Porphyromonas gingivalis,a periodontal pathogen,impairs post-infarcted myocardium by inhibiting autophagosome–lysosome fusion

While several previous studies have indicated the link between periodontal disease (PD) and myocardial infarction (MI), theunderlying mechanisms remain unclear. Autophagy, a cellular quality control process that is activated in several diseases, includingheart failure, can be suppressed by Porphyromonas gingivalis (P.g.). However, it is uncertain whether autophagy impairment byperiodontal pathogens stimulates the development of cardiac dysfunction after MI. Thus, this study aimed to investigate therelationship between PD and the development of MI while focusing on the role of autophagy. Neonatal rat cardiomyocytes(NRCMs) and MI model mice were inoculated with wild-type P.g. or gingipain-deficient P.g. to assess the effect of autophagyinhibition by P.g. Wild-type P.g.-inoculated NRCMs had lower cell viability than those inoculated with gingipain-deficient P.g. Thisstudy also revealed that gingipains can cleave vesicle-associated membrane protein 8 (VAMP8), a protein involved in lysosomalsensitive factor attachment protein receptors (SNAREs), at the 47th lysine residue, thereby inhibiting autophagy. Wild-type P.g.-inoculated MI model mice were more susceptible to cardiac rupture, with lower survival rates and autophagy activity thangingipain-deficient P.g.-inoculated MI model mice. After inoculating genetically modified MI model mice (VAMP8-K47A) with wildtype P.g., they exhibited significantly increased autophagy activation compared with the MI model mice inoculated with wild-typeP.g., which suppressed cardiac rupture and enhanced overall survival rates. These findings suggest that gingipains, which arevirulence factors of P.g., impair the infarcted myocardium by cleaving VAMP8 and disrupting autophagy. This study confirms thestrong association between PD and MI and provides new insights into the potential role of autophagy in this relationship.

Hypoxia induces PGC-la expression and mitochondrial biogenesis in the myocardium of TOF patients

PGC-1α, a potent transcriptional coactivator, is the major regulator of mitochondrial biogenesis and activity in the cardiac muscle. The dysregulation of PGC-la and its target genes has been reported to be associated with congenital and acquired heart diseases. By examining myocardium samples from patients with Tetralogy of Fallot, we show here that PGC-1α expression levels are markedly increased in patients compared with healthy controls and positively correlated with the severity of cyanosis. Furthermore, hypoxia significantly induced the expression of PGC-1α and mitochondrial biogenesis in cultured cardiac myocytes. Mechanistic studies suggest that hypoxia-induced PGC-1α expression is regulated through the AMPK signaling pathway. Together, our data indicate that hypoxia can stimulate the expression of PGC-1α and mitochondrial biogenesis in the cardiac myocytes, and this process might provide a potential adaptive mechanism for cardiac myocytes to increase ATP output and minimize hypoxic damage to the heart.

Efficient Gene Delivery to Myocardium with Ultrasound Targeted Microbubble Destruction and Polyethylenimine

The aim of present study was to evaluate the feasibility and efficiency of enhanced green fluorescent protein （EGFP） gene delivery to myocardium in vivo by ultrasound targeted microbubble destruction （UTMD） and polyethylenimine （PEI）. SonoVue/DNA and PEI/DNA/SonoVue complexes were prepared. Gel electrophoresis analysis was performed to determine the structural integrity of plasmid DNA or PEI/DNA after UTMD. Solutions of plasmid DNA, SonoVue/DNA, PEI/DNA complexes or PEI/DNA/SonoVue complexes were respectively transduced into BALB/c mice hearts by means of transthoracic ultrasound irradiation. Mice undergoing PBS injection, plasmid injection or PEI/DNA complexes injection without ultrasound irradiation served as controls. Gene expression in myocardium was detected 4 days after treatment. Cryosections and histological examinations were conducted. Electrophoresis gel assay showed no damage to DNA or PEI/DNA complexes after UTMD. When the heart was not exposed to ultrasound, the expression of EGFP was observed in the subendocardial myocardium obviously. The strongest expression was detected in the anterior wall of the left ventricle when the heart was exposed to ultrasound alone. Injection of PEI/DNA complexes and UTMD resulted in the highest transfection efficiency and the distributional difference of EGFP was not obvious. No tissue damage was seen histologically. In conclusion, a combination of UTMD and PEI was highly effective in transfecting mice hearts without causing any apparently adverse effect. It provides an alternative to current clinical gene therapy and opens a new concept of non-viral gene delivery for the treatment of cardiac disease.

