A strategy of retrograde injection of bone marrow mononuclear cells into the myocardium for the treatment of ischemic heart disease

OBJECTIVE: Bone marrow cells implantation (BMI) has been reported to efficiently improve ischemic heart disease. However, BMI strategies are generally invasive. To establish a BMI strategy for ischemic heart disease, we performed implantation of autologous cryopreserved mononuclear cells (MNCs) from bone marrow (BM) retrogradely into the myocardium via the coronary vein in pigs with acute myocardial infarction (AMI) and old myocardial infarction (OMI). METHODS: BM cells were harvested from the pigs' fumurs. MNCs were collected by centrifugation and were cryopreserved. Anterior myocardial infarction was induced by occlusion of the midportion of the left anterior descending coronary artery without surgical intervention. Frozen BM cells were quickly thawed and injected retrogradely via the coronary vein into the myocardium through a single balloon infusion catheter 6 h and 2 weeks after the induction of infarction. Four weeks after implantation, coronary arteriograms were obtained, cardiac function was analyzed with the use of a conductance catheter, and histopathologic analysis was performed with a confocal laser microscope. Plasma levels of natriuretic peptides and angiogenic growth factors were measured after BMI. RESULTS: Flow cytometric analysis revealed that 90% of cryopreserved BM cells were viable in vitro. Labeled BM cells were entirely distributed around in the infarcted area of maycardium in pigs. BMI increased collateral neovascuralization in infarcted hearts. BMI significantly improved cardiac function in AMI with BMI and OMI with BMI groups. BMI also increased the formation of microcapillary arteries in infarcted hearts. Levels of natriuretic peptides were significantly decreased, and levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2) were significantly increased after BMI. Confocal laser microscopy revealed the presence of proliferative and activated myocardial cells in infarcted hearts after BMI. CONCLUSION: The retrograde infusion of cryopreserved BM cells into myocardium efficiently induced angiogenesis and improved cardiac function in pigs with AMI or OMI. These results suggest that the present strategy of BMI will be safe and feasible as an angiogenic cell therapy for ischemic heart disease.     

Regulation of the isozymes of protein kinase C in the surviving rat myocardium after myocardial infarction: distinct modulation for PKC-alpha and for PKC-delta

Objective The goal of this study was to clarify the regulation of the isozymes of protein kinase C (PKC) in the process of remodeling after myocardial infarction. Methods An in vivo model of regional myocardial infarction induced by ligation of the left anterior coronary artery in rats was used. Hemodynamic parameters and the heart and lung weights were determined 1 week and 1, 2 and 3 months after operation. In transmural biopsies from the non-ischemic left ventricular wall of the infarcted heart, PKC activity (ELISA) and the expression of its major isozymes, PKC-α, PKC-δ and PKC-ε (Westernblot analysis) were determined. Results As early as one week after myocardial infarction, heart weight and left ventricular enddiastolic pressures were significantly increased. Lung weights increased after 2 – 3 months, indicating progressive pulmonary congestion. The activity of PKC was significantly increased about 1.8-fold after 1 week, decreasing progressively in the later time course. Whereas the expression of PKC-ε did not change, PKC-α was increased after 1 month (157 %) and then returned to baseline values. In contrast, PKC-δ expression was significantly augmented after 2 and 3 months of myocardial infarction (187 %). Conclusions These data demonstrate for the first time that in the remodeling heart after myocardial infarction, a subtype-selective regulation of the PKC isozymes occurs: The upregulation of PKC-α coincides with the development of hypertrophy, whereas the extensive upregulation of PKC-δ outlasts the process of developing hypertrophy and persists in the failing heart. The trigger mechanisms for this newly characterized process remains to be elucidated. Received: 25 October 2001, Returned for revision: 3 December 2001, Revision received: 19 December 2001, Accepted: 20 December 2001     

急性心肌梗塞大鼠心肌非缺血区脂质过氧化增强

结扎大鼠左冠状动脉主干造成急性心肌梗塞(AMI)。AMI后2小时,脂质过氧化(LPO)增强不仅发生于心肌缺血区,也发生于非缺血区及肝脏;AMI后24及72小时,心肌缺血区LPO产物已降至对照组水平以下,而非缺血区LPO产物仍高于对照组。AMI早期LPO增强与AMI所致的应激有关。     

