Reduction of heart rate by chronic beta1-adrenoceptor blockade promotes growth of arterioles and preserves coronary perfusion reserve in postinfarcted heart

Adequate growth of coronary vasculature in the remaining left ventricular (LV) myocardium after myocardial infarction (post-MI) is a crucial factor for myocyte survival and performance. We previously demonstrated that post-MI coronary angiogenesis can be stimulated by bradycardia induced with the ATP-sensitive K(+) channel antagonist alinidine. In this study, we tested the hypothesis that heart rate reduction with beta-blockade may also induce coronary growth in the post-MI heart. Transmural MI was induced in 12-mo-old male Sprague-Dawley rats by occlusion of the left anterior descending coronary artery. Bradycardia was induced by administration of the beta-adrenoceptor blocker atenolol (AT) via drinking water (30 mg/day). Three groups of rats were compared: 1) control/sham (C/SH), 2) MI, and 3) MI + AT. In the MI + AT rats, heart rate was consistently reduced by 25-28% compared with C/SH rats. At 4 wk after left anterior descending coronary ligation, infarct size was similar in MI and MI + AT rats (67.1 and 61.5%, respectively), whereas a greater ventricular hypertrophy occurred in bradycardic rats, as indicated by a higher ventricular weight-to-body weight ratio (3.4 +/- 0.1 vs. 2.8 +/- 0.1 mg/g in MI rats). Analysis of LV function revealed a smaller drop in ejection fraction in the MI + AT than in the MI group ( approximately 24 vs. approximately 35%). Furthermore, in MI + AT rats, maximal coronary conductance and coronary perfusion reserve were significantly improved compared with the MI group. The better myocardial perfusion indexes in MI + AT rats were associated with a greater increase in arteriolar length density than in the MI group. Thus chronic reduction of heart rate induced with beta-selective blockade promotes growth of coronary arterioles and, thereby, facilitates regional myocardial perfusion in post-MI hearts.     

Echocardiographic detection of congestive heart failure in postinfarction rats

In studies of congestive heart failure (CHF) treatment, it is essential to select animals with a similar degree of cardiac dysfunction. However, this is difficult to establish without hemodynamic evaluation in rat postinfarction-induced CHF. This study aimed to diagnose CHF in long-term follow-up postinfarction rats using only echocardiographic criteria through a J-tree cluster analysis and Fisher's linear discriminant function. Two sets of sham and infarcted rats were studied. The first was used to perform cluster analysis and the second to prospectively validate the results. Six months after inducing myocardial infarction (MI), rats were subjected to transthoracic echocardiography. Infarct size was measured by histological analysis. Six echocardiographic variables were used in the cluster analysis: left ventricular (LV) systolic dimension, LV diastolic dimension-to-body weight ratio, left atrial diameter-to-body weight ratio, LV posterior wall shortening velocity, E wave, and isovolumetric relaxation time. Cluster analysis joined the rats into one sham and two MI groups. One MI cluster had more severe anatomical and echocardiographic changes and was called MI with heart failure (MI/HF+, n = 24, infarct size: 42.7 ± 5.8%). The other had less severe changes and was called MI without heart failure (MI/HF-, n = 11, infarct size: 32.3 ± 9.9%; P < 0.001 vs. MI/HF+). Three rats with small infarct size (21.6 ± 2.2%) presenting mild cardiac alterations were misallocated in the sham group. Fisher's linear discriminant function was built using these groups and used to prospectively classify additional groups of sham-operated (n = 20) and infarcted rats (n = 57) using the same echocardiographic parameters. The discriminant function therefore detected CHF with 100% specificity and 80% sensitivity considering allocation in MI/HF+ and sham group, and 100% specificity and 58.8% sensitivity considering MI/HF+ and MI/HF- groups, taking into account pathological criteria of CHF diagnosis. Echocardiographic analysis can be used to accurately predict congestive heart failure in postinfarction rats.     

