Improvement In Fractional Shortening Research Articles

Left Ventricular Geometry and Angiogenesis Improvement in Rat Chronic Ischemic Cardiomyopathy following Injection of Encapsulated Mesenchymal Stem Cells.

Injection of hydrogel and cells into myocardial infarction (MI) patients is one of the emerging treatment techniques, however, it has some limitations such as a lack of electromechanical properties and neovascularization. We investigated the therapeutic potential of new electroactive hydrogel [reduced graphene oxide (rGO)/Alginate (ALG)] encapsulated human bone marrow mesenchymal stem cells (BMSCs). The experimental study involved ligating the left anterior descending coronary artery (LAD) in rat models of chronic ischemic cardiomyopathy. Echocardiograms were analyzed at 4 and 8 weeks after MI treatment. In the eighth week after injection in the heart, the rats were sacrificed. Histological and immunohistochemical analyses were performed using Hematoxylin and Eosin (H and E) staining, Masson's trichrome staining and anti-CD31 antibody to analyze tissue structure and detect neovascularization. In comparison to the control and other treatment groups, MSCs encapsulated in rGO-ALG showed significant improvements in fractional shortening (FS), ejection fraction (EF), wall thickness and internal diameters (P<0.05). The morphological observation showed several small blood vessels formed around the transplantation site in all treated groups especially in the MSC-ALG-rGO group 8 weeks after the transplantation. Also, Masson's trichrome staining indicated an increased amount of collagen fibers in rGO-ALG-MSC. Microvessel density was significantly higher using MSC-ALG-rGO compared to controls (P<0.01). This study demonstrates that intramyocardial injection of rGO/ALG, a bio-electroactive hydrogel, is safe for increasing LV function, neovascularization, and adjusting electrical characteristics following MI. The results confirm ALG promising capability as a natural therapeutic for cardiac regeneration.

Exercise Preconditioning Alters Markers of Cellular Senescence and Prevents DOX‐Induced Cardiorespiratory Dysfunction

Doxorubicin (DOX) is a highly effective chemotherapeutic agent; however its clinical use is limited due to cardiorespiratory complications, including the development of dyspnea, exercise intolerance, and cardiomyopathy. While the mechanisms responsible for DOX‐induced cardiorespiratory dysfunction remain unclear, evidence indicates that DOX administration causes DNA damage and promotes oxidative stress‐induced cellular senescence. Factors shown to contribute to DOX‐induced cellular senescence include cell cycle arrest, driven by signaling through the p16INK4a/Rb and p21/p53 pathways, impaired mitochondrial function, upregulation of pro‐survival pathways, increased lysosomal mass, and expression of the proinflammatory senescence‐associated secretory phenotype (SASP). In contrast, exercise has been shown to prevent cellular senescence in several tissues, leading to the hypothesis that endurance exercise preconditioning is sufficient to prevent senescence induced in the heart and diaphragm muscle following DOX chemotherapy treatment. To determine the relationship between DOX‐induced cardiorespiratory impairment, exercise preconditioning and the impact of accelerated cellular senescence, female Sprague‐Dawley rats underwent two weeks of endurance exercise training (treadmill running, 5 days/week, 60 min/day, 30m/min, 0% grade) before receiving a bolus dose of DOX (20mg/kg IP) or saline treatment. Functional measures were evaluated 48 hours after DOX or saline administration, followed by tissue collection for subsequent analysis of cellular senescence markers. Our results revealed that exercise preconditioning prior to DOX administration prevented diastolic and systolic dysfunction in the heart, with marked improvements in fractional shortening, myocardial performance index, and posterior wall shortening velocity compared to sedentary DOX‐treated animals. DOX‐induced weight loss, reduction in grip strength, and diaphragm weakness were partially rescued with exercise as well, with diaphragm specific force production preserved at frequencies of 60‐160Hz. Finally, both exercise and DOX appear to alter expression of proteins associated with the senescent phenotype in the heart and diaphragm. Therefore, the protective effects of exercise against DOX cardiorespiratory dysfunction may be related to changes in muscle cellular senescence.

Prospective study of dilated cardiomyopathy in dogs eating nontraditional or traditional diets and in dogs with subclinical cardiac abnormalities.