Post-ischemic myocardial fibrosis occurs independent of hemodynamic changes

OBJECTIVES: Myocardial fibrosis is a major component of ventricular remodeling after large myocardial infarction (MI). The present study tests the hypothesis that post-ischemic myocardial fibrosis can occur independent of hemodynamic changes. METHODS: A mouse model of distal left coronary artery ligation was established to induce a small infarct (less than 15% of the left ventricle) in order to avoid significant mechanical overload after permanent myocardial ischemia. Left heart catheterization was performed to evaluate the post-infarct hemodynamics. Tissues from both ischemic and non-ischemic myocardium were examined for mRNA and protein expression at 24, 72 h and 7 days after ligation. RESULTS: Heart/body weight ratio after ligation was increased by approximately 10% over sham control although there is no statistically significant difference in hemodynamic parameters between the two groups. Non-ischemic myocardium distant from the infarct site showed molecular evidence of myocardial fibrosis 72 h and 7 days after ligation. There was marked up-regulation of mRNAs for extracellular matrix (ECM) proteins and their cross-linking enzyme, such as collagens type I, III and VI, and lysyl oxidase. Immunohistochemical study confirmed that the expression of these ECM proteins was significantly increased in the non-ischemic myocardium after 7 days. TGF-beta1 was up-regulated after 72 h in both ischemic and non-ischemic myocardium. CONCLUSIONS: Molecular and histopathological findings demonstrate that abnormal myocardial fibrosis can be induced by a small infarct independent of secondary hemodynamic changes.     

Activation of mitogen-activated protein kinases in the non-ischemic myocardium of an acute myocardial infarction in rats

As one of the signal transduction pathways related to myocardial remodeling, mitogen-activated protein kinases (MAPKs) possibly play an important role in ischemic heart disease, but it is still unknown whether myocardial MAPKs are activated in the non-ischemic region of an acute myocardial infarction (AMI). Therefore, the present study investigated the myocardial activity of extracellular signal-regulated kinases (ERKs), c-Jun NH2 terminal kinases (JNKs) and p38MAPK during the acute phase of an infarction of the rat heart, and measured the geometrical ventricular changes by echocardiography. All MAPKs were significantly activated in the ischemic myocardium (IM), non-ischemic septal wall (SW), and right ventricular wall (RV). Furthermore, the activation patterns of MAPKs differed in each region. The activation of p44ERK, JNKs and p38MAPK in the IM occurred rapidly after myocardial ischemia, followed by those in the SW and RV. The activator protein-1 DNA binding activities of the IM, SW and RV increased significantly at I day after coronary ligation. Echocardiography showed increased SW motion and RV dilatation. In conclusion, this is the first in vivo evidence that myocardial MAPKs are activated in the non-ischemic region of an AMI. Echocardiographic results suggest that acceleration of workload and/or stretch may partially induce the activation of MAPKs.     

急性心肌缺血大鼠诱生型一氧化氮合酶的检测

目的 观察诱生型一氧化氮合酶(inducible nitric oxide sythase,iNOS)蛋白在心肌梗死后早期大鼠心脏的表达变化.方法 将12只大鼠随机分为心肌梗死组和假手术组.采用开胸结扎冠状动脉左前降支的方法建立心肌缺血模型,用免疫组织化学方法检测大鼠心肌梗死后24 h缺血心脏iNOS蛋白颗粒的表达.结果 在大鼠冠状动脉结扎后24h,梗死周围区心肌组织细胞出现大量iNOS蛋白表达,与假手术组相比差异有统计学意义(P＜0.01)假手术组未见iNOS蛋白表达.结论 正常心肌组织无iNOS蛋白表达,心肌梗死后早期缺血心肌组织检测到大量iNOS蛋白表达.     

