大鼠骨髓基质干细胞体外培养方法的实验研究

目的研究培养骨髓基质干细胞(MSCs)的简易方法和其在体外经5-氮胞苷诱导为心肌细胞,为心肌梗死后心力衰竭的干细胞移植治疗提供理想的种子细胞。方法采用传统的密度梯度离心分离加贴壁法和略加改进的Wakitani方法。具体操作是:取SD大鼠分离出股骨和胫骨,用10%DMEM培养基冲出骨髓液,以2×105/ml密度接种于培养瓶中,培养出的骨髓基质干细胞连续传3代,使细胞纯化后,用5-氮胞苷诱导,观察细胞形态改变。结果大鼠骨髓液接种于培养瓶中后,第2天大部分细胞已经贴壁,贴壁生长的细胞以分散、克隆集落的方式增殖,细胞大多呈梭形,7～10d后克隆集落逐渐增多,细胞融合,传代培养后细胞呈分布均匀的纺锤形细胞生长。MSCs经10!mol/L5-氮胞苷诱导3周后,发现细胞形态较前更加细长,呈梭形,类似心肌细胞样形态。结论应用略加改进的Wakitani方法,能较好地进行骨髓基质干细胞的培养和扩增,且操作简便;在体外培养的骨髓基质干细胞经诱导可转化为心肌细胞,有望成为心肌梗死后心力衰竭干细胞移植治疗的细胞材料。

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.

Effects of IQ_23 on the Repolarization and Both Components of the Delayed Rectifier Potassium Current in Guinea Pig Ventricular Myocytcs

Using patch clamp whole cell recording techiques, we examined the effects ofIQ_23, a benzyl-isoquinoline derivative with antiarrhythmic activities, on the action potential (AP) andpotassium currents in single guinea pig ventricular myocytes. The results showed that IQ_23 at 10, 30and 100 μmol ·L_-1 slowed the repolarization in AP dose-dependently. The APD_90 were prolonged by15%, 28% and 31% respectively. This effect did not depend on the extracellular Ca^2+. In voltageclamp mode, IQ_23 effectively blocked both the components of the delayed rectifier potassium current(I_k), i.e., I_ks and I_kr. At concentrations of 30 and 100 μmol· L^-1, IQ_23 suppressed I_ks by 21% and 26%and suppressed I_kr by 67% and 86% respectively. But even at 100 μmol·L^-1, IQ_23 had little effect onthe inward rectifier potassium current (I_k1). It is concluded: 1. IQ_23 can dose-dependently prolongAPD in the ventriculas myocytes of guinea pig, the effect does not depend on the extracellular Ca^2+; 2.IQ_23 blocks both I_ks and Ikr in the ventricular myocytes without obvious specificities between them.

Biological Characteristics of Endothelial Progenitor Cell and Its Influence on Angiogenesis Improvement

Endothelial progenitor cell （EPC） is a term that refers to multiple cell types that play roles in the regeneration of the endothelial lining of blood vessels. The EPCs in bone marrow will participate in the internal circulation in a body sub- jected to the stimulation by external factors such as injury, ischemia or drug. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels. Therefore, this paper reviews the sources, isolation and culture of EPCs, the factors influencing the proliferation and activity of EPCs, and the roles of EPCs in angiogenesis.

The Effect of Benzyltetrahydropalmatine (BTHP) on Action Potentials and the Two Components of Delayed Rectifying Potassium Currents in Guinea Pig Ventricular Myocytes

The effects of BTHP on Ca 2+ independent action potential and the two components of delayed rectifier potassium currents were studied in guinea pig single ventricular myocytes by using whole cell patch clamp technique. BTHP 30 μmol·L -1 significantly prolonged APD 90 from 143±16 ms to 184±21 ms ( P 【0.01, n=5) without affecting either the RP or APA, and the APD prolonging effects of BTHP were independent of extracellular Ca 2+ . BTHP inhibited both I kr (IC 50 =7 9 μmol·L -1 ) and I ks (IC 50 =22 4 μmol·L -1 ) in a concentration dependent fashion. The results demon strated that BTHP had no obvious selectivity for I kr and I ks .

