Late-Breaking Basic Science Abstracts From the American Heart Association's Scientific Sessions 2010, Chicago, Illinois, November 13–17, 2010
Late-Breaking Basic Science Oral Abstracts
Use of Cardiac Stem Cells for the Treatment of Heart Failure: Translation from Bench to the Clinical Setting
Roberto Bolli, Atul R Chugh, Univ of Louisville, Louisville, KY; Domenico D'Amario, Harvard Med Sch, Boston, MA; Marcus F Stoddard, Sohail Ikram, Stephen G Wagner, Garth M Beache, Univ of Louisville, Louisville, KY; Annarosa Leri, Harvard Med Sch, Boston, MA; Toru Hosoda, Harvard Med Sch, Boston, KY; John H Loughran, Univ of Louisville, Louisville, KY; Polina Goihberg, Claudia Fiorini, Harvard Med Sch, Boston, MA; Naresh K Solankhi, Ibrahim Fahsah, Arka Chatterjee, Julius B Elmore, D. G Rokosh, Mark S Slaughter, Univ of Louisville, Louisville, KY; Jan Kajstura, Piero Anversa; Harvard Med Sch, Boston, MA
Extensive preclinical studies have documented the presence in the adult heart of cardiac stem cells (CSCs) that express c-kit and are clonogenic, self-renewing, and multipotent. CSCs have been shown to improve postinfarction LV dysfunction in animal models, but their efficacy in humans has not been evaluated. In August 2008, we obtained FDA approval to begin SCIPIO (Stem Cell Infusion in Patients with Ischemic CardiOmyopathy). SCIPIO is a phase I, randomized, single-center, open-label trial of CSCs in patients with postinfarction LV dysfunction (EF 40%) who undergo CABG surgery. Autologous CSCs are isolated from the right atrial appendage (harvested during CABG), expanded, and re-infused intracoronarily 4 months after CABG; patients are followed for 2 years. We report here the initial results at 4 months after treatment in the first 8 CSC-treated patients. Of these, one was excluded (aortic stenosis), and in another one echo images were inadequate. In the remaining 6 CSC-treated patients, LVEF (3D echo) increased markedly from 32.4±2.3% before CSC infusion to 43.3±3.3% 4 months later (P=0.017) (Figure). This was associated with a significant improvement in regional wall motion score (echo) in the infused LV regions (1.91±0.18 vs. 1.63±0.13, P=0.03), LV synchrony (segmental 3D echo; 16-segment normalized SD: 13.08±3.23 vs. 3.83±0.96, P=0.05), and NYHA functional class (2.43±0.79 vs. 1.57±0.54, P=0.016). There was also a trend toward a reduction in infarct size (assessed by MRI score in 4 patients) and in rest perfusion defects (sestamibi SPECT score). In the 2 patients seen at 1 year post-CSC, functional improvement persisted. No adverse effects have been recorded. Prior to delivery, CSCs showed high telomerase activity and telomere length >7 kbp. These initial results of CSC therapy in humans are very encouraging, and suggest that infusion of autologous CSCs is effective in improving LV systolic function, reducing infarct size, and augmenting functional capacity.
Author Disclosures: R. Bolli: None. A.R. Chugh: None. D. D'Amario: None. M.F. Stoddard: None. S. Ikram: None. S.G. Wagner: None. G.M. Beache: None. A. Leri: None. T. Hosoda: None. J.H. Loughran: None. P. Goihberg: None. C. Fiorini: None. N.K. Solankhi: None. I. Fahsah: None. A. Chatterjee: None. J.B. Elmore: None. D.G. Rokosh: None. M.S. Slaughter: None. J. Kajstura: None. P. Anversa: None.
