Sarcolemmal fatty acid transfer in isolated cardiomyocytes governed by albumin/membrane-lipid partition

The mechanism of transfer of long chain fatty acids across the myocardial sarcolemmal membrane was investigated in isolated, calcium-resistant, rat cardiomyocytes. The initial rate of 14C-palmitate uptake was determined at constant and increasing palmitate/albumin ratios. The latter condition led to a saturable dependence of uptake rate on palmitate concentration. At a constant palmitate/albumin ratio however, there was an almost constant rate of uptake even though the absolute concentration of palmitate increased. The enhanced metabolic rate resulting from electrically induced contractions of the myocytes decreased the apparent Km of uptake from 62 to 23 microM. Thirty seconds after administration, there was no further increase in the [14C]palmitate content of the myocytes. Moreover, from experiments using ghost membrane vesicles the concentration of palmitate in membranes increased almost linearly with increasing palmitate/albumin ratios. This concentration remained virtually constant if vesicles were pre-treated with diamide. Our results do not support the concept of an albumin receptor-mediated uptake but rather suggest that fatty acids are incorporated into cardiomyocytes by a simple diffusion process which is not rate-limiting. The rate of uptake is influenced both by the metabolic rate and by the concentration of fatty acids in the membranes. The rate-limiting step of fatty acid uptake is probably either the formation of acyl-CoA catalyzed by the membrane associated acyl-CoA synthetase, or the transfer of fatty acid carnitine esters across the mitochondrial matrix membrane.     

Differential translocation or phosphorylation of alpha B crystallin cannot be detected in ischemically preconditioned rabbit cardiomyocytes

Alpha B Crystallin (alpha BC) is a putative effector protein of ischemic preconditioning (IPC), that is phosphorylated on Ser 45 by ERK1/2 and Ser 59 by the p38 MAPK substrate, MAPKAPK-2. Translocation and phosphorylation of alpha BC was determined in cytosolic and cytoskeletal fractions by 1D SDS-PAGE and IEF, or using Ser 45 and Ser 59 phospho-specific antibodies in: (1) control rabbit cardiomyocytes; (2) cells preconditioned by 10 min in vitro ischemia; or after pre-treatment with specific inhibitors of (3) Ser/Thr protein phosphatase 1/2A (calyculin A); (4) p38 MAPK (SB203580); or (5) ERK 1/2 (PD98059); all prior to 180 min ischemia. Ischemia induced a cytosolic to cytoskeletal translocation of alpha BC, which was similar in all the groups. Highly phosphorylated isoforms (D1/2) of alpha BC were present in cytosolic but not cytoskeletal fractions at 0 min ischemia. By 60-90 min ischemia, D1/2 isoforms had translocated to the cytoskeletal fraction. Calyculin A maintained D1/2 levels throughout prolonged ischemia. SB203580 decreased alpha BC phosphorylation. Neither PD98059 nor IPC altered alpha BC phosphorylation during prolonged ischemia. It is concluded that alpha BC phosphorylation during ischemia is regulated by p38 MAPK but not by ERK 1/2. The inability to detect a correlation between IPC protection and either alpha BC translocation or phosphorylation suggests that the proteins in the highly phosphorylated isoform bands of alpha BC quantitated in this study are not protective end effectors of classical IPC.     

高表达microRNA-22对缺氧心肌细胞的保护作用及机制

目的观察高表达microRNA-22对体外培养的缺氧心肌细胞的保护作用及机制。方法用携带microRNA-22的腺病毒载体和空载体转染体外缺氧条件下培养的原代心肌细胞,检测高表达microRNA-22后心肌细胞生物学特性的改变。采用MTT检测细胞活力,EdU检测细胞DNA合成能力,Caspase-3检测细胞凋亡情况。结果表达microRNA-22的腺病毒载体成功转染原代心肌细胞,流式细胞仪检测绿色荧光蛋白阳性细胞比例95%,高表达microRNA-22从蛋白水平显著下调PTEN表达。与空载腺病毒组相比,高表达microRNA-22组细胞核增殖明显增加,细胞生长更快,细胞活力增加,细胞凋亡减少。结论体外高表达microRNA-22能保护缺氧条件下的心肌细胞。     