Experimental study on the relationship of bile acids and myocardium damage in obstructive jaundice

AIM To investigate the morphologic changes of the myocardium and its relationship to serum bile acids in obstructive jaundice. METHODS Part Ⅰ: 35 rats were randomly assigned to three groups: Group Ⅰ (BDL1, n =11), the common bile duct (CBD) was ligated and severed and killed after one week. Group Ⅱ (BDL2, n =11), the CBD was ligated and severed and killed after two weeks. Group Ⅲ (SO, n =13), the CBD was simply isolated. The hearts were taken for morphologic studies and blood was taken to determine the total serum bile acids (TAB). Part Ⅱ: 13 rats received gastric intubation of 10% 4ml/kg of sodium cholate, and their serum TBA and the morphologic changes of the heart were examined. RESULTS One to two weeks after the CBD was ligated and severed, the mitochondrium of the myocardium was damaged and the serum TBA obviously increased. When the rats were administered sodium cholate to make their peak blood concentration close to the average blood concentration in BDL2, their myocardium was damaged in a similar degree. CONCLUSION The myocardium was damaged in obstructive jaundice and the endogenous bile acids was one of the factors.

Protective effects of erythropoietin pretreatment on myocardium with hypoxia/reoxygenation injury in rats

Objective: To establish the rat model with myocardial hypoxia/reoxygenation (H/R) injury, and investigate the protective effect of EPO pretreatment on the myocardium. Methods: Sixty male adult Wistar rats were randomly divided into 3 groups: control group, H/R group, and EPO group, 20 in each group. The rats in EPO group accepted injection of 5 000 U/kg recombinant human erythropoietin (RHuEPO) through vein, and the other rats accepted the injection of the same volume of saline. Twenty-four hours after the injection, rats in the EPO and H/R groups were put into the hypoxia environment for 12 h and then returned to the normoxic environment for 2 h, and then the samples of blood and myocardium were collected. Serum myocardial enzyme activity, apoptosis, ultrastructure, myocardial MDA contents, EPO receptor (EPOR) expression in cardiac myocytes and cardiac functions were tested. Results: EPOR expression was positive in cardiac myocytes of adult rat according to the result of immunonistochemitry assaying. Compared to those in H/R group, rats in EPO group presented lighter injury of myocardial ultrastructure, the reduction of serum myocardial enzyme activity, inhibition of apoptosis, the better recovery of cardiac functions, and the less production of oxygen-derived free radicals. Conclusion: Adult rat cardiac myocytes could express EPOR, and EPO pretreatment produced protective effects on myocardium with H/R injury.

Effects of PPAR-y in the Myocardium on the Development of Ventricular Septation

Ventricular septum defects(VSDs)are common types of congenital heart diseases caused by developmental defect;they contribute to 25%-30%of all adult congenital heart diseases.The peroxisome proliferator-activated receptor gamma(PPAR-y)is widely expressed in mammalian tissues and in the immune system,regulating cell differentiation and immune and inflammatory responses.The PPAR-y gene has recently been found crucial for heart development,but the mechanism of action is not clear.This study aims to investigate the effects of the PPAR-y gene in the myocardium on the development of ventricular septation.In this study,we applied Cre-loxP recombination enzyme(CRE)technology to downregulate the expression of the PPAR-y gene in different cardiac tissues,RT-PCR to examine the expression of the c-fos and TGF-B!genes,and histology staining to check the defect of embryonic heart at embryonic day 14.5(E14.5).We found that the downregulation of the PPAR-p gene resulted in a ventricular membranous septation defect of the embryonic heart at E14.5.Furthermore,only conversion of a Tnt:Cre,but not Mef2c:Cre,Tie2:Cre,or Wnt:Cre PPAR-T floxed allele to a null allele resulted in VSD.PPAR-/mi-Orv+embryos showed increascs in atrioventricular(AV)-cushion cells and the expression of c-fos gene but no change in the expression of TGF-B1 at E10.5.Our study demonstrates PPAR-N in the myocardium is required for ventricular septation through regulation of AV-cushion cell proliferation by a Tntc-fos signal.