BackgroundRecent studies have investigated dogs with presumed diet‐associated dilated cardiomyopathy (daDCM), but prospective studies of multiple breeds are needed.Hypothesis/ObjectivesTo evaluate baseline features and serial changes in echocardiography and cardiac biomarkers in dogs with DCM eating nontraditional diets (NTDs) or traditional diets (TDs), and in dogs with subclinical cardiac abnormalities (SCA) eating NTD.AnimalsSixty dogs with DCM (NTD, n = 51; TDs, n = 9) and 16 dogs with SCA eating NTDs.MethodsEchocardiography, electrocardiography, and measurement of taurine, cardiac troponin I, and N‐terminal pro‐B‐type natriuretic peptide were performed in dogs with DCM or SCA. Diets were changed for all dogs, taurine was supplemented in most, and echocardiography and cardiac biomarkers were reassessed (3, 6, and 9 months).ResultsAt enrollment, there were few differences between dogs with DCM eating NTDs or TDs; none had low plasma or whole blood taurine concentrations. Improvement in fractional shortening over time was significantly associated with previous consumption of a NTD, even after adjustment for other variables (P = .005). Median survival time for dogs with DCM was 611 days (range, 2‐940 days) for the NTD group and 161 days (range, 12‐669 days) for the TD group (P = .21). Sudden death was the most common cause of death in both diet groups. Dogs with SCA also had significant echocardiographic improvements over time.Conclusions and Clinical ImportanceDogs with DCM or SCA previously eating NTDs had small, yet significant improvements in echocardiographic parameters after diet changes.

Cardiac Revascularization Post Myocardial Infarction Enhances Remuscularization and Improves Function

BackgroundHuman induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) offer an unprecedented opportunity to remuscularize infarcted human hearts. However, studies show that the majority of hiPSC‐CMs die post transplantation into the ischemic environment, limiting their regenerative potential and clinical application. Death of transplanted CMs occurs in the first few days post‐transplantation due to ischemia. Thus, attempts have been made to promote blood perfusion (i.e. addition of endothelial cells and/or angiogenic factors). However, these approaches require weeks for new vessels to form and to carry blood compared to the rapid death of transplanted CMs (2–3 days).PurposeOur goal was to improve the vascularization of the ischemic hearts to improve the survival of hiPSC‐CMs and increase heart remuscularization and function.MethodsWe performed left anterior descending artery (LAD) ligation in immunocompromised rats to model myocardial infarction. To improve vascularization, we used an innovative strategy consisting of ready‐made microvessels isolated from adipose tissue that form a vasculature and carry blood within the first days post subcutaneous implantation. We have co‐implanted ready‐made microvessels with hiPSC‐CMs by intra‐myocardial injection 2 weeks post LAD ligation. Cardiac function was assessed at 0, 2 and 4 weeks post‐implantation by echocardiography and by pressure‐volume loop at the endpoint (4 weeks). Immunohistochemistry and blood perfusion studies were performed at 1 and 4 weeks post‐implantation.ResultsCompared to hiPSC‐CM transplantation alone, microvessels promoted a ~600% increase in hiPSC‐CM survival with significant reduction in scar size. Echocardiography and pressure–volume (PV) loop analysis performed 4 weeks post‐MI revealed that hiPSC‐CM transplantation attenuated post‐infarct ventricular dilation and enhanced left ventricular contractility by demonstrating a significant improvement in fractional shortening (FS), ejection fraction (EF) as well as other functional parameters (end‐systolic volume, end‐diastolic volume, dP/dt max and min, Tau). Remarkably, co‐transplantation of hiPSC‐CMs with microvessels showed significantly superior functional recovery compared to hiPSC‐CMs alone in all the parameters assessed. Microvessels showed remarkable persistence and integration at both early and late time points, resulting in significantly faster blood perfusion and higher vessel density in the grafts.ConclusionThese findings provide a novel approach to cell‐based therapies for myocardial infarction whereby incorporation of ready‐made microvessels can serve as a personalized delivery system to improve functional outcomes in cell replacement therapies post‐myocardial infarction.Support or Funding InformationCanadian Institutes of Health Research (CIHR), Institute of Circulatory and Respiratory Health grant #137352 and #PJT153160. Discovery grant from the Natural Sciences and Engineering Research Council (NESERC): RGPIN 06621‐2017.