Inhibition of apoptotic responses after ischemic stress in isolated hearts and cardiomyocytes

Recent findings on the induction of anti-apoptotic gene expression in ischemic/reperfused hearts encouraged us to investigate whether ischemic/reperfused hearts may be protected against apoptosis induction. To analyze this hypothesis we performed studies on isolated perfused hearts of rat. For apoptosis induction, hearts were perfused with the NO donor (±)-S-nitroso-N-acetylpenicillamine (SNAP, 10 μM) for 30 minutes. Four hours thereafter apoptosis was detected by DNA laddering and TUNEL assay. Under normoperfusion SNAP induced 5.5 ± 1.4 TUNEL-positive myocytes per tissue section (vs. 1.8 ± 0.5 in controls). But when hearts were subjected to 20 minutes of no flow ischemia, which was sufficient for energy depletion of the hearts without inducing severe necrotic or apoptotic cell death, reperfusion in the presence of SNAP did not induce apoptosis. To analyze if this mode of protection is a property of the cardiomyocytes, we performed corresponding experiments on ventricular cardiomyocytes of rat. Again, under normoxic conditions SNAP (100 (μM) increased the number of TUNEL-positive cells to 12.6 ± 4.9 % (vs. 5.4 ± 0.7 % in controls). But when SNAP was added after 3 h of simulated ischemia, which was sufficient for energy depletion of the cells without inducing apoptotic cell death, the number of apoptotic cells did not increase. The ischemia-induced protection of hearts and cardiomyocytes goes along with an increased expression of several anti-apoptotic genes, mainly of the bcl-2 family. This indicates that ischemic conditions induce an anti-apoptotic gene program in cardiomyocytes, which may also be responsible for the observed anti-apoptotic actions in the intact ischemic/reperfused myocardium. Received: 20 March 2002, Returned for 1. revision: 8 April 2002, 1. Revision received: 30 April 2002, Returned for 2. revision: 21 May 2002, 2. Revision received: 29 May 2002, Returned for 3. revision: 29 May 2002, 3. Revision received: 6 June 2002, Accepted: 12 June 2002 Correspondence to: Dr. G. Taimor     

Sarcolemmal fragility secondary to the degradation of dystrophin in dilated cardiomyopathy, as estimated by electron microscopy

A common gene deletion or mutation of delta-sarcoglycan (δ-SG) in dystrophin-related proteins (DRPs) is identified in both TO-2 strain hamsters and human families with dilated cardiomyopathy. We have succeeded in the long-lasting in vivo supplementation of a normal δ-SG gene by recombinant adeno-associated virus vector, restoration of the morphological and functional degeneration, and improvement in the prognosis of the TO-2 hamster. To evaluate the integrity of the sarcolemma (SL) and the subsequent change of organelles in cardiomyocytes of the TO-2 strain hamster, we examined electron microscopy (EM) images focusing on the sarcolemmal stability at the end stage of heart failure. Two types of sarcolemmal degradation were detected: the widened and locally thickened SL, and blurred and discontinuous SL. Bizarrely formed mitochondria of varying sizes were also observed. Immuno-EM revealed clear expression of dystrophin in the SL and intense expression at the costamere as well as at the T-tubules in the control F1B strain hearts, but a patchy deposition of dystrophin was observed along the SL without the transgene of δ-SG. In contrast to the previous reports that dystrophin’s integrity was intact, the present results suggest that the gene deletion of δ-SG and the loss of δ-SG protein in the SL cardioselectively cause the morphological and functional deterioration of dystrophin and the resultant instability of the SL. The sarcolemmal fragility may be similar to Duchenne-type progressive muscular dystrophy in skeletal muscle. In addition to the mechanical role, another aspect of DRPs for the intracellular signal transmission is also discussed.     

Evaluation of single-tube osmotic fragility as a screening test for thalassemia

A single-tube osmotic fragility test has been proposed for thalassemia screening with a range of different concentrations of saline having been employed. We have compared the sensitivity and specificity of 0.32%, 0.34%, and 0.36% buffered saline, and on the basis of our findings, recommend the use of 0.36% saline. This gave definitely positive or equivocal results in 81 of 85 patients with beta thalassemia trait and in 4 of 4 with alpha(0) thalassemia trait. There were 14% false positive results in hematologically normal patients and 81% of the samples from patients with various variant hemoglobins gave positive results. The sensitivity was 95% and specificity 86%. The single-tube osmotic fragility test is potentially useful in under-resourced laboratories although it cannot replace automated red cell indices using electronic counters.     

