Redox signaling in cardiac myocytes

The heart has complex mechanisms that facilitate the maintenance of an oxygen supply-demand balance necessary for its contractile function in response to physiological fluctuations in workload as well as in response to chronic stresses such as hypoxia, ischemia, and overload. Redox-sensitive signaling pathways are centrally involved in many of these homeostatic and stress-response mechanisms. Here, we review the main redox-regulated pathways that are involved in cardiac myocyte excitation-contraction coupling, differentiation, hypertrophy, and stress responses. We discuss specific sources of endogenously generated reactive oxygen species (e.g., mitochondria and NADPH oxidases of the Nox family), the particular pathways and processes that they affect, the role of modulators such as thioredoxin, and the specific molecular mechanisms that are involved-where this knowledge is available. A better understanding of this complex regulatory system may allow the development of more specific therapeutic strategies for heart diseases.     

Manganese-induced apoptosis in rat myocytes

Manganese can be toxic to the heart, causing dysfunction following long exposure. In our experiments, we examined the cytotoxicity of manganese in neonatal rat ventricular myocytes (NRVM) by MTT assays in vitro. Results showed that after incubation in the different concentrations of manganese for 24 h, apparent cytotoxicity was observed. At 500, 1000, and 1500 2 microM of manganese, the percentage of cell viability dropped to 82% +/- 6.13, 78% +/- 5.28, and 66% +/- 4.22, respectively. When cells were treated for 48 h, all concentrations tested exerted toxic effect; especially from 500 to 1500 microM the cell viability dropped from 67% +/- 4.84 to 37% +/- 3.25. Apoptosis in NRVM was then examined by flow cytometry. Results showed that the percentage of apoptotic cells treated with 500 microM of manganese for 24 h increased from 4% +/- 0.84 to 7% +/- 1.16. After 48 h of incubation, this percentage increased to 11% +/- 0.91. There was no significant difference between control groups (0 microM manganese) after 24 and 48 h incubation. The morphological changes of NRVM nuclei were visualized with the fluorescent DNA-binding dye Hoechst33342 after incubation in 500 microM of manganese for 48 h. Compared with normal nuclei, apoptotic nuclei showed the typical features of fragmentation and condensation. To investigate whether there are any apoptotic gene expression changes during apoptosis, we examined the expression level of Bcl-2, Bax, and P53 mRNAs after treatment with 500 microM of manganese for 48 h. The Bcl-2 mRNA expression decreased while the expression of Bax as well as P53 mRNAs increased. These results suggested that manganese cytotoxicity on NRVM could induce apoptosis in NRVM cells. The apoptosis process might involve, and be promoted by, the changes of the expression levels of P53, Bcl-2, and Bax proteins.     

Polyploidization of transplanted cardiac myocytes

Pieces of cardiac ventricular tissue of late embryonic or 1-day postnatal rats, implanted beneath the kidney capsule of adult syngeneic hosts, formed viable, beating transplants. these transplants were investigated over a 40-day postoperative course. In the transplants, cellular binucleation and nuclear polyploidization occurred according to the same schedule as in the heart in situ. The composition of the classes of myocytes was identical both in the hearts in situ and in transplants, but the number of non-diploid myocytes in the intact heart reached 90%, whereas in transplants it varied from 30 to 60%. In contrast to the heart in situ, myocytes in transplants grew feebly after the phase of polyploidization. From these data one can conclude that under conditions of transplantation the temporal sequence of cellular binucleation and nuclear polyploidization follows the normal course, but that a greater number of myocytes remain in a diploid state than is the case in the normal heart. The growth of cardiac myocytes seems to be related to their level of function.     

Cyclical mechanical stretch-induced apoptosis in myocytes from young rats but necrosis in myocytes from old rats

Mechanical load as stimulus for apoptosis and necrosis could be responsible for the loss of cardiomyocytes. Ventricular myocytes from young (3 mo) and old (14-24 mo) rats underwent cyclical mechanical stretch (CMS; 5% elongation, 1 Hz) for 24 h. Spontaneous apoptosis was in myocytes from young rats 0.33 +/- 0.12% and from old rats 1.05 +/- 0.35% [Tdt-mediated dUTP nick-end labeling (TUNEL) assay]; associated with a decrease of Bcl-2. CMS increased the apoptosis to 0.58 +/- 0.18% in myocytes from young rats. Western blot analysis showed that CMS reduced Bcl-2 and increased p53 (young rats). Bax was not changed by CMS. These were confirmed by cytochrome c release (31 +/- 13%) and by the enrichment of cytosolic nucleosomes (11 +/- 8%). CMS did not influence the apoptosis in myocytes from old rats (TUNEL assay, Bcl-2, Bax, or p53). CMS did not cause necrosis in myocytes from young rats. CMS increased the number of necrotic cells by showing the cell membrane rupture in myocytes from old rats (50 +/- 13% 5-hexadecanoylaminofluorescein-positive and 38 +/- 6% propidium iodide-positive cells) as well as by measuring the lactate dehydrogenase release. The results suggest that CMS-induced apoptosis in myocytes of young rats but necrosis in myocytes from old rats, which could be attributed to more stress sensitivity of cells from old rats.     

