Abstract-We previously reported that combined blockade of adenosine receptors and ATP-sensitive Kϩ channels (K ϩ ATP channels) blunted but did not abolish the response of coronary blood flow to exercise. This study tested the hypothesis that the residual increase in coronary flow in response to exercise after adenosine receptor and K ϩ ATP channel blockade is dependent on endogenous NO. Dogs were studied at rest and during a four-stage treadmill exercise protocol under control conditions, during K ϩ ATP channel blockade with glibenclamide (50 g ⅐ kg Ϫ1 ⅐ min Ϫ1 IC) in the presence of adenosine receptor blockade with 8-phenyltheophylline (8-PT, 5 mg/kg IV), and after the addition of the NO synthase inhibitor N G -nitro-L-arginine (LNNA, 1.5 mg/kg IC). During control conditions, coronary blood flow was 49Ϯ3 mL/min at rest and increased to 92Ϯ8 mL/min at peak exercise. LNNA alone or in combination with 8-PT did not alter resting coronary flow and did not impair the normal increase in flow during exercise, indicating that when K ϩ ATP channels are intact, neither NO nor adenosine-dependent mechanisms are obligatory for maintaining coronary blood flow. Combined K ϩ ATP channel and adenosine blockade decreased resting coronary flow to 27Ϯ3 mL/min (PϽ.05), but exercise still increased flow to 45Ϯ5 mL/min (PϽ.05). The subsequent addition of LNNA further decreased resting coronary flow to 20Ϯ2 mL/min and markedly blunted exercise-induced coronary vasodilation (coronary vascular conductance, 0.20Ϯ0.03 mL ⅐ min Ϫ1 ⅐ mm Hg Ϫ1 at rest versus 0.24Ϯ0.04 mL ⅐ min Ϫ1 ⅐ mm Hg Ϫ1 during the heaviest level of exercise; Pϭ.22), so that coronary flow both at rest and during exercise was below the control resting level. The findings suggest that K Key Words: blood flow Ⅲ endothelium Ⅲ K ϩ channel Ⅲ ischemia Ⅲ reactive hyperemia R ecent evidence indicates that hyperpolarization of the vascular smooth muscle cell membrane caused by opening of K ϩ ATP channels is an important mechanism for metabolic coronary vasodilation. 1 We previously reported that blockade of vascular smooth muscle K ϩ ATP channels in chronically instrumented dogs decreased resting coronary blood flow but did not attenuate the increase in flow that occurred in response to treadmill exercise.2 However, after K ϩ ATP channel blockade had been established, the subsequent addition of adenosine receptor blockade reduced the exercise-induced increase in coronary flow by more than half, 3 indicating increased importance of adenosine in causing metabolic vasodilation after K ϩ ATP channels are blocked. Nevertheless, even after the combination of K ϩ ATP channel blockade and adenosine receptor blockade, exercise still produced a substantial increase in coronary flow.Bernstein et al 4 have reported that exercise causes increased NO production across the coronary circulation. We have previously observed that NO contributes to coronary vasodilation distal to a coronary artery stenosis, which results in hypoperfusion during exercise. 5 It is possible that NO could similarly contr...

In this new model of heart failure/hypertrophy, the abnormal myocardial HEP metabolism is related to the decreased CK-Mt protein level, which in turn is related to the severity of the hypertrophy.

Stromal-derived factor 1alpha (SDF-1alpha) is a key stem cell homing factor that is crucial for mobilization of stem cells from bone marrow to peripheral blood and subsequent engraftment to the tissue of diseased organs. It has been reported that SDF-1alpha is transiently over-expressed in ischemic myocardium. Therefore, there may be a limited time window after acute myocardial infarction (AMI) during which stem cells are recruited to injured myocardium for repair. This study aimed at investigating whether controlled release of SDF-1alpha via a novel conjugated poly(ethylene glycol) (PEG) (PEGylated) fibrin patch at the infarct site would increase the rate of stem cell recruitment and offer potential therapeutic benefits. Recombinant mouse SDF-1alpha was covalently bound to the PEGylated fibrinogen as evidenced by immunoprecipitation and western blotting. The PEGylated fibrinogen, bound with recombinant mouse SDF-1alpha, was mixed with thrombin to form the PEGylated fibrin patch. The release kinetics of SDF-1alpha were detected in vitro using enzyme-linked immunosorbent assay. Using a mouse AMI model produced by a ligature on the left anterior descending coronary artery, a PEGylated fibrin patch bound with SDF-1alpha (100 ng) was placed on the surface of the infarct area of the left ventricle. Infarct size, left ventricular (LV) function, and the percentage of sca-1(+)/c-kit(+) cells within the infarct area were measured at days 7, 14, and 28 after AMI. In vitro results showed that SDF-1alpha was successfully bound to the PEGylated fibrin patch and can be released from the patch constantly for up to 10 days. Two weeks after infarction, the myocardial recruitment of c-kit(+) cells was significantly higher in the group treated with the SDF-1alpha PEGylated fibrin patch (n = 9) than in the AMI control group (n = 10) (p < 0.05; 11.20 +/- 1.71% vs. 4.22 +/- 0.96%, respectively). At day 28 post-AMI, unlike the control group, the group with the SDF-1alpha-releasing patch maintained stable release of SDF-1alpha concurrent with additional stem cell homing. Moreover, LV function was significantly better than in the control group. These data demonstrate that the PEGylated fibrin patch based SDF-1alpha delivery can improve the rate of c-kit(+) cell homing and improve LV function in hearts with postinfarction LV remodeling.

