Xue-Hai Chen

Acceleration of the Healing Process and Myocardial Regeneration May Be Important as a Mechanism of Improvement of Cardiac Function and Remodeling by Postinfarction Granulocyte Colony–Stimulating Factor Treatment

Background—We investigated whether the improvement of cardiac function and remodeling after myocardial infarction (MI) by granulocyte colony–stimulating factor (G-CSF) relates to acceleration of the healing process, in addition to myocardial regeneration. Methods and Results—In a 30-minute coronary occlusion and reperfusion rabbit model, saline (S) or 10 μg · kg−1 · d−1 of human recombinant G-CSF (G) was injected subcutaneously from 1 to 5 days after MI. Smaller left ventricular (LV) dimension, increased LV ejection fraction, and thicker infarct-LV wall were seen in G at 3 months after MI. At 2, 7, and 14 days and 3 months after MI, necrotic tissue areas were 14.2±1.5/13.4±1.1, 0.4±0.1/1.8±0.5*, 0/0, and 0/0 mm2 ·· slice−1 · kg−1, granulation areas 0/0, 4.0±0.7/8.5±1.0*, 3.9±0.8/5.7±0.7,* and 0/0 mm2 · slice−1 kg−1, and scar areas 0/0, 0/0, 0/0, and 4.2±0.5/7.9±0.9* mm2 slice−1 kg−1 in G and S, respectively (*P <0.05, G versus S). Clear increases of macrophages and of matrix metalloproteinases (MMP) 1 and 9 were seen in G at 7 days after MI. This suggests that G accelerates absorption of necrotic tissues via increase of macrophages and reduces granulation and scar tissues via expression of MMPs. Meanwhile, surviving myocardial tissue areas within the risk areas were significantly increased in G despite there being no difference in LV weight, LV wall area, or cardiomyocyte size between G and S. Confocal microscopy revealed significant increases of cardiomyocytes with positive 3,3,3′3′-tetramethylindocarbocyanine perchlorate and positive troponin I in G, suggesting enhanced myocardial regeneration by G. Conclusions—The acceleration of the healing process and myocardial regeneration may play an important role for the beneficial effect of post-MI G-CSF treatment.

The role of serotonin in ischemic cellular damage and the infarct size-reducing effect of sarpogrelate, a 5-hydroxytryptamine-2 receptor blocker, in rabbit hearts.

OBJECTIVE We aimed to clarify the relation between sarpogrelate (SG), a 5-hydroxytryptamine (5-HT)-2 receptor blocker, and myocardial interstitial serotonin or infarct size during ischemia and reperfusion. BACKGROUND In cardiac tissues serotonin is rich in vascular platelets, mast cells, sympathetic nerve endings, and the receptors are present in platelets and cardiomyocytes. METHODS The myocardial interstitial serotonin levels were measured using a microdialysis technique during 30-min ischemia with and without SG in in vivo as well as isolated rabbit hearts. Other rabbits underwent 30 min of ischemia and 48 h of reperfusion, and the effect of SG on the infarct size was investigated in the absence and presence of a selective protein kinase C (PKC) inhibitor, chelerythrine (5 mg/kg, intravenously), or a mitochondrial adenosine triphosphate sensitive potassium (KATP) channel blocker, 5-hydroxydecanoate (5-HD) (5 mg/kg, intravenously). In another series, the effect of SG on PKC isoforms in cytosol and membrane fraction was assessed after a 20-min global ischemia in isolated rabbit hearts. RESULTS Interstitial serotonin levels were markedly increased during 30-min ischemia in in vivo and isolated hearts, and the increases were inhibited by SG in each. The infarct size was reduced by SG (27 +/- 2% vs. 40 +/- 3% of control). This effect was blocked by chelerythrine and 5-HD, respectively. Sarpogrelate further enhanced the ischemia-induced translocation of PKC-epsilon to the membrane fraction. CONCLUSIONS Sarpogrelate reduces the myocardial infarct size by inhibiting the serotonin release followed by enhancement of PKC-epsilon translocation and opening of the mitochondrial KATP channel in ischemic myocytes.

Local overexpression of HB-EGF exacerbates remodeling following myocardial infarction by activating noncardiomyocytes

Insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) are cardiogenic and cardiohypertrophic growth factors. Although the therapeutic effects of IGF and HGF have been well demonstrated in injured hearts, it is uncertain whether natural upregulation of HB-EGF after myocardial infarction (MI) plays a beneficial or pathological role in the process of remodeling. To answer this question, we conducted adenoviral HB-EGF gene transduction in in vitro and in vivo injured heart models, allowing us to highlight and explore the HB-EGF-induced phenotypes. Overexpressed HB-EGF had no cytoprotective or additive death-inducible effect on Fas-induced apoptosis or oxidative stress injury in primary cultured mouse cardiomyocytes, although it significantly induced hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. Locally overexpressed HB-EGF in the MI border area in rabbit hearts did not improve cardiac function or exhibit an angiogenic effect, and instead exacerbated remodeling at the subacute and chronic stages post-MI. Namely, it elevated the levels of apoptosis, fibrosis, and the accumulation of myofibroblasts and macrophages in the MI area, in addition to inducing left ventricular hypertrophy. Thus, upregulated HB-EGF plays a pathophysiological role in injured hearts in contrast to the therapeutic roles of IGF and HGF. These results imply that regulation of HB-EGF may be a therapeutic target for treating cardiac hypertrophy and fibrosis.