Electroporation induced by internal defibrillation shock with and without recovery in intact rabbit hearts

Defibrillation shocks from implantable cardioverter defibrillators can be lifesaving but can also damage cardiac tissues via electroporation. This study characterizes the spatial distribution and extent of defibrillation shock-induced electroporation with and without a 45-min postshock period for cell membranes to recover. Langendorff-perfused rabbit hearts (n = 31) with and without a chronic left ventricular (LV) myocardial infarction (MI) were studied. Mean defibrillation threshold (DFT) was determined to be 161.4 ± 17.1 V and 1.65 ± 0.44 J in MI hearts for internally delivered 8-ms monophasic truncated exponential (MTE) shocks during sustained ventricular fibrillation (>20 s, SVF). A single 300-V MTE shock (twice determined DFT voltage) was used to terminate SVF. Shock-induced electroporation was assessed by propidium iodide (PI) uptake. Ventricular PI staining was quantified by fluorescent imaging. Histological analysis was performed using Masson's Trichrome staining. Results showed PI staining concentrated near the shock electrode in all hearts. Without recovery, PI staining was similar between normal and MI groups around the shock electrode and over the whole ventricles. However, MI hearts had greater total PI uptake in anterior (P < 0.01) and posterior (P < 0.01) LV epicardial regions. Postrecovery, PI staining was reduced substantially, but residual staining remained significant with similar spacial distributions. PI staining under SVF was similar to previously studied paced hearts. In conclusion, electroporation was spatially correlated with the active region of the shock electrode. Additional electroporation occurred in the LV epicardium of MI hearts, in the infarct border zone. Recovery of membrane integrity postelectroporation is likely a prolonged process. Short periods of SVF did not affect electroporation injury.     

Inhibition of cyclooxygenase-2 improves cardiac function after myocardial infarction in the mouse

Cyclooxygenase (COX)-2 is expressed in the heart in animal models of ischemic injury. Recent studies have suggested that COX-2 products are involved in inflammatory cell infiltration and fibroblast proliferation in the heart. Using a mouse model, we questioned whether 1). myocardial infarction (MI) in vivo induces COX-2 expression chronically, and 2). COX-2 inhibition reduces collagen content and improves cardiac function in mice with MI. MI was produced by ligation of the left anterior descending coronary artery in mice. Two days later, mice were treated with 3 mg/kg NS-398, a selective COX-2 inhibitor, or vehicle in drinking water for 2 wk. After the treatment period, mice were subjected to two-dimensional M-mode echocardiography to determine cardiac function. Hearts were then analyzed for determination of infarct size, interstitial collagen content, brain natriuretic peptide (BNP) mRNA, myocyte cross-sectional area, and immunohistochemical staining for transforming growth factor (TGF)-beta and COX-2. COX-2 protein, detected by immunohistochemistry, was increased in MI versus sham hearts. MI resulted in increased left ventricular systolic and diastolic dimension and decreased ejection fraction, fractional shortening, and cardiac output. NS-398 treatment partly reversed these detrimental changes. Myocyte cross-sectional area, a measure of hypertrophy, was decreased by 30% in the NS-398 versus vehicle group, but there was no effect on BNP mRNA. The interstitial collagen fraction increased from 5.4 +/- 0.4% in sham hearts to 10.4 +/- 0.9% in MI hearts and was decreased to 7.9 +/- 0.6% in NS-398-treated hearts. A second COX-2 inhibitor, rofecoxib (MK-0966), also decreased myocyte cross-sectional area and interstitial collagen fraction. TGF-beta, a key regulator of collagen synthesis, was increased in MI hearts. NS-398 treatment reduced TGF-beta immunostaining by 40%. NS-398 treatment had no effect on infarct size. These results suggest that COX-2 products contribute to cardiac remodeling and functional deficits after MI. Thus selected inhibition of COX-2 may be a therapeutic target for reducing myocyte damage after MI.     

Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats.

Massive loss of cardiac myocytes after myocardial infarction (MI) is a common cause of heart failure. The present study was designed to investigate the improvement of cardiac function in MI rats after embryonic stem (ES) cell transplantation. MI in rats was induced by ligation of the left anterior descending coronary artery. Cultured ES cells used for cell transplantation were transfected with the marker green fluorescent protein (GFP). Animals in the treated group received intramyocardial injection of ES cells in injured myocardium. Compared with the MI control group injected with an equivalent volume of the cell-free medium, cardiac function in ES cell-implanted MI animals was significantly improved 6 wk after cell transplantation. The characteristic phenotype of engrafted ES cells was identified in implanted myocardium by strong positive staining to sarcomeric alpha-actin, cardiac alpha-myosin heavy chain, and troponin I. GFP-positive cells in myocardium sectioned from MI hearts confirmed the survival and differentiation of engrafted cells. In addition, single cells isolated from cell-transplanted MI hearts showed rod-shaped GFP-positive myocytes with typical striations. The present data demonstrate that ES cell transplantation is a feasible and novel approach to improve ventricular function in infarcted failing hearts.     

