Myocyte Nuclear Density Research Articles

Nonocclusive multivessel intracoronary infusion of allogeneic cardiosphere-derived cells early after reperfusion prevents remote zone myocyte loss and improves global left ventricular function in swine with myocardial infarction.

Intracoronary cardiosphere-derived cells (icCDCs) infused into the infarct-related artery reduce scar volume but do not improve left ventricular (LV) ejection fraction (LVEF). We tested the hypothesis that this reflects the inability of regional delivery to prevent myocyte death or promote myocyte proliferation in viable myocardium remote from the infarct. Swine (n = 23) pretreated with oral cyclosporine (200 mg/day) underwent a 1-h left anterior descending coronary artery (LAD) occlusion, which reduced LVEF from 61.6 ± 1.0 to 45.3 ± 1.5% 30 min after reperfusion. At that time, animals received global infusion of allogeneic icCDCs (n = 8), regional infusion of icCDCs restricted to the LAD using the stop-flow technique (n = 8), or vehicle (n = 7). After 1 mo, global icCDCs increased LVEF from 44.8 ± 1.9 to 60.8 ± 3.8% (P < 0.05) with no significant change after LAD stop-flow icCDCs (44.8 ± 3.6 to 50.9 ± 3.1%) or vehicle (46.5 ± 2.5 to 47.7 ± 2.6%). In contrast, global icCDCs did not alter infarct volume (%LV mass) assessed at 2 days (11.2 ± 2.3 vs. 12.6 ± 2.3%), whereas it was reduced after LAD stop-flow icCDCs (7.1 ± 1.1%, P < 0.05). Histopathological analysis of remote myocardium after global icCDCs demonstrated a significant increase in myocyte proliferation (147 ± 32 vs. 14 ± 10 nuclei/106 myocytes, P < 0.05) and a reduction in myocyte apoptosis (15 ± 9 vs. 46 ± 10 nuclei/106 myocytes, P < 0.05) that increased myocyte nuclear density (1,264 ± 39 vs. 1,157 ± 33 nuclei/mm2, P < 0.05) and decreased myocyte diameter (13.2 ± 0.2 vs. 14.5 ± 0.3 μm, P < 0.05) compared with vehicle-treated controls. In contrast, remote zone changes after regional LAD icCDCs were no different from vehicle. These data indicate that changes in global LVEF after icCDCs are dependent upon preventing myocyte loss and hypertrophy in myocardium remote from the infarct. These arise from stimulating myocyte proliferation and reducing myocyte apoptosis indicating the importance of directing cell therapy to viable remote regions.NEW & NOTEWORTHY Administration of allogeneic cardiosphere-derived cells to the entire heart via global intracoronary infusion shortly after myocardial infarction favorably influenced left ventricular ejection fraction by preventing myocyte death and promoting myocyte proliferation in remote, noninfarcted myocardium in swine. In contrast, regional intracoronary cell infusion did not significantly affect remote zone myocyte remodeling. Global cell administration targeting viable myocardium remote from the infarct may be an effective approach to prevent adverse ventricular remodeling after myocardial infarction.

Panax Quinquefolium Saponins Attenuate Myocardial Dysfunction Induced by Chronic Ischemia

Background/Aims: Previous studies in rat models of myocardial ischemia showed that Panax quinquefolium saponins (PQS) could attenuate ischemic/reperfusion injury, increase vessel density and improve cardiac function. In the current study, we examined whether PQS could attenuate myocardial dysfunction in a swine model of chronic myocardial ischemia (CMI). Methods: CMI was established in Bama mini-pigs by placing amroid constrictor on the left anterior descending artery (LAD). Starting from 2 months after the surgery, pigs randomly received PQS (30 mg/kg/day), atorvastatin (1.5 mg/kg/day), or no drug for one month (n=6). A group of pigs receiving sham surgery was included as an additional control. Glucose utilization was assessed with positron emission tomography-computer tomography (PET-CT). Cardiac function was assessed with echocardiography. Myocyte size, nuclear density, and arteriolar density were examined in tissue section obtained from the ischemia area. Potential molecular targets of PQS were identified using proteomic analysis with isobaric tags for relative and absolute quantitation (iTARQ) and network pharmacology. Results: In comparison to the sham controls, pigs implanted with ameroid constrictor had decreased ventricular wall motion, left ventricular ejection fraction (LVEF), and glucose utilization. PQS significantly increased cardiac function and glucose utilization. Arteriole density and myocyte nuclear density were increased. Myocyte diameter was decreased. PQS also attenuated the CMI-induced change of protein expression profile. The effects of atorvastatin were generally similar to that of PQS. However, PQS attenuated the reduction of left ventricular systolic WT induced by CMI more robustly than atorvastatin. Conclusion: The results from the current study supports the use of PQS in patients with coronary artery disease.

Cortical Bone Stem Cell Therapy Preserves Cardiac Structure and Function After Myocardial Infarction.