Chronic stress causes protein kinase C epsilon-aldehyde dehydrogenase 2 signaling pathway perturbation in the rat hippocampus and prefrontal cortex,but not in the myocardium

Chronic stress is strongly associated with the occurrence and development of depression and cardiovascular disease.Stress can induce altered mitochondrial function and activation of apoptosis in the cardio-cerebral system.However,it is unknown whether the protein kinase C ε（PKCε）-aldehyde dehydrogenase 2（ALDH2） pathway is altered under chronic stress,and this study sought to address this question.A rat model of depression was established using a chronic unpredictable mild stress（CUMS） protocol.After experiencing CUMS for 4 weeks,the sucrose preference test and the forced swim test verified depressive-like behaviors.Enzyme linked immunosorbent assays showed that ALDH2 activity was decreased in the rat hippocampus and prefrontal cortex,but was not altered in the myocardium.Western blot assays demonstrated reduced levels of ALDH2 and PKCε,but increased levels of 4-hydroxy-2-nonenal（4 HNE） adducts.Caspase-3 expression did not obviously alter,but active forms of caspase-3 were increased in the hippocampus and prefrontal cortex.In the myocardium,expression of ALDH2,PKCε and 4 HNE adducts did not remarkably alter;while caspase-3 expression was reduced and the active forms of caspase-3 were upregulated.Pearson＇s correlation test demonstrated that expression of 4 HNE adducts was positively correlated with levels of the active forms of caspase-3 in the hippocampus and prefrontal cortex,but not in the myocardium.In conclusion,chronic stress can damage the PKCε-ALDH2 signaling pathway in the hippocampus and prefrontal cortex,but not in the myocardium.Moreover,4 HNE is associated with active forms of caspase-3 in the hippocampus and prefrontal cortex.

Parthenolide attenuates LPS-induced activation of NF-κB in a time-dependent manner in rat myocardium

Parthenolide （PTN）, a selective nuclear factor kappa B （NF-κB） inhibitor, has been used extensively to inhibit NF-κB activation. The duration of the inhibitory effect of PTN on NF-kB in vivo remains unclear. This study was to determine whether a lipopolysaccharide （LPS） challenge 6, 12 and 24 h after the administration of PTN could activate NF-κB. Rats were devided into five groups. The rats in the PTN, PTN＋LPS and DMSO groups were injected intraperitoneally with PTN or DMSO. After 6, 12 or 24 h, LPS was administered in LPS and PTN＋LPS groups. The expressions of NF-κB p50, IκBα and p-IκBα were inhibited in both PTN and PTN＋LPS group at end of 6 and 12 h and no effects at 24 h. In summary, myocardial NF-κB expression occurs 1 h after the administration of LPS. PTN blocks this effect given at 6 h and no inhibitory effect 24 h after administration in vivo.

In vivo mechanical study of helical cardiac pacing electrode interacting with canine myocardium

Cardiac pacing is a medical device to help human to overcome arrhythmia and to recover the regular beats of heart. A helical configuration of electrode tip is a new type of cardiac pacing lead distal tip. The helical electrode attaches itself to the desired site of heart by screwing its helical tip into the myocardium. In vivo experiments on anesthetized dogs were carried out to measure the acute interactions between helical electrode and myocardium during screw-in and pull-out processes. These data would be helpful for electrode tip design and electrode/myocardium adherence safety evaluation. They also provide reliability data for clinical site choice of human heart to implant and to fix the pacing lead. A special design of the helical tip using strain gauges is instrumented for the measurement of the screw-in and pull-out forces. We obtained the data of screw-in torques and pull-out forces for five different types of helical electrodes at nine designed sites on ten canine hearts. The results indicate that the screw-in torques increased steplike while the torque-time curves presente saw-tooth fashion. The maximum torque has a range of 0.3-1.9 Nmm. Obvious differences are observed for different types of helical tips and for different test sites. Large pull-out forces are frequently obtained at epicardium of left ventricle and right ventricle lateral wall, and the forces obtained at right ventricle apex and outflow tract of right ventricle are normally small. The differences in pull-out forces are dictated by the geometrical configuration of helix and regional structures of heart muscle.