STATINS MEDIATE RECOVERY FROM CHEMOTHERAPHY-INDUCED CARDIOTOXICITY

Doxorubicin (Dox) is a frequently used chemotherapeutic agent with heart failure as a major adverse event. While several studies have demonstrated prevention of Dox-induced cardiomyopathy (Dox-CM) in rodents, no translatable therapies have emerged to reverse Dox-CM. Statins have potential as a widely-prescribed, well-tolerated class of drugs with pleiotropic effects and a limited side-effect profile compared to current Dox-CM standard treatment strategies. To determine the potential of statins to reverse Dox-CM, C57BL/6 mice were administered Dox (10mg/kg bolus, IP), leading to a wide derangement of the cardiac transcriptome within 7 days , and reduced fractional shortening 14 days post Dox (27.4%±1.1 vs control: 40%±0.4). Dox-CM mice showed no evidence of cardiac hypertrophy or fibrosis, although there was significant activation of the DNA damage response pathway (γH2AX, ATM, and acetyl-p53) compared to controls. Interestingly, Dox-induced apoptosis was limited to non-cardiomyocyte cells. In vitro analysis of isolated neonatal rat cardiomyocytes shows a significant decrease in spontaneous contractile rate following Dox-treatment, while electrical pacing thresholds required to induce cardiac alternans were reduced in whole heart preparations from Dox-CM mice compared to controls. Accordingly, SERCA-2 protein expression was decreased while phospholamban and HAX-1 are upregulated in Dox-CM hearts vs control. Together, this data suggests that Dox-CM is a result of defective calcium handling. Dox-CM mice that then received daily rosuvastatin (10μg or 100μg/d, IP; figure 1) or atorvastatin treatment (20 μg/d, IP) showed significant improvement in fractional shortening (37.7%±1.8) after two weeks compared to vehicle controls (27.75%±1.9). Statin treatment significantly improved spontaneous cardiomyocyte contractile rate and increased the pacing threshold required to induce cardiac alternans. Analysis of whole heart lysates by Western blotting revealed activation of HSP27-Akt signaling along with resolution of Dox-induced reduction in SERCA-2 protein expression. Furthermore, statin treated mice show fewer CD68 positive cells in heart cross-sections compared to animals treated with Dox alone. Inflammatory pathways appeared to be differentially regulated in the Dox-CM and statin post-treatment groups as noted by increased expression of C5a, CXCL12, IL-7, IL-13, and IL-16 in the statin treated mice compared to Dox-CM. Our detailed analysis of Dox-CM suggests that contractile impairment is related to reversible changes in calcium handling and inflammation, as opposed to the long-held theory of cardiomyocyte loss. Accordingly, the recovery of heart function through statin treatment represents a major step forward in the treatment of Dox-CM and is a readily translatable therapy for cancer survivors suffering from heart failure.

Abstract 417: Targeting the NAD/NADH Ratio for Heart Failure Therapy

Energy deficiency and mitochondrial dysfunction are strongly implicated in the development of heart failure (HF). Yet, the exact mechanisms remain poorly understood. Recently, a causal relationship between loss of cardiac NAD(H) homeostasis and mitochondrial dysfunction have been proposed in HF. A number of studies have demonstrated the benefit of raising NAD levels prior to or at the time of inducing heart failure, but it is unknown whether this strategy benefits hearts with established or pre-existing dysfunction. Therefore, in the present study we increased NAD level in wild-type (WT) mice after pathological hypertrophy and dysfunction were induced by transverse aortic constriction (TAC) surgery. WT mice were treated daily for six-weeks with nicotinamide riboside (NR) starting four-weeks after TAC. The NR treatments significantly elevated NAD level in the TAC hearts (3.1 fold, n=4) compared to vehicle treated mice, accompanied by an improvement in fractional shortening (FS) (sham vehicle 51%±2% vs TAC Vehicle 31%±3% vs TAC NR 43%±5%, n=4 each group). Most evidence thus far suggest the benefit of raising NAD level in the failing heart involves activation of sirtuin 3 (Sirt3), a NAD dependent deacylase. We hypothesize there are Sirt3 independent mechanisms that are activated by increasing NAD levels to benefit the failing heart. To test this hypothesis, we treated Sirt3 deficient (Sirt3-/-) mice with NR starting four-weeks after TAC surgery and extended the study to ten-weeks post TAC. NR treatments elevated cardiac NAD level (2.9-fold, n=4) compared to vehicle treated controls. This correlated with lowered global protein acetylation in the hearts and an improvement in FS (sham vehicle 50%±5% vs TAC vehicle 24%±6% vs TAC NR 41%±8%). We also looked at mitochondrial function in Sirt3-/- mice ten-weeks after TAC ± NR treatments and observed an increase in state 3 ADP-stimulated respiration in the NR treated Sirt3-/- mice (sham vehicle 183±17 vs TAC vehicle 103±23 vs TAC NR 127±21 nmol/min/mg). These findings suggest increasing NAD can improve cardiac function through Sirt3 independent mechanisms including one that enhances mitochondrial respiration. Further elucidation of these mechanisms could identify novel NAD sensitive therapeutic targets.

Review of children with Takayasu's arteritis at a Southern African tertiary care centre.

The objective of this study was to determine the clinical presentation, demographic profile, vascular involvement, origins, management, and outcome of children diagnosed with Takayasu's arteritis at a Southern African tertiary care centre between 1993 and 2015. This is a retrospective analysis of all children with Takayasu's arteritis captured on a computerised electronic database during the study period. A total of 55 children were identified. The female:male ratio was 3.2:1, and the mean age was 9.7±3.04 years. Most originated outside the provincial borders of the study centre. The majority presented with hypertension and heart failure. In all, 37 (67%) patients had a cardiomyopathy with a mean fractional shortening of 15±5%. A positive purified protein derivative test was documented in 73%. Abdominal aorta and renal artery stenosis were the predominant angiographic lesions. A total of 23 patients underwent 30 percutaneous interventions of the aorta, pulmonary, and renal arteries: eight stents, 22 balloon angioplasties, and seven had nephrectomies. All patients received empiric tuberculosis treatment, immunosuppressive therapy, and anti-hypertensive agents as required. Overall, there was a significant reduction in systolic blood pressure and improvement in fractional shortening (p<0.05) with all treatments. Takayasu's arteritis is more common in girls and frequently manifests with hypertension and heart failure. The abdominal aorta and renal arteries are mostly affected. Immunosuppressive, anti-hypertensive, and vascular intervention therapies improve blood pressure control and cardiac function.