Lysosomal trafficking in rat cardiac myocytes

By immunolabeling of cryosections, we have characterized in rat cardiac myocytes the cation-independent mannose-6-phosphate receptor (MPR), a lysosomal membrane glycoprotein, lgp120, and a lysosomal enzyme, MEP (homologous to cathepsin L). Most of the MPR label was located in large membrane-filled structures (MPR structures) in large clusters of mitochondria adjacent to but distinct from the Golgi complex. Lpg120 and MEP showed typical lysosomal localization throughout the cell, often associated with regions that appeared to contain autophagosome-like structures. In addition, MEP and lgp120 co-localized within MPR structures. MEP and MPR were localized inside the lumen of MPR structures. MPR was associated mostly with inner membranes, whereas lgp120 was predominantly bound to the outer limiting membrane. MPR, lgp120, and MEP were not detected in Golgi stacks, but some labeling was seen in the putative TGN. Our data suggest that the MPR structures are prelysosomes involved in lysosomal enzyme targeting in rat cardiac myocytes.     

Passive stiffness of rat cardiac myocytes

Intact single cells were isolated from adult rat hearts by enzymatic digestion and suspended in 0.25 mM Ca++ Tyrode's solution. Quiescent, clearly striated rodlike cells were selected for study of the elastic properties of the cells at various stages of membrane and myofilament extraction. Selected cells were placed in a relaxing solution (pCa + 9, 10 mn EGTA) and then each end gently pulled into the tip of a closely fitting suction micropipette for attachment to a force transducer and length perturbation driver. This procedure was performed in low Ca++ to prevent Ca++ loading of the cell during attachment and at room temperature to prevent chemical skinning of the cell. Stiffness was measured by applying a 5-Hz sinusoidal length perturbation (5 percent L0) to one end of the cell while measuring the induced tension change at the other. The ratio of sinusoidal tension change to applied length change (stiffness) was determined for each cell over a length range of about 1-1.3 L0 before removal of the contractile filaments and up to 3.0 L0 after treatment with 0.6 M KI. The stiffness-length relation was measured first in relaxing solution and then in 0.25 mM Ca++ Tyrode's. If spontaneous contractions or contracture occurred the cell was rejected. If the cell remained quiescent and relaxed it was treated again with relaxing solution and 1 percent Triton X-100 to remove the membranes. The stiffness-length relation was again measured and then the cell was superfused with 0.47 M KCl/10 mM pyrophosphate solution to remove the myosin filaments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium flux in turtle ventricular myocytes

The relative contribution of the sarcoplasmic reticulum (SR), the L-type Ca(2+) channel and the Na(+)/Ca(2+) exchanger (NCX) were assessed in turtle ventricular myocytes using epifluorescent microscopy and electrophysiology. Confocal microscopy images of turtle myocytes revealed spindle-shaped cells, which lacked T-tubules and had a large surface area-to-volume ratio. Myocytes loaded with the fluorescent Ca(2+)-sensitive dye Fura-2 elicited Ca(2+) transients, which were insensitive to ryanodine and thapsigargin, indicating the SR plays a small role in the regulation of contraction and relaxation in the turtle ventricle. Sarcolemmal Ca(2+) currents were measured using the perforated-patch voltage-clamp technique. Depolarizing voltage steps to 0 mV elicited an inward current that could be blocked by nifedipine, indicating the presence of Ca(2+) currents originating from L-type Ca(2+) channels (I(Ca)). The density of I(Ca) was 3.2 +/- 0.5 pA/pF, which led to an overall total Ca(2+) influx of 64.1 +/- 9.3 microM/l. NCX activity was measured as the Ni(+)-sensitive current at two concentrations of intracellular Na(+) (7 and 14 mM). Total Ca(2+) influx through the NCX during depolarizing voltage steps to 0 mV was 58.5 +/- 7.7 micromol/l and 26.7 +/- 3.2 micromol/l at 14 and 7 mM intracellular Na(+), respectively. In the absence of the SR and L-type Ca(2+) channels, the NCX is able to support myocyte contraction independently. Our results indicate turtle ventricular myocytes are primed for sarcolemmal Ca(2+) transport, and most of the Ca(2+) used for contraction originates from the L-type Ca(2+) channel.     