Cortical bone stem cells (CBSCs) have been shown to reduce ventricular remodeling and improve cardiac function in a murine myocardial infarction (MI) model. These effects were superior to other stem cell types that have been used in recent early-stage clinical trials. However, CBSC efficacy has not been tested in a preclinical large animal model using approaches that could be applied to patients. To determine whether post-MI transendocardial injection of allogeneic CBSCs reduces pathological structural and functional remodeling and prevents the development of heart failure in a swine MI model. Female Göttingen swine underwent left anterior descending coronary artery occlusion, followed by reperfusion (ischemia-reperfusion MI). Animals received, in a randomized, blinded manner, 1:1 ratio, CBSCs (n=9; 2×107 cells total) or placebo (vehicle; n=9) through NOGA-guided transendocardial injections. 5-ethynyl-2'deoxyuridine (EdU)-a thymidine analog-containing minipumps were inserted at the time of MI induction. At 72 hours (n=8), initial injury and cell retention were assessed. At 3 months post-MI, cardiac structure and function were evaluated by serial echocardiography and terminal invasive hemodynamics. CBSCs were present in the MI border zone and proliferating at 72 hours post-MI but had no effect on initial cardiac injury or structure. At 3 months, CBSC-treated hearts had significantly reduced scar size, smaller myocytes, and increased myocyte nuclear density. Noninvasive echocardiographic measurements showed that left ventricular volumes and ejection fraction were significantly more preserved in CBSC-treated hearts, and invasive hemodynamic measurements documented improved cardiac structure and functional reserve. The number of EdU+ cardiac myocytes was increased in CBSC- versus vehicle- treated animals. CBSC administration into the MI border zone reduces pathological cardiac structural and functional remodeling and improves left ventricular functional reserve. These effects reduce those processes that can lead to heart failure with reduced ejection fraction.

Heart‐Derived Stem Cells in Miniature Swine with Coronary Microembolization: Novel Ischemic Cardiomyopathy Model to Assess the Efficacy of Cell‐Based Therapy

A major problem in translating stem cell therapeutics is the difficulty of producing stable, long-term severe left ventricular (LV) dysfunction in a large animal model. For that purpose, extensive infarction was created in sinclair miniswine by injecting microspheres (1.5 × 106 microspheres, 45 μm diameter) in LAD. At 2 months after embolization, animals (n = 11) were randomized to receive allogeneic cardiosphere-derived cells derived from atrium (CDCs: 20 × 106, n = 5) or saline (untreated, n = 6). Four weeks after therapy myocardial function, myocyte proliferation (Ki67), mitosis (phosphor-Histone H3; pHH3), apoptosis, infarct size (TTC), myocyte nuclear density, and cell size were evaluated. CDCs injected into infarcted and remodeled remote myocardium (global infusion) increased regional function and global function contrasting no change in untreated animals. CDCs reduced infarct volume and stimulated Ki67 and pHH3 positive myocytes in infarct and remote regions. As a result, myocyte number (nuclear density) increased and myocyte cell diameter decreased in both infarct and remote regions. Coronary microembolization produces stable long-term ischemic cardiomyopathy. Global infusion of CDCs stimulates myocyte regeneration and improves left ventricular ejection fraction. Thus, global infusion of CDCs could become a new therapy to reverse LV dysfunction in patients with asymptomatic heart failure.

Abstract 11714: Allogeneic Cardiosphere-Derived Cells Administered at the Time of Reperfusion Only Improve Ejection Fraction When Infused to Ischemic as well as Remote Myocardial Regions in Swine With Acute Myocardial Infarction

Introduction: While intracoronary infusion of cardiosphere-derived cells (CDCs) to myocardial infarcts using the stop-flow technique reduces scar volume, ejection fraction (EF) remains unchanged. Hypothesis: We hypothesized that this may relate to ineffective treatment of myocyte loss in normally-perfused remote regions which could negatively impact EF. We therefore compared CDC infusion to the entire heart with conventional regional infusion to the infarct-related artery. Methods: Swine (n=24) chronically treated with cyclosporine (100 mg/day) underwent a 1-hour LAD occlusion and EF decreased from 60 ± 2% at baseline to 46 ± 2% after reperfusion (p&lt;0.05 vs. baseline). We infused either 10x10^6 intracoronary allogeneic CDCs via stop-flow infusion (regional LAD infusion), 20x10^6 intracoronary allogeneic CDCs to the entire heart under flow (global infusion) or vehicle 30 minutes after reperfusion. Four weeks later we assessed left ventricular function (echo), myocyte proliferation (Ki67), infarct size (TTC), myocyte nuclear density and cell size. Data were compared to vehicle. Results: Regional LAD infusion increased wall thickening and reduced scar volume but did not affect EF (Table). In contrast, global infusion increased EF despite an insignificant reduction in infarct volume. This was accompanied by 2-fold increases in myocyte proliferation (Ki67) in border and remote regions which did not change with regional LAD infusion. Global infusion increased myocyte nuclear density by 12% and decreased cell size by 9% while there were no significant changes after regional LAD infusion. Conclusion: Although regional LAD infusion reduces scar size and improves regional function, only global infusion improves EF since it effectively stimulates endogenous myocyte regeneration throughout the entire left ventricle. Thus, global infusion may more effectively prevent deleterious post-infarction remodeling than regional administration alone.