The role and modulation of autophagy in experimental models of myocardial ischemia-reperfusion injury

A physiological sequence called autophagy qualitatively determines cellular viability by removing protein aggregates and damaged cyto-plasmic constituents, and contributes significantly to the degree of myocardial ischemia-reperfusion （I/R） injury. This tightly orchestrated cata-bolic cellular‘housekeeping’ process provides cells with a new source of energy to adapt to stressful conditions. This process was first described as a pro-survival mechanism, but increasing evidence suggests that it can also lead to the demise of the cell. Autophagy has been implicated in the pathogenesis of multiple cardiac conditions including myocardial I/R injury. However, a debate persists as to whether autophagy acts as a protec-tive mechanism or contributes to the injurious effects of I/R injury in the heart. This controversy may stem from several factors including the va-riability in the experimental models and species, and the methodology used to assess autophagy. This review provides updated knowledge on the modulation and role of autophagy in isolated cardiac cells subjected to I/R, and the growing interest towards manipulating autophagy to increase the survival of cardiac myocytes under conditions of stress-most notably being I/R injury. Perturbation of this evolutionarily conserved intracellular cleansing autophagy mechanism, by targeted modulation through, among others, mammalian target of rapamycin （mTOR） inhibitors, adenosine monophosphate-activated protein kinase （AMPK） modulators, calcium lowering agents, resveratrol, longevinex, sirtuin activators, the proapoptotic gene Bnip3, IP3 and lysosome inhibitors, may confer resistance to heart cells against I/R induced cell death. Thus, therapeutic ma-nipulation of autophagy in the challenged myocardium may benefit post-infarction cardiac healing and remodeling.

MicroRNA-15a/b are up-regulated in response to myocardial ischemia/reperfusion injury

Objective Several studies have indicated that miR-15a,miR-15b and miR-16 may be the important regulators of apoptosis.Since attenuate apoptosis could protect myocardium and reduce infarction size,the present study was aimed to find out whether these miRNAs participate in regulating myocardial ischemia reperfusion （I/R） injury.Methods Apoptosis in mice hearts subjected to I/R was detected by TUNEL assay in vivo,while flow cytometry analysis followed by Annexin V/PI double stain in vitro was used to detect apoptosis in cultured cardiomyocytes which were subjected to hypoxia/reoxygenation （H/R）.Taqman real-time quantitative PCR was used to confirm whether miR-15a/15b/16 were involved in the regulation of cardiac I/R and H/R.Results Compared to those of the controls,I/R or H/R induced apoptosis of cardiomyocytes was significantly iucreased both in vivo （24.4％ ± 9.4％ vs.2.2％ ± 1.9％,P ＜ 0.01,n =5） and in vitro （14.12％ ±0.92％ vs.2.22％ ± 0.08％）.The expression of miR-15a and miR-15b,but not miR-16,was increased in the mice I/R model,and the results were consistent in the H/R model.Conclusions Our data indicate miR-15 and miR-15b are up-regulated in response to cardiac I/R injury,therefore,down-regulation of miR- 15a/b may be a promising strategy to reduce myocardial apoptosis induced by cardiac I/R injury.

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.

Effect of Fufang Danshen Pill on Bone Marrow Stem Mobilization when Myocardial Scathe

Objective：To investigate the effect of Fufang Danshen pill on bone marrow stem mobilization during myocardial scathe. Methods：Rat models with expansionary myocardial disease were established by Pituitrin and Furazolidone. Experimental rats were divided into the contrast group, the myocardial scathe group （MS group）, the myocardial scathe and Fufang Dansben pill group （ MS ＋ FD group） and the myocardial scathe and fluvastatin group （ MS ＋ FT group）. The ratio of CD34^＋ cells was examined at the 1^st, 3^nl and 6^th weekend. Index of heart structure and function including LVESD, LVEDD. LYEF, LVEDP and dp/dtmax were evaluated at the 6^th weekend. The HW/BW index was calculated. Results：In the MS group, the index of HW/BW, LVESD, LVEDD and LVEDP were obviously increased （P 〈 0.01 ） and index of dp/ dtmax and LVEF were obviously decreased （P 〈 0.05 ）. The ratio of CD34^＋ cells was significantly improved at the 1^at weekend and then reduced slowly with no difference from that of the contrast group at the 6th weekend. Compared the MS ＋ FD group and the MS ＋ FT group with the MS group, the index of HW/BW, LYESD, LYEDD and LYEDP of were signifi cantly decreased （ P 〈 0.05 ） and index of dp/dtmax and LVEF were increased （P 〈 0.01 ）. The ratio of CD34^＋ cells was significantly higher at the 1^st, 3^nl and 6^th weekend, but had no statistic meaning at 3^nl and 6^th weekend （P 〉 0.05 ）. Conclusion：Pituitrin and Furazolidone can be used to establish rat models with expansionary myocardial disease. There has bone marrow stem mobilization during the early period of myocardial scathe. Fufang Danshen pill has effect on improving bone marrow stem mobilization, lightening the expansionary degree of heart and protecting the heart function. The effect of Fufang Danshen pill is as same as that of fluvastatin.