25 - Role of selenomethionine supplementation in the modulation of damage during inflammation and myocardial ischemia/reperfusion injury

Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury. This partly arises from the presence of activated phagocytes releasing the heme enzyme myeloperoxidase (MPO) and the associated production of hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). The dietary supplement selenomethionine (SeMet) has been shown to both bolster endogenous antioxidant processes and readily react with MPO-derived oxidants, yet its ability to protect against cardiac I/R injury is unknown. The aim of this study was to assess whether supplementation with SeMet could modulate the extent of cellular damage observed in an in vitro cardiac myocyte model exposed to (patho)-physiological levels of HOCl and HOSCN and an in vivo rat model of cardiac I/R injury. Exposure of cardiac myocytes to HOCl resulted in a dose-dependent increase in necrotic cell death, which could be prevented by SeMet supplementation (25 µM). This was attributed to SeMet preventing the HOCl-induced loss of mitochondrial inner trans-membrane potential, and the associated cytosolic calcium accumulation, together with bolstering the activity of the selenium-dependent antioxidant enzyme glutathione peroxidase. In contrast, no apparent protection of SeMet supplementation was evident upon exposure of cells to HOSCN, where higher oxidant concentrations (100 – 125 µM) were required to induce cell death. In vivo, a significant increase in selenium levels in the plasma, brain, kidney, and heart tissue were seen in rats fed a diet supplemented with 2 mg/kg SeMet compared to controls. Echocardiograph assessment of LV function at 4 weeks post-surgical intervention in the I/R injury cohort demonstrated limited improvement in fractional shortening and ejection fraction in SeMet-supplemented fed rats compared to controls. However, a more marked improvement was observed in the sham injury cohort, indicating SeMet supplementation’s potential cardiac health benefit.

Improvement in fractional shortening in aortic regurgitation rats: cardiac muscle network

Aortic regurgitation (AR), a volume overload to the heart, impairs systolic function. Paroxetine (parox) treatment, a selective serotonin reuptake inhibitor, improves systolic function in AR rats, probably due decreases in the expression of β-myosin heavy chain (βMyHC) gene. An intricate network regulate genes co-expressing microRNAs (miR-208a,-208b and -499) and transcriptional repressors which in turn controls MyHCisoforms content. Thus, we verify the gene expression of those microRNAs and the transcriptional repressor (Thrap1) in AR rats treated with parox. Male Wistar rats (280-300kg) were submitted to sham or AR surgery. Morphofunctional variables of the hearts were analyzed by echocardiograms. Parox (10mg/kg) was administered subcutaneously for 4 weeks. There were 4 groups: AR+parox, AR+saline, Sham+parox and Sham+saline. At week 8 the animals were euthanized for tissue collection and analysis of gene expression by RTq-PCR. Two way repeated measures ANOVA were used for comparisons. Parox treatment preserved the fractional shortening in AR rats. The expression of miR-208a and Thrap 1 were not changed. Though there was a decrease in miR-208b and miR-499 gene expression, which may regulate the decrease of β-MyHC isoform in AR rats treated with parox, explaining the improvement in systolic function. Financial support: FAPESP 2013/11372-6, FAPESP 2013/00742-7

MicroRNA induced cardiac reprogramming in vivo: evidence for mature cardiac myocytes and improved cardiac function.

A major goal for the treatment of heart tissue damaged by cardiac injury is to develop strategies for restoring healthy heart muscle through the regeneration and repair of damaged myocardium. We recently demonstrated that administration of a specific combination of microRNAs (miR combo) into the infarcted myocardium leads to direct in vivo reprogramming of noncardiac myocytes to cardiac myocytes. However, the biological and functional consequences of such reprogramming are not yet known. The aim of this study was to determine whether noncardiac myocytes directly reprogrammed using miRNAs in vivo develop into mature functional cardiac myocytes in situ, and whether reprogramming leads to improvement of cardiac function. We subjected fibroblast-specific protein 1-Cre mice/tandem dimer Tomato (tdTomato) mice to cardiac injury by permanent ligation of the left anterior descending coronary artery and injected lentiviruses encoding miR combo or a control nontargeting miRNA. miR combo significantly increased the number of reprogramming events in vivo. Five to 6 weeks after injury, morphological and physiological properties of tdTomato(-) and tdTomato(+) cardiac myocyte-like cells were analyzed ex vivo. tdTomato(+) cells expressed cardiac myocyte markers, sarcomeric organization, excitation-contraction coupling, and action potentials characteristic of mature ventricular cardiac myocytes (tdTomato(-) cells). Reprogramming was associated with improvement of cardiac function, as analyzed by serial echocardiography. There was a time delayed and progressive improvement in fractional shortening and other measures of ventricular function, indicating that miR combo promotes functional recovery of damaged myocardium. The findings from this study further validate the potential use of miRNA-mediated reprogramming as a therapeutic approach to promote cardiac regeneration after myocardial injury.