Abstract 154: Allogeneic Cardiosphere-derived Cells From an Aged Donor Elicit Long-term Improvements in Left Ventricular Function and Myocyte Proliferation in a Mini-swine Model of Chronic Myocardial Ischemia

Objective: Virtually all large animal studies of cell-based therapy have employed young donor-recipient pairs. Since aging may impact the reparative ability of allogeneic cardiosphere-derived cells (CDCs), we tested whether CDCs from an aged donor promote functional repair of ischemic myocardium using a mini-swine model that enabled serial assessment of left ventricular (LV) function over an extended follow-up period. Methods: Immunosuppressed mini-swine (cyclosporine 100 mg/day) with a chronic (4-months) LAD stenosis were untreated (n=8) or received 20 million allogeneic aged CDCs (n=10). Cells were cultivated from a 9-year old mini-pig and infused into the 3 major coronary arteries under continuous flow (1 million cells/min). LV function was assessed by echocardiography at baseline and over a 3-month follow-up period, at which time histological assessment of myocyte morphometry, proliferation (Ki67), and cell retention (Y-FISH) was performed. Results: Wall thickening (WT) of the ischemic LAD region was impaired at baseline (LAD: 42.7 ± 2.1% vs. Remote: 81.4 ± 4.8%, p&lt;0.01) and not different between treatment groups. One month later, LAD WT improved in aged CDC-treated animals (from 41.0 ± 3.1 to 53.0 ± 1.7%, p=0.01) but remained depressed in untreated animals (from 45.1 ± 2.8% to 44.9 ± 2.6%, p=0.97). Extended follow-up revealed that LAD WT continued to improve, reaching 60.2 ± 1.6% (p=0.01) 3-months after CDC treatment. Histological analyses demonstrated morphometric changes consistent with myocyte regeneration, including an increase in myocyte nuclear density (1231 ± 34 vs. 1094 ± 34 nuclei/mm 2 , p=0.02) and reduction in myocyte diameter (13.9 ± 0.2 vs. 14.5 ± 0.3 μm, p=0.05). Increases in Ki67 + myocytes (468 ± 92 vs. 199 ± 32 nuclei/10 6 myocyte nuclei, p=0.02) and rare Y + cells in sex-mismatched recipients (0.5 ± 0.2 cells/cm 2 ) supported endogenous myocyte proliferation as the primary source of new myocytes. Conclusion: CDCs from an aged donor retain their reparative capacity to promote functional improvement and increase myocyte number following global intracoronary infusion. The progressive improvement in LV function 3-months after treatment with ongoing myocyte proliferation supports a long-lasting benefit after a single infusion.

Revascularization of Chronic Hibernating Myocardium Stimulates Myocyte Proliferation and Partially Reverses Chronic Adaptations to Ischemia

BackgroundThe time course and extent of recovery after revascularization of viable dysfunctional myocardium are variable. Although fibrosis is a major determinant, myocyte structural and molecular remodeling may also play important roles. ObjectivesThis study sought to determine whether persistent myocyte loss and/or irreversibility of protein changes that develop in hibernating myocardium have an impact on functional recovery in the absence of infarction. MethodsSwine implanted with a chronic left anterior descending artery (LAD) stenosis to produce hibernating myocardium underwent percutaneous revascularization, with serial functional recovery evaluated for 1 month (n = 12). Myocardial tissue was evaluated to assess myocyte size, nuclear density, and proliferation indexes in comparison with those of normal animals and nonrevascularized controls. Proteomic analysis by 2-dimensional differential in-gel electrophoresis was used to determine the reversibility of molecular adaptations of hibernating myocytes. ResultsAt 3 months, physiological features of hibernating myocardium were confirmed, with depressed LAD wall thickening and no significant infarction. Revascularization normalized LAD flow reserve, with no immediate change in LAD wall thickening. Regional LAD wall thickening slowly improved but remained depressed 1 month post–percutaneous coronary intervention. Surprisingly, revascularization was associated with histological evidence of myocytes re-entering the growth phase of the cell cycle and increases in the number of c-Kit+ cells. Myocyte nuclear density returned to normal, whereas regional myocyte hypertrophy regressed. Proteomic analysis demonstrated heterogeneous effects of revascularization. Up-regulated stress and cytoskeletal proteins normalized, whereas reduced contractile and metabolic proteins persisted. ConclusionsDelayed recovery of hibernating myocardium in the absence of scar may reflect persistent reductions in the amounts of contractile and metabolic proteins. Although revascularization appeared to stimulate myocyte proliferation, the persistence of small immature myocytes may have contributed to delayed functional recovery.