Tritiated digoxin. XX. Tissue distribution in experimental myocardial infarction

The role and potential hazards of digitalis glycoside administration in acute myocardial infarction remain controversial. We investigated the concentration of tritiated digoxin in normal, ischemic, and infarcted left ventricular myocardium of the dog after ligation of the anterior interventricular coronary artery. The normal homogeneous distribution of tritiated digoxin in the normal canine left ventricle was altered following acute myocardial infarction. The ischemic and infarcted zones exhibited a marked diminution in digoxin concentration. Oxidative phosphorylation determinations confirmed tissue hypoxia in the infarcted zone. The gradient of digoxin concentration between normal, ischemic, and infarcted zones of myocardium may potentiate the development of an arrhythmia in the electrically unstable infarcted myocardium.     

Alterations in cardiac troponin subunits in myocardial infarction

1. Alterations in troponin subunits in myocardial infarction were studied in the dog heart by the analysis of troponin-tropomyosin complex, i.e, native tropomyoin, of total structural proteins in SDS gel electrophoresis, and by the measurement of degree of activation of actomyosin-ATPase by Ca++. 2. At 12 to 24 hours after coronary ligation, reductions in TN-C and tropomyosin were observed followed by a decrease in TN-I at 48 hours. The relative contents of these subunits were the lowest at 72 hours to 7 days falling to less than 10% of those in the non-ischemic myocardium. On the contrary, TN-T was preserved through the course of myocardial infarction. 3. Actomyosin-ATPase activity was increased at 12 to 24 hours after coronary ligation and then reduced rapidly at 24 to 48 hours together with the degradation of myosin. However, the activation of actomyosin-ATPase by Ca++-troponin-tropomyosin was reduced already at 12 hours simultaneously with the reduction in TN-C, and almost completely lost at 48 hours. 4. Troponin subunits and actomyosin-ATPase activity returned to those of the control myocardium at 28 days after coronary ligation indicating the recovery of infarcted tissue.     

心力衰竭大鼠心肌细胞凋亡及Bax蛋白表达的探讨

为了探讨心肌细胞凋亡在心力衰竭中的地位和作用,用结扎冠状动脉方法建立大鼠心力衰竭模型.采用末端脱氧核糖酸转移酶介导的dUTP缺口末端标记(TUNEL)染色法对心肌组织进行原位细胞凋亡检测,发现心衰组心肌细胞核有黄褐颗粒状改变.用SP免疫组化染色,心衰组Bax蛋白在膜及胞浆有阳性表达;而血管紧张素转换酶抑制剂(ACEI)治疗组,心肌凋亡细胞数和Bax蛋白表达明显减少,表明心肌细胞凋亡是心力衰竭发生原因之一,在心衰的发生中起着一定的作用,而ACEI能减轻凋亡的发生.     

High cardiac angiotensin-II-forming activity in infarcted and non-infarcted human myocardium

The aims of this study were to compare human cardiac angiotensin-II-forming activity (AIIFA) between the intact area of control autopsy hearts without cardiac disease (n = 10) and the infarcted or non-infarcted area of autopsy hearts with myocardial infarction (MI, n = 7) and to determine responsible angiotensin-II-forming enzymes. Cardiac total and chymase-dependent AIIFAs were significantly higher in the infarcted and non-infarcted myocardium than those in non-MI heart, while angiotensin-converting enzyme-dependent AIIFA increased only in the infarcted myocardium. The density of chymase antibody-positive mast cells in the non-infarcted area of MI heart correlated positively with total or chymase-dependent AIIFA. Augmented AIIFA was also detected in the left atrium of post-MI hearts. Our results indicated that cardiac angiotensin II formation could be activated in the infarcted as well as in non-infarcted myocardium of the post-MI human heart.     

Studies on pathophysiological significance of sympathetic activity in myocardial infarction.