Bone marrow-derived mononuclear cell (BMNC) transplantation provides the possibility of rescue or regeneration of myocardium lost during acute myocardial infarction (AMI). The extensive death of transplanted cells and the lack of sustained engraftment may limit its application. We investigated whether delivery of BMNCs by an injectable PEGylated fibrin biomatrix that covalently binds hepatocyte growth factor (HGF) would enhance the rate of cell engraftment and improve cardiac function. Balb/C female mice with AMI secondary to left anterior descending coronary ligation were randomly assigned to one of six groups: the Saline control group (n = 8) received a myocardial injection of saline (50 microL); the Cell group (n = 10) received a myocardial injection in the peri-infarct and infarct zones consisting of 500,000 murine BMNCs suspended in 50 microL saline; and the Biomatrix + HGF (n = 9) and Biomatrix + HGF + Cell (n = 9) group hearts received the HGF-loaded injectable biomatrix (identical volume) with or without entrapped BMNCs. Control groups consisting of the biomatrix alone (n = 9) and Biomatrix + Cells (n = 9) without HGF were also included for comparison. The left ventricular (LV) function was measured by echocardiography at days 14 and 28 post-MI. All animals were euthanized 4 weeks after AMI and transplantation for evaluation of angiogenesis, apoptosis, and fibrosis by immunohistochemistry. Cell prevalence rate at 4 weeks increased 15-fold in hearts receiving the Biomatrix + HGF + Cell delivery (p < 0.01), which was accompanied by the lowest levels of apoptosis and the highest LV function recovery among the treated groups.

It is unknown how to use hESC to effectively treat hearts with postinfarction LV remodeling. Using a porcine model of postinfarction LV remodeling, this study examined the functional improvement of enhanced delivery of combined transplantation of hESC-derived endothelial cells (ECs) and hESC-derived smooth muscle cells (SMCs) with a fibrin 3D porous scaffold biomatrix. To facilitate tracking the transplanted cells, the hESCs were genetically modified to stably express green fluorescent protein and luciferase (GFP/Luc). Myocardial infarction (MI) was created by ligating the 1st diagonal coronary artery for 60 min followed by reperfusion. Two million each of GFP/Luc hESC-derived ECs and SMCs were seeded in the 3D porous biomatrix patch and applied to the region of ischemia/reperfusion for cell group (MI+P+C, n=6), whereas biomatrix without cell (MI+P, n=5), or saline only (MI, n=5) were applied to control group hearts with same coronary artery ligation. Functional outcome (1 and 4 weeks follow-up) of stem cell transplantation was assessed by cardiac magnetic resonance imaging (MRI). The transplantation of hESC-derived vascular cells resulted in significant LV functional improvement. Significant engraftment of hESC-derived cells was confirmed by both in vivo and ex vivo bioluminescent imaging (BLI). The mechanism underlying the functional beneficial effects of cardiac progenitor transplantation is attributed to the increased neovascularization. These findings demonstrate a promising therapeutic potential of using these hESC-derived vascular cell types and the mode of patch delivery.