Abstract 15656: Global Intracoronary Infusion of Allogeneic Cardiosphere-Derived Cells (CDCs) Immediately After Reperfusion Stimulates Myocyte Regeneration in Remote Viable Myocardium in Swine With Acute Myocardial Infarction

Introduction: Previous studies evaluating regenerative strategies have focused on administering stem cells to the infarct related artery. Unfortunately, this does not stimulate cardiac regeneration nor prevent myocyte loss in viable remote myocardium that becomes dysfunctional due to LV remodeling. Hypothesis: We hypothesized that administrating allogeneic CDCs to the entire heart (global CDC infusion) at the time of reperfusion could improve function, stimulate myocyte regeneration and prevent myocyte loss throughout the LV. Methods: Yorkshire swine (n=12) underwent a one hour LAD occlusion. Ejection fraction (EF) decreased from 63 ± 2% at baseline to 44 ± 2% after infarction (p&lt;0.05 vs. baseline). Immediately after reperfusion animals were randomized to allogeneic intracoronary CDCs or vehicle in a blinded fashion (n=6 each). Allogeneic CDCs (20 x 10^6 CDCs, cyclosporine 100 mg/day) were infused into the 3 major coronary distributions without interrupting flow. Four weeks later we assessed myocardial function (echo), myocyte proliferation (Ki67), infarct size (TTC), myocyte nuclear density and cell size in comparison to vehicle. Results: After 4 weeks, global CDC infusion increased wall thickening and ejection fraction despite the fact that infarct volume was not different (Table). CDCs stimulated myocyte proliferation (Ki67) in border and remote regions by 3-fold. As a result, myocyte number (nuclear density) increased by 34 % and cell size decreased by 7% in viable infarct border and remote regions. Conclusion: Global infusion of allogeneic CDCs immediately after reperfusion 1.) stimulates myocyte regeneration in viable myocardium remote from the infarct and 2.) improves regional and global LV function without altering infarct size. Thus, treating viable remote myocardium as well as the infarct related artery with allogeneic CDCs may afford a widely available approach to reverse LV dysfunction in patients with acute myocardial infarction.

Global intracoronary infusion of allogeneic cardiosphere-derived cells improves ventricular function and stimulates endogenous myocyte regeneration throughout the heart in swine with hibernating myocardium.

BackgroundCardiosphere-derived cells (CDCs) improve ventricular function and reduce fibrotic volume when administered via an infarct-related artery using the “stop-flow” technique. Unfortunately, myocyte loss and dysfunction occur globally in many patients with ischemic and non-ischemic cardiomyopathy, necessitating an approach to distribute CDCs throughout the entire heart. We therefore determined whether global intracoronary infusion of CDCs under continuous flow improves contractile function and stimulates new myocyte formation.Methods and ResultsSwine with hibernating myocardium from a chronic LAD occlusion were studied 3-months after instrumentation (n = 25). CDCs isolated from myocardial biopsies were infused into each major coronary artery (∼33×106 icCDCs). Global icCDC infusion was safe and while ∼3% of injected CDCs were retained, they did not affect ventricular function or myocyte proliferation in normal animals. In contrast, four-weeks after icCDCs were administered to animals with hibernating myocardium, %LADWT increased from 23±6 to 51±5% (p<0.01). In diseased hearts, myocyte proliferation (phospho-histone-H3) increased in hibernating and remote regions with a concomitant increase in myocyte nuclear density. These effects were accompanied by reductions in myocyte diameter consistent with new myocyte formation. Only rare myocytes arose from sex-mismatched donor CDCs.ConclusionsGlobal icCDC infusion under continuous flow is feasible and improves contractile function, regresses myocyte cellular hypertrophy and increases myocyte proliferation in diseased but not normal hearts. New myocytes arising via differentiation of injected cells are rare, implicating stimulation of endogenous myocyte regeneration as the primary mechanism of repair.

Abstract 21: Quantification of Total Myocyte Regeneration Following Intracoronary Cardiosphere-Derived Cell (icCDC) Treatment in Swine with Chronic Myocardial Ischemia

Objective: icCDCs improve function of ischemic myocardium and increase cardiomyocyte nuclear density but the extent to which this is explained by an increase in nuclei per cell vs. the formation of new myocytes is unclear. We aimed to address this issue and quantify new myocyte formation throughout the left ventricle (LV) after global icCDC treatment in swine with hibernating myocardium. Methods: Swine with a chronic LAD stenosis (N=29) received either ~35 x 10 6 icCDCs or no treatment. Regional function (echocardiography) and myocyte morphometric indices were assessed 1 month later. Transverse and longitudinal tissue sections were used to measure regional myocyte nuclear density, diameter, length, and nuclei/cell, from which myocyte volume and myocytes per gram of myocardium were calculated. Results: icCDC-mediated improvements in regional function were accompanied by an increase in myocyte nuclear density without a change in the number of nuclei/cell (Table). Compared with untreated animals, icCDC-treated animals exhibited a reduction in myocyte volume and a reciprocal increase in the number of myocytes per gram of tissue. Anatomic LV hypertrophy did not occur, however, as LV mass/body mass ratio was not different between groups (untreated: 2.5 ± 0.1 vs. icCDCs: 2.3 ± 0.1). Conclusion: These data demonstrate that icCDCs produce significant myocyte regeneration throughout the regionally ischemic heart that is dissociated from alterations in LV mass. Changes in myocyte nuclear density do not reflect an altered number of nuclei/cell and quantitative estimates suggest that intracoronary delivery of CDCs to the entire LV increases the number of myocytes by ~25% in just 4 weeks.

