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Endothelial Progenitor Cell Targeting to Ischemic Myocardium Attenuates Hypoxia and Enhances Cardiomyocyte Viability and Function
S8 Journal of Cardiac Failure Vol. 12 No. 6 Suppl. 2006 020 Endothelial Progenitor Cell Targeting to Ischemic Myocardium Attenuates Hypoxia and Enhances Cardiomyocyte Viability and Function Pavan Atluri1, Kevin J. Morine1, George P. Liao1, Corinna M. Panlilio1, Y. Joseph Woo1; 1Div Cardiothoracic Surgery, Dept Surgery, U.Pennsylvania, Philadelphia, PA Introduction: Ischemic cardiomyopathy is a global health concern. Endothelial progenitor cell (EPC) upregulation with granulocyte-monocyte colony stimulating factor (GMCSF) followed by stromal-cell derived factor-1a (SDF) directed EPC chemokinesis is a means of myocardial revascularization.We recently reported upregulated EPCs, enhanced neovasculogenesis and increased myocardial perfusion with this therapy. To further investigate the biologic mechanisms we studied hypoxia attenuation, myocardial preservation, and function. Hypothesis: Neovasculogenic therapy with SDF/GMCSF enhances myocardial function by attenuating hypoxia and preserving myocardium. Methods: Lewis rats underwent LAD coronary ligation and progressed into heart failure. At 3wks the animals were randomized to intramyocardial SDF and subcutaneous GMCSF injections as therapy or saline as control. 6wks post-ligation borderzone vascular endothelial growth factor (VEGF) levels were quantified by western blot analysis. Endogenous VEGF is produced in response to tissue ischemia, thereby serving as a marker for hypoxia. Myocardial viability was determined by apoptosis as measured by caspase-3 activity levels and preservation of myocardial mass. Myocardial function was measured by echocardiography. Results: This therapy attenuated hypoxia as evidenced by decreased VEGF production. Enhanced myocardial preservation was noted by diminished apoptosis and enhanced myocardial to body mass ratio. There was a significant improvement in myocardial function with SDF/GMCSF therapy. Conclusions: GMCSF EPC upregulation and SDF mediated EPC chemokinesis enhances myocardial function by reversing myocardial ischemia and enhancing myocardial viability.
6 week post-ligation variables
GMCSF/SDF Therapy
Saline Control
Myocardial VEGF concentration 5420 6 2975 12211 6 1491 Caspase-3 activity level 718 6 80 887 6 106 3 Myocardial to body mass ratio 2.72 6 0.06 10 2.45 6 0.07 103 (n 5 6) Ejection fraction (%, n 5 5) 61.8 6 8.6 41.3 6 9.3 Fractional shortening (%, n 5 5) 38.3 6 8.4 23.5 6 4.5 End-systolic borderzone wall 3.37 6 0.27 1.76 6 0.78 thickness (mm, n 5 5) End-diastolic borderzone wall 2.92 6 0.15 1.63 6 0.86 thickness (mm, n 5 5) Cardiac output (ml/min, n 5 5) 33.2 6 7.4 20.6 6 5.9 Wall motion score index, 1.56 6 0.20 2.09 6 0.14 1 5 normal (n 5 5)
p5 0.015 0.030 0.0007 0.018 0.028 0.008 0.038 0.02 0.002
021 Erythropoietin Attenuates the Ischemic-Reperfusion Injury through Redistribution of bARK1 Dong-Ju Choi1, Eun-Ji Kim1, Seo-Yeong Kang1, Bon-Kwon Koo2, Cheol-Ho Kim1; 1 Cardiovacular Center, Seoul National University Bundang Hostpital, Seongnam, Gyeonggi-Do, Korea; 2Cardiovacular Center, Seoul National University Hospital, Seoul, Korea Background; Erythropoietin (EPO), known to stimulate the production of red blood and used to treat anemia in cancer and renal failure, has recently been shown to have a tissue protective effect on diabetic neuropathy, autoimmune encephalomyelitis, and stroke in experimental models. The goal of this study was to determine whether EPO could protect myocardium from ischemia/reperfusion injury and investigate the relation to bARK1. Methods: In anesthetized open-chest male Sprague-Dawley rats, receiving 5,000U/kg of erythropoietin (EPO) intravenously or saline 30 min prior to ischemia, area of necrosis (AN by TTC), the area at risk (AAR by Evans blue), % of infarct (AN/AAR and AN/LV), and Left ventricular hemodynamics by were measured after 30 min of coronary occlusion and 24 hours of reperfusion. LV hemodynamics was accessed by measuring the first derivative of left ventricular pressure (dP/dtMAX and dP/dtmin) and the end-systolic elastance, (Ees) by pressure-volume relationships with IV dobutamine infusion by using high fidelity microcatheter (Millar MikroTipÒ). To examine the mechanistic pathways involved in EPO-mediated myocardial protection, the distributions of bARK1 in membrane or cytosol and GRK activities were evaluated. Results: EPO administration reduced infarct size by 31% and 37%, respectively (%AN/LV; control, 40.1 6 8.8 vs. EPO, 25.2 6 4.3%: p ! 