[Three Different Digestion Methods to Separate Neonatal Rat Cardiomyocytes].

OBJECTIVES: To investigate the efficacy of three different digestion methods for the separation of neonatal rat cardiomyocytes. METHODS: We employed three different digestion methods to separate neonatal rat cardiomyocytes. Group A was 0.08% trypsin digestion alone, group B was 0.08% trypsin+0.08% type 2 collagenase mixed digestion, group C was 0.08% trypsin and 0.08% type 2 collagenase isolated digestion. The number of cells and cell viability after differential adhesion were recorded. The purity of cardiomyocytes was evaluated by immunofluorescence staining. The cell vitality was assessed by the detection of mitochondrial membrane potential with JC-1 staining, and the ratio of red to green fluorescence intensity by laser scanning confocal microscope. RESULTS: There was nostatistically significant difference in the number of cells between three groups (P >0.05).The rate of cell viability in group C was significantly higher than that in group A (P <0.01). No statistically significant difference was found in the purity of cardiomyocytes between three groups (P >0.05). The ratio of red to green fluorescence intensity in group A, B and C were 0.928±0.078, 0.943±0.099 and 1.160±0.089, respectively; the ratio in group C was significantly higher than that in group A and group B (P <0.01). CONCLUSION: The cell isolation method with 0.08% trypsin and 0.08% type 2 collagenase isolated digestion could be served as conventional method to separate neonatal rat cardiomyocytes.

Nkx2.5 enhances the efficacy of mesenchymal stem cells transplantation in treatment heart failure in rats.

AIMS: The aim of this study is to determine whether Nkx2.5 transfection of transplanted bone marrow mesenchymal stem cells (MSCs) improves the efficacy of treatment of adriamycin-induced heart failure in a rat model. MAIN METHODS: Nkx2.5 was transfected in MSCs by lentiviral vector transduction. The expressions of Nkx2.5 and cardiac specific genes in MSCs and Nkx2.5 transfected mesenchymal stem cells (MSCs-Nkx2.5) were analyzed with quantitative real-time PCR and Western blot in vitro. Heart failure models of rats were induced by adriamycin and were then randomly divided into 3 groups: injected saline, MSCs or MSCs-Nkx2.5 via the femoral vein respectively. Four weeks after injection, the cardiac function, expressions of cardiac specific gene, fibrosis formation and collagen volume fraction in the myocardium as well as the expressions of GATA4 and MEF2 in rats were analyzed with echocardiography, immunohistochemistry, Masson staining, quantitative real-time PCR and Western blot, respectively. KEY FINDINGS: Nkx2.5 enhanced cardiac specific gene expressions including α-MHC, TNI, CKMB, connexin-43 in MSCs-Nkx2.5 in vitro. Both MSCs and MSCs-Nkx2.5 improved cardiac function, promoted the differentiation of transplanted MSCs into cardiomyocyte-like cells, decreased fibrosis formation and collagen volume fraction in the myocardium, as well as increased the expressions of GATA4 and MEF2 in adriamycin-induced rat heart failure models. Moreover, the effect was much more remarkable in MSCs-Nkx2.5 than in MSCs group. SIGNIFICANCE: This study has found that Nkx2.5 enhances the efficacy of MSCs transplantation in treatment adriamycin-induced heart failure in rats. Nkx2.5 transfected to transplanted MSCs provides a potential effective approach to heart failure.

Chitosan hydrogel improves mesenchymal stem cell transplant survival and cardiac function following myocardial infarction in rats.

Myocardial infarction (MI) remains the leading cause of cardiovascular-associated mortality and morbidity. Improving the retention rate, survival and cardiomyocyte differentiation of mesenchymal stem cells (MSCs) is important in improving the treatment of patients with MI. In the present study, temperature-responsive chitosan hydrogel, an injectable scaffold, was used to deliver MSCs directly into the infarcted myocardium of rats following MI. Histopathology and immunohistochemical staining were used to evaluate cardiac cell survival and regeneration, and cardiac function was assessed using an echocardiograph. It was demonstrated that chitosan hydrogel increased graft size and cell retention in the ischemic heart, promoted MSCs to differentiate into cardiomyocytes and increased the effects of MSCs on neovasculature formation. Furthermore, chitosan hydrogel enhanced the effect of MSCs on the improvement of cardiac function and hemodynamics in the infarcted area of rats following MI. These findings suggest that chitosan hydrogel is an appropriate material to deliver MSCs into infarcted myocardium.