Chronic blockade of vasopressin type1a receptor and/or type2 receptor improves both cardiac and renal damage and dysfunction in hypertensive rats

Background: Vasopressin (AVP) type 1a receptor (V1aR) antagonist could theoretically benefit heart failure (HF) patients by decreasing afterload and ameliorating remodeling. However, the conflicting results have been reported. Also, there is a concern that increased AVP may have detrimental effects through V1aR in HF patients treated with oral type 2 receptor (V2R) antagonist, tolvaptan. Therefore, we examined whether the chronic administration of V1a antagonist may exert beneficial effects and also its additive effects to tolvaptan treatment in rat hypertensive HF model. Methods and results: In Dahl salt-sensitive hypertensive rats, not only circulating AVP level, but also myocardial AVP and V1a receptor (V1aR), and renal V1aR and V2R expressions were significantly activated during the transition to HF (18-21 weeks). First, they were chronically treated with vehicle, V1aR antagonist (OPC21268), tolvaptan, or OPC21268 plus tolvaptan (combination) from pre-hypertrophic stage (6 weeks). All three treatments did not affect blood pressure. In OPC21268 and tolvaptan groups, the median survival time was significantly improved from 112 day (vehicle) to 141 day and 160 day, respectively. It was improved to a greater extent with the combined treatment (175 day). Echocardiography showed the improvement of fractional shortening (FS) was observed in all three treatment groups and the suppression of LV hypertrophy (LVH) only in OPC 21268 and combination groups. Furthermore, renal histopathologic damage (glomerulosclerosis and tubulointerstitial fibrosis) was ameliorated, and renal function (creatinine clearance) was also improved in all three treatment groups at HF stage. When these treatments were started at the established LVH phase (11 weeks), the improvement of survival was observed only in tolvaptan group and no additive improvement in the combined treatment. Conclusions: These findings suggest that V1aR blockade may ameliorate the development of LVH and HF in the treatment of hypertensive HF with renal injuries, and have limited effects starting after the establishment of LVH. Also, the chronic administration of both V1a and V2 antagonists exerts further beneficial effects. The underlying mechanism may be related to protection of myocardial and renal damages independent of blood pressure.

2012 Late-Breaking Basic Science Oral Session

### Cardiac Myosin Binding Protein C is an Ultra-early and Cardiac-specific Biomarker of Myocardial Necrosis Diederik W Kuster1, Adriana Cardenas-Ospina2, Lawson Miller2, Christian Troidl3, Holger M Nef3, Christoph Liebetrau3, Mollmann Helge3, Karen S Pieper4, Kenneth W Mahaffey4, Neal S Kleiman5, Bruno D Stuyvers2, Ali J Marian6, Sakthivel Sadayappan1; 1Loyola Univ Chicago, Maywood, IL, 2Memorial Univ, St John’s, Canada, 3Kerckhoff Heart and Thorax Cntr, Bad Neuheim, Germany, 4Duke Univ Med Cntr, Durham, NC, 5The Methodist DeBakey Heart and Vascular Cntr, Houston, TX, 6Univ of Texas Health Sciences, Houston, TX Rationale: We recently demonstrated that proteolytic cleavage fragments of cardiac myosin binding protein-C (cMyBP-C) can be detected in the serum after myocardial infarction (MI) in a rat model and in patients with MI. The findings implied that serum cMyBP-C might be useful as cardiac-specific biomarker for MI. The release kinetics of cMyBP-C in the circulation post-MI remains to be elucidated. Objective: Determine the release kinetics of cMyBP-C as an ultra-early and cardiac-specific biomarker of myocardial necrosis. Method and Results: To determine the exact timing of cMyBP-C release in the bloodstream post-acute MI, left anterior descending (LAD) coronary artery was ligated in adult swine (n=6). ECG showed significant ST elevation. Infarct size represented 12.4 ± 1.9% of total ventricular mass. Blood samples were collected before and at predetermined time points between 30 min and 14 days after LAD ligation. Plasma cMyBP-C level was quantified using a highly sensitive and rapid sandwich enzyme-linked immunosorbent assay. Compared with baseline, cMyBP-C levels were increased in post-MI serum within 45 min (0.64 ± 0.52 ng/ml) after LAD ligation and declined after 16 hrs to the baseline level (0.01 ± 0.00 ng/ml). In contrast, cardiac troponin I (cTnI) level peaked after …

Impact of Chronic Kidney Disease (CKD) and Cardiovascular Risk Factors on Cardiac Function in Children after Renal Transplantation