Cilnidipine, an N+L-Type Dihydropyridine Ca Channel Blocker, Suppresses the Occurrence of Ischemia/Reperfusion Arrhythmia in a Rabbit Model of Myocardial Infarction

Dihydropyridine Ca channel blockers are widely prescribed for the treatment of hypertension and coronary artery diseases, but it remains unknown whether these agents protect against arrhythmias. We investigated whether cilnidipine, an N+L-type Ca channel blocker, reduces the incidences of ventricular premature beats (VPBs) and, if so, via what mechanisms. Japanese white rabbits underwent 30 min of ischemia and 48 h of reperfusion. Cilnidipine (0.5 or 1.0 μg/kg/min, i.v.) or saline (i.v.) was administered from 30 min before ischemia to 30 min after reperfusion. Electrocardiogram and blood pressure were monitored and the incidences of VPBs were measured. At 48 h after reperfusion, myocardial infarct was measured. Myocardial interstitial noradrenaline levels were determined before, during and after 30 min of ischemia with cilnidipine (0.5 and 1.0 μg/kg/min) or saline. The incidences of VPBs during ischemia and reperfusion were significantly attenuated in the cilnidipine 0.5 group (15.6±3.1 and 6.8±1.9 beats/30 min) and in the cilnidipine 1.0 group (10.4±4.9 and 3.5±1.0 beats/30 min) compared to the control group (27.2±4.5 and 24.2±3.1 beats/30 min), respectively. Myocardial interstitial noradrenaline levels were significantly reduced in the cilnidipine 0.5 and 1.0 groups compared to the control group during ischemia and reperfusion. The antiarrhythmic effect of cilnidipine may be related to the attenuation of cardiac sympathetic nerve activity. This finding may provide new insight into therapeutic strategies for hypertensive patients with ventricular arrhythmias.

Lindera strychnifolia is protective against post-ischemic myocardial dysfunction through scavenging hydroxyl radicals and opening the mitochondrial KATP channels in isolated rat hearts.

Lindera strychnifolia (tendai-uyaku), a medicinal plant, has long been used for the treatment of cardiac, renal and rheumatic diseases in Japan. We aim to clarify (1) whether L. strychnifolia is protective against post-ischemic myocardial dysfunction, and (2) whether its effect is related to scavenging hydroxyl radicals and opening the mitochondrial KATP channels in isolated rat hearts. Male Sprague-Dawley rats were orally given 1 ml/day of L. strychnifolia, which was extracted from 0.75 and 1.5 g/kg of roots of L. strychnifolia for 4 days. The rat hearts were excised and perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. The hearts were paced at 320 beats/min except during ischemia. Left ventricular developed pressure (LVDP, mmHg), +/- dP/dt (mmHg/sec) and coronary flow (ml/min) were continuously monitored. All hearts were perfused for a total of 120 minutes consisting of a 30-minute pre-ischemic period followed by 30 minutes of global ischemia and 60 minutes of reperfusion with or without 5-HD, a mitochondrial KATP channel blocker. The levels of lactate, LDH and 2,5-DHBA, an indicator of hydroxyl radicals, in the perfusate during reperfusion period were also measured. Treatment with L. strychnifolia significantly improved LVDP and +/- dP/dt without altering coronary flow during reperfusion. The 100 microM of 5-HD in Krebs-Henseleit solution was perfused during the 10 minutes of pre-ischemic periods. Pretreatment with 5-HD abolished the improvement of LVDP and +/- dP/dt by L. strychnifolia. L. strychnifolia significantly attenuated the levels of lactate, LDH and 2,5-DHBA during reperfusion, and which were restored by pretreatment with 5-HD. In conclusion, L. strychnifolia is protective against post-ischemic left ventricular dysfunction through scavenging hydroxyl radicals and opening the KATP channels in the isolated rat heart.

Benidipine reduces myocardial infarct size involving reduction of hydroxyl radicals and production of protein kinase C-dependent nitric oxide in rabbits.