Rationale The mechanism by which endogenous progenitor cells contribute to functional and beneficial effects in stem cell therapy remains unknown. Objective Utilizing a novel 31P magnetic resonance spectroscopy–2-dimensional chemical shift imaging method, this study examined the heterogeneity and bioenergetic consequences of postinfarction left ventricular (LV) remodeling and the mechanisms of endogenous progenitor cell contribution to the cellular therapy. Methods and Results Human embryonic stem cell–derived vascular cells (hESC-VCs) that stably express green fluorescent protein and firefly luciferase (GFP+/Luc+) were used for the transplantation. hESC-VCs may release various cytokines to promote angiogenesis, prosurvival, and antiapoptotic effects. Both in vitro and in vivo experiments demonstrated that hESC-VCs effectively inhibit myocyte apoptosis. In the mouse model, a fibrin patch–based cell delivery resulted in a significantly better cell engraftment rate that was accompanied by a better ejection fraction. In the swine model of ischemia-reperfusion, the patch-enhanced delivery of hESC-VCs resulted in alleviation of abnormalities including border zone myocardial perfusion, contractile dysfunction, and LV wall stress. These results were also accompanied by a pronounced recruitment of endogenous c-kit+ cells to the injury site. These improvements were directly associated with a remarkable improvement in myocardial energetics, as measured by a novel in vivo 31P magnetic resonance spectroscopy–2-dimensional chemical shift imaging technology. Conclusions The findings of this study demonstrate that a severely abnormal heterogeneity of myocardial bioenergetics in hearts with postinfarction LV remodeling can be alleviated by the hESC-VCs therapy. These findings suggest an important therapeutic target of peri-scar border zone and a promising therapeutic potential for using hESC-VCs together with the fibrin patch–based delivery system.

This study examined whether alterations in myocardial creatine kinase (CK) kinetics and high-energy phosphate (HEP) levels occur in postinfarction left ventricular remodeling (LVR). Myocardial HEP and CK kinetics were examined in 19 pigs 6 wk after myocardial infarction was produced by left circumflex coronary artery ligation, and the results were compared with those from 9 normal pigs. Blood flow (microspheres), oxygen consumption (MV˙o 2), HEP levels [31P magnetic resonance spectroscopy (MRS)], and CK kinetics (31P MRS) were measured in myocardium remote from the infarct under basal conditions and during dobutamine infusion (20 μg ⋅ kg−1 ⋅ min−1iv). Six of the pigs with LVR had overt congestive heart failure (CHF) at the time of study. Under basal conditions, creatine phosphate (CrP)-to-ATP ratios were lower in all transmural layers of hearts with CHF and in the subendocardium of LVR hearts than in normal hearts ( P < 0.05). Myocardial ATP (biopsy) was significantly decreased in hearts with CHF. The CK forward rate constant was lower ( P < 0.05) in the CHF group (0.21 ± 0.03 s−1) than in LVR (0.38 ± 0.04 s−1) or normal groups (0.41 ± 0.03 s−1); CK forward flux rates in CHF, LVR, and normal groups were 6.4 ± 2.3, 14.3 ± 2.1, and 20.3 ± 2.4 μmol ⋅ g−1 ⋅ s−1, respectively ( P < 0.05, CHF vs. LVR and LVR vs. normal). Dobutamine caused doubling of the rate-pressure product in the LVR and normal groups, whereas CHF hearts failed to respond to dobutamine. CK flux rates did not change during dobutamine in any group. The ratios of CK flux to ATP synthesis (from MV˙o 2) under baseline conditions were 10.9 ± 1.2, 8.03 ± 0.9, and 3.86 ± 0.5 for normal, LVR, and CHF hearts, respectively (each P < 0.05); during dobutamine, this ratio decreased to 3.73 ± 0.5, 2.58 ± 0.4, and 2.78 ± 0.5, respectively ( P = not significant among groups). These data demonstrate that CK flux rates are decreased in hearts with postinfarction LVR, but this change does not limit the response to dobutamine. In hearts with end-stage CHF, the changes in HEP and CK flux are more marked. These changes could contribute to the decreased responsiveness of these hearts to dobutamine.

Conventional protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into smooth-muscle cells (SMCs) can be inefficient and generally fail to yield cells with a specific SMC phenotype (i.e., contractile or synthetic SMCs). Here, we present two novel hiPSC-SMC differentiation protocols that yield SMCs with predominantly contractile or synthetic phenotypes. Flow cytometry analyses of smooth-muscle actin (SMA) expression indicated that ~45% of the cells obtained with each protocol assumed an SMC phenotype, and that the populations could be purified to ~95% via metabolic selection. Assessments of cellular mRNA and/or protein levels indicated that SMA, myosin heavy chain II, collagen 1, calponin, transgelin, connexin 43, and vimentin expression in the SMCs obtained via the Contractile SMC protocol and in SMCs differentiated via a traditional protocol were similar, while SMCs produced via the Sythetic SMC protocol expressed less calponin, more collagen 1, and more connexin 43. Differences were also observed in functional assessments of the two SMC populations: the two-dimensional surface area of Contractile SMCs declined more extensively (to 12% versus 44% of original size) in response to carbachol treatment, while quantification of cell migration and proliferation were greater in Synthetic SMCs. Collectively, these data demonstrate that our novel differentiation protocols can efficiently generate SMCs from hiPSCs.