Effect of berberine on Cdk9 and cyclin T1 expressions in myocardium of diabetic rats

Objective： To investigate the effect of berberine, one of the main alkaloids of Rhizoma coptidis, on myocardial morphology and the expressions of cyclin-dependent kinase 9 （Cdk9） and cyclin T1 protein in the myocardium of type 2 diabetic rats. Methods： Type 2 diabetes mellitus rats were induced by an injection of 35 mg/kg streptozotocin （STZ） and a high-carbohydrate/high-fat diet for 16 weeks. Diabetic rats were given low-, middle-, high-dose berberine （75, 150, 300 mg/kg）, fenofibrate （100 mg/kg） and rosiglitazone （4 mg/kg） for another 16 weeks, respectively. The myocardium structure was observed with hematoxylin ＆ eosin （H＆E） staining and Cdk9 and cyclin T1 protein expressions were detected by immunohistochemistry. Results： Middle-dose, high-dose berberine improved myocardial hypertrophy and interstitial fibrosis of diabetic rats. Cdk9 and cyclin T1 protein were significantly lower in diabetic myocardium than in control one （P〈0.01）, and middle-dose, high-dose berberine and fenofibrate obviously increased both Cdk9 and cyclin T1 expression to near control level （P〈0.01）. Conclusion： Berberine modulates Cdk9 and cyclin T1 protein expression in diabetic myocardium which may contribute to ameliorate myocardium damage.

IMPLANTATION OF AUTOLOGOUS BONE MARROW MONONUCLEAR CELLS INTO ISCHEMIC MYOCARDIUM ENHANCES CORONARY CAPILLARIES AND SYSTOLIC FUNCTION IN MINISWINE

Objective To investigate the therapeutic effectiveness of intracoronary implantation of autologous bone marrow mononuclear cells (BM-MNC) in miniswine model of reperfused myocardial infarction. Methods Sixteen miniswine myocardial ischemic reperfusion injury models made by ligation of the distal one third segment of left anterior descending artery for 90 minutes were randomized into 2 groups. In BM-MNC group (n = 9), (3.54±0.90)×108 BM-MNC were intracoronary injected, and in the control group (n = 7), phosphate buffered saline was injected by the same way. Echocardiographic and hemodynamic results, vessel density, and myocardial infarction size were evaluated and compared before and 4 weeks after cell transplantation. Results In BM-MNC group, there were no differences between before and 4 weeks after transplantation in aspects of left ventricular ejection fraction (LVEF), interventricular septal thickness, left ventricular lateral and anterior septal wall thickness, cardiac output, or +dp/dtmax. In control group, LVEF, interventricular septal thickness, left ventricular lateral and anterior septal wall thickness, cardiac output, and +dp/dtmax decreased significantly 4 weeks after transplantation (P < 0.05). Left ventricular end-diastolic pressure and –dp/dtmax did not change significantly before and after cell transplantation in both groups. Capillary density in BM-MNC group was greater than that in control group [(13.39 ± 6.96)/high power field vs. (3.50 ± 1.90)/high power field, P < 0.05]. Infarction area assessed by tetrazolium red staining and the infarction percentage decreased in BM-MNC group compared with those in control group (P < 0.05). Conclusions Transplantation of BM-MNC into myocardium with ischemic reperfusion injury increases capillary density and decreases infarction area. It has significantly beneficial effect on cardiac systolic function rather than on diastolic function.

Clinical study of applying fructose-1,6-diphosphate and captopril to enhance the protective effects of cardioplegia solution on ischemic myocardium

In the present experiment,fructose-1,6-diphosphate（FDP）and captopril（Cap）wereadded to the cold potassium cardioplegia solution and the levels of malondialdehyde（MDA）,cre-atine phosphokinase MB（CPK-MB）,thromboxane B（TXB2）and 6-keto-PGF1α in plasma weremeasured during open-heart surgery.Quantitative study of myocardial ultrastructure and obser-vation of cardiac resuscitation were also undertaken.The findings suggested that FDP,especiallywhen combined with Cap could significantly strengthen the protective effects of cold potassiumcardioplegia solution on ischemic myocardium.

