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Aim: Cardiac hypertrophy and myocardial apoptosis are two major factors in heart failure. As a classical regulator of apoptosis, apoptosis repressor with caspase recruitment domain (ARC) has recently also been found to have a protective effect against hypertrophy. However, the mechanism underlying this effect is still not fully understood. Methods: In the present study, we established animal and cellular models to monitor the changes in total and nuclear ARC during cardiac hypertrophic processes. The preventive effects of nuclear ARC in cellular hypertrophy were verified by ARC regulation and nuclear export inhibition. To further explore the mechanism for nuclear ARC superficially, we analysed proteins that interact with ARC in the nucleus via Co‐IP and mass spectrometry. Results: The expression of total ARC in hypertrophic myocardial tissue and H9C2 cells remained invariant, while the level of nuclear ARC decreased dramatically. By altering the content of ARC in H9C2 cells, we found that both nuclear ARC transfection and nuclear ARC export blockade attenuated norepinephrine or angiotensin II‐induced hypertrophy, while ARC knockdown had an inverse effect. Co‐IP data showed that ARC interacted with prohibitin (PHB) in the nucleus and might participate in maintaining the level of PHB in cells. Conclusions: These findings suggest a novel mechanism for ARC in cardiac hypertrophy prevention and also indicate that the anti‐hypertrophic roles of ARC are probably associated with its localization in nucleus, which imply the nuclear ARC as a potential therapeutic target for cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Na+, K+-ATPase β2 subunit (NKA1b2) is not only a regulator of Na+, K+-ATPase, but also functions in the interaction between neuron and glia cells as a Ca2+-dependent adhesion molecule. To further study the function of NKA1b2, the anti-NKA1b2 polyclonal antibody was prepared to recognize the outer-membrane carboxyl portion segment of NKA1b2. The coding region for amino acids 190–290 at the carboxyl portion of NKA1b2 (NKA1b2-CP) was sub-cloned into the vector pGEX-4T-2 and introduced into the Escherichia coli BL21(DE3) cell for efficient soluble expression. The amino acid sequence of expressed protein was determined using mass spectrometry following Mascot analysis. After purification, GST-NKA-β2-CP was used to immunize the adult rabbits following standard protocols. The produced antiserum could detect the NKA1b2 protein expressed not only in the prokaryotic cells (E. coli) but also in the eukaryotic cells (COS7) transfected with NKA1b2 expression vector (pEGFP-NKA1b2). Furthermore, the antiserum was used for determining the localization of NKA1b2 in primary culture of neonatal rat neurons using immunohistochemical technique. Results demonstrated that NKA1b2 was localized both in the cytoplasm and cellular membrane. The preparation of anti-NKA-β2-CP polyclonal antibody will facilitate further functional study on NKA1b2. [Copyright &y& Elsevier]

The stress is generated in response to changes in internal and external environment of the body is characterized by systemic reactions to neuroendocrine activation of the immune network. More and more evidence suggests that highly activated under stress conditions neuroendocrine immune network will have a strong and sustained influence on the body's metabolism. The effect of stress on the metabolism is not only confined to the glucose metabolism, fat metabolism and protein metabolism, and important role in regulating the metabolism of trace elements. Sustained high-load stress on the metabolism of the negative impact beyond the body's compensatory capacity will lead to a disease such as diabetes, atherosclerosis and secondary diseases.

Endotoxins, derived from cell walls of most Gram-negative bacteria and some cyanobacteria, are common pyrogen and highly immunogenic molecules, and related to many diseases. In this paper, a detection method for endotoxin activity in water environment using kinetic-turbid assay of Tachypleus Amebocyte Lysate (TAL) was established, the influence of pH and salts on TAL assay was investigated. Results showed that it was favorable for TAL assay in the pH range of 6.0-8.4, at low pHs, inhibition results were observed and opposite results were obtained at high pHs. The pH should be adjusted by Tris-HCl (pH = 7.4) buffer before the endotoxin detection. No significant interference was shown in the detection of water containing NaCl, Na2SO4, CaCl2, MgCl2 and KCl with a concentration of less than 50 mg x L(-1), however, the inhibition occurred at the concentration up to 1000-10,000 mg x L(-1). Only 2. 5 mg x L(-1) of FeCl, Fe2(SO4)3, AlCl3 and Al2 (SO4)3 caused significant inhibition. Endotoxin activities of ultrapure water, tap water and recreational water were detected by TAL assay, and their endotoxin activities were < 0.06 EU x mL(-1), 0.46 EU x mL(-1) and 432. 68 EU x mL(-1), respectively.