Background: Barriers to successful outcomes following pediatric transplantation have shifted from ischemic reperfusion injury and rejection to more long-term complications. Of particular concern is the high prevalence of CKD owing to preexisting damage and nephrotoxicity, as well as other CV complications such as hypertension and cardiomyopathy. On the other hand, the occurrence and progression of cardiomyopathy is well known in patients with end-stage renal disease (ESRD). However, the feasibility of renal transplantation in the setting of cardiac dysfunction and the effect of renal transplantation on this progression remain poorly studied in pediatric patients. Patients and methods: A single-center prospective examination of cardiac function in pediatric renal transplants was conducted. Fifty children with ESRD and severe systolic dysfunction were examined. Thirty patients underwent renal transplantation while 20 were used as a control group. Clinical data were collected and compared for the pretransplant and post transplant periods. Results: Dialysis, nutrition and chronic kidney disease and heart failure therapy led to improved cardiac function before renal transplantation however, normal cardiac function was not achieved before transplantation in most patients. After transplantation, there was a statistically significant improvement in all cardiac parameters There was a regression in left ventricular end diastolic volume(LVED) in children (4.7 ± 0.8 to 4.2 ± 0.5). There was a regression in left ventricular end systolic volume (LVES) (3.1 ± 0.6 to 2.4 ± 0.4). There was an improvement in fractional shortening (FS) (32.6 ± 5.3 to 41.7 ± 7.6). All patients experienced excellent allograft function in the peritransplant period with excellent mean estimated creatinine clearance 1 year post transplant. Increased hemoglobin percentage and improvements in renal function showed strong associations with different cardiac functions. Conclusions: Renal transplantation can be performed safely in children with ESRD and cardiac dysfunction. It improves cardiac morphology and function and should be done as early as possible to attain optimum results. The presence of systolic dysfunction in pediatric dialysis patients should not defer referral for renal transplantation.

Abstract 194: Increasing Expression of Connexin43 in Bone Marrow Stem Cells Enhances Their Protective Effects on the Infarcted Heart

Introduction: Coupling of stem cells (SCs) injected into the infarcted heart to endogenous cells that survived injury may enhance SC survival and engraftment and improve their protective effects. Connexin43 (Cx43) gap junctions are some of the first proteins expressed by differentiating SCs transplanted into the heart after myocardial infarction (MI), suggesting that cell coupling is critical for SC survival and differentiation. Hypothesis: Overexpression of Cx43 in SCs will enhance their ability to engraft, differentiate, and improve cardiac function and survival after transplant into the infarcted heart. Methods: Adult mouse SCs isolated from compact bone were sorted for c-kit surface marker using magnetic beads. SCs were transduced with PLL3.7 lentivirus encoding the Cx43 gene (GJA1) at MOI=300 to produce overexpression, which was confirmed by Western analysis on SC lysates. MI was induced in 12-week-old male C57BL/6 mice by ligating the left anterior descending coronary artery, and 40,000 SCs in 20 uL saline were administered immediately after MI via intramyocardial injection. A total of 69 animals were used: 18 received Cx43 SCs, 30 received non-transduced SCs (NTSC), 11 received only saline, and 10 underwent sham surgery. Echocardiography was performed 1 and 2 weeks post-MI. Results: Animals receiving Cx43 SCs had improved survival (90.0%) versus NTSC (76.7%) and saline controls (70.0%). The Cx43 SC group displayed improved ejection fraction versus NTSC and saline controls at 1 week (48.7% v. 43.3%, p=0.04; 30.1%, p&lt;0.0001) and 2 weeks post-MI (45.7% v. 40.4%, p=0.08; 26.8%, p=0.0004). Similar improvements in fractional shortening, stroke volume, and cardiac output were observed. Administration of Cx43 SC and NTSC attenuated remodeling with no difference between the two groups. Both decreased end-diastolic and systolic volumes, increased thickness of the infarct-related anterior wall, and decreased heart weight (HW), HW/body weight and HW/tibia length ratios at 1 and 2 weeks post-MI. Conclusion: All SCs improved survival and cardiac function, and attenuated remodeling. Transplantation of Cx43 SCs improved survival and cardiac function even more than NTSCs, suggesting that Cx43 may play a crucial role in engraftment and differentiation.

Effect of bone marrow-derived extracellular matrix on cardiac function after ischemic injury

Ischemic heart disease is a leading cause of death, with few options to retain ventricular function following myocardial infarction. Hematopoietic-derived progenitor cells contribute to angiogenesis and tissue repair following ischemia reperfusion injury. Motivated by the role of bone marrow extracellular matrix (BM-ECM) in supporting the proliferation and regulation of these cell populations, we investigated BM-ECM injection in myocardial repair. In BM-ECM isolated from porcine sternum, we identified several factors important for myocardial healing, including vascular endothelial growth factor, basic fibroblast growth factor-2, and platelet-derived growth factor-BB. We further determined that BM-ECM serves as an adhesive substrate for endothelial cell proliferation. Bone marrow ECM was injected in a rat model of myocardial infarction, with and without a methylcellulose carrier gel. After one day, reduced infarct area was noted in rats receiving BM-ECM injection. After seven days we observed improved fractional shortening, decreased apoptosis, and significantly lower macrophage counts in the infarct border. Improvements in fractional shortening, sustained through 21 days, as well as decreased fibrotic area, enhanced angiogenesis, and greater c-kit-positive cell presence were associated with BM-ECM injection. Notably, the concentrations of BM-ECM growth factors were 103–108 fold lower than typically required to achieve a beneficial effect, as reported in pre-clinical studies that have administered single growth factors alone.