0.01) and (%AN/AAR; control, 57.5 6 10 vs. EPO, 38.5 6 6.9%: p ! 0.01)., whereas AAR was not changed in two group (%AAR/LV; control, 69.6 6 6.5 vs. EPO, 65.7 6 5.1%). EPO also improved ischemia/reperfusion-induced myocardial contractile dysfunction and responsiveness to dobutamine. Basal state LV function and) were increased in EPO (dP/dtMAX; 8418.6 6 1521 vs. 6390 6 978 mmHg/sec and dP/dtmin; 6586.7 6 1128 vs. 5375.6 6 797mmHg/sec, p ! 0.05) and myocardial responsiveness was also enhanced in EPO (Ees; 0.750 6 0.197 vs. 0.454 6 0.138, p ! 0.01). Expression of bARK1 in whole myocardium was not changed, but the level of bARK1 and GRK activities of membrane were increased about w3 folds after injection of EPO. Conclusions: EPO, administered at the point of reperfusion, reduced infarct size in whole animal model with a bARK1 dependent manner. These results suggest that EPO may be able to
directly protect the myocardium against ischemic-reperfusion injury and suggest the clinical implications in the situations as acute coronary syndrome.
022 Circadian Variation of Central AT1 Receptor Expression and Hemodynamics in Heart Failure Tarek M. Mousa1, Li Yu1, Irving H. Zucker1; 1Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE The 24 hour circadian variability of cardiovascular parameters is well accepted eg. blood pressure (BP), heart rate (HR), and baroreflex sensitivity (BRS). Loss of these circadian rhythms has been described in chronic heart failure (CHF). We have shown a disrupted circadian variability of HR, BP, and BRS in mice with CHF following myocardial infarction (MI). This disrupted rhythm was associated with an up-regulation of central angiotensin II type 1 receptors (AT1) as early as 2 weeks post MI. It is not clear however whether these phenomena are related. We hypothesized that blockade of AT1 receptors in mice post MI would restore circadian rhythm. Hemodynamics were recorded in C57BL/6 mice using radiotelemetry units in the conscious unrestrained state. BRS was assessed using the spontaneous sequence technique. Day-time (lights on 6:00-18:00) and night-time (lights off 18:00-6:00) averages were taken during the middle 6 hour periods by 5-min recordings every hour. Systemic administration of the AT1 receptor blocker, losartan for1 week at a dose of 1 mg/g in mice 8 weeks post MI failed to restore the lost circadian variability of HR, BP, & BRS. Losartan infusion enhanced the day-time BRS (1.26 6 0.2 vs 1.85 6 0.08 bpm/mmHg, p ! 0.05); with a trend towards an enhanced night-time BRS that did not achieve statistical significance (1.14 6 0.1 vs 1.69 6 0.14 bpm/mmHg, p O 0.05). Losartan infusion also significantly reduced AT1 protein receptor expression in the brainstem in the day-time (1.18 6 0.11 vs 0.85 6 0.06, p ! 0.05) with a trend towards a reduction in the AT1 expression in the night-time that did not reach statistical significance (1.2 6 0.11 vs 1.02 6 0.08, p O 0.05) without restoring circadian rhythm. Furthermore, there were no changes in the brainstem, NAD(P)H subunit gp91phox following losartan infusion. This study is, to our knowledge, the first to demonstrate a circadian pattern of AT1 receptor expression in the brain that coincides with the peripherally recorded hemodynamic changes. We conclude that AT1 receptor expression is mediated by angiotensin II binding but that the circadian variability of hemodynamics and receptor expression is angiotensin II independent.