Abstract 185: Global Infusion of Allogeneic Cardiosphere-Derived Cells Proportionally Stimulates Angiogenesis and Myocyte Regeneration in Swine With Chronic Myocardial Infarction

Background: Previously we reported that global infusion of CDCs in infarcted and non-infarcted remote regions improved LV function in swine with chronic myocardial infarction. The effect of global infusion of CDCs on angiogenesis and myocyte regeneration in infarcted and non-infarcted remote regions is unknown. Methods: Swine (n=11) with chronic infarction in the left anterior descending (LAD) coronary artery region were studied 8 weeks after microsphere embolization (1x10^6 particles, 45μm diameter). CDCs were isolated from myocardial atrium and 20 x 10^6 CDCs were infused into the 3 major coronary distributions after cyclosporine (100mg/day) immunosuppression. Data were compared to untreated animals. To assess the factors related to angiogenesis, VEGF and SDF-1 expression on cultured CDCs were quantified by RT-PCR. Angiogenesis was quantified by assessing capillary density (von Willebrand Factor) with immunohistochemistry. Myocyte regeneration was quantified by assessing myocyte nuclear density. The ratio of capillary density to myocyte nuclear density was evaluated. Results: icCDCs improved infarcted LAD (13±6 vs. 36±6%, p&lt;0.05) and remote (47±8 vs. 99±10%, p&lt;0.05) wall thickening with increased ejection fraction (32±3 vs. 45±4%, p&lt;0.05) compared to untreated. RT- PCR from cultured CDCs showed VEGF expression and to a less extent SDF-1 expression. icCDCs increased capillary density and myocyte nuclear density in infarcted LAD and remote regions by 40% (Table). Thus, the ratio of capillary density to myocyte nuclear density was maintained in untreated and icCDCs animals. Conclusion: These data indicate that in a model of infarction, global infusion of icCDCs stimulates angiogenesis and myocyte regeneration to the same extent in infarcted and non-infarcted regions resulting in improved global function. Thus, global infusion of icCDCs may afford a widely available approach to regenerate myocardium in patients with ischemic cardiomyopathy.

Abstract 9912: Global Intracoronary Infusion of Allogeneic Atrial Cardiosphere-Derived Cells (CDCs) Stimulates Myocyte Regeneration and Reverses LV Remodeling in Miniature Swine With Chronic Myocardial Infarction

Background: Intracoronary infusion of autologous CDCs (icCDCs) into the infarct-related artery using the “stop flow” technique improves regional function in patients but global function remains unchanged. We tested the hypothesis that this reflects the inability to regress cellular hypertrophy and replace myocytes lost in remote remodeled regions that can be overcome by global intracoronary infusion of CDCs. Methods: Miniswine (n=10) were studied 8 weeks after chronic infarction was produced by LAD microembolization (1x10 6 microspheres, 45μm diameter). Ejection fraction decreased from 63 ± 2% at baseline to 31 ± 2% four weeks after infarction (p&lt;0.05 vs. baseline) and remained unchanged at 8 weeks (32 ± 2%, p-ns vs. 4 weeks). Allogeneic CDCs (20 x 10 6 CDCs, cyclosporine 100 mg/day) isolated from donor atrial biopsies were infused into the 3 major coronary distributions without interrupting flow. We assessed myocardial function (echo), myocyte proliferation (Ki67) and infarct size (TTC) in comparison to untreated controls. Regional myocyte regeneration was quantified by assessing myocyte nuclear density and size. Results: Infusing icCDCs into infarcted and remodeled remote myocardium increased regional function and ejection fraction (Table). icCDCs stimulated myocyte proliferation (Ki67) in LAD and remote regions by three-fold. As a result, myocyte number (nuclear density) increased by 35% and myocyte cell diameter decreased by 33% in both LAD and remote myocardium. The reduction in infarct volume after icCDCs was not significant. Conclusion: We conclude that global infusion of allogeneic icCDCs without “stop flow” 1.) stimulates myocyte proliferation throughout the LV and 2.) in a model of infarction, improves regional function with a substantial increase in left ventricular ejection fraction. Thus, allogeneic icCDCs may afford a widely available platform to reverse LV dysfunction in patients with chronic post infarction LV remodeling.

Abstract 8851: Intracoronary Infusion of Cardiosphere-Derived Cells (icCDCs) Improves Cardiac Function by Stimulating Myocyte Proliferation in Non-Infarcted Hibernating Myocardium with No Effect in Normal Myocardium