The Relative Contribution of Paracine Effect versus Direct Differentiation on Adipose-Derived Stem Cell Transplantation Mediated Cardiac Repair

Background

Recent studies have demonstrated that transplantation of adipose-derived stem cell (ADSC) can improve cardiac function in animal models of myocardial infarction (MI). However, the mechanisms underlying the beneficial effect are not fully understood. In this study, we characterized the paracrine effect of transplanted ADSC and investigated its relative importance versus direct differentiation in ADSC transplantation mediated cardiac repair.

Methodology/Principal Findings

MI was experimentally induced in mice by ligation of the left anterior descending coronary artery. Either human ADSC, conditioned medium (CM) collected from the same amount of ADSC or control medium was injected into the peri-infarct region immediately after MI. Compared with the control group, both ADSC and ADSC-CM significantly reduced myocardial infarct size and improved cardiac function. The therapeutic efficacy of ADSC was moderately superior to ADSC-CM. ADSC-CM significantly reduced cardiomyocyte apoptosis in the infarct border zone, to a similar degree with ADSC treatment. ADSC enhanced angiogenesis in the infarct border zone, but to a stronger degree than that seen in the ADSC-CM treatment. ADSC was able to differentiate to endothelial cell and smooth muscle cell in post-MI heart; these ADSC-derived vascular cells amount to about 9% of the enhanced angiogenesis. No cardiomyocyte differentiated from ADSC was found.

Conclusions

ADSC-CM is sufficient to improve cardiac function of infarcted hearts. The therapeutic function of ADSC transplantation is mainly induced by paracrine-mediated cardioprotection and angiogenesis, while ADSC differentiation contributes a minor benefit by being involved in angiogenesis. Highlights 1 ADSC-CM is sufficient to exert a therapeutic potential. 2. ADSC was able to differentiate to vascular cells but not cardiomyocyte. 3. ADSC derived vascular cells amount to about 9% of the enhanced angiogenesis. 4. Paracrine effect is the major mechanism of ADSC therapeutic function for MI.     

Functional and bioenergetic modulations in the infarct border zone following autologous mesenchymal stem cell transplantation

Preclinical and clinical studies have demonstrated that stem cell transplantation can improve the left ventricular (LV) contractile performance, yet the underlying mechanisms remain unknown. We examined whether mesenchymal stem cell (MSC) transplantation-induced beneficial effects are secondary to paracrine-associated improvements in LV contractile performance, wall stress, and myocardial bioenergetics in hearts with postinfarction LV remodeling. Myocardial contractile function and bioenergetics were compared 4 wk after acute myocardial infarction in normal pigs (n = 6), untreated pigs with myocardial infarction (MI group; n = 6), and pigs receiving autologous MSC transplantation (MI + MSC group; n = 5). A distal occlusion of the left anterior descending coronary artery instigated significant myocardial hypertrophy. Ejection fraction decreased from 55.3 +/- 3.1% (normal) to 30.4 +/- 2.3% (MI group; P < 0.01) and to 45.4 +/- 3.1% (MI + MSC group; P < 0.01 vs. MI). Hearts in the MI group developed severe contractile dyskinesis in the infarct zone and border zone (BZ). MSC transplantation significantly improved contractile performance from dyskinesis to active contraction (P < 0.01 vs. MI). BZ systolic wall stress was severely increased in MI hearts but significantly improved after MSC transplantation (P < 0.01 vs. MI). The BZ demonstrated profound bioenergetic abnormalities in MI pigs; this was significantly improved after MSC transplantation (P < 0.01 vs. MI). Patchy spared myocytes were found in the infarct zone of hearts receiving MSC transplantation but not in control hearts. These data demonstrate that MSC transplantation into the BZ causes significant improvements in myocardial contractile performance and reduction in wall stress, which ultimately results in significant bioenergetic improvements. Low cell engraftment indicates that MSCs did not provide a structural contribution to the damaged heart and that the observed beneficial effects likely resulted from paracrine repair mechanisms.     

Exercise training preserves coronary flow and reduces infarct size after ischemia-reperfusion in rat heart.