023 Older Dogs with Whole Body and Myocardial Insulin Resistance Develop an Accelerated Course of Dilated Cardiomyopathy Following Rapid Pacing Pratik Parikh1, Siva Bhashyam1, Indu G. Poornima1, Hakki Bolukoglu1, You-Tang Shen2, Richard P. Shannon1; 1Department of Medicine, Allegheny General Hospital, Pittsburgh, PA; 2Department of Cell Biology & Molecular Medicine, University of Medicine and Dentistry of New Jersey, Newark, NJ Background: Aging is associated with insulin resistance (IR) and also predisposes to worsening heart failure. We sought to determine if IR in the absence of hyperglycemia predisposed to an accelerated course of LV dysfunction following rapid pacing (RVP) in old dogs with documented IR. Methods: Nine young (Y: 4years) and 7 old (O: 11 years) dogs were instrumented with LV pressure gauges (LVP, LVEDP, LVdP/dt), aortic (systolic and diastolic pressure) and coronary sinus catheters, and flow probes on left circumflex (CBF). Dogs underwent measurements of hemodynamics and plasma substrates and a two- hour hyperinsulinemic-euglycemic clamp to measure WB and myocardial glucose uptake (MGU) at Baseline. Dogs then underwent RVP (240 min-1) until symptomatic DCM developed. Results: At baseline, there was no difference in body weight (Y: 15 6 0.6; O: 15 6 0.4 kg) or plasma glucose (Y: 92 6 4; O: 95 6 4 mg/dl) but O had higher (p ! 0.02) NEFA (Y: 389 6 45; O: 796 6 77 mmol/L); and plasma insulin (Y: 38 6 6; O: 119 6 20 pmol/L). Old dogs had impaired WB glucose disposition (Y: 11.5 6 1; O: 8.0 6 0.5 mg/kg/min, p ! 0.05). While basal MGU was not different (Y: 0.38 6 0.1; O: 0.34 6 0.08 mg/min), insulin stimulated MGU was depressed in old dogs (Y: 2.5 6 0.5; O: 1.4 6 0.4 mg/min, p ! 0.05) consistent with both WB and myocardial IR. Myocardial mitochondria show vaculozation and disruption in old compared to young dogs. Old dogs developed severe symptomatic DCM within 7 days, while severe DCM took 28 days in young dogs (see table). Conclusion: Aging is associated with WB and myocardial IR that predisposes to accelerated course of DCM following RVP in conscious dogs. IR may explain the predisposition of older patients to DCM even in the absence of hyperglycemia. Young (n 5 9) Baseline Pacing (days) LVP (mmHg) LVEDP (mmHg) LVdP/dt (mmHg/sec) HR (min1) MAP (mmHg) LVEDD (mm)
118 9 2890 86 90 38
6 6 6 6 6 6
6 1 165 4 3 2
*p ! 0.05 compared to baseline. y p ! 0.01 Y vs. O.
Old (n 5 7)
DCM 28 101 30 1416 112 83 44
6 6 6 6 6 6 6
2 4* 2* 118* 5* 3* 2*
Baseline 130 13 2792 95 104 40
6 6 6 6 6 6
3y 2 212 8 4y 1
DCM 7 108 29 1363 114 85 44
6 6 6 6 6 6 6
3y 3* 2* 132* 9* 2* 3
6 week post-ligation variables
GMCSF/SDF Therapy
Saline Control
Myocardial VEGF concentration 5420 6 2975 12211 6 1491 Caspase-3 activity level 718 6 80 887 6 106 3 Myocardial to body mass ratio 2.72 6 0.06 10 2.45 6 0.07 103 (n 5 6) Ejection fraction (%, n 5 5) 61.8 6 8.6 41.3 6 9.3 Fractional shortening (%, n 5 5) 38.3 6 8.4 23.5 6 4.5 End-systolic borderzone wall 3.37 6 0.27 1.76 6 0.78 thickness (mm, n 5 5) End-diastolic borderzone wall 2.92 6 0.15 1.63 6 0.86 thickness (mm, n 5 5) Cardiac output (ml/min, n 5 5) 33.2 6 7.4 20.6 6 5.9 Wall motion score index, 1.56 6 0.20 2.09 6 0.14 1 5 normal (n 5 5)
p5 0.015 0.030 0.0007 0.018 0.028 0.008 0.038 0.02 0.002
021 Erythropoietin Attenuates the Ischemic-Reperfusion Injury through Redistribution of bARK1 Dong-Ju Choi1, Eun-Ji Kim1, Seo-Yeong Kang1, Bon-Kwon Koo2, Cheol-Ho Kim1; 1 Cardiovacular Center, Seoul National University Bundang Hostpital, Seongnam, Gyeonggi-Do, Korea; 2Cardiovacular Center, Seoul National University Hospital, Seoul, Korea Background; Erythropoietin (EPO), known to stimulate the production of red blood and used to treat anemia in cancer and renal failure, has recently been shown to have a tissue protective effect on diabetic neuropathy, autoimmune encephalomyelitis, and stroke in experimental models. The goal of this study was to determine whether EPO could protect myocardium from ischemia/reperfusion injury and investigate the relation to bARK1. Methods: In anesthetized open-chest male Sprague-Dawley rats, receiving 5,000U/kg of erythropoietin (EPO) intravenously or saline 30 min prior to ischemia, area of necrosis (AN by TTC), the area at risk (AAR by Evans blue), % of infarct (AN/AAR and AN/LV), and Left ventricular hemodynamics by were measured after 30 min of coronary occlusion and 24 hours of reperfusion. LV hemodynamics was accessed by measuring the first derivative of left ventricular pressure (dP/dtMAX and dP/dtmin) and the end-systolic elastance, (Ees) by pressure-volume relationships with IV dobutamine infusion by using high fidelity microcatheter (Millar MikroTipÒ). To examine the mechanistic pathways involved in EPO-mediated myocardial protection, the distributions of bARK1 in membrane or cytosol and GRK activities were evaluated. Results: EPO administration reduced infarct size by 31% and 37%, respectively (%AN/LV; control, 40.1 6 8.8 vs. EPO, 25.2 6 4.3%: p ! 0.01) and (%AN/AAR; control, 57.5 6 10 vs. EPO, 38.5 6 6.9%: p ! 0.01)., whereas AAR was not changed in two group (%AAR/LV; control, 69.6 6 6.5 vs. EPO, 65.7 6 5.1%). EPO also improved ischemia/reperfusion-induced myocardial contractile dysfunction and responsiveness to dobutamine. Basal state LV function and) were increased in EPO (dP/dtMAX; 8418.6 6 1521 vs. 6390 6 978 mmHg/sec and dP/dtmin; 6586.7 6 1128 vs. 5375.6 6 797mmHg/sec, p ! 0.05) and myocardial responsiveness was also enhanced in EPO (Ees; 0.750 6 0.197 vs. 0.454 6 0.138, p ! 0.01). Expression of bARK1 in whole myocardium was not changed, but the level of bARK1 and GRK activities of membrane were increased about w3 folds after injection of EPO. Conclusions: EPO, administered at the point of reperfusion, reduced infarct size in whole animal model with a bARK1 dependent manner. These results suggest that EPO may be able to
directly protect the myocardium against ischemic-reperfusion injury and suggest the clinical implications in the situations as acute coronary syndrome.
022 Circadian Variation of Central AT1 Receptor Expression and Hemodynamics in Heart Failure Tarek M. Mousa1, Li Yu1, Irving H. Zucker1; 1Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE The 24 hour circadian variability of cardiovascular parameters is well accepted eg. blood pressure (BP), heart rate (HR), and baroreflex sensitivity (BRS). Loss of these circadian rhythms has been described in chronic heart failure (CHF). We have shown a disrupted circadian variability of HR, BP, and BRS in mice with CHF following myocardial infarction (MI). This disrupted rhythm was associated with an up-regulation of central angiotensin II type 1 receptors (AT1) as early as 2 weeks post MI. It is not clear however whether these phenomena are related. We hypothesized that blockade of AT1 receptors in mice post MI would restore circadian rhythm. Hemodynamics were recorded in C57BL/6 mice using radiotelemetry units in the conscious unrestrained state. BRS was assessed using the spontaneous sequence technique. Day-time (lights on 6:00-18:00) and night-time (lights off 18:00-6:00) averages were taken during the middle 6 hour periods by 5-min recordings every hour. Systemic administration of the AT1 receptor blocker, losartan for1 week at a dose of 1 mg/g in mice 8 weeks post MI failed to restore the lost circadian variability of HR, BP, & BRS. Losartan infusion enhanced the day-time BRS (1.26 6 0.2 vs 1.85 6 0.08 bpm/mmHg, p ! 0.05); with a trend towards an enhanced night-time BRS that did not achieve statistical significance (1.14 6 0.1 vs 1.69 6 0.14 bpm/mmHg, p O 0.05). Losartan infusion also significantly reduced AT1 protein receptor expression in the brainstem in the day-time (1.18 6 0.11 vs 0.85 6 0.06, p ! 0.05) with a trend towards a reduction in the AT1 expression in the night-time that did not reach statistical significance (1.2 6 0.11 vs 1.02 6 0.08, p O 0.05) without restoring circadian rhythm. Furthermore, there were no changes in the brainstem, NAD(P)H subunit gp91phox following losartan infusion. This study is, to our knowledge, the first to demonstrate a circadian pattern of AT1 receptor expression in the brain that coincides with the peripherally recorded hemodynamic changes. We conclude that AT1 receptor expression is mediated by angiotensin II binding but that the circadian variability of hemodynamics and receptor expression is angiotensin II independent.