Key Words: Stem cell therapy, Ischemic heart disease, Regenerative medicine stem cells
Intravenous Gene Therapy with Pim-1 via a Cardiotropic Viral Vector Halts the Progression of Diabetic Cardiomyopathy Through Promotion of Pro-survival Signaling
Rajesh Katare, Andrea Caporali, Univ of Bristol, Bristol, United Kingdom; Lorena Zentilin, International Cntr for Genetic Engineering and Biotechnology, Padriciano, Italy; Graciella S Newby, Costanza Emanueli, Univ of Bristol, Bristol, United Kingdom; Mauro Giacca, International Cntr for Genetic Engineering and Biotechnology, Padriciano, Italy; Paolo Madeddu; Univ of Bristol, Bristol, United Kingdom
Background: Studies in transgenic mice showed the key role of moloney murine leukemia virus 1 (Pim-1) in the control of cardiomyocyte function and viability. Objective: To investigate if Pim-1 is a therapeutic target for the cure of diabetic cardiomyopathy (DCM), a steadily increasing cause of non-ischemic heart failure. Methods and Results: Western blot analysis on hearts of streptozotocin-induced type-1 (T1D) mice showed a time-dependent reduction in Pim-1 (8-fold at 20 wks from T1D induction), a parallel decline in the Pim-1 activators STAT-3 (6-fold) and Akt (7-fold) and an increase of the Pim-1 direct inhibitor miR-1 (6-fold) (P<0.01 vs. age-matched non-diabetic (ND) mice for all comparisons). Moreover, diabetic hearts showed low levels of anti-apoptotic Bcl-2, high levels of pro-apoptotic Bad and increased caspase 3/7 activity (P<0.01 vs. ND for all comparisons). Studies on murine cardiomyocytes challenged with high glucose (HG) confirmed the in vivo expressional changes. In in vitro rescue studies, anti-miR-1 boosted Pim-1 and Bcl-2 expression and promoted cardiomyocytes survival under HG. Similarly, transfection with Pim-1 plasmid prevented cardiomyocyte apoptosis. Finally, at 4 wks from T1D induction, mice were randomly assigned to receive an i.v. injection of human (h) Pim-1 via cardiotropic serotype-9 adeno-associated virus (1×1010 or 5×1010 pfu, doses decided on pilot titration studies) or empty vector. Expression of hPim-1 was confirmed by Western blot and immunohistochemistry in cardiomyocytes and to a less extent in skeletal muscles (Fig.1a), but not in other organs.Echocardiography showed that hPim-1 gene therapy attenuates diastolic dysfunction and prevents the development of left ventricle dilatation and failure in T1D mice (Fig.1b). Conclusion: Down-regulation of pro-survival Pim-1 contributes in the pathogenesis of DCM. Systemic delivery of hPim-1 via cardiotropic AAV9 represents a novel and effective approach to treat DCM.
Author Disclosures: R. Katare: Employment; Significant; Salary is paid by British Heart Foundation. Research Grant; Significant; British Heart Foundation. A. Caporali: None. L. Zentilin: None. G.S. Newby: None. C. Emanueli: None. M. Giacca: None. P. Madeddu: British Heart Foundation.
Key Words: Cardiomyopathy, Gene therapy, Diastolic function
Reduced Complex I Function of the Mitochondria Accelerates Heart Failure in Mice
Georgios Karamanlidis, Lorena Garcia Menendez, Stephen Kolwicz, Richard Palmiter, Rong Tian; Univ of Washington, Seattle, WA
Impaired mitochondrial function is closely associated with heart failure. To define the causal role of mitochondrial dysfunction in the development of heart failure we generated a cardiac-specific model of impaired Complex I function by deleting a subunit encoded by Ndusf4 in the mouse heart (cNdufs4−/−). Complex I activity decreased by ∼70% and complex I substrates supported respiration in permeabilized myofibers decreased by ∼50% in cNdufs4−/−, whereas complex II activity and complex II substrate supported respiration remained unaltered. Mitochondrial volume density and morphology were also similar in cNdufs4−/− and control (CON) hearts. Using 31P NMR spectroscopy of isolated perfused hearts, we found that high energy phosphate content and contractile function of the cNdufs4−/− hearts were normal suggesting that the deficiency of Complex I was fully compensated. In-vivo cardiac function assessed by echocardiography as well as the survival of cNdufs4−/− mice were also normal in up to one year. However, when cNdufs4−/− mice were subjected to pressure overload by transverse-aortic constriction (TAC) for 4 weeks the heart weight was increased by 2.5±0.1 folds compared to 1.6±0.1 folds in CON mice (n=5-6 per group; p<0.05). LV fractional shortening (FS) was significantly reduced in TAC-cNdufs4−/− (12±1%; n=5) mice compared to TAC-CON (36±6%; n=6; p<0.05). LVFS was 55±4% in sham-operated hearts for both genotypes. LV end-diastolic dimension increased 63% at 4 weeks post TAC in cNdufs4−/− (from 3.0±0.01 to 4.9±0.2 mm; p<0.05) while it did not change in TAC-CON (from 3.0±0.01 to 3.5±0.2 mm). Thus, our results show that reduced complex I function is well compensated both energetically and functionally under non-stressed conditions but renders the heart highly vulnerable to pathological stimuli. Therefore, despite a large reserve, mitochondrial dysfunction plays a critical role in the pathogenesis of heart failure in response to pathological stresses.
Author Disclosures: G. Karamanlidis: None. L. Garcia Menendez: None. S. Kolwicz: None. R. Palmiter: None. R. Tian: None.