The effect of endurance training on the resistance of the heart to left ventricular (LV) functional deficit and infarction after a transient regional ischemia and subsequent reperfusion was examined. Female Sprague-Dawley rats were randomly assigned to an endurance exercise training (Tr) group or a sedentary (Sed) control group. After 20 wk of training, hearts were excised, perfused, and instrumented for assessment of LV mechanical function, and the left anterior descending coronary artery was occluded to induce a transient regional ischemia (1 h) that was followed by 2 h of reperfusion. Throughout much of the regional ischemia-reperfusion protocol, coronary flow rates, diastolic function, and LV developed pressure were better preserved in hearts from Tr animals. During the regional ischemia, coronary flow to myocardium outside the ischemic zone at risk (ZAR) was maintained in Tr hearts, whereas it progressively fell in Sed hearts. On release of the coronary artery ligature, flow to the ZAR was greater in Tr than in Sed hearts. Infarct size, expressed as a percentage of the ischemic ZAR, was significantly smaller in hearts from Tr rats (24 +/- 3 vs. 32 +/- 2% of ZAR, P < 0.05). Mn- and CuZn-SOD protein expression were higher in the LV myocardium of Tr animals (P < 0.05 for both isoforms). Our data indicate that long-term exercise training leads to infarct sparing and better maintenance of coronary flow and mechanical function after ischemia-reperfusion.     

The role of NO in ischemia/reperfusion injury in isolated rat heart

Nitric oxide (NO) is an important regulator of myocardial function and vascular tone under physiological conditions. However, its role in the pathological situations, such as myocardial ischemia is not unequivocal, and both positive and negative effects have been demonstrated in different experimental settings including human pathology. The aim of the study was to investigate the role of NO in the rat hearts adapted and non-adapted to ischemia. Isolated Langendorff-perfused hearts were subjected to test ischemic (TI) challenge induced by 25 min global ischemia followed by 35 min reperfusion. Short-term adaptation to ischemia (ischemic preconditioning, IP) was evoked by 2 cycles of 5 min ischemia and 5 min reperfusion, before TI. Recovery of function at the end of reperfusion and reperfusion-induced arrhythmias served as the end-points of injury. Coronary flow (CF), left ventricular developed pressure (LVDP), and dP/dt(max) (index of contraction) were measured at the end of stabilization and throughout the remainder of the protocol until the end of reperfusion. The role of NO was investigated by subjecting the hearts to 15 min perfusion with NO synthase (NOS) inhibitor L-NAME (100 mmol/l), prior to sustained ischemia. At the end of reperfusion, LVDP in the controls recovered to 29.0 +/- 3.9 % of baseline value, whereas preconditioned hearts showed a significantly increased recovery (LVDP 66.4 +/- 5.7 %, p < 0.05). Recovery of both CF and dP/dt(max) after TI was also significantly higher in the adapted hearts (101.5 +/- 5.8 % and 83.64 +/- 3.92 % ) as compared with the controls (71.9 +/- 6.3 % and 35.7 +/- 4.87 %, respectively, p < 0.05). NOS inhibition improved contractile recovery in the non-adapted group (LVDP 53.8 +/- 3.1 %; dP/dt(max) 67.5 +/- 5.92 %) and increased CF to 82.4 +/- 5.2 %. In contrast, in the adapted group, it abolished the protective effect of IP (LVDP 31.8 +/- 3.1 %; CF 70.3 +/- 3.4 % and dP/dt(max) 43.25 +/- 2.19 %). Control group exhibited 100 % occurrence of ventricular tachycardia (VT), 57 % incidence of ventricular fibrillation (VF) - 21 % of them was sustained VF (SVF); application of L-NAME attenuated reperfusion arrhythmias (VT 70 %, VF 20 %, SVF 0 %). Adaptation by IP also reduced arrhythmias, however, L-NAME in the preconditioned hearts increased the incidence of arrhythmias (VT 100 %, VF 58 %, SVF 17 %). In conclusion: our results indicate that administration of L-NAME might be cardioprotective in the normal hearts exposed to ischemia/reperfusion (I/R) alone, suggesting that NO contributes to low ischemic tolerance in the non-adapted hearts. On the other hand, blockade of cardioprotective effect of IP by L-NAME points out to a dual role of NO in the heart: a negative role in the non-adapted myocardium subjected to I/R, and a positive one, due to its involvement in the mechanisms of protection triggered by short-term cardiac adaptation by preconditioning.     

