Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression

Intermittent hypoxia has been shown to provide myocardial protection against ishemia/reperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts compared with normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reduce myocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl-2/Bax, especially in membrane fraction.

Antioxidant Effect of Human Selenium-containing Single-chain Fv in Rat Cardiac Myocytes

Reactive oxygen species（ROS） plays a key role in human heart diseases. Glutathione peroxidase（GPX） functions as an antioxidant as it catalyzes the reduction of hydroperoxide. In order to investigate the antioxidant effect of human selenium-containing single-chain Fv（Se-scFv-B3）, a new mimic of GPX, a model system of hydrogen peroxide（H202）-induced rat cardiac myocyte damage was established. The cardiac myocyte damage was characte- rized in terms of cell viability, lipid peroxidation, cell membrane integrity, and intracellular H202 level. The Se-scFv-B3 significantly reduced H2O2-induced cell damage as shown by the increase of cell viability, the decline of malondialdehyde（MDA） production, lactate dehydrogenase（LDH） release, and intracellular H2O2 level. So Se-scFv-B3 may have a great potential in the treatment of human heart diseases induced by ROS.

EFFECT OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ACTIVATORS ON TUMOR NECROSIS FACTOR-αEXPRESSION IN NEONATAL RAT CARDIAC MYOCYTES

Objective To investigate the effect of peroxisome proliferator-activated receptor-α(PPARα) and PPARγactivators on tumor necrosis factor-α(TNFα) expression in neonatal rat cardiac myocytes. Methods Primary cultures of cardiac myocytes from 1- to 3-day-old Wistar rats were prepared, and myocytes were ex-posed to lipopolysaccharide (LPS) and varying concentrations of PPARαor PPARγactivator (fenofibrate or pioglitazone).RT-PCR and ELISA were used to measure TNFα, PPARα, and PPARγexpression in cultured cardiac myocytes. Transient tr-ansfection of TNFαpromoter with or without nuclear factor-kappaB (NF-κB) binding site to cardiac myocytes was performed. Results Pretreatment of cardiac myocytes with fenofibrate or pioglitazone inhibited LPS-induced TNFαmRNA and protein expression in a dose-dependent manner. However, no significant changes were observed on PPARαor PPARγmRNA expression when cardiac myocytes were pretreated with fenofibrate or pioglitazone. Proportional suppression of TNFαpromoter activity was observed when myocytes was transiently transfected with whole length of TNFαpromoter (-721/+17) after being stimulated with LPS and fenofibrate or pioglitazone, whereas no change of promoter activity was observed with transfection of TNFαreporter construct in deletion of NF-κB binding site (-182/+17). Conclusions PPARαand PPARγactivators may inhibit cardiac TNFαexpression but not accompanied by change of PPARαor PPARγmRNA expression. Therefore PPARαand PPARγactivators appear to play a role in anti-inflammation. The mechanism may partly be involved in suppression of the NF-κB pathway.

Effect of Cholic Acid on Fetal Cardiac Myocytes in Intrahepatic Choliestasis of Pregnancy

This study examined the effect of cholic acid （CA） on cultured cardiac myoeytes （CMs） from neonatal rats with an attempt to explore the possible mechanism of sudden fetal death in intra- hepatic cholestasis of pregnancy （ICP）. Inverted microscopy was performed to detect the impact of CA on the beating rates of rat CMs. MTT method was used to study the effect of CA on the viability of CMs. CMs cultured in vitro were incubated with 10 ~maol/L Ca2＋-sensitive fluorescence indicator fluo-3/AM. The fluorescence signals of free calcium induced by CA were measured under a laser scanning confocal microscope. The results showed that CA decreased the beating rates of the CMs in a dose-dependent manner. CA could suppress the activities of CMs in a time- and dose-dependent manner. CA increased the concentration of intracellular free calcium in a dose-dependent manner. Our study suggested that CA could inhibit the activity of CMs by causing calcium overload, thereby leading to the sudden fetal death in ICP.

