonic stem cells (hESCs) form bona fide cardiomyocytes in vitro which are readily generated in mass culture and are being tested in animal models of heart damage. The early results, while encouraging, underscore that much remains to be done. This review focuses on the many challenges that remain before hESCs-mediated repair of the human heart becomes a reality.     

Visualization of collagenase-sensitive acetylcholinesterase in isolated cardiomyocytes and in heart tissue

Summary Previous studies have indicated that the asymmetric form of acetylcholinesterase (collagen-tailed) is localized in the basal lamina of the neuromuscular junction of skeletal muscle. The present study shows localization of the asymmetric acetylcholinesterase in the heart of the rat. Antiserum to 14+18 S acetylcholinesterase of the electric eel was raised in rabbits. The purified antibody did not react with collagen type I or laminin. Collagenase reduced the immunoreactivity of the enzyme with the purified antibody. Isolated cardiomyocytes and frozen sections of the heart were stained for acetylcholinesterase with the antibody. Diffuse immunofluorescence appeared over the surface of the cardiomyocytes. In the frozen sections, the immunofluorescence was most intense at the cell boundaries. These data suggest that collagenase-sensitive acetylcholinesterase in the heart is present in the myocytes and occurs in the vicinity of the basal lamina.Abbreviations AChE acetylcholinesterase - BSA bovine serum albumin - PBS phosphate-buffered saline - DME Dulbecco's Modified Eagle Medium     

Depressant effects of prostacyclin in human atrial fibers and cardiomyocytes

The aim of this study was to characterize the electropharmacological effects of prostacyclin (PGI₂) in human atrial fibers and cardiomyocytes. Atrial tissues obtained from the hearts of 28 patients undergoing corrective cardiac surgery were used. Transmembrane action potentials were recorded using a conventional microelectrode technique, and twitch force by a transducer. Effects of PGI₂ (1 nM–10 µM) on action potential characteristics and contraction of atrial fibers were evaluated in normal [K]_o (4 mM) and high [K]_o (27 mM) in the absence and presence of cardiotonic agents. In addition, atrial and ventricular myocytes were isolated enzymatically from atrial tissues and hearts of 4 patients undergoing cardiac transplant. The effects of PGI₂ on Na- and Ca-dependent inward currents (I_Na and I_Ca) of cardiomyocytes were tested. In 9 human atrial fibers showing fast-response action potentials (mean dV/dt_max = 101 ± 15 Vs^–1) in 4 mM [K]_o, PGI₂ did not influence dV/dt_max of phase 0 depolarization even at 1 µM. However, at a concentration as low as 10 nM, PGI₂ depressed spontaneous rhythms or slow-response action potentials in high-K-depolarized fibers. PGI₂ also depressed delayed afterdepolarizations and aftercontractions induced by cardiotonic agents. In isolated cardiomyocytes, PGI₂ reduced I_Ca but not I_Na. The present findings show that, in human atrial fibers and cardiomyocytes, PGI₂ induces greater depressant effects on the slow-response action potential, I_Ca and triggered activity than on the fast-response action potential. It is suggested that PGI₂ may act through a selective reduction of transmembrane Ca influx.     

Overexpression of Slc30a7/ZnT7 increases the mitochondrial matrix levels of labile Zn2+ and modifies histone modification in hyperinsulinemic cardiomyoblasts