Objective: To investigate the dynamic expression of Heat shock protein 70 (Hsp70) in the lungs and plasma of rats with pulmonary fibrosis induced by silicon dioxide (SiO2)., Methods: Forty-eight Wistar rats were randomly divided into the control group exposed to normal solution and group exposed to SiO2 (50 mg/ml) with intratracheal injection. Each group was divided into four subgroups. The animals of SiO2 group and control group were sacrificed and lungs were collected on the 7th, 14th and 28th days after exposure, respectively. The left lung tissues were examined with the histopathologic HE staining. The expression and localization of Hsp70 protein in the lung tissues were examined with western blot assay and immunohistochemistry, respectively. The expression levels of Hsp70 protein in the plasma were measured by ELISA., Results: The expression of Hsp70 in lung tissues of SiO2 group increased on the 7th day and reached the peak value on the 14th day then decreased, but still was significantly higher than that of the control group, the expression of Hsp70 in plasma of SiO2 group still was significantly higher than that of the control group (P < 0.05). The maximum expression level of Hsp70 in plasma of SiO2 group on the 21st day after exposure was 0.216 ± 0.027 µg/ml., Conclusion: The expression levels of Hsp70 protein in the lung tissues and plasma of the group exposed to SiO2 significantly increased, which were associated with the process of pulmonary fibrosis. It was suggested that Hsp70 protein may play an important biological role in the pulmonary fibrosis induced by SiO2.

Objective: To investigate the changes of expression and subcellular location of nuclear growth-induced protein-B(NGFI-B) in cardiomyocytes of stressed rats and its biological effect and to provide scientific evidences for exploring the mechanism underlying myocardium injury induced by stress., Methods: The cell model of stress-induced cardiomyocyte injury were established. Western blot method and confocal microscopy method were used to investigate the subcellular location of NGFI-B in cardiomyocytes under stress. The flow-cytometry was selected to detect the apoptotic rate in cardiomyocytes in vitro. Western blot method was used to determine the content of cytochrome C protein in mitochondria and cytoplasm respectively., Results: Stress induced the increase of NGFI-B content in the mitochondria of cardiomyocytes and the translocation of NGFI-B from the nucleus to the mitochondria. The translocation of NGFI-B promoted the release of cytochrome C from the mitochondria and the cardiomyocyte apoptosis. Treatment of stressed cardiomyocytes with leptomycin B, a non-specific blocker of nuclear export, resulted in nuclear retention of NGFI-B and abrogated its ability to induce the release of cytochrome C from the mitochondria., Conclusion: Stress could induce NGFI-B translocation from the nucleus to the mitochondria in cardiomyocytes, which activated the mitochondrial pathway of cell apoptosis.

Aim: To explore the level of anti-HSP70 antibody in plasma during atherosclerosis procedure induced by high-fat diet in rat and the relationship of them., Methods: Twenty eight rat were divided into high-fat diet group (H) and control group (C). The total cholesterol (TC), Glyceride (TG), low density lipoprotein cholesterol (LDL-C) in serum, pathology change of rat Arch of the aorta were determined, the level of anti-HSP70 antibody and their Phenotype were evaluated by ELISA., Results: After two weeks, the serum concentrations of TC and LDL-C in rat supplemented by high-fat diet were significantly higher than those in control group (P < 0.01), the serum TG were much lower than those in control group (P < 0.01). Four weeks later the level of anti-HSP70 antibody, IgM, IgG phenotype were significantly higher than those in control group (P < 0.01). There were lipin deposition and mottling formation in rat Arch of the aorta in rat supplemented by high-fat diet in 12th week., Conclusion: Atherosclerosis could be induced by high-fat diet in rat. Accompany with the atherosclerosis procession, the level of anti-HSP70 antibody was continuously elevated, the level of anti-HSP70 antibody was related to atherosclerosis. The level of anti-HSP70 antibody was closely associated with atherosclerosis.