Role of NF-κB in protection of EPO pretreatment on neonatal rat cardiac myocytes with hypoxia/reoxygenation injury

Objective：To observe the protective effects of erythropoietin （EPO） pretreatment on cardiac myocyte with hypoxia/reoxygenation （H/R） injury and the role of NF-κBin this effects. Methods：After the H/R model of cardiac myocytes of neonatal rats was established, the cultured cardiac myocytes were divided into 4 groups, including EPO pretreatment group （ EPO 10 U/ml 24 h before H/R）, EPO pretreatment ＋ PDTC group（EPO 10 U/ml and PDTC 5 μg/ml 24 h before H/R）, PDTC group （PDTC 5 μg /ml 24 h before H/R） and eomrolgroup. Before and after the H/R, assay of LDH concentration in the culture medium, the survival rate of the myocytes tested by MTT chromatometry and the apoptosis by flow cytometry were undertaken. Activation of NF-κB was determined by EMSA before and after H/R. Results：EPO pretreatment markedly reduced the LDH concentration in the medium, elevated the survival rate of myocytes and inhibited the apoptosis after H/R. Addition of PDTC during the pretreatment abol- ished the protective effects of EPO pretreatment. NF-κB was markedly activated during EPO pretreatment and PDTCinhibited the activation. However, after H/R, the activity of NF-κB in myocytes with EPO pretreatment was significantly inhibited compared to the other myocytes. Conclusion：NF-κB is significantly activated during EPO pretreatment, but is inhibited after H/R, which is correlated with the protective effects of EPO pretreatment on cardiac myocytes with H/R. This phenomenon can be explained as the negative feedback mechanism of the activation of NF-κB.

Effects of simvastatin on hypertrophy and PTEN expression of rat cardiac myocytes

Objective To study the effects of simvastatin on the hypertrophy of cultured rat cardiac myocytes induced by serum and the role ofphosphatase and tensin homolog deleted on chromosome ten （PTEN） in the signal pathway. Methods Cultured neonatal Sprague- Dawley （SD） rat cardiac myocytes were treated with 15% fetal bovine serum, or without serum, or different consentrations of simvastatin. Image analysis system was used to measure the cardiac myocytes surface area. Protein synthesis of myocytes was measured via [3H]-leucine incorporation method. The expression level of atrial natriuretic peptide （ANP） mRNA in myocytes was determined with reverse transcription polymerase chain reaction （RT-PCR）. The mRNA and protein expression levels of PTEN in cardiac myocytes were investigated with RT-PCR and Western blot respectively. Results At 24 hours, cardiac myocytes surface area was significantly higher in 15% serum group （1611.16± 160.75 lam2） than in serum-free group （538.04±118.60 ±tm2, P〈0.01）. Simvastatin decreased the cell surface area in a concentration dependent manner. The cell surface area in 10-5 and 10-6 mol/L simvastatin groups were 799.84＋ 167.70 ±tm2 and 1076.88± 199.28 um2 respectively, which were both significantly lower than that in 15% fetal bovine serum group （P〈0.01）. Incorporation rate of [3H]-leucine was significantly higher in 15% fetal bovine serum group （2360± 106cpm/well） than that in serum-free group （1305±92 cpm/well, P〈0.01）. Incorporation rate of [3H]-leucine in 10.5 and 10.6 mol/L simvastatin groups were 1707±101 clam/well and 1962±125 cpm/well respectively, which were both lower than that in serum group （P〈0.01）. With the increase of simvastatin concentration, the expression level ofANP mRNA in cardiac myocytes was decreased gradually, which were 0.29±0.03 and 0.40-±0.03 respectively in 10.5 and 10-6 mol/L simvastatin groups, and significantly lower than that in serum group（0.60-±.03, P〈0.01）. Simvastatin increased the expressions of PTEN mRNA and protein in cardiac myocytes in a concentration dependent manner. PTEN mRNA expression level in 10-7, 10-6and 105mol/L simvastatin groups were 0.38±0.03, 0.83±0.04 and 0.85±0.05, respectively, which were all higher than that in 15% fetal bovine serum group （0.29±0.04, P〈0.05）. Similarly, PTEN protein level in 10-7, 10-6 and 10.5 mol/L simvastatin groups （39.25±3.41, 46.35±1.78 and 47.22±2.39 respectively） were also significantly higher than that in 15% fetal bovine serum group （32.21±4.06, P〈0.05）. Conclusion Simvastatin can inhibit the hypertrophy of cultured rat cardiac myoeytes induced by serum, and the increase of expression level of PTEN might be involved in the mechanism （J Geriatr Cardio12010; 7：47-51）.