Characterization of a Chinese KCNQ1 mutation (R259H) that shortens repolarization and causes short QT syndrome 2.

OBJECTIVES: To evaluate the association between a KCNQ1 mutation, R259H, and short QT syndrome (SQTS) and to explore the electrophysiological mechanisms underlying their association. METHODS: We performed genetic screening of SQTS genes in 25 probands and their family members (63 patients). We used direct sequencing to screen the exons and intron-exon boundaries of candidate genes that encode ion channels which contribute to the repolarization of the ventricular action potential, including KCNQ1, KCNH2, KCNE1, KCNE2, KCNJ2, CACNA1c, CACNB2b and CACNA2D1. In one of the 25 SQTS probands screened, we discovered a KCNQ1 mutation, R259H. We cloned R259H and transiently expressed it in HEK-293 cells; then, currents were recorded using whole cell patch clamp techniques. RESULTS: R259H-KCNQ1 showed significantly increased current density, which was approximately 3-fold larger than that of wild type (WT) after a depolarizing pulse at 1 s. The steady state voltage dependence of the activation and inactivation did not show significant differences between the WT and R259H mutation (P > 0.05), whereas the time constant of deactivation was markedly prolonged in the mutant compared with the WT in terms of the test potentials, which indicated that the deactivation of R259H was markedly slower than that of the WT. These results suggested that the R259H mutation can effectively increase the slowly activated delayed rectifier potassium current (I Ks) in phase 3 of the cardiac action potential, which may be an infrequent cause of QT interval shortening. CONCLUSIONS: R259H is a gain-of-function mutation of the KCNQ1 channel that is responsible for SQTS2. This is the first time that the R259H mutation was detected in Chinese people.

Role of the lung in the progression of multiple organ dysfunction syndrome in ageing rat model.

BACKGROUND: Multiple organ dysfunction syndrome in the elderly (MODSE) is a problem with high mortality in the critical care of elderly patients. The pathogenesis of MODSE remains elusive. This study aimed to establish rat models of MODSE and to investigate the pathogenetic mechanism responsible for the development of MODSE in the rat models. METHODS: Twenty-four-month old rats (elderly) received intravenous injection of lipopolysaccharide (LPS) to induce rat model of MODSE. In the model, we observed the physical responses, biochemical indices changes, histopathological features of vital organs, including lung, liver, heart, and kidney. We also investigated the sequence of individual organ dysfunction and changes of proinflammatory factors. Three-month-old rats, serving as young rat controls, received parallel procedures. Besides, normal saline injection was also performed on elderly and young control rats. RESULTS: All rats displayed different degree of physical response after LPS injection, preceded by deterioration of respiratory status. At 6 hours, lung injury was observed, which started earlier than other organ injury that was observed in about 24 hours. Furthermore, all vital organ injury was more severe in elderly rats than in young rats at the same time points. After LPS injection, pulmonary alveolar macrophages apoptosis rate increased obviously, and was more significant in elderly rats ((43.4 ± 8.4)%) than in young rats ((24.2 ± 3.0)%). LPS injection also enhanced tumor necrosis factor a (TNF-a) concentration significantly in these organs. Its peak concentration appeared at 6 hours in lung tissue and at 24 hours in other organs after LPS injection. TNF-a level was higher in elderly rats than in young rats at the same time points. The increase was most significant in lung tissue. After intravenous administration of LPS, toll-like receptor 4 (TLR4) expression in lung tissue was upregulated markedly, and peaked at 6 hours. In contrast, upregulation of TLR4 expression in liver peaked at 24 hours, lagging behind that in the lung. CONCLUSION: Lung is the first and most seriously injured organ in rat model of MODSE and it may play an "initiating" role in the development of MODSE.

Mutational analysis of KCNJ11 in Chinese elderly essential hypertensive patients.

OBJECTIVE: To compare the distribution of KCNJ11 polymorphisms between elderly Chinese population with and without hypertension. METHODS: We examined the mutation of KCNJ11 gene by directly sequencing. Data for the present study were obtained from 250 hypertensive subjects (60 to 83 years old) as well as 250 normotensive subjects (60 to 86 years old). RESULTS: We found nine different mutations in KCNJ11, including six novel mutations (I131M, L147I, L147V, L147L, Q235H, G245C). None of the novel mutations were found in the normotensive subjects, and all the residues were conserved in other species. These sequence variants in Chinese population indicate the diversity of the human library and the complexity of hypertension. CONCLUSIONS: The consistent finding of our present study provided a basis for the development of new strategies to diagnosis and treat hypertension in the elderly.