Effects of calsequestrin over-expression on excitation-contraction coupling in isolated rabbit cardiomyocytes

OBJECTIVE: This study investigated the role of calsequestrin (CSQ) in the control of excitation-contraction (E-C) coupling in the heart. METHODS: CSQ over-expression was induced in isolated rabbit ventricular cardiomyocytes using an adenovirus coding for rabbit CSQ (Ad-CSQ). After 24 h of culture, CSQ protein expression was increased by 58+/-18% (n=10). An adenovirus coding for beta-galactosidase (Ad-LacZ) was used as a control. RESULTS: In voltage-clamped, Fura-2-loaded cardiomyocytes, L-type Ca2+ current (I(Ca,L)) and Ca2+ transient amplitude were both increased in the Ad-CSQ group by approximately 78%. Doubling the external Ca2+ concentration in the control group (Ad-LacZ) increased the LTCC amplitude to a similar degree (85+/-6%), but increased the Ca2+ transient amplitude by 149+/-13%. This suggests that SR Ca2+ release may be inhibited upon CSQ over-expression. Alternatively, nifedipine (0.5 microM) was used to reduce I(Ca,L) in Ad-CSQ-transfected cells to values comparable to control (Ad-LacZ). Under these conditions, Ca2+ transient amplitude was not different from Ad-LacZ, but the SR Ca2+ content was approximately 60% higher as assessed by both the caffeine-induced Ca2+ release and the accompanying Na+/Ca2+ exchanger current (I(NCX)). The cause of the increased I(Ca,L) is unknown. No change in the expression level of the alpha1-subunit of the L-type Ca channel was observed. beta-Escin-permeabilized cardiomyocytes were used to study Ca2+ sparks imaged with Fluo-3 at 145-155 nmol/L [Ca2+]. Spontaneous Ca2+ spark frequency, duration, width, and amplitude were unchanged in the Ad-CSQ group, but SR Ca2+ content was 48% higher than Ad-LacZ. CONCLUSIONS: CSQ over-expression increased SR Ca2+ content but reduced the gain of E-C coupling in rabbit cardiomyocytes.     

Interaction of ischemic and antibiotic-induced injury in the rabbit kidney

The tubular necrosis produced by transient unilateral ischemia, three toxic cephalosporins, and the aminoglycoside neomycin were studied separately and in different combinations in the rabbit kidney. It was found that (1) mildly damaging transient ischemia (25 min) and a minimally toxic dose of the rapidly secreted cephalosporin cephaloglycin (60 mg/kg of body weight) are synergistically damaging; (2) there is no synergy between ischemia and the nonsecreted cephalosporin cephaloridine (90 mg/kg); and (3) ischemia and neomycin (100 mg/kg per day for three days) are not additively damaging, but the aminoglycoside has an additive effect with the combined insults of ischemia and cefazolin (500 mg/kg). Studies of transport showed that ischemia potentiates cephalosporin toxicity probably because it increases postischemic antibiotic concentrations in proximal tubular cells and that this increased uptake is the result of transiently augmented tubular secretion. Although this ischemic protocol reduced inulin clearance by 40%, it increased cephaloglycin secretion by an amount more than sufficient to overcome the decrease in filtration.     

