Abdul Mansoor

Bioenergetic and Functional Consequences of Bone Marrow–Derived Multipotent Progenitor Cell Transplantation in Hearts With Postinfarction Left Ventricular Remodeling

Background— The present study examined whether transplantation of adherent bone marrow-derived stem cells, termed pMultistem, induces neovascularization and cardiomyocyte regeneration that stabilizes bioenergetic and contractile function in the infarct zone and border zone (BZ) after coronary artery occlusion. Methods and Results— Permanent left anterior descending artery occlusion in swine caused left ventricular remodeling with a decrease of ejection fraction from 55±5.6% to 30±5.4% (magnetic resonance imaging). Four weeks after left anterior descending artery occlusion, BZ myocardium demonstrated profound bioenergetic abnormalities, with a marked decrease in subendocardial phosphocreatine/ATP (31P magnetic resonance spectroscopy; 1.06±0.30 in infarcted hearts [n=9] versus 1.90±0.15 in normal hearts [n=8; P<0.01]). This abnormality was significantly improved by transplantation of allogeneic pMultistem cells (subendocardial phosphocreatine/ATP to 1.34±0.29; n=7; P<0.05). The BZ protein expression of creatine kinase-mt and creatine kinase-m isoforms was significantly reduced in infarcted hearts but recovered significantly in response to cell transplantation. MRI demonstrated that the infarct zone systolic thickening fraction improved significantly from systolic “bulging” in untreated animals with myocardial infarction to active thickening (19.7±9.8%, P<0.01), whereas the left ventricular ejection fraction improved to 42.0±6.5% (P<0.05 versus myocardial infarction). Only 0.35±0.05% donor cells could be detected 4 weeks after left anterior descending artery ligation, independent of cell transplantation with or without immunosuppression with cyclosporine A (with cyclosporine A, n=6; no cyclosporine A, n=7). The fraction of grafted cells that acquired an endothelial or cardiomyocyte phenotype was 3% and ≈2%, respectively. Patchy spared myocytes in the infarct zone were found only in pMultistem transplanted hearts. Vascular density was significantly higher in both BZ and infarct zone of cell-treated hearts than in untreated myocardial infarction hearts (P<0.05). Conclusions— Thus, allogeneic pMultistem improved BZ energetics, regional contractile performance, and global left ventricular ejection fraction. These improvements may have resulted from paracrine effects that include increased vascular density in the BZ and spared myocytes in the infarct zone.

A fibrin patch-based enhanced delivery of human embryonic stem cell-derived vascular cell transplantation in a porcine model of postinfarction left ventricular remodeling

It is unknown how to use human embryonic stem cell (hESC) to effectively treat hearts with postinfarction left ventricular (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 three‐dimensional (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 first diagonal coronary artery for 60 minutes 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. 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. 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. STEM CELLS 2011;29:367–375

Direct comparison of an intravascular and an extracellular contrast agent for quantification of myocardial perfusion

A direct comparison of extracellular and intravascular contrast agents for the assessment of myocardial perfusion was carried out in a porcine model (N = 5) with a flow-limiting occluder on the left anterior descending coronary artery. Rapid imaging during the first pass of an extracellular or intravascular contrast agent with a saturation-recovery-prepared TurboFLASH sequence showed comparable peak contrast-to-noise enhancements in myocardial tissue regions with flows averaging 1.1 ± 0.2 at baseline to 4.8 ± 0.6 ml/min/g during hyperemia. The coefficient of variation between the MR estimates of blood flow with Gadomer-17 and the microsphere blood flow measurements was 11 ± 11, while the corresponding coefficient of variation for blood flow estimates with the extracellular CA was 23 ± 11. Blood volume differences between rest and hyperemia observed with the intravascular tracer were significant (Vvasc(rest) = 0.078 ± 0.013 ml/g, versus Vvasc(hyperemia) = 0.102 ± 0.019 ml/g; p < 0.05). The effects of water exchange were minimized through the choice of pulse sequence parameters to provide blood volume estimates consistent with the changes expected between rest and hyperemia. This study represents the first application of multiple indicators in first pass imaging studies for the assessment of myocardial perfusion. The use of an intravascular instead of an extracellular contrast agent allows a reduction of the degrees of freedom for modeling tissue residue curves and results in improved accuracy of blood flow estimates.