BackgroundCellular free Zn²⁺ concentrations ([Zn²⁺]) are primarily coordinated by Zn²⁺-transporters, although their roles are not well established in cardiomyocytes. Since we previously showed the important contribution of a Zn²⁺-transporter ZnT7 to [Zn²⁺]_i regulation in hyperglycemic cardiomyocytes, here, we aimed to examine a possible regulatory role of ZnT7 not only on [Zn²⁺]_i but also both the mitochondrial-free Zn²⁺ and/or Ca²⁺ in cardiomyocytes, focusing on the contribution of its overexpression to the mitochondrial function.MethodsWe mimicked either hyperinsulinemia (by 50-μM palmitic acid, PA-cells, for 24-h) or overexpressed ZnT7 (ZnT7OE-cells) in H9c2 cardiomyoblasts.ResultsOpposite to PA-cells, the [Zn²⁺]_i in ZnT7OE-cells was not different from untreated H9c2-cells. An investigation of immunofluorescence imaging by confocal microscopy demonstrated a ZnT7 localization on the mitochondrial matrix. We demonstrated the ZnT7 localization on the mitochondrial matrix by using immunofluorescence imaging. Later, we determined the mitochondrial levels of [Zn²⁺]_Mit and [Ca²⁺]_Mit by using the Zn²⁺ and Ca²⁺ sensitive FRET probe and a Ca²⁺-sensitive dye Fluo4, respectively. The [Zn²⁺]_Mit was found to increase significantly in ZnT7OE-cells, similar to the PA-cells while no significant changes in the [Ca²⁺]_Mit in these cells. To examine the contribution of ZnT7 overexpression on the mitochondria function, we determined the level of reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) in these cells in comparison to the PA-cells. There were significantly increased production of ROS and depolarization in MMP and increases in marker proteins of mitochondria-associated apoptosis and autophagy in ZnT7-OE cells, similar to the PA-cells, parallel to increases in K-acetylation. Moreover, we determined significant increases in trimethylation of histone H3 lysine27, H3K27me3, and the mono-methylation of histone H3 lysine36, H3K36 in the ZnT7OE-cells, demonstrating the role of [Zn²⁺]_Mit in epigenetic regulation of cardiomyocytes under hyperinsulinemia through histone modification.ConclusionsOverall, our data have shown an important contribution of high expression of ZnT7-OE, through its buffering and muffling capacity in cardiomyocytes, on the regulation of not only [Zn²⁺_]i but also both [Zn²⁺]_Mit and [Ca²⁺]_Mit affecting mitochondria function, in part, via histone modification.     

心肌细胞钾通道的研究进展

从分子水平上看,所有钾通道都是由一个基因家族中的基因所编码的４个亚基组成,通道的失活门控机制在Ｎ型、Ｃ型和Ｐ型三种,通道的孔道结构均在跨膜片段Ｓ５至Ｓ６之间,尽管各种钾通道在分子结构上的共处远多于不同之处,但每种钾通道的个性表现却有非常重要的生理意义,迄今为止在心肌细胞上发现的８种钾通道的电导值,门控动力学特征、离子动力学特征,通道激动剂,阻断剂和调制剂均不同。     

PKC-Dependent Phosphorylation May Regulate the Ability of Connexin43 to Inhibit DNA Synthesis

Phosphorylation affects several biological functions of connexin43 (Cx43), although its role on Cx43-mediated inhibition of DNA synthesis is not known. Previous studies showed increased Cx43 phosphorylation on serine in response to growth factor stimulation of cardiomyocytes, mediated by protein kinase C-epsilon (PKCε). Here we report that activation of PKCε is also necessary for stimulation of cardiomyocyte DNA synthesis and mitosis. We have investigated the participation of specific serine residues that are putative PKC targets in producing phosphorylated Cx43 species and also in regulating DNA synthesis in cardiomyocytes. Interference with the PKC signaling system and/or the phosphorylation of specific amino-acids of Cx43 may allow regulation of the mitogenic response.     

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1^−/−) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca²⁺ cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1^−/− compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca²⁺ transient (Δ[Ca²⁺]_i) responses in NOS1^−/− cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca²⁺]_i across the range of pacing frequencies and increased myofilament Ca²⁺ sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca²⁺ reuptake, and restored SR Ca²⁺ content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca²⁺ leak, HYD improves Ca²⁺ cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.     

乙醛脱氢酶2可减少氧化损伤诱导的心肌细胞凋亡

乙醛脱氢酶2 (aldehyde dehydrogenase 2, ALDH2)是线粒体特异性酶,已被证明参与氧化应激诱导的细胞凋亡,而在心肌细胞中的作用知之甚少。本研究旨在通过用特异性ALDH2抑制剂大豆苷抑制ALDH2活性来研究ALDH2在抗霉素A诱导的心肌细胞凋亡中的作用。应用抗霉素A和大豆苷诱导小鼠心肌细胞,然后测定ALDH2酶活性、细胞内活性氧(reactive oxy gen species, ROS)含量和细胞凋亡,应用RT-PCR和蛋白质印迹法(Western blotting)检测ALDH2 m RNA和蛋白表达。结果表明,抗霉素A (40μg/mL)可诱导新生心肌细胞凋亡,而大豆苷(50μmol/L)能有效地抑制ALDH2活性而对细胞凋亡没有影响,并且可显著增强抗霉素A诱导的心肌细胞凋亡(53.72%～71.33%, p<0.05)。与单独用抗霉素A处理的细胞相比,抗霉素A和大豆苷共处理的心肌细胞中活化的丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号传导途径(p38-MAPK)的磷酸化也显著增加。本研究初步表明,改变线粒体ALDH2活性可能是减少氧化损伤诱导的心肌细胞凋亡的潜在选择。