Effects of adenovirus-mediated sorcin overexpression on excitation-contraction coupling in isolated rabbit cardiomyocytes

To evaluate the effect of sorcin on cardiac excitation-contraction coupling, adult rabbit ventricular myocytes were transfected with a recombinant adenovirus coding for human sorcin (Ad-sorcin). A beta-galactosidase adenovirus (Ad-LacZ) was used as a control. Fractional shortening in response to 1-Hz field stimulation (at 37 degrees C) was significantly reduced in Ad-sorcin-transfected myocytes compared with control myocytes (2.10+/-0.05% [n=311] versus 2.42+/-0.06% [n=312], respectively; P<0.001). Action potential duration (at 20 degrees C) was significantly less in the Ad-sorcin group (458+/-22 ms, n=11) compared with the control group (520+/-19 ms, n=10; P<0.05). In voltage-clamped, fura 2-loaded myocytes (20 degrees C), a reduced peak-systolic and end-diastolic [Ca2+]i was observed after Ad-sorcin transfection. L-type Ca2+ current amplitude and time course were unaffected. Caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) and the accompanying inward Na+-Ca2+ exchanger (NCX) current revealed a significantly lower SR Ca2+ content and faster Ca2+-extrusion kinetics in Ad-sorcin-transfected cells. Higher NCX activity after Ad-sorcin transfection was confirmed by measuring the NCX current-voltage relationship. beta-Escin-permeabilized rabbit cardiomyocytes were used to study the effects of sorcin overexpression on Ca2+ sparks imaged with fluo 3 at 145 to 160 nmol/L [Ca2+] using a confocal microscope. Under these conditions, caffeine-mediated SR Ca2+ release was not different between the two groups. Spontaneous spark frequency, duration, width, and amplitude were lower in sorcin-overexpressing myocytes. In summary, sorcin overexpression in rabbit cardiomyocytes decreased Ca2+-transient amplitude predominantly by lowering SR Ca2+ content via increased NCX activity. The effect of sorcin overexpression on Ca2+ sparks indicates an effect on the ryanodine receptor that may also influence excitation-contraction coupling.     

Erythrocyte osmotic fragility and oxidative stress in experimental hypothyroidism

The present study was planned to explain the relation between erythrocyte osmotic fragility and oxidative stress and antioxidant statue in primary hypothyroid-induced experimental rats. Twenty-four Spraque Dawley type female rats were divided into two, as control (n=12) and experimental (n=12), groups weighing between 160 and 200 g. The experimental group animals have received tap water methimazole added standard fodder to block the iodine pumps for 30 d (75 mg/100 g). Control group animals were fed tap water and only standard fodder for the same period. At the end of 30 d blood samples were drawn from the abdominal aorta of the rats under ether anesthesia. T₃, T₄, and TSH levels were measured and the animals that had relatively lower T₃, T₄, and higher TSH levels were accepted as hypothyroid group. Hormone levels of the control group were at euthyroid conditions. Osmotic fragility, as a lipid peroxidation indicator malondialdehyde (MDA), antioxidant defense system indicators superoxide dismutase (SOD) and glutathione (GSH) levels were measured in the blood samples. Osmotic fragility test results: There was no statistically significant difference found between maximum osmotic hemolysis limit values of both group. Minimum osmotic hemolysis limit value of hypothyroid group was found to be higher than that of control group values (p<0.02). The standard hemolysis and hemolytic increment curve of the hypothyroid group drawn according to osmotic fragility test results was found to be shifted to the right when compared to control group’s curve. This situation and hemolytic increment value, which shows maximum hemolysis ratio, is the proof of increased osmotic fragility of the erythrocytes in hypothyroidism. There is no statistically significant difference found between hypothyroid and control groups in the lipid peroxidation indicator MDA and antioxidant indicators SOD and GSH levels. As a result of our study it may be concluded that hypothyroidism may lead to an increase in osmotic fragility of erythrocytes. But the increase in erythrocyte osmotic fragility does not originate from lipid peroxidation.     

Effects of CCCP-induced mitochondrial uncoupling and cyclosporin A on cell volume,cell injury and preconditioning protection of isolated rabbit cardiomyocytes