Aim: To assay the transcriptional regulation effect of hHSF1 on prohibitin gene promoter., Methods: The total length of hHSF1 exon was amplified by PCR method and cloned into pcDNA3. 1(+) vector. pcDNA3. 1(+)-hHSF1 and pGI3 prohibitin were co-transfected into HEK293 cells. The luciferase activity was detected by Dual-Luciferase Reporter Assay System to evaluate the transcriptional regulation effect of hHSF1 on prohibitin gene promoter., Results: The pcDNA3.1(+)-hHSF1 vector was constructed successfully. The assay of luciferase activity showed that the transcription of pGL3-prohibitin was dramatically upregulated by hHSF1., Conclusion: hHSF1 can transcriptionally regulate prohibitin expression.

Aim: To study the effects of stress on the cognitive function and physical fitness and its biological mechanisms, provide scientific basis for seeking protective measures to reduce stress-induced damage., Methods: The model of restraint stress was adopted in our experiment. Step-down test and exhaustive swimming test were used to measure the learning and memory function and physical fitness in mice, respectively. The contents of GC in plasma, hippocampus LTP, ECG, myocardial ultra-structure, cell apoptosis rate and the level of Hsp70 expression of myocardium in rats were detected., Results: Compared with control group, the impairment of learning and memory function and decline of exercise tolerance were observed in restraint stress group. The elevation in plasma GC levels, ECG abnonrmality, and cell apoptosis rate were also observed under restraint stress. Furthermore, myocardial structure was damaged, and myocardial Hsp70 expression and hippocampus LTP were suppressed in restraint stress group than those in the control., Conclusion: Stress may cause the neuro-endocrine dysfunction and homeostasis disorder, and then, lead to the cognitive function and physical fitness damage consequently.

Aim: To construct the RNAi eukaryotic vector of inhibitory member of the prohibitin (PHB-1) gene and observe the interfering effect in HEK293 cell line after the vector transfection., Methods: The specific Mi RNA sequence was designed according to the PHB-1 sequence in GenBank, complementary single-strand DNA oligonucleotides were designed and synthesized, and annealed the single-stranded oligonucleotides to generate a double strands oligonucleotides , cloned the oligonucleotides into pcDNATM6.2-GW/EmGFP-MiR-PHB to obtain an entry clone and then sequence analysis was performed. The recombinant plasmid was transfected into HEK293 cell by liposome. PHB-1 expression was detected by Western blotting., Results: The DNA sequence of interest clone to the vector was constructed to generate an entry clone and an expression clone successfully, which were proved by sequence determination. Western blotting analysis demonstrated that PHB-1 MiR RNA expression construction could suppress the expression of PHB-1., Conclusion: A RNAi eukaryotic vector containing prohibitin gene was successfully constructed.

Aim: To assay the transcriptional activation effect of TR3 and it's deletion mutation in yeast two hybrid system., Methods: The total length of TR3 and TR3/delta1-690 gene was amplified by PCR method and cloned into pGBKT7 vector. Bait vector of pGBKT7-TR3 and pGBKT7-TR3/delta1-690 was transformed into AH109 competence yeast. Then self activation of the recombination vector was tested by assay the activity of beta-galactosidae., Results: The pGBKT7-TR3 and pGBKT7-TR3/AM 690 vector was successfully constructed. The filter paper containing beta-galactosidae didn't changed to blue showed that the reporter gene wasn't activationed., Conclusion: TR3 and TR3/delta1-690 hadn't the activity of transcriptional activation.

Aim: To approach the expression of prohibitin in oxidative stressed cardiomyocytes., Methods: The oxidative stress model was established by treating neonatal cardiomyocytes with H2O2. Injury of cardiomyocytes were evaluated by detecting the LDH activity and MTT cell survival rate.The expression level of prohibitin was examined via the Western-blotting. The ability of mitochondrial oxidative phosphorylation was determined by measuring ATP synthesis via H+ -ATPase. Mitochondrial membrane potential was detected by flow cytometry using Rhl23., Results: LDH activity increased significantly after exposure to H202, while the cell survival rate decreased by 34.51%-65.5%. The contents of mitochondrial prohibitin in stress group was much higher than that in control group. At the same time, the ability of ATP synthesis decreased by 60% and mitochondrial transmembrane potential decreased too., Conclusion: Express of prohibitin in oxidative stress cardiomyocytes was compensated increase. Prohibitin translocated to mitochondria after oxidative stress. Oxidative stress led to mitochondrial dysfunction.