Changes of Expression of Stretch-activated Potassium Channel TREK-1 mRNA and Protein in Hypertrophic Myocardium

The expression of stretch activated potassium channel TREK-1 mRNA and protein of hypertrophic myocardium was measured. Using a model of hypertrophy induced by coarctation of abdominal aorta in male Wistar rats, the expression of TREK-1 mRNA and protein was detected by using semi quantitative RT-PCR and Western blot respectively. At 4th and 8th week after constriction of the abdominal aorta , rats developed significant left ventricular hypertrophy. As compared to sham-operated group, stretch-activated potassium channel TREK-1 mRNA was strongly expressed and protein was up regulated in operation groups （P〈0.05）. It was concluded that the expression of TREK-1 was up-regulated in hypertrophic myocardium induced by chronic pressure overload in Wistar rats.

Effect of protective myocardium by allitridum from decreasing apoptosis in rats with ischemia/reperfusion injury

Objective: Previous studies have shown t hat allitridum can protect myocardium from ischemia/reperfusion (I/R) injury, bu t whether allitridum had the effect of anti-apoptosis is unclear. The aim of th is study was to investigate whether allitridum had the effects of pharmacologica l preconditioning and decreasing myocardium apoptosis after ischemic insult. Methods: Pentobarbital sodium-anesthetized Sprague-Dawley (SD) rats underwent 30 min of left anterior descending (LAD) coronary occlusion foll owed by 120 min of reperfusion. Thirty-six rats were divided into three groups randomly: Control group, I/R group and allitridum (G) group. The control and I/R groups with saline, G group with allitridum were administrated 24 h before oper ation. Control group underwent only sham operation; the other two groups underwe nt I/R operation. Infarcted size (IS/AAR %) was measured in I/R and G groups. Ma londialdehyde (MDA), Creatine kinase isoenzyme-MB (CK-MB ), Superoxide dismuta se (SOD)and the apoptosis index (AI) by TUNEL staining were measured in each gro up. In addition, DNA fragmentation by agarose gel electrophoresis was conducted on DNA isolated from these groups. Results: Allitridum p retreatm ent decreased the infarcted size compared with I/R group in IS/ AAR%[(21.85±1. 49)% vs. ( 44.65±4.65)%, P<0.01], CK-MB [(986.40±94.01) vs. (2044.2 5±1 07.28) U/L, P<0.01] and MDA [(3.26±0.35) vs. (4.96±0.46) nmol/mg pro, P<0.01], and SOD level in G group was higher than that of I/R group [(140 .20±12.89)vs. (73.16±11.22) U/mg pro, P<0.01]. AI of I/R group was highe r than that of G group [(13.99±3.05)% vs. (6.97±1.23)%, P<0.01], which was consistent with that in DNA fragmentation by agarose gel electrophoresis. Conclusion: This study indicated that allitridum had the effect of protecting myocardium against I/R injury and decreasing infarcted zone. The e ffect was probably through decreasing myocardium apoptosis in I/R injury.

Synthesis Hexadecyl 3-{4-[2-Hydroxy-3(isopropylamino)propoxy] phenyl}propionate as Potential Ligand for Liposome Targeting to Ischemic Myocardium

Novel hexadecyl 3-{4-[2-hydroxy-3(isopropylamino)propoxy]phenyl}propionate(HPP)was synthesized and its effect on delivery of liposomes into cultured cardiomyocytes was examined.The structure of HPP was characterized by IH NMR,1R and MS.The amount of cardiomyocytes uptake of HPP-liposome was 3.9-fold higher than plain-liposome,and the increase was 6.2-fold when hypoxia happens.It indicated that HPP was a potential ligand for liposome targeting to ischemic myocardium.