Myocardial Oxygenation During High Work States in Hearts With Postinfarction Remodeling

BACKGROUND Postinfarction left ventricular remodeling (LVR) is associated with reductions in myocardial high-energy phosphate (HEP) levels, which are more severe in animals that develop overt congestive heart failure (CHF). During high work states, further HEP loss occurs, which suggests demand-induced ischemia. This study tested the hypothesis that inadequate myocyte oxygen availability is the basis for these HEP abnormalities. METHODS AND RESULTS Myocardial infarction was produced by left circumflex coronary artery ligation in swine. Studies were performed in 20 normal animals, 14 animals with compensated LVR, and 9 animals with CHF. Phosphocreatine (PCr)/ATP was determined with 31P NMR and deoxymyoglobin (Mb-delta) with 1H NMR in myocardium remote from the infarct. Basal PCr/ATP tended to be decreased in postinfarct hearts, and this was significant in animals with CHF. Infusion of dobutamine (20 microg x kg-1 x min-1 IV) caused doubling of the rate-pressure product in both normal and LVR hearts and resulted in comparable significant decreases of PCr/ATP in both groups. This decrease in PCr/ATP was not associated with detectable Mb-delta. In CHF hearts, rate-pressure product increased only 40% in response to dobutamine; this attenuated response also was not associated with detectable Mb-delta. CONCLUSIONS Thus, the decrease of PCr/ATP during dobutamine infusion is not the result of insufficient myocardial oxygen availability. Furthermore, in CHF hearts, the low basal PCr/ATP and the attenuated response to dobutamine occurred in the absence of myocardial hypoxia, indicating that the HEP and contractile abnormalities were not the result of insufficient oxygen availability.

Stem Cells for Myocardial Repair With Use of a Transarterial Catheter

Background— Using a swine model of postinfarction left ventricle (LV) remodeling, we investigated marrow-derived, multipotent progenitor cell (MPC) transplantation into hearts with acute myocardial infarction (AMI) via a novel transarterial catheter. Methods and Results— The left anterior descending coronary artery was balloon-occluded after percutaneous transluminal angiography to generate AMI (60-minute no-flow ischemia). The transarterial catheter was then placed in the same coronary artery, and either 50×106 MPCs (cell group, n=6) or saline (control, n=6) was injected into the border zone (BZ) myocardium. LV function was assessed by magnetic resonance imaging before AMI and at 1 and 4 weeks after AMI, whereas myocardial energy metabolism was assessed by 31P-magnetic resonance spectroscopy at week 4. One week after AMI, the ejection fraction was significantly reduced in both groups from a baseline of ≈50% to 31.3±3.9% (cell group) and 33.3±3.1% (control). However, at week 4, the cell group had a significant recovery in ejection fraction. The functional improvements were accompanied by a significant improvement in myocardial bioenergetics. Histologic data demonstrated a 0.55% cell engraftment rate 4 weeks after MPC transplantation. Only 2% of engrafted cells were costaining positive for cardiogenic markers. Vascular density in the BZ was increased in the cell group. Conditioned medium from cultured MPCs contained high levels of vascular endothelial growth factor, which was increased in response to hypoxia. MPCs cocultured with cardiomyocytes inhibited changes in cardiomyocyte mitochondrial membrane potential and cytochrome c release induced by tumor necrosis factor-&agr;. Conclusions— Thus, a paracrine effect may contribute significantly to the observed therapeutic effects of MPC transplantation.

Long-term functional improvement and gene expression changes after bone marrow-derived multipotent progenitor cell transplantation in myocardial infarction.