Aim: To elucidate the mechanism of depression induced by chronic unpredictable mild stress (CUMS), the effects of CUMS on serotonin (5-HT), tryptophan, stress hormones and behaviour were investigated in rats., Methods: Depression was induced by for 8 weeks CUMS and confirmed by behavioral tests, the brain and plasma levels of monoamine neurotransmitters were analyzed by HPLC-ECD techniques, the content of plasma corticosterone was evaluated by I125 cortisol radioactivity immunoassay and the serum tryptophan content was measured by HTTACHI L-8800 amino acid analyzer., Results: (1) Rats exposed to a series of mild, unpredictable stressors for 8 weeks displayed the decreased body weight, reduced scores of open-field test and preference of sucrose solution (P < 0.05). (2) Plasma and brain 5-HT contents in rats after exposure to CUMS 8 weeks decreased significantly (P < 0.05). While serum tryptophan content increased at the same time (P < 0.05). (3) Plasma norepinephrine and epinephrine in rats were increased after CUMS 8 weeks, but there was no difference between control and CUMS group in plasma corticosterone., Conclusion: The behavioral changes induced by CUMS for 8 weeks are similar to the features of human depression, which may be related to the disturbances of tryptophan metabolism induced by increased norepinephrine and epinephrine in CUMS rat.

Aim: To establish a method for detection of plasma total homocysteine with HPLC., Methods: The chromatography analysis was carried out using a Symmetry Shield RP18. The mobile phase was sodium acetate (0.08 mol/L) and methanol (1%) and we utilized a HPLC system with fluorescence detection of plasma homocysteine derivatized from reaction with 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F)., Results: The average recoveries were 95.8 - 100.8% and the relative standard deviations were 1.2-2.0%., Conclusion: The results showed it to be a rapid and accurate method for the determination of homocysteine level in plasma.

Aim: To probe the related proteins to stress-induced myocardium injury., Methods: After establishment of a myocardium injury model induced by restraint stress in rats, myocardium proteins of restraint stress-treated and untreated rats were extracted, and the two-dimensional polyacrylamide gel electrophoresis (2-DE) maps of the extracted proteins were established by using the immobilized pH gradient (IPG) and SDS-PAGE two-dimensional electrophoresis respectively. The alterative protein spots were analyzed by Image Master 3.01 software and identified with assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching., Results: Proteomics analysis showed that there were 10 proteins were significantly influenced by restraint stress in rat myocardium. After stress, proteins, including cardiac myosin heavy chain, dihydrolipoamide succinyltransferase component of 2-oxoglutarate dehydrogenase complex, similar to dihydrolipoamide S-succinyltransferase, mitochondrial aldehyde dehydrogenase, H (+)-transporting ATP synthase, albumin, myosin heavy chain and apolipoprotein A-I precursor showed increased expression. Mitochondrial aconitase and uncoupling protein UCP-3 showed decreased expression., Conclusion: These differential expressive proteins might be involved in stress-induced injury to myocardium.

Aim: To study the distribution and efficiency of pEGFP-N1 plasmid after its transfection in cardiomyocytes., Methods: The neonatal rat cardiomyocytes were cultured. According to the different grow period of neonatal rat cardiomyocytes, the distribution and efficiency of pEGFP-N1 plasmid after its transfection in cardiomyocytes were studied., Results: The efficiency was significantly increased after pEGFP-N1 plasmid transfection in one-day cardiomyocytes. The distribution of EGFP was in the cytoplasm and nucleus., Conclusion: The efficiency of pEGFP-N1 plasmid after its transfection in cardiomyocytes was related to the grow period of neonatal rat cardiomyocytes. The distribution of EGFP was in the cytoplasm and nucleus.