Pyridostigmine Restores Cardiac Autonomic Balance after Small Myocardial Infarction in Mice

The effect of pyridostigmine (PYR) - an acetylcholinesterase inhibitor - on hemodynamics and cardiac autonomic control, was never studied in conscious myocardial infarcted mice. Telemetry transmitters were implanted into the carotid artery under isoflurane anesthesia. Seven to ten days after recovery from the surgery, basal arterial pressure and heart rate were recorded, while parasympathetic and sympathetic tone (ΔHR) was evaluated by means of methyl atropine and propranolol. After the basal hemodynamic recording the mice were subjected to left coronary artery ligation for producing myocardial infarction (MI), or sham operation, and implantation of minipumps filled with PYR or saline. Separate groups of anesthetized (isoflurane) mice previously (4 weeks) subjected to MI, or sham coronary artery ligation, were submitted to cardiac function examination. The mice exhibited an infarct length of approximately 12%, no change in arterial pressure and increased heart rate only in the 1st week after MI. Vagal tone decreased in the 1^st week, while the sympathetic tone was increased in the 1^st and 4^th week after MI. PYR prevented the increase in heart rate but did not affect the arterial pressure. Moreover, PYR prevented the increase in sympathetic tone throughout the 4 weeks. Concerning the parasympathetic tone, PYR not only impaired its attenuation in the 1^st week, but enhanced it in the 4^th week. MI decreased ejection fraction and increased diastolic and systolic volume. Therefore, the pharmacological increase of peripheral acetylcholine availability by means of PYR prevented tachycardia, increased parasympathetic and decreased sympathetic tone after MI in mice.     

Post-infarct treatment with [Pyr1]-apelin-13 reduces myocardial damage through reduction of oxidative injury and nitric oxide enhancement in the rat model of myocardial infarction

Apelin is a newly discovered peptide that has been recently shown to have cardioprotective effects in the animal model of myocardial infarction (MI) and ischemia/reperfusion (I/R) injuries. The aim of the present study was to investigate the long term cardioprotective effect of [Pyr1]-apelin-13 in the rat model of MI. Male Wistar rats (n = 22) were randomly divided into three groups: (1) sham operated group (2) control MI group and (3) MI treated with apelin (MI-AP group). MI animals were subjected to 30 min of left anterior descending coronary artery (LAD) ligation and 14 days of reperfusion. 24 h after LAD ligation, apelin (10 nmol/kg/day) was administered i.p. for 5 days. Blood sampling was performed at days 1, 3, 5 and 7 after MI for determination of serum changes of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), malondialdehyde (MDA) and nitric oxide (NO). Myocardial infarct size (IS) and hemodynamic function were also measured at the end of the study at day 14. We found out that post infarct treatment with apelin decreases infarct size, serum levels of LDH, CK-MB and MDA and increases heart rate and serum level of NO in the consecutive days, but there were no significant differences in blood pressure in the MI-AP group in comparison with MI. In conclusion, apelin has long term cardioprotective effects against myocardial infarction through attenuation of cardiac tissue injury and lipid peroxidation and enhancement of NO production.     

Cardiac overexpression of A1-adenosine receptor protects intact mice against myocardial infarction

Previous studies have shown that high-level (300-fold normal) cardiac overexpression of A1-adenosine receptors (A1-ARs) in transgenic (TG) mice protects isolated hearts against ischemia-reperfusion injury. However, this high level of overexpression is associated with bradycardia and increased incidence of arrhythmia during ischemia in intact mice, which interfered with studies to determine whether this line of TG mice might also be protected against myocardial infarction (MI) in vivo. For these studies, we therefore selected a line of TG mice that overexpresses the A1-AR at more moderate levels (30-fold normal), which affords cardioprotection in the isolated heart while minimizing bradycardia and arrhythmia during ischemia in intact mice. Wild-type (WT; n = 10) and moderate-level A1-AR TG (n = 10) mice underwent 45 min of left anterior descending coronary artery occlusion, followed by 24-h reperfusion. Infarct size and region at risk were determined by triphenyltetrazolium chloride and phthalo blue staining, respectively. Infarct size (% region at risk) in WT mice was 52 +/- 3%, whereas overexpression of A1-ARs in the TG mice markedly reduced infarct size to 31 +/- 3% (P < 0.05). Furthermore, contractile function (left ventricular ejection fraction) as determined by cardiac magnetic resonance imaging 24 h after MI was better preserved in TG vs. WT mice. Cardiac overexpression of A1-ARs reduces infarct size by 40% and preserves cardiac function in intact mice after MI.     