Cell swelling may contribute to acute cell injury subsequent to ischemia/reperfusion. The potential role of mitochondrial uncoupling and the resultant mitochondrial swelling, due to opening of the mitochondrial permeability transition pore (MPTP), were examined in an in vitro ischemically pelleted isolated rabbit cardiomyocyte model using the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) to uncouple mitochondria. Cyclosporin A (CsA) was employed to inhibit MPTP opening. Cell volume was determined by a cell-flotation, density-gradient assay, using bromododecane. Cell viability, subsequent to an osmotic stress, was determined by trypan blue permeability. Ischemic preconditioning (IPC) facilitated volume regulation following an osmotic stress. Ischemic-cell swelling was reduced by IPC. IPC protected ischemically pelleted cells, but CsA had no significant effects on injury or IPC protection. CCCP ischemia accelerated rates of ischemic contracture and injury, and abolished IPC protection. IPC protection was restored by CsA. In CCCP-ischemic-uncoupled cells, subjected to a reduced (170 mOsm) osmotic stress, CsA and IPC afforded independent and additive protection. Chelerythrine and 5-hydroxydecanoate (5-HD) blocked IPC, but did not reduce CsA protection. Electron microscopy confirmed that CCCP ischemia induced mitochondrial matrix swelling that was reduced by CsA. Cardioprotection by IPC and CsA was accompanied by proportional reductions in cell swelling. Morphometric analysis of the electron photomicrographs demonstrated that the mitochondrial volume fractions were significantly reduced in the CsA/CCCP (29.8 +/- 2.3%, P < 0.004) and IPC/CsA/CCCP (31.5 +/- 1.7%, P < 0.0008) groups as compared to the CCCP-ischemic group (40.5 +/- 1.7%) The IPC/CCCP group (39.5 +/- 4.2%) was not significantly different from the CCCP-ischemic group. NIM 811, a CsA analogue MPTP blocker with no calcineurin inhibitory activity, afforded protection similar to CsA. The results suggest that CsA protection may, in part, be mediated by reduction of mitochondrial swelling.     

The effect of EDTA as an anticoagulant on the osmotic fragility of erythrocytes

The osmotic fragility test is used to determine the extent of red blood cell haemolysis produced by osmotic stress. Since the quality of this test may easily be influenced by environmental and technical factors we have determined osmotic fragility reference values in our own conditions. The results show significantly increased osmotic resistance of erythrocytes in our conditions vs the published values for blood samples anticoagulated with heparin. Furthermore, the use of EDTA as an anticoagulant increased the osmotic fragility of red blood cells as compared with heparin. We conclude that EDTA can be used as an anticoagulant for the osmotic fragility test in order to simplify routine procedures. However, every laboratory should determine its own reference values which would reflect the local environmental and technical factors.     

Landiolol enhances effect of ischemic preconditioning in isolated rabbit hearts

The effect of the ultra-short-acting beta blocker, landiolol, on ischemic preconditioning was examined in isolated rabbit hearts. Ischemic preconditioned hearts received 2 episodes of 5 min each of global ischemia and reperfusion. The left anterior descending coronary artery was occluded for 1 hour and reperfused for 1 hour. Left ventricular end-systolic and end-diastolic pressures and infarct size were measured. Seven control hearts had no drug infused. Four groups of 6 hearts each were pretreated with 1 or 3 microM of landiolol or a combination of 1 or 3 microM landiolol and ischemic preconditioning. A further group of 6 hearts had ischemic preconditioning without landiolol. Ischemic preconditioning significantly reduced left ventricular end-diastolic pressure and infarct size compared to the controls. Landiolol alone did not change left ventricular end-diastolic pressure or infarct size, but landiolol 3 microM and ischemic preconditioning decreased left ventricular end-diastolic pressure more than preconditioning alone. These data suggest that pre-ischemic landiolol infusion may enhance the cardioprotective effect of ischemic preconditioning.     

T-type calcium current in electrical activity of cardiomyocytes isolated from rabbit pulmonary vein