Na+, K+-ATPase beta2 subunit (NKA1b2) is not only a regulator of Na+, K+-ATPase, but also functions in the interaction between neuron and glia cells as a Ca2+-dependent adhesion molecule. To further study the function of NKA1b2, the anti-NKA1b2 polyclonal antibody was prepared to recognize the outer-membrane carboxyl portion segment of NKA1b2. The coding region for amino acids 190-290 at the carboxyl portion of NKA1b2 (NKA1b2-CP) was sub-cloned into the vector pGEX-4T-2 and introduced into the Escherichia coli BL21(DE3) cell for efficient soluble expression. The amino acid sequence of expressed protein was determined using mass spectrometry following Mascot analysis. After purification, GST-NKA-beta2-CP was used to immunize the adult rabbits following standard protocols. The produced antiserum could detect the NKA1b2 protein expressed not only in the prokaryotic cells (E. coli) but also in the eukaryotic cells (COS7) transfected with NKA1b2 expression vector (pEGFP-NKA1b2). Furthermore, the antiserum was used for determining the localization of NKA1b2 in primary culture of neonatal rat neurons using immunohistochemical technique. Results demonstrated that NKA1b2 was localized both in the cytoplasm and cellular membrane. The preparation of anti-NKA-beta2-CP polyclonal antibody will facilitate further functional study on NKA1b2.

To investigate the effect of stress on homocysteine metabolism in the rat and explore the mechanism as well as the key regulatory link of stress-induced hyperhomocysteinemia, male Wistar rats were treated with restraint stress while control rats received routine treatment. By HPLC-fluorometry, the homocysteine level in rat plasma was determined. Cystathionine beta-synthase (CBS) activity in blood, heart, liver and kidney was measured by radioisotope assay using [(14)C]-serine as the labeled substrate. Total RNA was isolated from rat liver after restraint stress. RT-PCR and Northern blot were used to estimate the level of CBS mRNA. The results showed that hyperhomocysteinemia was induced by restraint stress. The highest CBS enzyme activity was seen in rat livers. A decrease in hepatic activities of CBS was found in restraint stress rats. The 29.4% +/-2.5% reduction in the activity of CBS was accompanied by a 44.1% +/-3.4% decrease in its mRNA level. CBS enzyme activity was slightly elevated in the kidney of stressed rats while it was almost undeterminable in the cardiovascular system. The study suggests that stress leads to an inhibition of the transsulfuration pathway in homocysteine metabolism. The hepatic CBS influenced by stress at the level of transcription exerts a profound effect on the circulating levels of homocysteine. The liver is the key organ where stress affects homocysteine metabolism.

The pre-transformed human fetal brain cDNA library was used to screen the protein interacting with neuroglobin by using yeast two hybrid system III from ClonTech Inc. The protein encoded by one of the clones interacting with neuroglobin (NGB) was confirmed to be the C terminus of the Na(+), K(+)-ATPase beta2 subunit (NKA1b2) based on amino acid sequences. Then the full-length coding region cDNA sequence of NKA1b2 was obtained from human fetal brain cDNA library by PCR. A set of experiments were designed to test the interaction between NGB and NKA1b2. Interaction between NGB and NKA1b2 was confirmed by binding assay in vitro. Furthermore, the interaction was also proved by co-immunoprecipitation test in vivo. Moreover, the structure integrity of neuroglobin was found to be essential for the interaction between NGB and NKA1b2 by yeast two hybrid method with a series of neuroglobin truncated mutants.

Aim: To observe the effects of stress on Ica-L, steady-state activation curves and steady-state inactivation curves., Methods: Use NE to construct stress cell model, then the whole-cell patch-clamp recording technique was used to record the Ica-L, the steady-state activation curves and the steady-state inactivation curves. With FCM technique, we observed the rate of apoptosis of cardiomyocytes. By dying cells with Fura-2 and fluorometry, we determined [Ca2+]i., Results: The amplitude of peak current of Ica-L increased significantly, and by analyzing the steady-state activation curve, we found that the curve was shifted to left, the V1/2 of stress group was (-14.59 +/- 0.24 ) mV vs (-0.69 +/- 0.36) mV of control group. The rate of apoptosis was increased from 0.36% to 2.17% (P < 0.01). The [Ca2+]i increased by 16.7%., Conclusion: Stress can bring on increasing of Ica-L, and the channels are easy to be activated. These changes can cause "calcium overload" and then induce apoptosis which lead to injury of myocytes in stress.