Background: icCDCs have been shown to improve function and reduce fibrotic area when regionally infused into infarcted myocardium. Their ability to improve flow and function in viable dysfunctional myocardium in the absence of scar is unclear. Methods: Swine (n=12) with collateral-dependent hibernating myocardium from a chronic LAD occlusion were studied 4-months after instrumentation in the closed-chest propofol sedated state. CDCs were isolated from myocardial biopsies at the time of initial instrumentation. We assessed regional flow, LAD wall thickening (%LADWT) and global EF before and 4 weeks after icCDCs (∼40x10 6 cells) into each of the 3 major coronary arteries. Autologous (n=3) as well as non-autologous CDCs with cyclosporine treatment (200mg/day; n=3) had similar effects and results were pooled and compared to untreated swine (n=6). Histological analysis was performed to assess the frequency of Ki-67 and phospho-Histone H3 (pHH3) positive myocytes along with myocyte nuclear density and myocyte diameter. Results: Infusion of icCDCs in normal animals (icCDCs; n=5 vs. untreated; n=6) did not affect regional (%LADWT 51±1% vs. 48±3%, p-ns) or global function (EF: 70±2% vs. 70±3%, p-ns). In contrast, in hibernating myocardium, %LADWT increased from 23±6 to 51±5 % (p&lt;0.01) and EF increased from 57±1 to 71±4 % (p&lt;0.01) after icCDCs. There was no change in LAD coronary flow reserve (adenosine vasodilated flow/ resting flow 1.88 ± 0.20 to 2.03 ± 0.31, p-ns). icCDCs increased the number of Ki-67 and pHH3 positive myocytes (Table). This was accompanied by a prominent increase in myocyte nuclear density with reduced myocyte size suggesting the formation of new myocytes. Conclusion: We conclude that icCDCs result in a robust improvement in function in hibernating myocardium. This is not secondary to increases in myocardial perfusion but is secondary to pronounced myocyte proliferation and regeneration with newly formed myocytes in diseased but not normal myocardium.

Heart failure therapy mediated by the trophic activities of bone marrow mesenchymal stem cells: a noninvasive therapeutic regimen

Heart failure carries a poor prognosis with few treatment options. While myocardial stem cell therapeutic trials have traditionally relied on intracoronary infusion or intramyocardial injection routes, these cell delivery methods are invasive and can introduce harmful scar tissue, arrhythmia, calcification, or microinfarction in the heart. Given that patients with heart failure are at an increased surgical risk, the development of a noninvasive stem cell therapeutic approach is logistically appealing. Taking advantage of the trophic effects of bone marrow mesenchymal stem cells (MSCs) and using a hamster heart failure model, the present study demonstrates a novel noninvasive therapeutic regimen via the direct delivery of MSCs into the skeletal muscle bed. Intramuscularly injected MSCs and MSC-conditioned medium each significantly improved ventricular function 1 mo after MSC administration. MSCs at 4 million cells/animal increased fractional shortening by approximately 40%, enhanced capillary and myocyte nuclear density by approximately 30% and approximately 80%, attenuated apoptosis by approximately 60%, and reduced fibrosis by approximately 50%. Myocyte regeneration was evidenced by an approximately twofold increase in the expression of cell cycle markers (Ki67 and phosphohistone H(3)) and an approximately 13% reduction in mean myocyte diameter. Increased circulating levels of hepatocyte growth factor (HGF), leukemia inhibitory factor, and macrophage colony-stimulating factor were associated with the mobilization of c-Kit-positive, CD31-positive, and CD133-positive progenitor cells and a subsequent increase in myocardial c-Kit-positive cells. Trophic effects of MSCs further activated the expression of HGF, IGF-II, and VEGF in the myocardium. The work highlights a cardiac repair mechanism mediated by trophic cross-talks among the injected MSCs, bone marrow, and heart that can be explored for noninvasive stem cell therapy.

Pravastatin Improves Function in Hibernating Myocardium by Mobilizing CD133 + and cKit + Bone Marrow Progenitor Cells and Promoting Myocytes to Reenter the Growth Phase of the Cardiac Cell Cycle

3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibitors have been reported to increase circulating bone marrow progenitor cells and variably improve global function in heart failure. The potential role of improved perfusion versus direct effects of statins on cardiac myocytes has not been established. We chronically instrumented swine with a left anterior descending artery (LAD) stenosis to produce chronic hibernating myocardium with regional contractile dysfunction in the absence of heart failure. Hemodynamics, function, perfusion, and histopathology were assessed in pigs treated for 5 weeks with pravastatin (n=12) versus untreated controls (n=10). Regional LAD wall thickening was depressed under baseline conditions (LAD 3.7+/-0.3 versus 6.6+/-0.3 in remote regions, P<0.01). It remained unchanged in untreated animals but increased from 3.8+/-0.6 to 5.2+/-0.5 mm after pravastatin (P<0.01). There was no increase in myocardial perfusion at rest or during vasodilation. Pravastatin mobilized circulating CD133(+)/cKit(+) bone marrow progenitor cells and increased myocardial tissue levels (LAD CD133(+) cells from 140+/-33 to 884+/-167 cells/10(6) myocyte nuclei and cKit(+) cells from 223+/-49 to 953+/-123 cells/10(6) myocyte nuclei). Pravastatin increased myocytes in mitosis (phospho-histone-H3; 9+/-5 to 43+/-7 nuclei/10(6) myocyte nuclei, P<0.05) and the growth phase of the cell cycle (Ki67; 410+/-82 to 1261+/-235 nuclei/10(6) myocyte nuclei, P<0.05) in diseased but not normal hearts. As a result, pravastatin increased LAD myocyte nuclear density from 830+/-41 to 1027+/-55 nuclei/mm(2) (P<0.05). These data indicate that, in the absence of impaired endothelial function and heart failure, dysfunctional hibernating myocardium improves after pravastatin. This effect is independent of myocardial perfusion and related to mobilization of CD133(+)/cKit(+) bone marrow progenitor cells which stimulate myocyte proliferation resulting in quantitative increases in myocyte nuclear density.