In an attempt to elucidate the pathophysiological significance of the sympathetic hyperactivity in the acute stage of myocardial infarction, the author observed changes in the urinary excretion of CA, the CA content in the myocardium and the hemodynamics in both clinical and experimental myocardial infarction, and the following were found: 1) In clinical myocardial infarction, the urinary excretion of CA was markedly increased immediately after an attack, and the assay of myocardial specimens form the autopsied patients of acute myocardial infarction revealed that the CA content in the non-infarcted area was lower than that in the infarcted area. 2) In the experiments on rabbits with ligated coronary artery, the increase in cardiac contractility and rise in blood pressure in response to CA was supressed after the ligation of coronary artery. In the early stage of experimental myocardial infarction, the decrease of myocardial CA content in the non-infarcted area was, as in autopsied patients, predominant over the decrease of that in the infarcted area. In the chronic stage (more than one week after the coronary ligation), the CA content in the infarcted area showed further decrease, but in the non-infarcted area it was recovered to the level in the control animals. The uptake of exogenous NA into the non-infarcted area decreased in the acute stage, and in the infarcted area it showed marked decreased in the chronic stage. The urinary excretion of CA was increased in the acute stage of myocardial infarction. 3) The administration of betamethasone suppressed the decrease in the CA content in the myocardium following the ligation of coronary artery. Based on these findings, the author came to a postulation that the sympathetic hyperactivity which is suggested by increased urinary excretion of CA and decreased CA content in the myocardium results from the reasonable biophylactic reaction so as to supplement the cardiac hypofunction derived from myocardial infarction.     

Tumor necrosis factor-alpha at acute myocardial infarction in rats and effects on cardiac fibroblasts

Tumor necrosis factor-alpha (TNF-alpha) biosynthesis by the myocardium in response to several diseases has not been solely associated with activation of the immune system but may also serve as a stress response in the context of neurohumoral gene activation. In this regard, beneficial as well as adverse effects of the cytokine on injured myocardium have been reported. TNF-alpha has been suggested to modulate myocyte and fibroblast cell growth and function. Now, in a rat model of acute myocardial infarction TNF-alpha expression and effects on cardiac fibroblast were determined. TNF-alpha was detected in rat hearts with acute myocardial infarction, parallel to the presence of proliferating fibroblasts, at the border zone of the infarct region, to a lesser degree in the infarct zone and was still present in the surviving myocardium. Similarly, the TNF-alpha mRNA level was, compared to sham-operated heart, higher in the infarct area. In the remote myocardium, a trend to an elevated TNF-alpha mRNA level was observed. TNF-alpha stimulated proliferation and expression of fibronectin in fibroblasts isolated from the infarct, non-infarct-region and sham-operated hearts. Angiotensin II is mitogenic for fibroblasts post-myocardial infarction and effects might be mediated indirectly by TNF-alpha. Addition of a neutralising anti-TNF-alpha antibody inhibited angiotensin II stimulated proliferation of fibroblasts only from the infarcted myocardium. The regional differences in TNF-alpha protein and mRNA levels, parallel to proliferating fibroblasts and proliferative effects may foster the reparative, reactive and adverse post-infarct remodeling of the heart.     

Induction of left ventricular remodeling and dysfunction in the recipient heart following donor heart myocardial infarction: new insights into the pathological role of tumor necrosis factor-alpha from a novel heterotopic transplant-coronary ligation model

BACKGROUND: Neurohormonal and cytokine activation after acute myocardial infarction contribute to cardiac remodeling. This study aimed to examine the effects of tumor necrotic factor (TNF)-alpha and angiotensin II on cardiac remodeling and dysfunction after acute myocardial infarction. METHODS AND RESULTS: We performed isogenic heterotopic cardiac transplantation and simultaneous coronary ligation to produce myocardial infarction in the donor heart, and to evaluate the hearts of both donors and recipients in Lewis rats. The recipients in the ligation group showed significant body-weight loss, hyperthermia, tachycardia, hypotension and leukocytosis at day 7. A significant decrease in left ventricular fractional shortening and + dP/dt, and a significant increase in left ventricular enddiastolic dimension/body weight and left ventricular enddiastolic pressure were also observed in the recipient hearts in the ligation group at day 7. With the exception of the increased perivascular fibrosis, these recipient responses were no longer seen at day 21. TNF-alpha was significantly elevated not only in the plasma but also in the recipient hearts in the ligation group at day 7. In contrast, angiotensin II was significantly increased only in the infarct region of the donor hearts, but not in the plasma. Further, the recipients' transient left ventricular remodeling and dysfunction were completely abolished by the intravenous administration of chimeric TNF-alpha soluble receptor. CONCLUSIONS: We developed a novel heterotopic cardiac transplantation-coronary ligation model capable of inducing myocardial infarction in the absence of downstream hemodynamic effects, and allowing differential quantification of indexes of cardiac remodeling in vivo, such as the local and remote effects of angiotensin II and TNF-alpha on cardiac remodeling. Modification of activated cytokines, such as TNF-alpha induced by cardiac ischemic stress, might be a beneficial strategy for the treatment of cardiac dysfunction and subsequent cardiac remodeling after acute myocardial infarction.     