Effects of IQ23 on the Repolarization and Both Components of the Delayed Rectifier Potassium Current in Guinea Pig Ventricular Myocytcs

Using patch clamp whole cell recording techiques, we examined the effects ofIQ23, a benzyl-isoquinoline derivative with antiarrhythmic activities, on the action potential （AP） andpotassium currents in single guinea pig ventricular myocytes. The results showed that IQ23 at 10, 30and 100 μmol ·L-1 slowed the repolarization in AP dose-dependently. The APD90 were prolonged by15%, 28% and 31% respectively. This effect did not depend on the extracellular Ca2+. In voltageclamp mode, IQ23 effectively blocked both the components of the delayed rectifier potassium current（Ik）, i.e., Iks and Ikr. At concentrations of 30 and 100 μmol· L-1, IQ23 suppressed Iks by 21% and 26%and suppressed Ikr by 67% and 86% respectively. But even at 100 μmol·L-1, IQ23 had little effect onthe inward rectifier potassium current （Ik1）. It is concluded: 1. IQ23 can dose-dependently prolongAPD in the ventriculas myocytes of guinea pig, the effect does not depend on the extracellular Ca2+; 2.IQ23 blocks both Iks and Ikr in the ventricular myocytes without obvious specificities between them.

Human bone marrow-derived mesenchymal stem cells transplanted into damaged rabbit heart to improve heart function

Objective: The present study was designed to test whether transplantation of human bone marrow-derived mesen- chymal stem cells (hMSCs) in New Zealand rabbits with myocardial infarction can improve heart function; and whether engrafted donor cells can survive and transdifferentiated into cardiomyocytes. Methods: Twenty milliliters bone marrow was obtained from healthy men by bone biopsy. A gradient centrifugation method was used to separate bone marrow cells (BMCs) and red blood cells. BMCs were incubated for 48 h and then washed with phosphate-buffered saline (PBS). The culture medium was changed twice a week for 28 d. Finally, hematopoietic cells were washed away to leave only MSCs. Human MSCs (hMSCs) were premarked by BrdU 72 h before the transplantation. Thirty-four New Zealand rabbits were randomly divided into myocardial infarction (MI) control group and cell treated group, which received hMSCs (MI+MSCs) through intramyocardial injection, while the control group received the same volume of PBS. Myocardial infarction was induced by ligation of the left coronary artery. Cell treated rabbits were treated with 5×106 MSCs transplanted into the infarcted region after ligation of the coronary artery for 1 h, and the control group received the same volume of PBS. Cyclosporin A (oral solution; 10 mg/kg) was provided alone, 24 h before surgery and once a day after MI for 4 weeks. Echocardiography was measured in each group before the surgery and 4 weeks after the surgery to test heart function change. The hearts were harvested for HE staining and immunohistochemical studies after MI and cell transplantation for 4 weeks. Results: Our data showed that cardiac function was significantly improved by hMSC transplan- tation in rabbit infarcted hearts 4 weeks after MI (ejection fraction: 0.695±0.038 in the cell treated group (n=12) versus 0.554±0.065 in the control group (n=13) (P<0.05). Surviving hMSCs were identified by BrdU positive spots in infarcted region and transdifferentiated into cardiomyocytes characterized with a positive cardiac phenotype: troponin I. Conclusion: Transplan- tation of hMSCs could transdifferentiate into cardiomyocytes and regenerate vascular structures, contributing to functional im- provement.

Mechanical stretch induces mitochondria-dependent apoptosis in neonatal rat cardiomyocytes and G_(2)/M accumulation in cardiac fibroblasts

Heart remodeling is associated with the loss of cardiomyocytes and increase of fibrous tissue owing to abnormal mechanical load in a number of heart disease conditions. In present study, a well-described in vitro sustained stretch model was employed to study mechanical stretch-induced responses in both neonatal cardiomyocytes and cardiac fibroblasts. Cardiomyocytes, but not cardiac fibroblasts, underwent mitochondria-dependent apoptosis as evidenced by cytochrome c (cyto c) and Smac/DIABLO release from mitochondria into cytosol accompanied by mitochondrial membrane potential (△ψ_m) reduction, indicative of mitochondrial permeability transition pore (PTP) opening. Cyclosporin A, an inhibitor of PTP, inhibited stretch-induced cyto c release, △ψ_m reduction and apoptosis, suggesting an important role of mitochondrial PTP in stretch-induced apoptosis. The stretch also resulted in increased expression of the pro-apoptotic Bcl-2 family proteins, including Bax and Bad, in cardiomyocytes, but not in fibroblasts. Bax was accumulated in mitochondria following stretch. Cell permeable Bid-BH3 peptide could induce and facilitate stretch-induced apoptosis and △ψ_m reduction in cardiomyocytes. These results suggest that Bcl-2 family proteins play an important role in coupling stretch signaling to mitochondrial death machinery, probably by targeting to PTP. Interestingly, the levels of p53 were increased at 12 h after stretch although we observed that Bax upregulation and apoptosis occurred as early as 1 h. Adenovirus delivered dominant negative p53 blocked Bax upregulation in cardiomyocytes but showed partial effect on preventing stretch-induced apoptosis, suggesting that p53 was only partially involved in mediating stretch-induced apoptosis. Furthermore, we showed that p21 was upregulated and cyclin B1 was downregulated only in cardiac fibroblasts, which may be associated with G_2/M accumulation in response to mechanical stretch.