In Vitro Study on Mesenchymal Stem Cells:Anti-apoptotic Effects on Cardiac Myocytes

Objectives To investigate the anti-apoptotic effects of mesenchymal stem cells （MSCs） on hypoxic injured cardiac myocytes in vitro. Methods MSCs were isolated from bone marrow of Sprague-Dawley （SD） rats, and cardiac myocytes from neonatal rats. The rat cardiac myocytes were co-cultured with MSCs or MSC-conditioned media in anoxia （95% N2 ±5% CO2） for 72 hours. Cell apoptosis was measured by Hoechst 33258 staining. The expression of Bcl-2 and Bax in cardiac myocytes was tested by Western Blot. Results The apoptotic rate was 51.6% ± 2.4% when cardiac myocytes were cultured in continuous hypoxia and was significantly decreased when cardiac myocytes were cocultured with MSCs or MSC-conditioned media （ 15.1% ± 5.4% and 24. 0% ± 4.2% respectively, P 〈 0. 001 ）. The decreased expression of Bax in the cardiac myocytes was greatly related to the decreasing of apoptosis, but there was no difference in Bcl-2 expression among these groups. Conclusions Co-cultured MSCs showed significant anti-apoptotic effects on cardiac myocytes in continuous hypoxia. The mechanism may be the interact of cell to cell and paracrine of cytokines which effected the expression of Bax in the cardiac myocytes.

Isolation of Ca^(2+) Tolerant Cardiomyocytes from Aadult Rats for Patch Clamp Studies

Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion apparatus with normal Tyrode's solution, then with Ca 2+-free Tyrode's solution and subsequently with low Ca 2+ enzyme solution containing collagenase 0.1-0.2 g/L. All the solutions were saturated with oxygen and the perfusion temperature was kept at 37 ℃. Finally hearts were washed by Ca 2+-free Tyrode's solution, after which the ventricles were minced into small pieces in KB solution, dispersed and filtered. The isolated myocytes were stored in KB solution at room temperature for 1 h and recovered to normal calcium concentration before patch clamp experiments.Results: When all the factors such as water, enzyme, Ca 2+,pH, and oxygen were well controlled, the well constructed and rod-like cardiac myocytes with a yielding rate of 30%-50% came out.Conclusion: All the factors should be well controlled, which ensured the isolated cells Ca 2+ tolerant and appropriate for patch clamp experiments.

The Effects of Captopril and Cicaprost on Changes of Cardiac Membrane Fluidity and Lipid Peroxidation

The main purpose of this study was to investigate the protective actions of captopril and cicaprost on changes of membrane fluidity of cultured neonatal rat myocardial cells exposed to anoxia and sugar deprivation.Lipid peroxidation level estimated by determining the thiobarbituric acid reactive substance(TBARS)content and lactate dehydrogenase(LDH)released in culture medium was also observed in order to examine other membrane-related changes due to anoxia.Membrane fluidity was monitored by measuring changes in the steady state fluorescence anisotropy(r_s)by fluorescence spectroscopy.The r_s value,TBARS level and LDH release were significantly increased after 3 h anoxia.Captopril(180 μmol/L),cicaprost(30 nmol/L)and indomethacin(1μmol/L)did not alter r_s, TBARS level and LDH activity of normal cultured neonatal rat myocardial cells.However,both captopril and cicaprost significantly prevented the increases of r_s,TBARS content and LDH release in those cells exposed to anoxia and sugar deprivation.lndomethacin abolished the actions of captopril on TBARS production and LDH release,but maintained its membrane fluidity protection.These results indicate that captopril and cicaprost protect membrane fluidity and lipid peroxidation changes in anoxia- injured myocardial cells.The action mechanism of captopril may be due,in part,to stimulation of prostacyclin synthesis and/or release.

STUDY ON MECHANICAL INTERACTIONS BETWEEN SINGLE CARDIAC MYOCYTE AND ELASTIC SUBSTRATE

Quantitative investigation on mechanical characteristics of cardiac myocytes has important physiological significance. Based on elastic substrate technique, this paper develops a set of algorithms for high-efficiency cellular traction recovery. By applying a gradient-based digital image correlation method to track randomly distributed fluorescence microbeads on the deformed substrate induced by single cardiac myocyte, high-resolution substrate displacement field can readily be obtained. By using a numerical algorithm based on the integral Boussinesq solution, cell-substrate tractions are reconstructed in a stable and reliable manner. Finally, spatiotemporal dynamics of a single cardiac myocyte is investigated as it adheres to a polyacrylamide elastic substrate.