INTRODUCTION: Pulmonary veins (PVs) are known to initiate paroxysmal atrial fibrillation. T-type calcium current (I(Ca-T)) has a role in normal and abnormal automaticity of cardiomyocytes. The aim of this study was to evaluate whether I(Ca-T) contributes to PV electrical activity. METHODS AND RESULTS: By whole-cell clamp techniques in rabbit myocytes, I(Ca-T) was identified in 12 (39%) of 31 PV cardiomyocytes with pacemaker activity, 2 (9%) of 23 PV cardiomyocytes without pacemaker activity, and 2 (15%) of 13 atrial myocytes (P < 0.05). Maximum I(Ca-L) and I(Ca-T) densities from PV cardiomyocytes with pacemaker activity were 6.87 +/- 2.17 pA/pF and 1.38 +/- 0.69 pA/pF, respectively. Nickel (40 microM) decreased the spontaneous activity in 5 (36%) of 14 PV cardiomyocytes (3.1 +/- 0.6 Hz vs 2.2 +/- 0.5 Hz, P < 0.05), reduced the amplitudes of delayed after depolarization from 13 +/- 1 mV to 7 +/- 1 mV (n = 4, P < 0.05) and inhibited transient inward currents from 1.2 +/- 0.2 pA/pF to 0.7 +/- 0.1 pA/pF (n = 11, P < 0.01). CONCLUSIONS: We conclude that I(Ca-T) contributes to PV pacemaker activity and triggered activity, which are of functional importance in PV arrhythmogenesis.     

Disruption of microtubules as an early sign of irreversible ischemic injury. Immunohistochemical study of in situ canine hearts.

Structural disruption of the cytoskeleton may be involved in irreversible ischemic injury. In the present study, ischemic changes in microtubules during various periods of myocardial ischemia were studied with an immunohistochemical technique in open-chest dogs. In intact myocardium, microtubules were stained as a filamentous network throughout cytoplasm and a circular network around the nucleus, which disappeared with colchicine treatment. In brief ischemia of less than 15 minutes, microtubule patterns were unaltered. After 20 minutes, however, characteristic microtubule stains were partly lost in patchy lesions. As an increase in ischemic period, lesions of loss of microtubule stains were increased in number and size. After 120 minutes of reperfusion following 60 minutes of ischemia, the lesions with intact actin filaments but with disrupted microtubules were replaced by the severely injured cells in which the regular myofibrillar registrations were distinctly disrupted. After 24 hours of reperfusion following 40 minutes of occlusion of the left circumflex artery, the percent area of disrupted microtubules at 40 minutes of ischemia was replaced by that of irreversibly injured lesions in the posterior papillary muscle. These results indicate that disruption of microtubules during ischemia heralds irreversible ischemic injury. However, in in vitro study, the myocardium incubated in hypoxic solution for 60-120 minutes demonstrated earlier disruption of the microtubules than the vinculin. Electron microscopic study also showed minimal irreversible changes in the lesions with disrupted microtubules. Thus, taken together, we conclude that microtubules that support the structural integration of myofibrils and other organelles are disrupted in severe myocardial ischemia before the irreversible injury, promoting the irreversible change after reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapamycin confers preconditioning-like protection against ischemia-reperfusion injury in isolated mouse heart and cardiomyocytes

Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian target of rapamycin (mTOR) signaling and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients. Recently, the antigrowth properties of rapamycin have been utilized for cardiovascular benefit as stents impregnated with rapamycin effectively reduce coronary restenosis. We report here a novel role of this drug in protection against ischemia/reperfusion (I/R) injury. Adult male ICR mice were treated with rapamycin (0.25 mg/kg, IP) or volume-matched DMSO (solvent for rapamycin). The hearts were subjected to 20 min of global ischemia and 30 min of reperfusion in Langendorff mode. The blocker of mitochondrial KATP channel, 5-hydroxydecanoate (5-HD, 100 microM) was given 10 min before ischemia. Infarct size in the DMSO treated group was 28.2 +/- 1.3% and was reduced to 10.1 +/- 2.8% in the rapamycin-treated mice (64% decrease, P < 0.001). 5-HD blocked the protective effect (infarct area 32.2 +/- 1.8%, P < 0.001 vs. rapamycin). The infarct limiting effect of rapamycin was not associated with improved recovery of ventricular function. We further examined the effect of rapamycin in protection against necrosis and apoptosis in adult cardiomyocytes subjected to simulated ischemia and reoxygenation. Myocytes treated with rapamycin in doses from 25-100 nM demonstrated significantly lower trypan blue-positive necrotic cells and TUNEL-positive apoptotic nuclei, supporting the protective role of drug in the intact heart. These data suggest that rapamycin induces potent preconditioning-like effect against myocardial infarction through opening of mitochondrial KATP channels. We propose that rapamycin may be a novel therapeutic strategy to limit infarction, apoptosis, and remodeling following I/R injury in the heart.