STUDY ON LIQUID-LIQUID EXTRACTION OF RESVERATROL FROM GIANT KNOTWEED AND ITS PROTECTIVE EFFECT ON MYOCARDIUM INJURY

Objective An efficient extraction and separation method of resveratrol from a Chinese herb giant knotweed was developed and the protective effect of resveratrol on myocardium injury was investigated.Methods An orthogonal experiment was utilized to optimize the extraction conditions and the pure white crystal obtained utilizing the proposed method was used for the investigation of myocardium ischemic injury.Results Resveratrol was found to have many beneficial activities including the protective effect on the heart and the scavenging of free radical.Conclusion The protective effect of resveratrol on myocardium injury is related to the quenching of lipid peroxidation.

THE EFFECT OF ACUTE MYOCARDIUM ISCHEMIC ON HEART FUNCTION OF PREGNANCY RAT

Objective To investigate the effect of acute myocardium ischemic on heart function of pregnancy rat. Methods 13 female SD rats and 6 early pregnancy rats were divided into normal group, unpregnant group with acute myocardial infarction and early pregnant group with acute myocardial infarction. The anterior branch of the left coronary artery was ligated. 3 weeks later, Image 1.31 software was used to measure areas of myocardial infarction, and to evaluate hemodynamics of heart with powerLAB4.12, and cardiac tissues were stained with Massion. Results Compared with unpregnant group with acute myocardial infarction , the early pregnant group with acute myocardial infarction had less myocardial infarction area (28.86% vs. 36.8%),and had a higher left ventricle end systolic pressure, ±dp/dt _ max , and lower left ventricle end diastolic pressure. Massion stain showed the amount of collagen of the lesion was less in the early pregnant group with acute myocardial infarction than that in unpregnant group. Conclusion The early pregnant group with acute myocardial infarction had better heart contractive and diastolic function .

The Effects of Radix Ginseng Rubra on cAMP of Experimental Ischemic Myocardium in Rats

The model of SD rats ligated at the proximate left anterior descend (LAD) of coronary was used. The number and dissociation constant of β receptor were studied by using receptor autoradiography to observe the changes in β receptor and the effects of Radix Ginseng Rubra on cAMP in experimental ischemic myocardium. The result showed that the number of binding site in simple ligation group (B max =0.279) was obviously higher than that in sham operation group (B max =0.093) and the dissociation constant of simple ligation group (Kd=12.431) was higher than that of sham operation group (k d=1.319). There was a significant difference between the two groups ( P <0.05). It proved that the number of β receptor was increased and the activity was elevated in myocardial cell membranes after ligation of LAD. The myocardial cAMP level in simple ligation group (1293.96±519.36)×10 -3 nmol/g was much higher than that in sham operation group (774.44±210.55)×10 -3 nmol/g ; but the level of cAMP in ligation group after receiving Radix Ginseng Rubra treatment (805.02±362.48 pm/g) was obviously lower than that in simple ligation group ( P <0.01), which was close to the result of sham operation. The results indicated that Radix Ginseng Rubra could decrease the cAMP level in ischemic myocardium.

Study on the Cultivation of the Mycardium and Myocardium Self-disciplining Cell Mass of Mouse Embryonic Bodies with their Own Feeder Layer

Myocardium tissue of Kunming mouse embryonic bodies was cultured with the feeder layer of their embryonic fibroblasts in TCM199.The results indicate that some piece of myocardium tissue can be cultured in the feeder layer;Their contracting frequency changed with the temperature and the morphology changed with the time;It was not all pieces of tissue with the same appearance that could contract,even though some of them grow well.

Intactness of membranous structure in canine stunned myocardium shown with colloidal lanthanum nitrate

A comparative study was performed of the permeability of cell membranous structures in canine stunned myocardium and that of infarct myocardium by using colloidal lanthanum nitrate as an ultrastructure probe. Stunned myocardium was characterized by mitochondrial swelling, vacuolar formation, a few lanthanum granules deposrting inside cells and intact structure of such membranes as cell membrane, mitochondrial membrane and lysosome membrane.Infarct myocardium was characterized by a large number of lanthanum granules passing through ruptured cell membrane and depositing in the cytoplasm and mitochondria. These results suggest that membranous structure remained intact during the occurrence of stunned myocardium.