Abstract 5497: Chronic Stenosis of the Proximal Left Anterior Descending Coronary Artery in Juvenile Rats Results in a Critical Impairment in Coronary Flow Reserve and Leads to the Development of Hibernating Myocardium without Necrosis

Background: Previous studies in adult rats have shown that a chronic coronary artery stenosis can produce variable degrees of heart failure and global LV dysfunction with concomitant fibrosis and necrosis. In contrast, placement of a fixed LAD stenosis in juvenile swine leads to the gradual development of hibernating myocardium with regional dysfunction in the absence of infarction. We hypothesized that a similar approach could be used to produce hibernating myocardium in growing rats. Methods: Sprague-Dawley rat pups (3 wk old, 40±2g, n=24) were instrumented with a 200μm stenosis using an 8 – 0 suture around the proximal LAD and compared to sham controls (n=7). At 12-wks post-instrumentation (399±6g), ejection fraction (EF) and regional wall thickening (%WT) were determined by echocardiography and myocardial perfusion was measured using fluorescent microspheres (n=8). Histology was used to assess necrosis, fibrosis and regional myocyte loss. Results: Mortality was low (16%) and survivors (n=20) had no gross evidence of infarction. LAD end-diastolic wall thickness was normal (1.2 ± 0.04 vs. 1.2 ± 0.04 mm in controls, p=ns) and EF was preserved (65 ± 2% vs.70±1% in controls, p=0.07). After 12 weeks, LAD %WT was significantly reduced (49 ± 5% vs. 74 ± 5% in remote, p&lt;0.01). In LAD subendocardium, vasodilated flow was decreased (3.1 ± 0.7 vs.7.1±1.2 ml/min/g in remote, p&lt;0.01) with a critical impairment in coronary flow reserve (adenosine/rest 1.2 ± 0.3 vs.1.8 ± 0.2 in remote, p&lt;0.05). Although fibrosis and necrosis were absent, regional myocyte nuclear density in LAD subendocardium decreased (489 ± 27 vs. 628 ± 24 nuclei per mm 2 in remote, p&lt;0.01) and relative reductions were closely correlated with LAD flow reserve (r =0.76, p&lt;0.05) and LAD %WT (r =0.54, P&lt;0.05). Conclusion: Like swine, a chronic LAD stenosis in growing rats leads to a critical impairment in coronary flow reserve with reduced regional myocardial perfusion, wall thickening and regional myocyte loss without infarction. Our findings establish a model of hibernating myocardium in rodents, facilitating high-throughput studies to identify novel mechanisms and therapies for chronic ischemic LV dysfunction.

Intracoronary Administration of AdvFGF-5 (Fibroblast Growth Factor-5) Ameliorates Left Ventricular Dysfunction and Prevents Myocyte Loss in Swine With Developing Collaterals and Ischemic Cardiomyopathy

Fibroblast growth factor (AdvFGF-5) improves regional function by stimulating myocyte hypertrophy without increasing myocardial perfusion in swine with hibernating myocardium. We performed the present study to determine whether AdvFGF-5 could prevent the progression of LV dysfunction in swine with ischemic cardiomyopathy. Swine were chronically instrumented with LAD and LCX stenoses to produce viable dysfunctional myocardium and studied 1 month after instrumentation in the closed-chest sedated state. Flow and regional function before and 30 days after intracoronary AdvFGF-5 (2x10(12) vp, n=9) were compared with animals receiving intracoronary AdvEGFP (2x10(12) vp, n=6). Histological analysis was performed to quantify myocyte size, myocyte nuclear density, apoptosis (TUNEL), and the frequency of myocytes in the proliferative phase of the cell cycle (Ki-67 staining). LAD wall-thickening (27+/-3 to 46+/-6%, P<0.05) and EF (39+/-4 to 56+/-3%, P<0.05) increased after AdvFGF-5. AdvFGF-5 increased maximal perfusion during adenosine vasodilation despite no differences in baseline flow or stenosis severity. After AdvFGF-5, TUNEL-positive myocytes decreased 6-fold and Ki-67 positive myocyte nuclei increased 2-fold. As a result, AdvFGF-5 produced a marked increase in myocyte nuclear density (957+/-54 to 1447+/-40 nuclei/mm2, P<0.05). These data indicate that AdvFGF-5 increases regional function and maximal perfusion distal to stenotic arteries when administered before the development of collaterals. This was associated with a reduction in myocyte apoptosis, an increase in Ki-67-positive myocytes, and an increase in myocyte number. Thus, AdvFGF-5 offers a potential therapeutic approach to prevent the progression of ischemic cardiomyopathy and heart failure.