Different effects of a high-cholesterol diet on ischemic cardiac dysfunction and remodeling induced by coronary stenosis and coronary occlusion

OBJECTIVES: The aim of the study was to assess whether and how the high-cholesterol diet (HCD)-related worsening of heart failure differs between coronary stenosis (CS)-induced myocardial ischemia and coronary occlusion-induced myocardial infarction (MI). BACKGROUND: An HCD, a risk factor for coronary artery disease, also worsens ischemic heart failure. Although accelerated coronary plaque formation may be a cause of this, other mechanism(s), such as its effects through the coronary microcirculation, remain to be clarified. METHODS: In rats fed a normal chow diet or HCD, CS or MI was created surgically, and we assessed left ventricular (LV) function by echocardiography and myocardial inflammation by histopathology. In the CS groups, CS severity by histopathology, myocardial perfusion by microspheres, myocardial protein kinase C (PKC) translocation by Western blotting, and myocardial endothelial nitric oxide (NO) function were also investigated by the in vitro myocardial oxygen consumption method. RESULTS: Coronary stenosis impaired myocardial endothelial NO function and reduced coronary flow reserve, evoking myocardial ischemia, as shown by PKC- activation, myocardial inflammation, fibrosis, cardiac dysfunction, and remodeling. By itself, HCD greatly augmented such CS-induced myocardial abnormalities without modulating the CS severity. Such detrimental effects of HCD were ameliorated by supplying a cofactor of endothelial NO synthase-tetrahydrobiopterin. In contrast, MI-induced heart failure was not aggravated by HCD. CONCLUSIONS: The CS-induced ischemic myocardium seems to be more susceptible to the pro-inflammatory effect of HCD than infarcted myocardium, leading to aggravation of LV dysfunction and remodeling via modification of the coronary circulation downstream of the epicardial CS site, partly through impairment of endothelial NO.     

President lecture. Observations on myocardial ischemia

Several problems in myocardial ischemia are discussed based on our experimental and clinical studies. Histochemical changes of the myocardium of adult mongrel dogs with a coronary artery ligation were as follows: The most noticeable findings were a decrease of phosphorylase activity in the pericapillary region of the infarcted area and an enlargement of the discoloration area due to fusion according to the prolongation of the ligation period. This may be explained by the fact that ischemic process takes place around the pericapillary zone in the early stage due to an arborescent distribution of the vascular system in the myocardium. This is presumed as one of the factors which induces heterogeneity of the ischemic myocardium. The reversibility of the injured myocardium due to ischemia was investigated electron-microscopically. The time limit of the myocardial reversibility from ischemic damage was 30 to 50 min. Characteristic changes of LDH isoenzymes and the pathohistological changes of the myocardium after ligation and reperfusion of the coronary artery were as follows: a) A decrease of the LD5 (H subunit) fraction degeneration of the myocardial fibers, fibrosis of the connective tissue and scar formation took place. b) The minimum value of LD5 (H subunit) was observed in cases with a 7-day ligation in the ligation group and in cases with a 12-hour ligation which was followed by a 7-day reperfusion in the reperfusion group. c) The time to reach the minimum LD5 value differed between these 2 groups. The most probable cause of this difference is the protection of the myocardium by reperfusion. 4) In 24 dogs 20 mCi 99mTc-PYP was injected intravenously one and a half hours before the reopening of the chest. Then, Tc-PYP images were obtained at frontal and left lateral views. Tc-PYP images of each cross-sectional surface were also obtained. In the ligation group, 99mTc-PYP uptake in the ischemic myocardium was demarcated clearly in cases with a 1-7 day ligation. In the reperfusion group, 99mTc-PYP uptake in the ischemic area was clearly noticed in cases with a 12-hour to 6-day ligation, which was followed by a 1- to 7-day reperfusion. When the period of the reperfusion was prolonged, it became more difficult to obtain a positive image. In 14 patients with acute myocardial infarction Tc-PYP scintigrams were studied with reference to their ECG.(ABSTRACT TRUNCATED AT 400 WORDS)     