Abstract 9306: Site-Specific Reporter Gene Imaging of Stem Cell Transplantation: Early Engraftment Predicts Late Improvement in Cardiac Function

Stable genetic modification of cardiac progenitor cells (CPCs) enables quantitative assessment of their engraftment and proliferation using reporter gene imaging. However, random lentiviral integration techniques carry the concomitant risk of introducing inadvertent mutations, including the unacceptable risk of proto-oncogene activation. Here, we demonstrate a safe, clinically applicable method for site-specific PET reporter gene imaging of transplanted CPCs. A triple fusion reporter gene was integrated specifically into chromosome 19 of Sca-1+ human CPCs using the ΦC31 integrase. Targeted integration into this genomic locus was confirmed using nested PCR and Southern blotting. Stably-integrated CPCs (1×10 6 ) were then transplanted into ischemic murine myocardium (n=40), and monitored noninvasively using PET, bioluminescence, cardiac MR, and echocardiography for four weeks. hCPC-treated hearts demonstrated a statistically significant improvement in fractional shortening (27.2±2.3% vs. 21.5±1.4%; P=0.04) as well as ejection fraction (48.3±2.6% vs. 42.2±1.7%; P=0.03) at day 14 post-transplant compared to PBS controls, as assessed by echocardiography and cardiac MRI, respectively. Initial PET signal intensity, as a surrogate marker of cellular engraftment, predicted the degree of functional improvement at week 2 (R 2 =0.73) in a “dose-effect” relationship. To investigate the mechanism of hCPC benefit on myocardial function, we isolated the transplanted cells from recipient myocardium by laser capture microdissection and found direct evidence for upregulated expression of the VEGF-α and CTGF pro-angiogenic growth factors in vivo. Here, we demonstrate the first use of site-specific reporter gene imaging to enable prognostic prediction of the ultimate success of CPC therapy. Given the encouraging but variable results of recent cell therapy trials, our results provide valuable insight into the determinants of successful cell therapy.

Induced Pluripotent Stem (iPS) Cells Inhibit Apoptosis and Fibrosis in Streptozotocin-Induced Diabetic Rats

Recent data suggests that transplanted bone marrow stem cells improve cardiac function in streptozotocin induced diabetic cardiomyopathy (SIDC). However, whether modified embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, or factors released from these cells can inhibit apoptosis and fibrosis remains completely unknown. Therefore, we hypothesized that transplanted ES cells overexpressing pancreatic transcription factor 1 a (Ptf1a), a propancreatic endodermal transcription factor, iPS cells, or their respective conditioned media (CM) will attenuate cardiac remodeling and improve cardiac function in SIDC. Experimental diabetes was induced in male Sprague-Dawley rats (8-10 weeks old) by intraperitoneal injections of streptozotocin (STZ) (65 mg/kg body weight). Animals were divided into different groups including control, STZ, stem cells, and CM. Histology, TUNEL, caspase-3 activity, sarcomeric α-actin, and DHE stainings were performed to assess cardiac apoptosis, fibrosis, and oxidative stress. Animals transplanted with ES cells, iPS cells, or both CM showed a significant (p < 0.05) reduction in apoptosis compared with STZ treated animals. Furthermore, our data also shows that active apoptosis was present in cardiac myocytes as confirmed with combined stainings with TUNEL, sarcomeric α-actin, and active caspase-3 antibodies. Increased oxidative stress as evidenced by DHE staining was significantly (p < 0.05) reduced following stem cell or CM transplantation. Moreover, stem cells or CM also attenuated increased interstitial and vascular fibrosis in SIDC hearts. Echocardiography analysis showed a significant (p < 0.05) improvement in fractional shortening in stem cell and CM transplanted groups compared with respective controls. In conclusion, our data suggest that transplanted stem cells or their CM inhibit apoptosis, reduce fibrosis, and improve cardiac function in STZ-treated diabetic rats.