The study examined the long-term outcome of cardiac stem cell transplantation in hearts with postinfarction left ventricular (LV) remodeling. Myocardial infarction (MI) was created by ligating the first and second diagonal branches of the left anterior descending coronary artery in miniature swine. Intramyocardial injections of 50 million LacZ-labeled bone marrow-derived multipotent progenitor cells (MPC) were performed in the periscar region (Cell, n = 7) immediately after MI, whereas, in control animals (Cont, n = 7), saline was injected. Functional outcome was assessed monthly for 4 mo with MRI and (31)P-magnetic resonance spectroscopy. Engraftment was studied on histology, and gene chip (Affymetrix) array analysis was used to study differential expression of genes in the two groups. MPC treatment resulted in improvement of ejection fraction as early as 10 days after MI (Cell, 43.4 +/- 5.1% vs. Cont, 32.2 +/- 5.5%; P < 0.05). This improvement was seen each month and persisted to 4 mo (Cell, 51.2 +/- 4.8% vs. Cont, 35.7 +/- 5.0%; P < 0.05). PCr-to-ATP ratio (PCr/ATP) improved with MPC transplantation, which was most pronounced at high cardiac work states (subendocardial PCr/ATP was 1.70 +/- 0.10 vs. 1.34 +/- 0.14, P < 0.05). There was no significant difference in scar size (scar/LV area * 100) at 10 days postinfarction. However, at 4 mo, there was a significant decrease in scar size in the Cell group (Cell, 4.6 +/- 1.0% vs. Cont, 8.6 +/- 2.4%; P < 0.05). No significant engraftment of MPC was observed. MPC transplantation was associated with a downregulation of mitochondrial oxidative enzymes and increased levels of myocyte enhancer factor 2a and zinc finger protein 91. In conclusion, MPC transplantation leads to long-term functional and bioenergetic improvement in a porcine model of postinfarction LV remodeling, despite no significant engraftment of stem cells in the heart. MPC transplantation reduces regional wall stresses and infarct size and mitigates the adverse effects of LV remodeling, as seen by a reduction in LV hypertrophy and LV dilatation, and is associated with differential expression of genes relating to metabolism and apoptosis.

Novel strategy for measuring creatine kinase reaction rate in the in vivo heart

In the heart, the creatine kinase (CK) system plays an important role in the cascade of ATP production, transportation, and utilization. The forward pseudo-first-order rate constant for the CK reaction can be measured noninvasively by the (31)P-magnetic resonance (MR) spectroscopy magnetization saturation transfer (MST) techniques. However, the measurement of MST in the in vivo heart is limited by the lengthy data acquisition time, especially for studies requiring spatial localization. This technical report presents a new method for measuring ATP production rate via CK that can reduce the MST data acquisition time by 82%. This method is validated using an in vivo pig model to evaluate the forward pseudo-first-order rate constant of myocardial CK reaction noninvasively.

ATP sensitive K+ channels are critical for maintaining myocardial perfusion and high energy phosphates in the failing heart

Congestive heart failure (CHF) is associated with intrinsic alterations of mitochondrial oxidative phosphorylation which lead to increased myocardial cytosolic free ADP. ATP sensitive K(+) channels (KATP) act as metabolic sensors that are important for maintaining coronary blood flow (MBF) and in mediating the response of the myocardium to stress. Coronary adenosine receptors (AdR) are not normally active but cause vasodilation during myocardial ischemia. This study examined the myocardial energetic response to inhibition of KATP and AdR in CHF. CHF (as evidenced by LVEDP>20mmHg) was produced in adult mongrel dogs (n=12) by rapid ventricular pacing for 4weeks. MBF was measured with radiolabeled microspheres during baseline (BL), AdR blockade with 8-phenyltheophylline (8-PT; 5mg/kg iv), and KATP blockade with glibenclamide (GLB; 20μg/kg/min ic). High energy phosphates were examined with (31)P magnetic resonance spectroscopy (MRS) while myocardial oxygenation was assessed from the deoxymyoglobin signal (Mb-δ) using (1)H MRS. During basal conditions the phosphocreatine (PCr)/ATP ratio (1.73±0.15) was significantly lower than in previously studied normal dogs (2.42±0.11) although Mb-δ was undetectable. 8-PT caused ≈21% increase in MBF with no change in PCr/ATP. GLB caused a 33±0.1% decrease in MBF with a decrease in PCr/ATP from 1.65±0.17 to 1.11±0.11 (p<0.0001). GLB did not change the pseudo-first-order rate constant of ATP production via CK (kf), but the ATP production rate via CK was reduced by 35±0.08%; this was accompanied by an increase in Pi/PCr and appearance of a Mb-δ signal indicating tissue hypoxia. Thus, in the failing heart the balance between myocardial ATP demands and oxygen delivery is critically dependent on functioning KATP channels.