[Mitochondrial tRNAMet mutation in Chinese Han essential hypertensive individuals].

The objective of the present study was to explore the relationship between mitochondrial tRNAMet mutation and development of essential hypertension in Chinese Han individuals. A total of 990 patients with essential hypertension were involved. The general data (sex, age, body mass index, onset age, and family history) and information on routine blood test, blood biochemical examination, and color Doppler echocardiography of these patients were collected. All subjects under-went venous blood drawing for seperating white blood cells and DNA extraction. Then, mitochondrial tRNAMet was amplified and sequenced after purification. The patients who carried the tRNAMet mutation were taken as the indicative cases and the controls were the patients with essential hypertension who did not carry the mutation. We performed a comparative analysis on the routine blood test, blood biochemical examination, color Doppler echocardiography, and other data between the indicative cases and control cases. Among the 990 essential hypertensive patients, there were 8 who carried the tRNAMet mutation, and 6 mutation sites were confirmed, including A4401G, C4410A, U4418C, A4435G, U4454C, and C4456U. Compared with the control cases, the indicative cases developed essential hypertension at earlier ages. The average levels of high density of lipoprotein cholesterol, left ventricular end diastolic diameter, stroke volume, and cardiac index were higher in the indicative cases than in the controls. While the average levels of hemoglobin and left ventricular ejection fraction were lower in the indicative cases than in the control cases. Among the 8 indicative cases, 5 had maternally inherited hyper-tension; one had paternally inherited hypertension; and two denied any family history of hypertension. These results indicated that the mitochondrial tRNAMet mutations might induce the changes in structure and function, which was involved in the progress of the essential hypertension by disturbing the blood metabolism, the steady-state of the blood cells, and the cardiac structure and function.

The protective roles of mitochondrial ATP-sensitive potassium channels during hypoxia-ischemia-reperfusion in brain.

The role of ATP-sensitive potassium (K(ATP)) channels in cerebral ischemia-reperfusion has been well documented. K(ATP) channel openers protect neuron by mimicking ischemic preconditioning. However, the different protection between the mitochondrial and sarcolemma K(ATP) openers has been seldom studied. In the experiment, we investigated the effects of K(ATP) channel openers diazoxide and pinacidil on the hypoxia-ischemia-reperfusion in cultured hippocampal neurons and gerbil brain. The cultured hippocampal neurons and gerbil brain were pretreated with diazoxide or pinacidil before oxygen-glucose deprivation (OGD) and cerebral ischemia-reperfusion, respectively. Survival rate, apoptosis rate and lactate dehydrogenase (LDH) releasing after the reperfusion were subsequently detected. Then the subunits mRNA was detected by RT-PCR. The survival rate and LDH content in diazoxide group increased more than that in pinacidil group (86.21±2.73% vs. 78.59±1.94%, P<0.05; 133.29±15.00 U/L vs. 193.47±3.39 U/L, P<0.01). The apoptosis rate in diazoxide group decreased significantly more than that in pinacidil group (23.82±0.14% vs. 37.05±0.67%, P<0.01). Diazoxide pretreatment increased the expression of Kir6.1 mRNA obviously. The results suggested that mitoK(ATP) channels opener diazoxide played a major protective role on cerebral ischemia-reperfusion. Furthermore, diazoxide might become a new treatment for cerebral ischemia diseases through increasing the expression of Kir6.1 mRNA.

[Construction of pSG5/TRIF and its expression in Huh7 cells].