Histological remodeling in an ovine heart failure model resembles human ischemic cardiomyopathy

Staged coronary embolization, causing myocardial microinfarctions, has been shown in dogs and sheep to cause chronic ischemic heart failure (HF) that resembles the hemodynamics of the human condition. However, its histopathological basis remains unclear. We examined the hypothesis that the ventricular remodeling seen in such sheep resembles the histopathology of human ischemic cardiomyopathy (ICM). Understanding the pathophysiology of this model will determine its place in the development of treatment strategies for HF. Global left ventricular (LV) damage resulting in HF was induced by staged coronary embolization in 11 sheep. Six others served as controls (normal control, NC). In HF sheep, the heart was harvested 6 months after LV ejection fraction (EF) had stabilized at <35%. Histopathological profiles were compared in biventricular transverse sections at midpapillary level using computed image analysis. LV end-diastolic volume increased in the HF group from 84.9±29 to 122.4±30.3 ml ( n=11, P<.05), but myocytes across the LV wall in noninfarcted zones decreased (435.7±38.2 NC; 297.8±48.4/unit area HF; n=11, P<.0001) as did myocyte nuclear density (990.5±51.5 NC; 677.5±121.1/mm 2 HF, n=11, P<.0001). In contrast, LV replacement and interstitial fibrosis increased as did myocyte diameter in noninfarcted zones: 0.1±0.1 to 6.2±4.5% ( P=.0049); 2.0±1.0 to 7.6±4.9% ( P=.0149); and 10.0±0.5 to 15.9±2.2 μm ( P<.0001), respectively. Although LV myocyte nuclear length increased (10.2±1.0 NC; 12.2±0.9 μm HF, n=11, P=.0006), right ventricular (RV) myocyte nuclear density and length did not alter. In this ovine chronic HF model, LV dilation and interstitial and myocyte remodeling resemble human ICM.

Profound apoptosis-mediated regional myocyte loss and compensatory hypertrophy in pigs with hibernating myocardium.

Myocyte apoptosis is seen in ischemic heart disease, but whether it can occur after reversible ischemia or independent of necrosis and replacement fibrosis is unknown. Pigs were instrumented with a stenosis of the left anterior descending coronary artery to chronically reduce coronary flow reserve over a period of 3 months. At this time, there was viable dysfunctional myocardium having the physiological features of hibernating myocardium. Resting subendocardial perfusion was reduced to 0.65+/-0.08 (mean+/-SEM) mL. min(-1). g(-1) in hibernating myocardium of instrumented pigs compared with 0.98+/-0.14 mL. min(-1). g(-1) in myocardium of sham-operated pigs (P<0.05). There was a critical limitation in subendocardial flow during vasodilation to 0.78+/-0.20 mL. min(-1). g(-1) in instrumented pigs versus 3. 24+/-0.50 mL. min(-1). g(-1) in sham-operated pigs (P<0.001). Histology revealed a regional reduction in myocyte nuclear density to 995+/-100 nuclei/mm(2) in hibernating myocardium from the instrumented group versus 1534+/-65 nuclei/mm(2) in myocardium from the sham-operated group (P<0.05), regional myocyte hypertrophy (myocyte volume per nucleus, 14 183+/-2594 in the instrumented group versus 9130+/-1301 microm(3) in the sham group; P<0.05), and minimal increases in connective tissue (5.8+/-0.9% in the instrumented group versus 3.0+/-0.2% in the sham group, P<0.05). Necrosis was not identified, but apoptosis was increased from 30+/-9 myocytes per 10(6) myocyte nuclei in myocardium from the sham group to 220+/-77 myocytes per 10(6) myocyte nuclei in hibernating myocardium (P<0.05). These findings indicate that reversible ischemia in an area of chronically reduced coronary flow reserve induces regional myocyte loss via an apoptotic mechanism. This may contribute to the progression of chronic coronary disease to heart failure and explain the lack of complete functional recovery after revascularization in hibernating myocardium.

Long-Term Treatment with Trandolapril Opposes Cardiac Remodeling and Prolongs Survival After Myocardial Infarction in Rats

Long-term treatment with angiotensin I converting enzyme inhibitors (ACEIs) prolongs survival in rats developing congestive heart failure after myocardial infarction (MI). In this experimental model, we investigated at regular intervals the effects of a 1-year oral treatment with trandolapril, a new ACEI, on (a) survival rate and duration and (b) hemodynamic, biological, and cardiac and vascular histomorphological parameters. Control MI rats and sham-operated trandolapril-treated and control rats were simultaneously studied. In MI rats, trandolapril significantly increased the survival rate and duration, increasing the life expectancy by approximately 6 months. It also significantly decreased the arterial blood pressure and increased diuresis. These effects occurred as soon as the treatment was started and persisted throughout the study. Trandolapril significantly limited the development of myocardial hypertrophy, decreasing the heart weight and left ventricular hypertrophic area and increasing the left ventricular myocyte nuclear density, but these effects, starting after 3-6 months, were delayed compared to the hemodynamic ones. Finally, trandolapril limited the development of myocardial (both subendocardial and subepicardial) and aortic fibrosis. It is concluded that the early pre- and afterload effects of trandolapril are initially responsible for its beneficial action on survival, whereas later the drug's antihypertrophic and antifibrotic properties together with persistent hemodynamic effects account for the prolonged survival improvement.