Reduction of infarct size by intravenous injection of uncultured adipose derived stromal cells in a rat model is dependent on the time point of application

Stem cell therapy is a promising tool to improve outcome after acute myocardial infarction (AMI), but needs to be optimized since results from clinical applications remain ambiguous. A potent source of stem cells is the stromal vascular fraction of adipose tissue (SVF), which contains high numbers of adipose derived stem cells (ASC). We hypothesized that: 1) intravenous injection can be used to apply stem cells to the heart. 2) Uncultured SVF cells are easier and safer when cultured ASCs. 3) Transplantation after the acute inflammation period of AMI is favorable over early injection. For this, AMI was induced in rats by 40min of coronary occlusion. One or seven days after AMI, rats were intravenously injected with vehicle, 5×10(6) uncultured rat SVF cells or 1×10(6) rat ASCs. Rats were analyzed 35 days after AMI. Intravenous delivery of both fresh SVF cells and cultured ASCs 7 days after AMI significantly reduced infarct size compared to vehicle. Similar numbers of stem cells were found in the heart, after treatment with fresh SVF cells and cultured ASCs. Importantly, no adverse effects were found after injection of SVF cells. Using cultured ASCs, however, 3 animals had shortness of breath, and one animal died during injection. In contrast to application at 7 days post AMI, injection of SVF cells 1 day post AMI resulted in a small but non-significant infarct reduction (p=0.35). Taken together, intravenous injection of uncultured SVF cells subsequent to the acute inflammation period, is a promising stem cell therapy for AMI.     

Hyperosmotic pretreatment reduces infarct size in the rat heart

Preconditioning of the heart can be achieved by an ischemia/reperfusion stimulus, but also by stretching of the heart by an acute volume overload. Since manipulations of the extracellular osmolality affects cell size, we hypothesized that hyperosmotic pretreatment of the isolated perfused rat heart could reduce infarct size following regional ischemia (RI). Langendorff perfused rat hearts were subjected to 30 min RI by ligature of the main branch of the left coronary artery followed by 120 min reperfusion (control group). Ischemic preconditioning (IP-5') was achieved by 5 min total global ischemia and 5 min reperfusion prior to RI. Hyperosmotic pretreatment was accomplished by perfusion with a hyperosmotic buffer (600 mOsm/kg H2O by adding mannitol) for 1 min, 2 min or 5 min. At the end of the experiments, the hearts were cut into 2 mm slices, incubated with triphenyltetrazoliumchloride before scanning and computerized for estimation of infarct size. The average infarct size (as percentage of area at risk) in the control group was 42% and was significantly reduced to 16% by ischemic preconditioning and to 17% by 2 min hyperosmotic pretreatment. Neither 1 min nor 5 min hyperosmotic pretreatment reduced infarct size as compared to the controls. The infarct reducing effect of 2 min hyperosmotic pretreatment was not blunted by inhibition of protein kinase C (chelerytrine chloride), the Na+/H+-exchanger (HOE 694) or stretch-activated anion channels (gadolinium chloride). The results indicate that short-lasting hyperosmotic perturbations of the extracellular environment may precondition the heart to a subsequent ischemic insult.     

Nitrate tolerance aggravates postischemic myocardial apoptosis and impairs cardiac functional recovery after ischemia

Objectives: This study examined the effects of nitrate tolerance (NT) on myocardial ischemia reperfusion (MI/R) injury and elucidated the potential mechanisms involved. Furthermore, the effects of GSH on postischemic myocardial apoptosis in NT rats were investigated. Methods and results: Male Sprague–Dawley rats were randomized to receive nitroglycerin (60 μg/kg/h) or saline for 12 h followed by 40 min of MI and 4 h of reperfusion. Myocardial apoptosis, infarct size, nitrotyrosine formation, plasma CK and LDH activity, and cardiac function were determined. MI/R resulted in significant apoptotic cell death, which was further increased in animals with NT. In addition, NT further increased plasma CK and LDH activity, enlarged infarct size, and impaired cardiac functional recovery after ischemia. Myocardial nitrotyrosine, a footprint for cytotoxic reactive nitrogen species formation, was further enhanced in the NT heart after MI/R. Treatment of NT animals with exogenous GSH inhibited nitrotyrosine formation, reduced apoptosis, decreased infarct size, and improved cardiac functional recovery. Conclusion: Our results demonstrate that nitrate tolerance markedly enhances MI/R injury and that increased peroxynitrite formation likely plays a role in this pathologic process. In addition, our results suggest that GSH could decrease peroxynitrite formation and reduce MI/R injury in nitrate tolerant hearts.     

