| BackgroundCardiovascular disease still stands on the first place among the diseases influencingmortality in the world. With the development of medical technique, the mortality of various heart diseases, especially the acute myocardial infarction (MI), is decreasing obviously. However, all the methods including drugs, thrombolysis and revascularization are not able to influence the dead myocardium. Many researchers devote to studies in the field of myocardium regeneration and repairing insulted myocardium with stem cells.Orlic et al. reported that after myocardial infarction the local injection of hematopoietic stem cells in the border zone of infarction may induce the regenerationof cardiomyocytes and improve heart function in mice. Similarly, the bone marrow mobilization (BMM) by G - CSF and SCF has the same effects on heart repairing. However, the finding is not testified by other research group and the concept of cardiac regeneration is still in debating. The influences of local microenvironment are believed to be pivotal on the differentiation of stem cells. Given that bone marrow stem cells can engraft in the infarcted heart after myocardial infarction, whether they can differentiate into fibroblasts and promote cardiac fibrosis and scar formation, which is not reported. Moreover, some findings imply that regenerated cardiomyocytes may have arrhythmic potential.Currently, the clinical trials using adult stem cell to treat heart diseases tend to be developed widely, however, some basic issues about stem cells involved are still not clarified. In this study, the mouse model of myocardial infarction and BMM with G - CSF and SCF were used to test whether the bone marrow stem cells can engraft into infarcted or ischemic myocardium and differentiate cells other than blood cells in infarcted heart. The influence of BMM on the heart function and the cardiac electrophysiology were also observed.Materials and Methods1.Animals: 90 CD - 1 wild mice, male, 12 - 14 weeks old and body weight 32 -45 grams; 60 C57/BL6 wild mice,female, 6-8 weeks old and body weight 20 - 26 grams. The transplanted bone marrow donor is EGFP transgenic mouse with the C57/BL6 background. The CD-1 mice and C57/BL6 mice were randomly separated into 2 groups: BMM and control group.2.Spleenectomy, BMM, bone marrow transplantation (BMT) and establishment of mouse model of myocardial infarction: The spleenectomy of mouse was performed 2 weeks before BMM or BMT. Solutions of SCF and rh G - CSF were seriously injected cutaneously (i. c. ) into CD-I mice for 6 days. In order to trace the bone marrow stem cells (BMSCs) , the C57/BL6 mice underwent EGFP labeled bone marrow cells transplantation 2 weeks after spleenectomy, and at least 8 weeks later, BMM was performed for C57/BL6. On day 3 of BMM, all the mice were anesthetized and underwent left anterior descending (LAD) coronary artery, MI was induced.3.Echocardiography and surface electrocardiogram (ECG) recordings; Echocardiography and ECG were performed before and 3 weeks after MI to evaluate the heart function and infarction region. The left ventricle (LV) function and the motion of ventricular wall were measured by LV fractional shortening in M mode recordings at levels of LV apex, papillary muscles and the mid - third of LV. Infarct size was estimated in short - and long - axis views using the circumference of akinetic segments, and by estimating the area of the akinetic zone. Trans - aortic Doppler flow signals were used to assess stroke volume. Cardiac output was calculated from stroke volume and heart rate. 4. Electrophysiological study in isolated hearts: The heart was excised rapidly and the aorta was cannulated. Then the heart was mounted on a vertical Langendorff apparatus. Mouse electrophysiologic catheter was placed in the right atrium and ventricle for atrial and ventricular pacing. A tissue ECG was recorded from Ag AgCl electrodes.Three murine monophasic action potentials (MAPs) were recorded from the right and left ventricular (LV) epicardium. One MAP was positioned within the border zone of the infarct to record afterdepolarizations. Atrial pacing was performed at different pacing cycle lengths to measure steady - state action potential durations (APD). The AV node was ablated and the spontaneous ventricular rhythm was observed for 10 minutes, to provoke ventricular arrhythmias initiated by afterdepolarizations. Thereafter, to test the propensity to reentry programmed ventricular stimulation was performed using up to two premature stimuli (S2, S3) at different pacing cycle lengths (80 140 ms).5.Histology and immuno -fluorescence; After completion of the electrophysiological procedure, hearts were fixed and stained using Goldner's Trichrom- staining and immuno - fluorescence technique. Sections were examined under a confocal fluorescence microscope. For quantification of different cell populations , the number of EGFP - positive cells and of EGFP - positive cells co - localizing the immunohistological marker (anti - CD45 or anti - troponin T / anti-β- MHC / antiα-actinin / anti - CD31 / anti - vWF / anti - SMα- actin antibody / anti - fibroblast serum) was determined. Cell populations were counted in 3 randomly selected fields of view at the same magnification (400×) in the infarcted area, the border - zone and in the non - infarcted area of the left ventricle.6. Statistic analysis. All data were expressed by SE. Treatment groups were compared using unpaired t - test or one - way ANOVA. Chi square test and log rank test were used to compare nominal parameters. P <0. 