Review:Cell therapy in congestive heart failure

Congestive heart failure （CHF） has emerged as a major worldwide epidemic and its main causes seem to be the aging of the population and the survival of patients with post-myocardial infarction. Cardiomyocyte dropout （necrosis and apoptosis） plays a critical role in the progress of CHF; thus treatment of CHF by exogenous cell implantation will be a promising medical approach. In the acute phase of cardiac damage cardiac stem cells （CSCs） within the heart divide symmetrically and/or asymmetrically in response to the change of heart homeostasis, and at the same time homing of bone marrow stem cells （BMCs） to injured area is thought to occur, which not only reconstitutes CSC population to normal levels but also repairs the heart by differentiation into cardiac tissue. So far, basic studies by using potential sources such as BMCs and CSCs to treat animat CHF have shown improved ventricular remodelling and heart function. Recently, however, a few of randomized, double-blind, placebo-controlled clinical trials demonstrated mixed results in heart failure with BMC therapy during acute myocardial infarction.

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.

Cardiotrophin-1 promotes cardiomyocyte differentiation from mouse induced pluripotent stem cells via JAK2/STAT3/Pim-1 signaling pathway

Background The induced pluripotent stem cell （iPSC） has shown great potential in cellular therapy of myocardial infarction （MI）, while its application is hampered by the low efficiency of cardiomyocyte differentiation. The present study was designed to investigate the effects of cardiotrophin-1 （CT-1） on cardiomyocyte differentiation from mouse induced pluripotent stem cells （miPSCs） and the underlying mechanisms involved. Methods The optimal treatment condition for cardiomyocyte differentiation from miPSCs was established with ideal concentration （10 ng/mL） and duration （from day 3 to day 14） of CT-1 administration. Up-regulated expression of cardiac specific genes that accounted for embryonic cardiogenesis was observed by quantitative RT-PCR. Elevated amount of a-myosin heavy chain （ct-MHC） and cardiac troponin I （cTn I） positive cells were detected by immunofluorescence staining and flow cytometry analysis in CT- 1 group. Results Transmission electron microscopic analysis revealed that cells treated with CT- 1 showed better organized sacromeric structure and more mitochondria, which are morphological characteristic of matured cardiomyocytes. Western blot demonstrated that CT-1 promotes cardiomyocyte differentiation from miPSCs partly via JAK2/STAT3/Pim-1 pathway as compared with control group. Conclusions These findings suggested that CT-1 could enhance the cardiomyocyte differentiation as well as the maturation of mouse induced pluripotent stem cell derived cardiomyocytes by regulating JAK2/STAT3/Pim-1 signaling pathway.

Regulation of swelling-activated chloride channels in embryonic chick heart cells

Swelling-activated Cl- currents, I(ci,swell), were measured during hyposmotic shock in white Leghorn embryonic chick heart cells using the whole-cell recording of patch-clamp technique. Genistein, an inhibitor of protein tyrosine kinase (PTK), suppressed I(ci,swell), Under isosmotic condition phorbol 12-myristate 13-acetate (PMA), an activator of PKC, elicited the Cl~ current similar to that in hyposmotic solution, whereas hyposmotic shock did not elicit I(ci,swell) in chelerythrine chloride(an inhibitor of PKC)-treated cells. Con-focal microscopy experiments using FITC-phalloidin as a fluorescent label of F-actin showed that the actin network was moved from cortical region of the cell to the center after hyposmotic shock as compared with the image under isosmotic condition. When the cells were treated with cytochalasin B (CB) or cytochalasin D (CD) under isosmotic condition the disruption of the F-actin integrity was observed, and I(ci,Sweii) was not elicited. With combination treatment of CB with PMA, hyposmotic solution could not elicited I(Ci,swell), The results suggested that the role of PTK, probably receptor tyrosine kinase, for regulation of I(ci,sweii) appeared to be at upstream site related to the role of F-actin. Then PKC signal pathway was activated somehow and finally change in the polymerization state of cytoskeleton led to activate the swelling-activated Cl- channels. These results demonstrate clearly that PTK, PKC and F-actin are important factors for regulation of I(Ci,swell) in embryonic chick heart cells as compared with often controversial results reported in different cell types.