Japanese white rabbits underwent 30 minutes of ischemia and 48 hours of reperfusion. Benidipine (3 or 10 μg/kg, i.v.) was administered 10 minutes before ischemia with and without pretreatment with L-NAME (10 mg/kg, i.v., a NOS inhibitor), chelerythrine (5 mg/kg, i.v., a PKC blocker) or 5-HD (5 mg/kg, i.v. a mitochondrial KATP channel blocker), genistein (5 mg/kg, i.v. a protein tyrosin kinase blocker). SNAP (2.5 mg/kg/min × 70 minutes, i.v., an NO donor) was also administered 10 minutes before ischemia. Benidipine significantly reduced the infarct size in a dose-dependent manner (3 μg/kg: 29.0 ± 2.7%, n = 8, 10 μg/kg: 23.0 ± 2.4%, n = 10) compared with the control (41.6 ± 3.3%, n = 10). This effect was completely blocked by L-NAME (39.9 ± 3.6%, n = 8) and chelerythrine (35.5 ± 2.4%, n = 8) but not by 5-HD (23.0 ± 2.4%, n = 10) or genistein (24.6 ± 3.1%, n = 10). SNAP also reduced the infarct size (24.6 ± 3.1%, n = 8). Benidipine significantly increased the expression of eNOS mRNA at 30 minutes after reperfusion and significantly increased the expression of eNOS protein at 3 hours after reperfusion in the ischemic area of the left ventricle. Benidipine and SNAP significantly decreased myocardial interstitial 2,5-DHBA levels, an indicator of hydroxyl radicals, during ischemia and reperfusion. Benidipine increased myocardial interstitial NOx levels, which effect was blocked by chelerythrine, during 0 to 30 minutes and 150 to 180 minutes after reperfusion. Benidipine reduces the infarct size through PKC-dependent production of nitric oxide and decreasing hydroxyl radicals but not through involving protein tyrosine kinase or mitochondrial KATP channels in rabbits.

Edaravone reduces myocardial infarct size and improves cardiac function and remodelling in rabbits

1 In the present study, we investigated the effect of 3‐methyl‐1‐phenyl‐2‐pyrazolin‐5‐one (edaravone), a free radical scavenger, on myocardial infarct (MI) size and cardiac function in an in vivo model of MI in rabbits. We further investigated the contribution of hydroxyl radicals, superoxide and nitric oxide (NO) to its effects. 2 Anaesthetized open‐chest Japanese white male rabbits were subjected to 30 min coronary occlusion and 48 h reperfusion. The control group (n = 10) was injected with saline 10 min before reperfusion. The edaravone group (n = 10) was injected with a bolus of 3 mg/kg edaravone 10 min before reperfusion. The edaravone + NG‐nitro‐l‐arginine methyl ester (l‐NAME) group (n = 5) was given 10 mg/kg, i.v., l‐NAME 10 min before the administration of 3 mg/kg edaravone. The l‐NAME group (n = 5) was given 10 mg/kg, i.v., l‐NAME 20 min before reperfusion. Infarct size was measured using the triphenyl tetrazolium chloride method and is expressed as a percentage of area at risk. Cardiac function was assessed by echocardiography 14 days after infarction. 3 In another series of experiments, rabbits were subjected to 30 min coronary occlusion and 30 min reperfusion and myocardial interstitial 2,3‐dihydroxybenzoic acid (DHBA) and 2,5‐DHBA levels, indicators of hydroxyl radical, were measured using a microdialysis technique. 4 Infarct size in the edaravone group was significantly reduced compared with that in the control group (27.4 ± 6.8 vs 43.4 ± 6.8%, respectively; P < 0.05). The edaravone‐induced reduction of infarct size was abolished by pretreatment with l‐NAME. Myocardial interstitial levels of 2,3‐DHBA and 2,5‐DHBA increased 20 and 30 min after ischaemia and peaked at 10 min reperfusion in the control group. Edaravone significantly inhibited the increase in 2,3‐DHBA and 2,5‐DHBA levels seen during reperfusion. Dihydroethidium staining showing in situ detectoion of superoxide was less intense in ischaemic myocardium in the edaravone‐treated group compared with the control group. Edaravone improved cardiac function and left ventricular remodelling 14 days after infarction. 5 In conclusion, edaravone significantly reduces MI size and improves cardiac function and LV remodelling by decreasing hydroxyl radicals and superoxide in the myocardium and increasing the production of NO during reperfusion in rabbits.