OBJECTIVE: To construct the plasmid pSG5/TRIF and investigate its expression in Huh7 cells. METHODS: The plasmid pCX4pur/Myc-TRIF was digested with Not I and the digestion product was blunted followed by further digestion with EcoR I to obtain the insert Myc-TRIF. pSG5 was digested sequentially with Sma I and EcoR I. All the digested products were analyzed with agarose gel electrophoresis. The products with the expected size were extracted and ligated, and the positive clones were screened by ampicillin and amplified. The recombinant pSG5/TRIF was extracted, purified, and identified by restriction endonuclease BamH I and agarose gel electrophoresis. The recombinant plasmids were transfected into Huh7 cells with FuGene 6 reagents and into Huh7 cells previously infected with recombinant vaccinia virus (rVV) via Lipofectin. Immunofluorescence and Western blotting were performed to detect the expression of the recombinant plasmids, and the transfection efficiency with different transfection reagents was compared. RESULTS: BamH I digestion resulted in a fragment with the expected size. Immunofluorescence staining showed successful expression of Myc-TRIF protein in Huh7 cells, and the transfection efficiency was enhanced in Huh7 cells previously infected with rVV. SDS-PAGE analysis showed that the relative molecular mass of the expressed product by pSG5/Myc-TRIF was about 100 ku, and prior infection of the cells with rVV obviously increased transfection efficiency, as was consistent with the results of immunofluorescence. CONCLUSION: pSG5/Myc-TRIF is successfully constructed and expressed in Huh7 cells. The expression efficiency can be increased by prior infection of the cells with rVV.

[Apoptosis of pulmonary alveolar macrophages in aged and adult rats: a comparative study].

OBJECTIVE: To investigate the role of alveolar macrophages (AM) in the initiation of multiple organ failure in the elderly (MOFE). METHODS: Three-month-old (adult) and 24-month-old (aged) males SD rats were used as experimental animals. Zymosan 0.5 g/kg was used to establish animal model of MOFE, normal saline were used among the control rats. The rats were divided into 4 groups: aged model group, aged control group, adult model group, and adult control group. Twenty-four hours after the establishment of model, 6 surviving rats from each group were killed. The trachea, bronthi, and lungs were isolated and lavaged with normal saline. One minute later the bronchi-alveolar lavage fluid (BALF) was re-extracted. Cells were collected from the fluid and put into 24-well cell culture plate. The alveolar macrophages (AMs) adhered to the wall were collected, suspended again, cultured, re-collected, centrifuged, and isolated. Apoptosis of the enriched AM was measured by propidium staining and flow cytometry. Fluo-3.AM staining and flow cytometry were used to detect the intracellular free calcium. Mitochondrial membrane electric potential was detected by rhodamine 123 staining with flow cytometry. RESULTS: The apoptotic rate (APO) of AM in the aged rat models was 43.4% +/- 8.4%, significantly higher than that of adult model rats (24.2% +/- 3.0%, P < 0.01). Compared with the controls, the intracellular calcium increased, but mitochondrial Dgr;Psim decreased in the 2 model groups. CONCLUSION: The AM APO% of aged MOFE model increases. It may be one of the causes of difficulty to control the inflammation in the lung with MOFE and easiness to induce MOFE in elderly when their lungs are infected or injured. Changes of intracellular calcium and mitochondrial Dgr;Psim may play pivotal roles in apoptosis of AM.

[Effect of manshuailing oral liquid on left ventricular diastolic dysfunction in patients with heart disease of Xin-Shen Yang deficiency type].

OBJECTIVE: To investigate the effect of Manshuailing Oral Liquid (MSL) on left ventricular diastolic dysfunction (LVDD) in patients with heart disease. METHODS: Ninety patients with LVDD were randomly divided into the conventional treated group (Group A, treated by conventional treatment with western drugs of cardiotonic, diuretic, coronary dilator, etc.) and the Chinese drug treated group (Group B, treated by conventional treatment plus MSL 2 times a day, 100 ml each time), 45 in each group. After 4 months treatment, the total heart failure coefficient (HFC) and cardiac functions were re-determined. RESULTS: After treatment, in both groups, the HFC lowered significantly (P < 0.05 or P < 0.01), the left ventricular peak velocity of early diastolic rapid filling (Emas) quickened, the left atrial systolic peak velocity (Amas) slowed down and Emas/Amas (E/A) enhanced, the isovolumetric relaxation time shortened. However, comparison between the two groups showed significant difference (P < 0.05) in either item, Group B was superior to Group A (P < 0.05). In the 62 patients with mixed heart failure, i.e. both systolic and diastolic dysfunction of left ventricle, Group B was superior to Group A in increasing ejection fraction, cardiac output and thickening rate of left ventricular posterior wall (P < 0.05). CONCLUSION: MSL could improve the heart function of patients with LVDD, and alleviate their clinical symptoms.