Improvement in Cardiac Function With Small Intestine Extracellular Matrix Is Associated With Recruitment of C-Kit Cells, Myofibroblasts, and Macrophages After Myocardial Infarction

This study tested the hypothesis that modulation of angiogenesis and cardiac function by injecting small intestine extracellular matrix emulsion (EMU) into myocardium is associated with recruitment of c-kit cells, myofibroblasts, and macrophages after myocardial infarction. Degradation of native extracellular matrix has been associated with adverse cardiac remodeling after infarction. Sixty-four rats were subjected to 45 min ischemia followed by 3, 7, 21, and 42 days of reperfusion, respectively. Saline or EMU (30 to 50 microl) was injected into the area at risk myocardium after reperfusion. Histological examination was performed by immunohistochemical staining, and cardiac function was analyzed using echocardiography. The population of c-kit-positive cells in infarcted myocardium with the EMU injection increased significantly relative to the saline control at 7 days of reperfusion. Along with this change, alpha-smooth muscle actin expressing myofibroblasts and macrophages accumulated to a significant extent compared with the saline control. Increased vascular endothelial growth factor protein level and strong immunoreactivity of vascular endothelial growth factor expression were observed. Angiogenesis in the EMU area was significantly enhanced relative to the saline control, evidenced by increased density of alpha-smooth muscle actin positive vessels. Furthermore, echocardiography showed significant improvements in fractional shortening, ejection fraction, and stroke volume in the EMU group. The wall thickness of the infarcted middle anterior septum in the EMU group was significantly increased relative to the saline control. We show for the first time that injection of EMU into the infarcted myocardium increases neovascularization and preserves cardiac function, potentially mediated by enhanced recruitment of c-kit-positive cells, myofibroblasts, and macrophages.

Surgical ventricular reconstruction in mice: Elucidating potential targets for combined molecular/surgical intervention

We hypothesize that persistent alterations in molecular signaling may drive recurrent pathologic remodeling even after the reduction of mechanical stress achieved via surgical ventricular reconstruction. We developed a murine model of surgical ventricular reconstruction that would facilitate molecular analysis of the postreconstruction myocardium and allow future exploitation of genetic models. C57/B6 mice underwent coronary artery ligation. For surgical ventricular reconstruction at 4 weeks after myocardial infarction, a purse-string suture (7-0 polypropylene) achieved at least partial exclusion of the apical aneurysm. Serial echocardiography was correlated to measurements of apoptosis and to Western blot analysis of key signaling cascades. An immediate 21.7% +/- 2.6% improvement in fractional shortening was seen in the remaining myocardium after surgical ventricular reconstruction. Reduction in left ventricular volume and improved function persisted at 1 week, but recurrent dilatation at 4 weeks (left ventricular end-diastolic volume of 63.5 +/- 2.5 vs 42.1 +/- 5.4 microL immediately after reconstruction; P < .05) was associated with a loss of functional improvement (fractional shortening 41.2% +/- 2% vs 46% +/- 0.9%; P < .01). At 1 week after surgical ventricular reconstruction, there was a transient reduction in myocardial apoptosis. A steady reduction in cardioprotective myocardial Akt activation, however, was not affected by ventricular reconstruction. This murine model recapitulates both the immediate benefits of surgical ventricular reconstruction and the longer-term recurrence of dilated cardiomyopathy seen previously in some animal models and human studies. Early analysis has begun to implicate persistent signaling changes in the postinfarction myocardium that may be responsible for recurrent dilatation after surgical ventricular reconstruction and that may become targets for combined surgical and molecular interventions.

Long-Term but Not Short-Term p38 Mitogen-Activated Protein Kinase Inhibition Improves Cardiac Function and Reduces Cardiac Remodeling Post-Myocardial Infarction

p38 mitogen-activated protein kinase (p38 MAPK) inhibition exerts beneficial effects on left ventricular (LV) remodeling and dysfunction. p38 MAPK activity is transiently increased soon after myocardial infarction (MI), suggesting brief inhibition may afford the same benefit as long-term inhibition. We examined chronic 12-week p38 MAPK inhibition compared with short-term (7-day) inhibition, and then we discontinued inhibition after MI. Post-MI rats at day 7 received either vehicle, 4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol (RWJ67657; RWJ) for 12 weeks (long term; LT-RWJ), RWJ for 1 week and discontinued for 11 weeks (1-week RWJ), or continuous ramipril for 12 weeks. In separate groups of animals, 24 h after MI, vehicle or RWJ was administered for 7 days. Cardiac function was assessed by echocardiography and hemodynamic measurements. Percentage of fractional shortening improved after LT-RWJ and ramipril, but not after 1-week RWJ treatment. Likewise, LV contractility and maximal first derivative of left ventricular pressure (dP/dt(max)) was improved (12.5 and 14.4%) and LV end diastolic pressure (LVEDP) was reduced (49.4 and 54.6%) with both treatments. Functional outcomes were accompanied by regression of interstitial collagen I and alpha-smooth muscle actin expression in LV noninfarct, border, and infarct regions with LT-RWJ and ramipril treatment. Hypertrophy was reduced in noninfarct (18.3 and 12.2%) and border regions (16.3 and 12.0%) with both treatments, respectively. Animals receiving RWJ 24 h after MI for 7 days showed similar improvements in fractional shortening, dP/dt(max), LVEDP, including reduced fibrosis and hypertrophy. In vitro experiments confirmed a dose-dependent reduction in hypertrophy, with RWJ following tumor necrosis factor-alpha stimulation. Continuous but not short-term p38 MAPK blockade attenuates post-MI remodeling, which is associated with functional benefits on the myocardium.