023 Older Dogs with Whole Body and Myocardial Insulin Resistance Develop an Accelerated Course of Dilated Cardiomyopathy Following Rapid Pacing Pratik Parikh1, Siva Bhashyam1, Indu G. Poornima1, Hakki Bolukoglu1, You-Tang Shen2, Richard P. Shannon1; 1Department of Medicine, Allegheny General Hospital, Pittsburgh, PA; 2Department of Cell Biology & Molecular Medicine, University of Medicine and Dentistry of New Jersey, Newark, NJ Background: Aging is associated with insulin resistance (IR) and also predisposes to worsening heart failure. We sought to determine if IR in the absence of hyperglycemia predisposed to an accelerated course of LV dysfunction following rapid pacing (RVP) in old dogs with documented IR. Methods: Nine young (Y: 4years) and 7 old (O: 11 years) dogs were instrumented with LV pressure gauges (LVP, LVEDP, LVdP/dt), aortic (systolic and diastolic pressure) and coronary sinus catheters, and flow probes on left circumflex (CBF). Dogs underwent measurements of hemodynamics and plasma substrates and a two- hour hyperinsulinemic-euglycemic clamp to measure WB and myocardial glucose uptake (MGU) at Baseline. Dogs then underwent RVP (240 min-1) until symptomatic DCM developed. Results: At baseline, there was no difference in body weight (Y: 15 6 0.6; O: 15 6 0.4 kg) or plasma glucose (Y: 92 6 4; O: 95 6 4 mg/dl) but O had higher (p ! 0.02) NEFA (Y: 389 6 45; O: 796 6 77 mmol/L); and plasma insulin (Y: 38 6 6; O: 119 6 20 pmol/L). Old dogs had impaired WB glucose disposition (Y: 11.5 6 1; O: 8.0 6 0.5 mg/kg/min, p ! 0.05). While basal MGU was not different (Y: 0.38 6 0.1; O: 0.34 6 0.08 mg/min), insulin stimulated MGU was depressed in old dogs (Y: 2.5 6 0.5; O: 1.4 6 0.4 mg/min, p ! 0.05) consistent with both WB and myocardial IR. Myocardial mitochondria show vaculozation and disruption in old compared to young dogs. Old dogs developed severe symptomatic DCM within 7 days, while severe DCM took 28 days in young dogs (see table). Conclusion: Aging is associated with WB and myocardial IR that predisposes to accelerated course of DCM following RVP in conscious dogs. IR may explain the predisposition of older patients to DCM even in the absence of hyperglycemia. Young (n 5 9) Baseline Pacing (days) LVP (mmHg) LVEDP (mmHg) LVdP/dt (mmHg/sec) HR (min1) MAP (mmHg) LVEDD (mm)
118 9 2890 86 90 38
6 6 6 6 6 6
6 1 165 4 3 2
*p ! 0.05 compared to baseline. y p ! 0.01 Y vs. O.
Old (n 5 7)
DCM 28 101 30 1416 112 83 44
6 6 6 6 6 6 6
2 4* 2* 118* 5* 3* 2*
Baseline 130 13 2792 95 104 40
6 6 6 6 6 6
3y 2 212 8 4y 1
DCM 7 108 29 1363 114 85 44
6 6 6 6 6 6 6
3y 3* 2* 132* 9* 2* 3