Adaptation in properties of skeletal muscle to coronary artery occlusion/reperfusion in rats

The present study was designed to determine if changes in function and metabolism of heart muscle induce alterations in characteristics of skeletal muscle. We investigated the histochemical and biochemical properties of soleus (SOL) and extensor digitorum longus (EDL) muscles in Wistar rats at the chronic phase after coronary artery occlusion/reperfusion. The size of myocardial infarct region was evaluated using a high resolution pinhole single photo emission computed tomography (SPECT) system. 4 wk after left coronary artery occlusion/reperfusion, the SOL and EDL of hindlimb were dissected out and immersed in isopentane cooled with liquid nitrogen for subsequent histochemical and biochemical analysis. From SPECT imaging, the blood circulation was recovered, but the recovery of fatty acid metabolism was not observed in infarct region of heart. Citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HAD) activities in infarct region of heart were lower in the myocardial infarction (MI, n = 6) group compared with that of age-matched sham-operated (Sham, n = 6) group. In addition, heart muscle hypertrophy caused by the dysfunction in MI group was observed. In skeletal muscle, the atrophy and transition of fiber type distribution in MI group, reported in previous studies of heart failure, were not observed. However, the succinate dehydrogenase (SDH) activity in the slow twitch oxidative (SO) from SOL of MI group decreased by 9.8% and in the fast twitch oxidative glycolytic fibers (FOG), 8.0% as compared with sham group. Capillary density of the SO fibers from SOL of MI group also reduced by 18.5% and in the FOG fibers, 18.2% as compared with Sham group. Decreased capillary density in this study related significantly to decreased SDH activity of single muscle fibers in chronic phase of perfusion after surgical infarction. Our results make it clear that there is a difference in the reaction of skeletal muscle to coronary artery occlusion/reperfusion compared with chronic heart failure. However, our data would support the notion that there is a linkage between the function of heart and physiological properties of skeletal muscle.     

Interstitial purine metabolites in hearts with LV remodeling

The myocardial ATP concentration is significantly decreased in failing hearts, which may be related to the progressive loss of the myocardial total adenine nucleotide pool. The total myocardial interstitial purine metabolites (IPM) in the dialysate of interstitial fluid could reflect the tissue ATP depletion. In rats, postmyocardial infarction (MI) left ventricular (LV) remodeling was induced by ligation of the coronary artery. Cardiac microdialysis was employed to assess changes of IPM in response to graded beta-adrenergic stimulation with isoproterenol (Iso) in myocardium of hearts with post-MI LV remodeling (MI group) or hearts with sham operation (sham group). The dialysate samples were analyzed for adenosine, inosine, hypoxanthine, xanthine, and uric acid. LV volume was greater in the MI group (2.2 +/- 0.2 ml/kg) compared with the sham group (1.3 +/- 0.2 ml/kg, P < 0.05). Infarct size was 28 +/- 4%. The baseline dialysate level of uric acid was higher in the MI group (18.9 +/- 3.4 micromol) compared with the sham group (4.6 +/- 0.7 micromol, P < 0.01). During and after Iso infusion, the dialysate levels of adenosine, xanthine, and uric acid were all significantly higher in the MI group. Thus the level of IPM is increased in hearts with postinfarction LV remodeling both at baseline and during Iso infusion. These results suggest that the decreased myocardial ATP level in hearts with post-MI LV remodeling may be caused by the chronic depletion of the total adenine nucleotide pool.     

Postinfarction healing dynamics in the mechanically unloaded rat left ventricle

The healing process is a key determinant for postinfarction left ventricular (LV) remodeling and the development of heart failure, which could be influenced by mechanical (pressure and/or volume) load. So far, limited information exists regarding an indepth characterization of the postinfarct healing process in the mechanically unloaded state. In the present work, we performed isogenic Lewis-to-Lewis rat abdominal heterotopic heart transplantation, which is characterized by hemodynamic unloading in the left ventricle, and simultaneously ligated the left anterior descending coronary artery (T-infarct group). Pathological evolution was dynamically compared with that of in situ infarcted Lewis hearts (I-infarct group) on days 3, 7, 14, and 35. There was a remarkable myocardial salvage in the unloaded heart, as shown by the improvement in infarct size (T-infarct group: 25.47% ± 4.31% vs. I-infarct group: 38.46% ± 4.82%, P < 0.01) and the smaller fraction of fibrosis in infarct segments (T-infarct group: 42.12% ± 8.40% vs. I-infarct group: 75.65% ± 10.51%, P < 0.01). In addition, there was a progressive disorganization of the two-dimensional collagen fiber alignment as well as retarded collagen fiber maturation in the T-infarct group. We also observed enhanced angiogenesis, lymphangiogenesis, and inflammatory cell retention in the infarct region during mechanical unloading. Moreover, capillary density and collagen deposition were significantly increased in the noninfarcted area of the unloaded heart compared with the same region in the in situ infarcted heart. In conclusion, ischemic insult in the mechanically unloaded heart elicits an altered inflammatory and healing response, which is characterized by myocardial salvage, delayed resolution of inflammation, and disorganization of the collagen orientation in the infarcted region. These findings could provide novel insights into the contribution of hemodynamic load in the postinfarction healing process. Further studies are warranted to elucidate its potential mechanism.     