Antioxidant Effect of Selenium-containing Glutathione S-Transferase in Rat Cardiomyocytes

As one of the most important antioxidant enzymes, glutathione peroxidase（GPX） protects cells and tissues from oxidative damage, and plays an important role in cardiovascular and cerebrovascular injuries induced by oxida- tive stress. The antioxidant effect of selenium-containing glutathione S-transferase（Se-GST）, a mimic of GPX was investigated on rat cardiomyocytes. To explore the protection function of Se-GST in hydrogen peroxide（H202） chal- lenged rat cardiomyocytes, we examined malondialdehyde（MDA）, lactate dehydrogenase（LDH）, superoxide dismu- tase（SOD） and cell apoptosis. The results demonstrate exposure of rat cardiomyocytes to H202 for 6 and 12 h induced the significant increases of MDA, LDH and apoptosis rate of cardiomyocytes, but pretreatment of rat cardiomyocytes with Se-GST at 0.0005 or 0.001 unit/mL prevents oxidative stress induced by H202 with the decreases of cell apopto- sis. All the results hint Se-GST has antioxidant activity for oxidative stress challenged rat cardiomyocvtes.

Safety and effect of umbilical cord blood -derived mesenchymal stem cells on apoptosis of human cardiomyocytes

Objective To study the safety and effect of the umbilical cord blood （UCB）-derived mesenchymal stem cells （MSCs） on apoptosis of human cardiomyocytes （HCM）. Methods UCB was collected at the time of delivery with informed consent obtained from 10 donors. The UCB-derived MSCs were treated with 5-azaserube （5-AZA） and were further induced to differentiate into cardiomyocytes. Telomerase activity, G-banding patterns of chromosomal karyotypes, tumor formation in nude mice, RT-PCR, and the effect of inhibiting apoptosis of HCM were investigated. Results MSCs derived from UCB were differentiated into cardiomyocytes in vitro, which possessed telomerase activity after 5-AZA induction, and no abnormal chromosomal karyotypes were observed. Expression of p53, cyclin A, cdk2, ~3 -actin, C-fos, h-TERT and c-myc were similar in MSCs before and after 5-AZA treatment. There was no tumor formation in nude mice after injection of UCB-derived MSCs. UCB-derived MSCs significantly inhibited apoptosis of HCM. Conclusion UCB-derived MSCs are a valuable, safe and effective source of cell-transplantation treatment .

Gene Product Expression of Cyclin D_2 and p16 During the Transition from Cardiac Myocyte Hyperplasia to Hypertrophy

The current study was to investigate mRNA expression of cyclin D 2 and p16 during the transition from cardiac myocyte hyperplasia to hypertrophy. Cultured cardiac myocytes (CM) and fibroblasts (FC) obtained from 1 day old Sparague Dawley rats were used in this study. We have determined (1) hyperplasia by cell growth curve and fluorescence activated cell sorting (FACS); and (2) ultrastructure by electron microscope observation; and (3) expressions of cyclin D 2 mRNA and p16 mRNA by using in situ hybridization and image analysis. The results were shown (1) Results of cell growth curve and FACS analysis showed CM could proliferate in the first 3 cultured days (4 days in postnatal development). But the ability decreased quickly, concomitant with the differentiation. (2) The ultrastructure of CM showed the large amount of myofilaments and mitochondrion and FC showed moderate amount of rough endoplasmic reticulum. (3) The expression of cyclin D 2 mRNA in 3 , 4 , 5 day CM group was 0.89 times(p<0.05), 0.80 times (p<0.05)and 0.56 times (p<0.01)of that in 1 day group respectively. P16 mRNA in 2 , 3 , 4 , 5 day CM group were 1.63 times(p<0.01),1.72 times(p<0.01),1.99 times (p<0.01)and 2.84 times (p<0.01) of that in 1 day group respectively. It can be concluded that cultured neonatal rat cardiac myocytes could proliferate during the first 3 cultured days, but the ability of proliferation decreased, from the fourth day, concomitant with differentiation. Cyclin D 2 and p16 have the key roles during the transition from myocyte hyperplasia to hypertrophy.