05 was defined as significant difference. Results1. The influence of BMM on murine mortality after MI; Total 124 mice survived to the date of MI induction. 70 mice were dead after MI, within 24 hours, the total mortality is 56.45%. The mortality of BMM and control CD-I mice were 51. 16% ,51. 61% ; 82. 61% BMM and 48. 15% control C57/BL6 mice were dead after MI (P < 0.05), respectively.2. The influence of BMM on the scope of infarction and hemodynamics: the ligation of LAD induced abnormality of LV anterior wall motion and decreasing of LV fractional shortening seen in echocardiography, typical ECG changes of anterior wall MI and histological changes: some LV myocardial tissues were replaced by scar tissue. There is no significant difference on the scope of MI and LV contractility between BMM group and control group, however, the cardiac output were higher in BMM mice than in control mice.3. Engraftment and differentiation of BMSGs in murine infarcted heart; Most EGFP positive cells were localized in infarction or border zone. Compared with control group, BMM increased the engraftment of BMDCs into infarction and border zone of murine heart (P < 0. 05). About 99% EGFP positive cells also expressed CD45 antigen, implying their white blood cell (WBC) phenotype. Only 1-3 EGFP and troponin T double positive cells were found in the infarcted area and border zone independent on BMM or control mice, whereas in non - ischemic area 0. 1 - 5 de novo differentiated cardiomyocytes per section can be found. A few EGFP positive fibroblast and vessel endothelial cells were detected in non - ischemic area rather than in infarcted area.4. The influence of BMM on cardiac electrical steadiness: after the blockage of AV node, no significant difference was found in afterdepolarization induced spontaneous arrhythmia between BMM and control group. APD were not prolonged in BMM mice. Programmed ventricular stimulation provoked less ventricular tachycardia (VT) in BMM mice: S1S2 induced VT in 11/25 (44%) BMM hearts vs. 18/26 (69% ) controls; S1S2S3 induced VT in 5/7 (71% ) BMM vs. 8/8 (100% ) controls, p < 0.05. Two thirds of arrhythmia episodes were polymorphic.DiscussionIn the mouse model of myocardial infarction, BMM can promote the engraftment of bone marrow derived cells (BMDCs) into infarcted myocardium, however, the most new differentiated cells engrafted in infarcted or non - infarcted area were white blood cells, only a few cardiomyocyte or myocardial - like cells and vessel endothelial cells were derived from BMSCs. Although fibroblasts are the main component of cells in the infarcted area, they do not come from BMSCs because all of these cells are EGFP negative, only a few fibroblasts in non - ischemic area was derived from BMSCs. The finding implies that the BMSCs do not take part in the pathological process of scar formation. Except white blood cells, other BMDCs can be rarely found, suggesting that the differentiating potential and survival ability of BMSCs may be influenced in the infarcted area because of shortage of blood supply.BMM improved the function of infarcted heart and enhanced cardiac output to some extent in this study, despite the failure of BMM to induce regeneration of cardiomyocytes. AV node was blocked to provoke afterdepolarization, it was not found that the BMM mice have tendency of increasing spontaneous arrhythmia and afterdepolarization. The decreased inducibility of malignant ventricular arrhythmia and shortage of APD imply that the increasing engraftment of BMDCs in infarcted heart may have some anti - arrhythmic effects.It was found that BMM may increase the mortality of MI in C57/B16 mice, whereas the mortalities of CD - 1 mice are similar in both BMM and control group. The possible reasons are that the C57/B16 mice underwent more treatments , eapecially the lethal FFR - ray irradiation. BMM enhanced the engraftment of WBCs in the infarct region, thereby worsened the inflammation of this area. A recent clinical trial reported that BMM by G - CSF with or without coronary arterial injection of hematopoietic stem cells isolated from autologous peripheral blood improved heart function of MI patients, however, the higher rate of restenosis after percutaneous coronary intervention was accompanied. This result, combined with the finding of higher mortality of MI mice in the present study, suggests that the clinical trial using BMM to treat MI should be developed prudently.Conclusion1.BMM with G - CSF and SCF can promote the BMDCs engraftment into infarcted region of mouse heart, and most of these cells are WBCs; under some conditions, BMM may increase the mortality of MI mouse.2. BMM induces cardiomyocyte regeneration to the least extent, which is not significant for the cardiac repair and improvement of heart function.3. BMSCs can differentiate into vessel endothelial cells and fibroblasts, however, they do not take part in the process of scar formation.4.BMM did not increase the incidence of ventricular arrhythmia in infarcted mice, and decreased re - entry related VT.
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