内皮祖细胞移植治疗大鼠急性心肌梗死的实验研究

目的探讨大鼠内皮祖细胞(EPCs)移植于梗死心肌的增殖分化情况及对心功能的影响。方法分离培养大鼠EPCs,免疫荧光法检测其CD34+、CD133+和Flk-1+的表达。将SD大鼠冠状动脉左前降支结扎制造急性心肌梗死模型后,在梗死心肌处植入DAPI标记的EPCs(实验组)或M 199培养液(对照组)。移植后1周及4周,心脏超声检查心功能,并对梗死区心肌组织进行移植细胞形态学检查及毛细血管密度测定,逆转录聚合酶链反应及酶联免疫吸附测定梗死周边区血管内皮生长因子(VEGF)的表达。结果培养获得EPCs,其表型为CD34+、CD133+和Flk-1+。植入梗死区的EPCs可以分化为血管内皮细胞,实验组梗死心肌处血管密度较对照组明显增高(P<0.01),并且VEGF基因及蛋白表达在移植后1周均较对照组明显增高(P<0.01)。移植后4周,实验组大鼠左心室射血分数及左心室短轴缩短率较对照组明显提高(P<0.01);而移植后1周,两组大鼠超声检测心功能各指标变化不明显。结论同种异体EPCs移植到梗死心肌大鼠缺血心肌能分化为毛细血管内皮细胞,促进梗死后心肌血管新生,改善心功能。     

Studies on the Na+-K+-ATPase in myocardial infarction

Changes in the cardiac sarcolemma in myocardial infarction were studied by both determination of Na+-K+-ATPase activity and SDS gel electrophoretic analysis of sarcolemmal proteins in the canine heart. Ninety minutes after coronary ligation, Na+-K+-ATPase activity in ischemic myocardium was decreased significantly to approximately 36% of that of non-ischemic myocardium, and it remained at the lower level for 28 days. By SDS gel electrophoresis, reduction of the protein band with molecular weight of 111,000, which is suggestive of the main component of ATPase, was observed simultaneously with the reduction of Na+-K+-ATPase activity. These results indicate that ischemia for 90 minutes produces substructural changes in the sarcolemma indicating irreversible myocardial changes.     

Heme oxygenase-1 promotes neovascularization in ischemic heart by coinduction of VEGF and SDF-1

Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with multiple protective functions in cardiovascular systems. Studies have shown that the timely cardiac HO-1 overexpression at acute phase of ischemic infarction (MI) provides protection via its anti-apoptotic and anti-inflammatory effects. Here we demonstrate that a delayed HO-1 transduction mediated by a recombinant adeno-associated virus in ischemic hearts of mice with permanent coronary artery ligation significantly attenuated left ventricular fibrosis and cardiac dysfunctions examined at 4 weeks post MI. HO-1-mediated protection was correlated with enhanced vascularization in the ischemic myocardium. HO-1 gene transfer resulted in a notable increase in the number of c-kit⁺- stem cells recruited to the infarcted area at 10 days after ligation. HO-1-mediated stem cell recruitment was also demonstrated in the heart of non-ischemic mice receiving intravenous infusion of green fluorescent protein-bearing bone marrow stem cells. Additional experiments revealed that vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) were highly induced in HO-1 transduced myocardium. Mononuclear cell infiltration was evident and colocalized with angiogenic factors in the same region. Flow cytometry analysis of the mononuclear cells isolated from HO-1-transduced left ventricles revealed that over 50% of cells expressed CD34, a marker of hematopoietic stem cells and endothelial progenitor cells. VEGF and SDF-1 blockade by neutralizing antibodies significantly attenuated HO-1-mediated neovascularization and protection in infarcted mice. These data suggest that cardiac HO-1 gene transfer post MI provides protection at least in part by promoting neovascularization through inducing angiogenic factors and the recruitment of circulating progenitor/stem cells.