Regulations of Thyroid Hormone on Cardiac Protein Kinase C Signal Pathway in vitro

The experiments were conducted to assess the influences of thyroid hormone on cardiac protein kinase C(PKC) signal pathway with cultured cardiac myocytes and fibroblasts as the models. Cells were pretreated with 1% newborn calf serum (NCS) or angiotensin II (Ang II), and then following by a triiodothyronine (T3) treatment. The PKC activity, PKCα and PKCε expressions were analyzed and compared. In 1% NCS pretreatment, T3 could inhibit PKC activity and PKCε expression in cardiac myocytes. The AngII pretreatment led to an increase of PKC activity and PKCε expression in cardiac myocytes, and an increase of PKC activity in cardiac fibroblasts. Following by T3 treatment, the increased PKC activity and PKCε expression in cardiac myocytes were markedly decreased. In conclusion, whether in 1% NCS or in Ang II pretreatment, T3 could inhibit PKC activity and PKCε expression in cardiac myocytes. Key words thyroid hormone - cardiac myocytes - cardiac fibroblasts - protein kinase C CLC number Q 572 Foundation item: Supported by the Natural Science Foundation of Hubei Province (98091)Biography: WANG Bao-hua (1974-), female, Ph. D, research direction: cardiovascular pathophysiology.

GRK5 is an essential co-repressor of the cardiac mineralocorticoid receptor and is selectively induced by finerenone

BACKGROUND In the heart,aldosterone(Aldo)binds the mineralocorticoid receptor(MR)to exert damaging,adverse remodeling-promoting effects.We recently showed that G protein-coupled receptor-kinase(GRK)-5 blocks the cardiac MR by directly phosphorylating it,thereby repressing its transcriptional activity.MR antagonist(MRA)drugs block the cardiac MR reducing morbidity and mortality of advanced human heart failure.Non-steroidal MRAs,such as finerenone,may provide better cardio-protection against Aldo than classic,steroidal MRAs,like spironolactone and eplerenone.AIM To investigate potential differences between finerenone and eplerenone at engaging GRK5-dependent cardiac MR phosphorylation and subsequent blockade.METHODS We used H9c2 cardiomyocytes,which endogenously express the MR and GRK5.RESULTS GRK5 phosphorylates the MR in H9c2 cardiomyocytes in response to finerenone but not to eplerenone.Unlike eplerenone,finerenone alone potently and efficiently suppresses cardiac MR transcriptional activity,thus displaying inverse agonism.GRK5 is necessary for finerenone’s inverse agonism,since GRK5 genetic deletion renders finerenone incapable of blocking cardiac MR transcriptional activity.Eplerenone alone does not fully suppress cardiac MR basal activity regardless of GRK5 expression levels.Finally,GRK5 is necessary for the antiapoptotic,anti-oxidative,and anti-fibrotic effects of both finerenone and eplerenone against Aldo,as well as for the higher efficacy and potency of finerenone at blocking Aldo-induced apoptosis,oxidative stress,and fibrosis.CONCLUSION Finerenone,but not eplerenone,induces GRK5-dependent cardiac MR inhibition,which underlies,at least in part,its higher potency and efficacy,compared to eplerenone,as an MRA in the heart.GRK5 acts as a co-repressor of the cardiac MR and is essential for efficient MR antagonism in the myocardium.

FINITE ELEMENT ANALYSIS OF CARDIAC MYOCYTE DEBONDING AND REORIENTATION DURING CYCLIC SUBSTRATE STRETCH EXPERIMENTS

The substrate stretch experiment, which is carried out on several kinds of adherent cells, is usually used to catch the physiological variation and morphological response to cyclic substrate deformation. In this paper, stretch loading was exerted on cardiac myocytes cultured on silica substrates using a custom-made substrate stretch device. The effect of stretch on the alignment orientation of cardiac myocytes was studied through morphocytological statistics. Under cyclic stretch stimulus, the long axes of cardiac myocytes oriented perpendicularly to the stretch direction for continuous stretch acting. However, the mechanism underlying these behaviors is not well understood from such in vitro tests. Finite element （FE） model was developed in the analysis to investigate these behaviors. Xu-Needleman formulation was used to define the interaction behavior for contact surfaces between cell and substrate. The role of cell viscoelasticity nature is studied in adherent cell debonding with the substrate and aligning perpendicular to the stretch direction during long time cyclic stretch stimulation. There were four different strain magnitudes considered in the simulation to find out the cell debonding affected by the cyclic strains. The potential role of cyclic strain frequency in regulating cell debonding and alignment was also studied using FE analysis.