心肌细胞的钙致钙释放

心肌细胞兴奋－收缩偶联由胞内钙变中介和调控。去极化进进入细胞的少量钙通过钙下释放（ＣＩＣＲ）过程发肌质多（ＳＲ）释放更多的钙,使胞浆钙浓度升高,导致收缩近年来证明,ＳＲ钙放呈梯级特征,提出了局部控制模型,以解释这种现象。钙火花的发现,直观地证硒钙释放单位的存在,进一步支持了局部控制模型。此外,钙释放通道的适应现象,可能是ＣＩＣＲ这一正反馈过程的负调节机制。     

心肌细胞Na^＋—Ca^2＋交换电流

心肌细胞上存在Ｎａ＋Ｃａ２＋交换系统,以３Ｎａ＋：１Ｃａ２＋方式交换,产生Ｎａ＋Ｃａ２＋交换电流（ＩＮａＣａ）。分子生物学实验证明：Ｎａ＋Ｃａ２＋交换体有１１个跨膜片段,其功能受多种因素的调节。膜片钳制技术研究表明Ｎａ＋Ｃａ２＋交换电流与心肌细胞动作电位形成和心律失常的产生有关。通过对Ｎａ＋Ｃａ２＋交换系统的深入研究,将有助于我们研制和开发作用于Ｎａ＋Ｃａ２＋交换电流的特异性较强的药物,对治疗心律失常和保护心肌细胞,减少心肌细胞的损伤有重要意义。     

Hormone-stimulated lipolysis in cardiac myocytes.

Type L hormone-sensitive lipase (HSL) activity was increased approx. 35% above control in cardiac myocytes incubated for 15 min with 5 nM-adrenaline. Concomitantly. adrenaline-stimulated myocytes had a lower triacylglycerol content, released more non-esterified fatty acid and had a higher intracellular concentration of cyclic AMP than did myocytes incubated without hormone. The lipase activity measured in adrenaline-stimulated and non-stimulated myocytes was stable in acetone/diethyl ether, stimulated by serum and inhibited by NaCl. These properties are consistent with the type L designation of this HSL. The finding that type L HSL is stimulated by adrenaline indicates that the enzyme that is being activated is found in the cell and not associated with an extracellular compartment of the myocardium.     

Calcium handling in zebrafish ventricular myocytes

The zebrafish is an important model for the study of vertebrate cardiac development with a rich array of genetic mutations and biological reagents for functional interrogation. The similarity of the zebrafish (Danio rerio) cardiac action potential with that of humans further enhances the relevance of this model. In spite of this, little is known about excitation-contraction coupling in the zebrafish heart. To address this issue, adult zebrafish cardiomyocytes were isolated by enzymatic perfusion of the cannulated ventricle and were subjected to amphotericin-perforated patch-clamp technique, confocal calcium imaging, and/or measurements of cell shortening. Simultaneous recordings of the voltage dependence of the L-type calcium current (I(Ca,L)) amplitude and cell shortening showed a typical bell-shaped current-voltage (I-V) relationship for I(Ca,L) with a maximum at +10 mV, whereas calcium transients and cell shortening showed a monophasic increase with membrane depolarization that reached a plateau at membrane potentials above +20 mV. Values of I(Ca,L) were 53, 100, and 17% of maximum at -20, +10, and +40 mV, while the corresponding calcium transient amplitudes were 64, 92, and 98% and cell shortening values were 62, 95, and 96% of maximum, respectively, suggesting that I(Ca,L) is the major contributor to the activation of contraction at voltages below +10 mV, whereas the contribution of reverse-mode Na/Ca exchange becomes increasingly more important at membrane potentials above +10 mV. Comparison of the recovery of I(Ca,L) from acute and steady-state inactivation showed that reduction of I(Ca,L) upon elevation of the stimulation frequency is primarily due to calcium-dependent I(Ca,L) inactivation. In conclusion, we demonstrate that a large yield of healthy atrial and ventricular myocytes can be obtained by enzymatic perfusion of the cannulated zebrafish heart. Moreover, zebrafish ventricular myocytes differed from that of large mammals by having larger I(Ca,L) density and a monophasically increasing contraction-voltage relationship, suggesting that caution should be taken upon extrapolation of the functional impact of mutations on calcium handling and contraction in zebrafish cardiomyocytes.     