Metformin improves cardiac function in a nondiabetic rat model of post-MI heart failure

Metformin is the first choice drug for the treatment of patients with diabetes, but its use is debated in patients with advanced cardiorenal disease. Epidemiological data suggest that metformin may reduce cardiac events, in patients both with and without heart failure. Experimental evidence suggests that metformin reduces cardiac ischemia-reperfusion injury. It is unknown whether metformin improves cardiac function (remodeling) in a long-term post-MI remodeling model. We therefore studied male, nondiabetic, Sprague-Dawley rats that were subjected to either myocardial infarction (MI) or sham operation. Animals were randomly allocated to treatment with normal water or metformin-containing water (250 mg·kg(-1)·day(-1)). At baseline, 6 wk, and 12 wk, metabolic parameters were analyzed and oral glucose tolerance tests (OGTT) were performed. Echocardiography and hemodynamic parameters were assessed 12 wk after MI. In the MI model, infarct size was significantly smaller after 12-wk metformin treatment (29.6 ± 3.2 vs. 38.0 ± 2.2%, P < 0.05). Moreover, metformin resulted in less left ventricular dilatation (6.0 ± 0.4 vs. 7.6 ± 0.6 mm, P < 0.05) and preservation of left ventricular ejection fraction (65.8 ± 3.7% vs. 48.6 ± 5.6%, P < 0.05) compared with MI control. The improved cardiac function was associated with decreased atrial natriuretic peptide mRNA levels in the metformin-treated group (50% reduction compared with MI, P < 0.05). Insulin resistance did not occur during cardiac remodeling (as indicated by normal OGTT) and fasting glucose levels and the pattern of the OGTT were not affected by metformin. Molecular analyses suggested that altered AMP kinase phosphorylation status and low insulin levels mediate the salutary effects of metformin. Altogether our results indicate that metformin may have potential to attenuate heart failure development after myocardial infarction, in the absence of diabetes and independent of systemic glucose levels.     

一氧化氮和线粒体ATP敏感性钾通道介导血管紧张素转换酶抑制剂加强阈下预处理的作用

实验采用离体大鼠心脏Langendorff灌流模型,观察含巯基（卡托普利）和不含巯基（培哚普利拉）的两种血管紧张素转换酶抑制剂（angiotensin-converting enzyme inhibitors,ACEI）对抗心肌缺血的作用,并探讨一氧化氮（nitric oxide,NO）和线粒体ATP敏感性钾通道（mimchondrial ATP-sensitive potassium channel,mitoKATP channel）是否参与ACEI的心肌保护作用。结果表明：（1）给予大鼠心脏2min全心停灌和10min复灌作为闽下缺血预处理（subthreshold preconditioning,sPC）、卡托普利或培哚普利拉单独使用,均不能改善长时间缺血复灌（缺血30min＋复灌120min）引起的心肌损伤。（2）当两种ACEI分别和sPC联合使用时,与sPC组相比,缺血心脏在长时间缺血后的复灌期问左室舒张末压（left ventricular end-diastolic pressure,LVEDP）明显降低,左宦发展压（left ventricular developed pressure,LVDP）和冠脉流量明显增高,乳酸脱氢酶（lactate dehydrogenase,LDH）的释放量和心肌梗死面积明显低于sPC组。（3）利用NOS抑制剂L-NAME和mitoKATP通道的抑制剂5-HD灌流10min后,可明显抑制卡托普利／培哚普利拉和sPC联合使用引起的LVEDP降低,并使LVDP和冠脉流量降低,LDH的释放量和心肌梗死面积明显增高（P〈0．05）。（4）sPC、卡托普利或培哚普利拉单独使用,心脏NO的产生增加。ACEI和sPC联合使用,与三者单独使用相比NO的浓度亦明显增高（P〈0．05）。结果提示：含与不含巯基的ACEI与闽下缺血预处理联合使用均可使大鼠心脏功能明显改善,其心肌保护作用的机制可能通过NO途径,并和mitoKATP通道的激活有关。