Long-term preservation of myocardial energetic in chronic hibernating myocardium

We previously reported that the myocardial energetic state, as defined by the ratio of phosphocreatine to ATP (PCr/ATP), was preserved at baseline (BL) in a swine model of chronic myocardial ischemia with mild reduction of myocardial blood flow (MBF) 10 wk after the placement of an external constrictor on the left anterior descending coronary artery. It remains to be seen whether this stable energetic state is maintained at a longer-term follow-up. Hibernating myocardium (HB) was created in minipigs (n = 7) by the placement of an external constrictor (1.25 mm internal diameter) on the left anterior descending coronary artery. Function was assessed with MRI at regular intervals until 6 mo. At 6 mo, myocardial energetic in the HB was assessed by (31)P-magnetic resonance spectrometry and myocardial oxygenation was examined from the deoxymyoglobin signal using (1)H-magnetic resonance spectrometry during BL, coronary vasodilation with adenosine, and high cardiac workload with dopamine and dobutamine (DpDb). MBF was measured with radiolabeled microspheres. At BL, systolic thickening fraction was significantly lower in the HB compared with remote region (34.4 ± 9.4 vs. 50.1 ± 10.7, P = 0.006). This was associated with a decreased MBF in the HB compared with the remote region (0.73 ± 0.08 vs. 0.97 ± 0.07 ml · min(-1) · g, P = 0.03). The HB PCr/ATP at BL was normal. DpDb resulted in a significant increase in rate pressure product, which caused a twofold increase in MBF in the HB and a threefold increase in the remote region. The systolic thickening fraction increased with DpDb, which was significantly higher in the remote region than HB (P < 0.05). The high cardiac workload was associated with a significant reduction in the HB PCr/ATP (P < 0.02), but this response was similar to normal myocardium. Thus HB has stable BL myocardial energetic despite the reduction MBF and regional left ventricular function. More importantly, HB has a reduced contractile reserve but has a similar energetic response to high cardiac workload like normal myocardium.

Transmural distribution of metabolic abnormalities and glycolytic activity during dobutamine-induced demand ischemia

The heterogeneity across the left ventricular wall is characterized by higher rates of oxygen consumption, systolic thickening fraction, myocardial perfusion, and lower energetic state in the subendocardial layers (ENDO). During dobutamine stimulation-induced demand ischemia, the transmural distribution of energy demand and metabolic markers of ischemia are not known. In this study, hemodynamics, transmural high-energy phosphate (HEP), 2-deoxyglucose-6-phosphate (2-DGP) levels, and myocardial blood flow (MBF) were determined under basal conditions, during dobutamine infusion (DOB: 20 microg x kg(-1) x min(-1) iv), and during coronary stenosis + DOB + 2-deoxyglucose (2-DG) infusion. DOB increased rate pressure products (RPP) and MBF significantly without affecting the subendocardial-to-subepicardial blood flow ratio (ENDO/EPI) or HEP levels. During coronary stenosis + DOB + 2-DG infusion, RPP, ischemic zone (IZ) MBF, and ENDO/EPI decreased significantly. The IZ ratio of creatine phosphate-to-ATP decreased significantly [2.30 +/- 0.14, 2.06 +/- 0.13, and 2.04 +/- 0.11 to 1.77 +/- 0.12, 1.70 +/- 0.11, and 1.72 +/- 0.12 for EPI, midmyocardial (MID), and ENDO, respectively], and 2-DGP accumulated in all layers, as evidenced by the 2-DGP/PCr (0.55 +/- 0.12, 0.52 +/- 0.10, and 0.37 +/- 0.08 for EPI, MID, and ENDO, respectively; P < 0.05, EPI > ENDO). In the IZ the wet weight-to-dry weight ratio was significantly increased compared with the normal zone (5.9 +/- 0.5 vs. 4.4 +/- 0.4; P < 0.05). Thus, in the stenotic perfused bed, during dobutamine-induced high cardiac work state, despite higher blood flow, the subepicardial layers showed the greater metabolic changes characterized by a shift toward higher carbohydrate metabolism, suggesting that a homeostatic response to high-cardiac work state is characterized by more glucose utilization in energy metabolism.