Mitochondrial membrane potential in cardiac myocytes

Mitochondria are involved in cellular functions that transcend the traditional role of these organelles as the energy factory of the cell. Their relative inaccessibility and the difficulties involved in attempts to study them in their natural environment -- the cytosol -- has delayed much of this understanding and they still have many secrets to yield. One of the relatively new fields in this respect is undoubtedly the analysis of mitochondrial membrane potential. The realization that its alteration may have important pathophysiological consequences has led to an increased interest in measuring this variable in a variety of biological settings, including cardiovascular diseases. Measurements of mitochondrial membrane potential tell us much about the role of mitochondria in normal cell function and in processes leading to cell death. However, we must be aware of the limitations of using isolated mitochondria, single cells and different fluorescent indicators.     

Monoacylglycerol lipase activity in cardiac myocytes

Monoacylglycerol lipase activity in homogenates of isolated myocardial cells (myocytes) from rat hearts was recovered in both particulate and soluble subcellular fractions. The activity present in the microsomal (100,000 X g pellet) fraction was solubilized by treatment with Triton X-100 and combined with the 100,000 X g supernatant fraction; the properties of monoacylglycerol lipase were investigated with this soluble enzyme preparation. The Km for the hydrolysis of a 2-monoolein substrate was 16 microM. The rates of hydrolysis of 1-monoolein and 2-monoolein were identical, and 1-monoolein was a competitive inhibitor (Ki = 20 microM) of the hydrolysis of 2-monoolein. Monoacylglycerol lipase activity was regulated by product inhibition according to the following order of potency: fatty acyl CoA greater than free fatty acids greater than fatty acyl carnitine.     

Beauvericin-induced channels in ventricular myocytes and liposomes

The antibiotic Beauvericin (BEA) was previously shown to express ionophoric properties under simple experimental systems. Its channel-forming activity was examined in inside-out patches of ventricular myocytes and synthetic membranes with the patch clamp and fluorescence imaging techniques. Current transitions to several open state levels were evident after wash-in. The BEA channel is cation-selective. Conductance and kinetics are presented for K(+) and Na(+) substates and main states. The pore was blocked by La(3+). In myocytes, the [K(+)](i) was reduced, while [Na(+)](i) and [Ca(2+)](i) increased, leading to cytolysis. These results indicate that BEA forms cation-selective channels in lipid membranes, which can affect the ionic homeostasis.     

Signal transduction of flumazenil-induced preconditioning in myocytes

The objective of this study was to examine the role of oxygen radicals, protein kinase C (PKC), and ATP-sensitive K(+) (K(ATP)) channels in mediating flumazenil-produced preconditioning. Chick cardiomyocyte death was quantified using propidium iodide, and oxygen radical generation was assessed using 2',7'-dichlorofluorescin oxidation. Preconditioning was initiated with 10 min of ischemia followed by 10 min of reoxygenation. Alternatively, flumazenil was infused for 10 min and removed 10 min before ischemia. Flumazenil (10 microM) and preconditioning increased oxygen radicals [1,693 +/- 101 (n = 3) and 1,567 +/- 98 (n = 3), respectively, vs. 345 +/- 53 (n = 3) in control] and reduced cell death similarly [22 +/- 3% (n = 5) and 18 +/- 2% (n = 6), respectively, vs. controls 49 +/- 5% (n = 8)]. Protection and increased oxygen radicals by flumazenil were abolished by pretreatment with the antioxidant thiol reductant 2-mercaptopropionyl glycine (800 microM; 52 +/- 10%, n = 6). Specific PKC inhibitors Go-6976 (0.1 microM) and chelerythrine (2 microM), given during ischemia and reoxygenation, blocked flumazenil-produced protection (47 +/- 5%, n = 6). The PKC activator phorbol 12-myristate 13-acetate (0.2 microM), given during ischemia and reoxygenation, reduced cell death similarly to that with flumazenil [17 +/- 4% (n = 6) and 22 +/- 3% (n = 5)]. Finally, 5-hydroxydecanoate (1 mM), a selective mitochondrial K(ATP) channel antagonist given during ischemia and reoxygenation, abolished the protection of flumazenil and phorbol 12-myristate 13-acetate. Thus flumazenil mimics preconditioning to reduce cell death in cardiomyocytes. Oxygen radicals activate mitochondrial K(ATP) channels via PKC during the process.