Antidiabetic drug miglitol inhibits myocardial apoptosis involving decreased hydroxyl radical production and Bax expression in an ischaemia/reperfusion rabbit heart

We examined whether antidiabetic drug miglitol could reduce ischaemia/reperfusion‐induced myocardial apoptosis by attenuating production. Japanese white rabbits were subjected to 30‐min coronary occlusion followed by 4‐h reperfusion with miglitol (10 mg kg−1, i.v., n=20) or saline (n=20). The infarct area was determined by myoglobin staining, and the infarct size (IS) was expressed as a percentage of the area at risk. DNA fragmentation was assessed by TUNEL method and DNA ladder formation. The expression of Bcl‐XL and Bax was detected by immunohistochemical analysis and Western blot analysis. Myocardial interstitial 2,5‐DHBA levels, an indicator of hydroxyl radicals, were measured during 30‐min ischaemia and 30‐min reperfusion in the absence (n=10) or presence of miglitol (10 mg kg−1, i.v., n=10) using a microdialysis technique. The IS was significantly reduced in the miglitol group (22.4±3.4%, n=10) compared to the control group (52.8±3.5%, n=10). Miglitol significantly decreased the 2,5‐DHBA level during ischaemia and reperfusion and suppressed the incidence of TUNEL‐positive myocytes in the ischaemic region (from 10.7±3.4 to 4.1±3.0%) and the intensity of DNA ladder formation. Miglitol significantly decreased the incidence of Bax‐positive myocytes in the ischaemic region (7.4±1.7 vs 13.7±1.9% of the control) and significantly attenuated the upregulation of Bax protein in the ischaemic regions (from 179±17 to 90±12% of sham). There was no difference in the expression of Bcl‐XL between the two groups. These data suggest that miglitol reduces myocardial apoptosis by attenuating production of hydroxyl radicals and suppressing the upregulation of the expression of Bax protein.

Autologous bone marrow cell transplantation improves left ventricular function in rabbit hearts with cardiomyopathy via myocardial regeneration-unrelated mechanisms

Recent studies suggest transplanted bone marrow cells (BMCs) can be used to reconstitute coronary vessels and myocardium following acute myocardial infarction, thereby improving cardiac function. We sought to investigate the therapeutic potential of BMC transplantation in the treatment of nonischemic cardiomyopathy. Experimental cardiomyopathy was produced by treating rabbits for 8 weeks with doxorubicin (2 mg/kg per week). Two weeks after the treatment was finished, freshly aspirated BMCs or an equivalent volume of phosphate-buffered saline was injected directly into the left ventricular free wall. Four weeks later, heart function was examined during perfusion on a Langendorff apparatus. Left ventricular developed pressure and ±dp/dt were significantly better in the transplantation group, among which echocardiography also showed significantly better ejection fractions. In addition, left ventricular weights as a fraction of body weight and left ventricular wall thicknesses were both lower in rabbits transplanted with BMCs than in controls. Immunohistochemical analyses carried out 2 weeks after transplantation showed no new myocardium and a very small number of endothelial cells originating from BMCs. On the other hand, immunoblotting revealed upregulated expression of transforming growth factor-β1 and downregulated expression of matrix metalloproteinase-1 and tumor necrosis factor-α following BMC transplantation. In conclusion, autologous BMC transplantation into cardiomyopathic rabbit hearts ameliorates the decline in ventricular function without regenerating cardiomyocytes, most likely by altering expression of various cytokines.

Quinaprilat reduces myocardial infarct size involving nitric oxide production and mitochondrial KATP channel in rabbits.

This study examined whether quinaprilat, an angiotensin-converting enzyme inhibitor, reduces the infarct size, and investigated the mechanisms for its infarct size–reducing effect, in rabbits. Japanese white rabbits underwent 30 min of ischemia and 48 h of reperfusion. Quinaprilat (100 &mgr;g/kg/h or 300 &mgr;g/kg/h for 70 min, IV) was administered 20 min before ischemia with or without pretreatment with N ω-nitro-l-arginine methyl ester (l-NAME) (10 mg/kg, IV, a nitric oxide synthase inhibitor), 5-hydroxydecanoic acid sodium salt (5-HD) or posttreatment with 5-HD (5 mg/kg, IV, a mitochondrial KATP channel blocker). The area at risk as a percentage of the left ventricle was determined by Evans blue dye and the infarct size was determined as a percent of the area at risk by triphenyl tetrazolium chloride staining. Using a microdialysis technique, myocardial interstitial levels of 2,5-dihydroxybenzoic acid (2,5-DHBA), an indicator of hydroxyl radicals, and NOx, an indicator of nitric oxide, were measured before, during, and after 30 min of ischemia. Quinaprilat significantly reduced the infarct size in a dose-dependent manner (30.1 ± 3%, n = 10, and 27.6 ± 2%, n = 7, respectively) compared with the control (46.5 ± 4%, n = 10). The infarct size–reducing effect of quinaprilat was completely blocked by pretreatment with l-NAME (43.8 ± 2%, n = 8) and 5-HD (50.1 ± 3%, n = 8) and posttreatment with 5-HD (50.3 ± 2%, n = 8), respectively. Quinaprilat did not affect the myocardial interstitial 2,5-DHBA level but significantly increased the NOx level during ischemia and reperfusion. Quinaprilat reduces myocardial infarct size involving NO production and mitochondrial KATP channels in rabbits without collateral circulation.