Reduced cardiac output is associated with decreased mitochondrial efficiency in the non-ischemic ventricular wall of the acute myocardial-infarcted dog

Cardiogenic shock is the leading cause of death among patients hospitalized with acute myocardial infarction （MI）. Understanding the mechanisms for acute pump failure is therefore important. The aim of this study is to examine in an acute MI dog model whether mitochondrial bio-energetic function within non-ischemic wall regions are associated with pump failure. Anterior MI was produced in dogs via ligation of left anterior descending （LAD） coronary artery, that resulted in an infract size of about 30% of the left ventricular wall. Measurements ofhemodynamic status, mitochondrial function, free radical production and mitochondrial uncoupling protein 3 （UCP3） expression were determined over 24 h period. Hemodynamic measurements revealed a 〉 50% reduction in cardiac output at 24 h post infarction when compared to baseline. Biopsy samples were obtained from the posterior non-ischemic wall during acute infarction. ADP/O ratios for isolated mitochondria from non-ischemic myocardium at 6 h and 24 h were decreased when compared to the ADP/O ratios within the same samples with and without palmitic acid （PA）. GTP inhibition of （PA）-stimulated state 4 respiration in isolated mitochondria from the non-ischemic wall increased by 7% and 33% at 6 h and 24 h post-infarction respectively when compared to sham and pre-infarction samples. This would suggest that the mitochondria are uncoupled and this is supported by an associated increase in UCP3 expression observed on western blots from these same biopsy samples. Blood samples from the coronary sinus measured by electron paramagnetic resonance （EPR） methods showed an increase in reactive oxygen species （ROS） over baseline at 6 h and 24 h post-infarction. In conclusion, mitochondrial bio-energetic ADP/O ratios as a result of acute infarction are abnormal within the non-ischemic wall. Mitochondria appear to be energetically uncoupled and this is associated with declining pump function. Free radical production may be associated with the induction of uncoupling proteins in the mitochondria.

Effects of Matrine on Aconitine-Induced Electrophysiological Changes in Rat Ventricular Myocytes

Aim To explore the reason that the antiarrhythmic effect of the extract oftraditional Chinese medicinal herb, matrine, is weaker than quinidine and verapamil by comparison ofthe effect and efficacy of matrine on various kinds of transmembrane ionic currents with those ofquinidine and verapamil; and to demonstrate the best targets for antiarrhythmic drugs. MethodsWhole-cell patch-clamp techniques were used to record the action potential and ionic currents insingle cells of rat ventricular myocytes. Aconitine was used to induce the changes of ioniccurrents, then study the effects of matrine and quinidine, verapamil on aconitine-induced unbalancedchannel currents and action potential. Results Aconitine 1 μmol·L^(-1) induced significantchanges in transmembrane currents and action potential in single cells of rat ventricular myocytes.APD was significantly prolonged by aconitine. Simultaneously, aconitine increased sodium, L-typecalcium and inward rectifier potassium currents. Matrine 100 μmol· L^(-1) reversed theaconitine-induced changes of sodium current (I_(Na)) from (-70.2+- 10.5) pA/pF to ( - 39.6+-4.0)pA/pF(n = 5, P < 0.05 vs aconitine); L-type calcium current (I_(Ca-L)) from (20.4+- 3.8) pA/pF to (- 12.9+- 2.9) pA/pF ( n = 6, P < 0.01); the inward rectifier potassium current (I_(k1) ) from (-32.2+- 1.08) pA/pF to ( -24.0+-3.4) pA/pF (n = 6, P < 0.01), and action potential duration. Thereversal effects of quinidine and verapamil on aconitine-induced changes of APD and ionic currentswere more marked than matrine. Conclusion Aco-nitine significantly disturbs the normal equilibriumof ion channels in ventricular myocytes. It induces changes of I_(Na), I_(Ca-L), I_(K1) andprolongation of action potential duration. Matrine at concentration 50 or 100 μmol·L^(-1)statistically significantly suppresses aconitine-induced changes of APD and ionic currents. Thepotency and efficacy of inhibitory effect of matrine are markedly weaker than those of commonly usedverapamil and quinidine.