PROTECTIVE ROLE OF SOPHOCARPINE IN COXSACKIEVIRUS B3 INFECTION IN CULTURED RAT CARDIOMYOCYTES

Objective To observe the anti-CVB3 （ Coxsackievirus B3 ） effect of sophocarpine （SC） extracted from Sophora flavescens, a traditional Chinese herb in vitro. Methods Cardiomyocytes from the neonatal rat were cultured to establish the viral myocarditis model The cells were divided into four groups： infected group （ infected by CVB3 ） , SC treated group （ added SC 100 μg/mL after viral infection ）, SC control group （ added SC 100 μg/mL only）, and normal control group. The cytopathic effect （CPE） and the beating frequency of the myocardial cells were observed and the LDH levels in the supernatant were measured at day 2,3, and 5. The cultured myocytes were added different concentrations of SC （ 12. 5 -400 μg/mL ） after infection with CVB3, the CPE was observed and the concentrations of LDH were measured and compared at day 2, 3, and 5. Results In the SC treated group （ 100 μg/mL ） , the cytopathic effect was lighter and the LDH level was lower than the infected group. SC in a concentration of 12. 5 - 300 μg/mL could relieve the CPE and lower the LDH level, while in a higher concentration （400 μ/m ） , it exacerbated the CPE caused by the virus, and the LDH levels were higher than the infected cells. Conclusion SC in certain concentration could protect the cultured rat cardiomyocytes from CVB3 infection.

Effects of adiponectin on oxidative stress and apoptosis in human cardiac myocytes cultured with high glucose

Background Diabetic cardiomyopathy is the major cause of morbidity and mortality in diabetic patients. Oxidative stress plays an important role in diabetic cardiomyopathy. This study aimed to investigate the effects of adiponectin on oxidative stress and apoptosis in human cardiac myocytes （HCM） cultured with high glucose. Methods The cells were assigned to three group： control group, high glucose group and high glucose plus adiponectin group. After culture for 24, 48, 72 hours, oxidative stress was evaluated by detecting levels of malondialdehyde （MDA） and superoxide dismutase （SOD） in the supernatant of culture media. The expression of p66Shc and Heme oxygenase-1 （HO-1） was detected by real-time polymerase chain reaction （PCR）. Flow cytometry was designed to observe and detect cellular apoptosis. Results Our findings showed significant increase in MDA levels and decrease in SOD activity in the high glucose group compared with the control group （P 〈0.05）. However, MDA levels were significantly decreased and SOD activity was significantly increased in the adiponectin group compared with those in the high-glucose group （P 〈0.05）. The mRNA expression of HO-1 in the high glucose group was significantly increased in a time-dependent manner compared with that in the control group （P 〈0.05）. Adiponectin further increased the mRNA expression of HO-1 induced by high glucose in a time-dependent manner （P 〈0.05）.The expression of p66Shc was significantly increased in high glucose group compared with that in the control group （P 〈0.05）. Adiponectin significantly suppressed the upregulation of p66Shc induced by high glucose （P 〈0.05）. The apoptotic rate of cardiomyocytes was significantly increased in the high glucose group compared with that in the control group while the apoptotic rate in the adiponectin group was remarkably declined in comparison with that in the high glucose group. Conclusion Adiponectin reduces high glucose-induced oxidative stress and apoptosis and plays a protective role in myocardial cells by upregulating the HO-1 expression and downregulating p66Shc expression.

Ultrasonic destruction of albumin microbubbles enhances gene transfection and expression in cardiac myocytes

Background It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial.This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes.Methods The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin （ASDA） and perfluoropropane-exposed sonicated dextrose albumin （PESDA） were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed.Results The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection （（51.95±2..41） U/g or （29.28±3.65） U/g vs. （0.84-0.21） U/g, P ＜0.01）, and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA （（51.95e2.41） U/g vs. （29.28±3.65）U/g, P ＜0.05）. Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced 3-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone （（111.35±11.21） U/g protein vs. （14.13±2.58） U/g protein, P＜0.01）. Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression （（35.63±7.65）U/g vs. （14.13±2.58） U/g, P＜0.05 ）.Conclusions Ultrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into cardiac myocytes, and the gene tranfection is related to the characteristics of microbubbles.