To investigate the upregulated genes associated with cold acclimation, a cold acclimation model was established based on Balb/C mouse. mRNA of muscle and liver were isolated, and the upregulated genes of these tissues were studied by representational differential analysis (RDA). The upregulated genes then were sequenced and searched by Blast software in GenBank database. The results showed that some genes were upregulated and possibly associated with cold acclimation. Three of these genes, transferrin, fibrinogen B-beta-chains and a new gene fragment (Genbank ID: AF454762), were confirmed to be upregulated by RNA slot-blot analysis. The finding of these genes might contribute to further understanding of the molecular mechanisms of cold acclimation.

Aim: To discuss the effects of restrained stress on cardiac myocyte apoptosis of rats in the whole body, and the effect of anti-stress-induced cardiac myocyte injury treated by Chinese herbs yixinning., Methods: Agarose gel electrophoresis and TUNEL are used to detect cardiac myocyte apoptosis., Results: (1) When restrained stress 1,2,4 week, the DNA ladder of stress groups was negative, while in situ apoptosis of stress groups increased apparently compared with control group (P < 0.01). (3) The DNA ladder of yixinning groups was negative too, while in situ apoptosis of yixinning groups decreased compared with stress group (P < 0.05). (3) Whereas in distilled water group, the above indices had no statistical significance compared with stress model group (P > 0.05)., Conclusion: The restrained stress can induce cardiac myocyte apoptosis in rats. Chinese herbs "yixnning" can inhibit cardiac myocyte apoptosis, and have functions of anti-stress injury.

Aim: To observe the injured effect of homocysteine (HCY) on cardiomyocytes and investigate its signal transduction mechanism as well as the key regulatory link., Methods: Cardiomyocytes were isolated from neonatal Wistar rats. After incubation with HCY, the survival rate of cardiomyocytes was determined by trypan blue stained assay, while the apoptosis rate was measured by TUNEL and FCM. Western blot and EMSA were used to tested ERK2 protein phosphorylation and NF-kappaB active expression in cardiomyocytes, respectively., Results: The survival rate of cardiomyocytes treated with HCY was reduced significantly in dose- and time- dependent manner. It was found that 10(-3) mol/L HCY could increase the apoptosis rate of cardiomyocytes to the peak (7.65%) at 4 h stress. Several HCY levels revealed the strong inhibitory effect on ERK2 protein phosphorylation, especially, 10(-3) mol/L HCY decreased the level of active ERK2 expression to 3.04% of control at 4 h (P < 0.01). NF-kappaB activation was also inhibited significantly by several HCY level for different time in cardiomyocytes., Conclusion: HCY plays an important role in injury of cardiomyocytes and apoptosis is a form of HCY-induced injury to cardiomyocytes. HCY can block ERK2 protein phosphorylation and NF-kappaB activation, which contribute to the injury of cardiomyocytes.

Objective: To investigate the effect of constrainting stress on biological characters and function of mitochondrial membrane in rat heart and to explore the possible mitochondrial membrane mechanism underlying stress-induced heart injury., Method: Stress animal model was established. After constrained for different times, all rats were killed and several indexes were examined., Result: Constrainting stress can induce mitochondrial permeability transition, decrease of mitochondrial membrane fluidity, increase of the production of membrane lipid peroxidation and injury of mitochondrial respiratory function which is in time-dependent manner., Conclusion: Mitochondrial membrane impairment and its effects are the important mechanism of stress-induced heart injury.

Aim: To study the mechanisms of protection of bcl-2 gene transfection against heat-stressed cardiomyocytes., Methods: Cardiomyocytes were isolated and cultured. bcl-2 was transfected into cardiomyocytes with Lipofectamine transfection methods. The cardiomyocytes were stressed by heat. The change of H+ -ATPase synthesis activity of cardiomyocytes mitochondria caused by bcl-2 transfection was measured by chemical radiation method. The changes of Caspase 3 activity of cardiomyocytes caused by bcl-2 transfection was measured by fluorometric analysis., Results: bcl-2 transfection could increase the H+ -ATPase synthesis activity of cardiomyocytes mitochondria under heat stress at 41 degrees C and 43 degrees C and could decrease the Caspase 3 activity of cardiomyocytes under heat stress at 41 degrees C and 43 degrees C., Conclusion: The protection effect of bcl-2 transfection on heat-stressed cardiomyocytes may be associated with preserved H+ ATPase synthesis activity of cardiomyocytes mitochondria and the activity of Caspase 3 of cardiomyocytes.