Key Words: Heart failure, Mitochondria, Metabolism
Ryanodine Receptor Phosphorylation by CaMKII Promotes Ventricular Arrhythmias and Arrhythmogenic Death in Mice with Heart Failure
Mark D McCauley, Ralph J van Oort, Sayali S Dixit, Baylor College of Medicine, Houston, TX; Laetitia Pereira, Yi Yang, Univ of California, Davis, Davis, CA; Jonathan L Respress, Qiongling Wang, Angela C de Almeida, Darlene G Skapura, Baylor College of Medicine, Houston, TX; Mark E Anderson, Univ of Iowa, Iowa City, IA; Donald M Bers, Univ of California, Davis, Davis, CA; Xander H Wehrens; Baylor College of Medicine, Houston, TX
Background: Approximately half of all heart failure patients die as a result of lethal ventricular arrhythmias. In these patients, diastolic release of Ca2+ from the sarcoplasmic reticulum (SR) through type 2 ryanodine receptors (RyR2) has been linked to sudden arrhythmogenic death. However, the molecular mechanisms behind this release remain unknown. We hypothesized that increased RyR2 phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII) is both necessary and sufficient to promote lethal ventricular arrhythmias. Methods and Results: We generated knock-in mice in which the CaMKII phosphorylation site on RyR2 is constitutively activated (S2814D). In lipid bilayer preparations there was increased RyR2 open probability in S2814D (Po 54.7%±8.0%, n=14) vs. WT mice (Po 1.3%±0.4%, n=14, p<0.001). Also, intact S2814D myocytes had higher diastolic SR Ca2+ release events on confocal microscopy as evinced by increased spark frequency (9.8±0.5, n=8) vs. WT mice (6.4±0.3, n=10, p<0.05). At baseline, young S2814D mice had structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia with increased risk for sudden cardiac death upon catecholaminergic provocation by caffeine/epinephrine (71% vs. 13%; n=8,7; p<0.05) or programmed electrical stimulation (53% vs. 6%; n=15,16; p<0.01). Transverse aortic constriction (TAC) surgery in S2814D and WT mice caused a significant decline in survival due to arrhythmogenic death on ECG telemetry (Survival 40% vs. 90%; n=10,11; p<0.05). Conversely, genetic inhibition of the CaMKII site on RyR2 (S2814A) rescues mutant mice from pacing-induced arrhythmias versus WT mice after TAC surgery (NSVT 14% vs. 75%, n=7,8; p<0.05). Conclusions: Abnormal CaMKII phosphorylation of RyR2 Ca2+ release channels plays a critical role in arrhythmogenesis and sudden cardiac death in heart failure. Inhibition of CaMKII phosphorylation of RyR2 may reduce the incidence of death from arrhythmias in patients with heart failure.
Author Disclosures: M.D. McCauley: None. R.J. van Oort: None. S.S. Dixit: None. L. Pereira: None. Y. Yang: None. J.L. Respress: None. Q. Wang: None. A.C. de Almeida: None. D.G. Skapura: None. M.E. Anderson: None. D.M. Bers: None. X.H.T. Wehrens: None.
Key Words: Calcium, Sudden cardiac death, Ventricular arrhythmia
Functional High Throughput Chemical Screen Identifies Two Small Molecule Suppressors of Long QT Syndrome
David S Peal, Robert Mills, Stacey N Lynch, Janet M Mosley, Patrick T Ellinor, Randall T Peterson, David J Milan; Massachusetts General Hospital, Boston, MA
Introduction: Long QT syndrome (LQTS) is a life-threatening disorder characterized prolongation of cardiac repolarization. The zebrafish mutant breakdance is a LQTS type 2 model with an I59S KCNH2 mutation, that faithfully recapitulates many features of human LQTS: ventricular action potential duration (APD) prolongation, spontaneous early afterdepolarizations, and 2:1 AV block in early stages of development. While many molecules prolong QT interval and ventricular APDs, very few compounds are known to shorten APD, and their effect sizes are small. We conducted a small molecule screen for rescue of the “breakdance” phenotype to identify compounds that shorten its ventricular APD as candidates for treatment of LQTS. Methods: 48 hour breakdance embryos were placed 3/well in 96-well plates, exposed to test compounds from commercially available small molecule libraries (Prestwick and Chembridge) for 24 hours and scored for rescue of 2:1 AV block. Optical voltage mapping using di-4 ANEPPS was performed to measure cardiac APDs. Glucocorticoid receptor and androgen receptor gene knockdown was accomplished by antisense morpholino injection at the single cell stage. Results: Screening of 1200 small molecules yielded 2 compounds, flurandrenolide and 2-MMB, that rescued zebrafish LQT2 breakdance embryos in a dose dependent fashion. Optical voltage mapping confirmed that treatment with each compound caused shortening of ventricular APDs in LQT fish. Time course experiments and structure activity analyses are consistent with the hypothesis that 2-MMB inhibits late sodium current to shorten ventricular APDs. In contrast, rescue with flurandrenolide was abrogated by knockdown of the glucocorticoid receptor, but not the androgen receptor, suggesting it acts at a transcriptional level via the glucocorticoid receptor to shorten APDs. Conclusions: This chemical screen in a zebrafish model of LQT2 syndrome has identified two compounds, flurandrenolide and 2-MMB, as previously unsuspected small molecules that shorten ventricular action potential duration and rescue zebrafish LQTS. Further study of these molecules will provide novel insights into the biology of repolarization and may lead to useful therapeutics for human LQT patients.
Author Disclosures: D.S. Peal: None. R. Mills: None. S.N. Lynch: None. J.M. Mosley: None. P.T. Ellinor: None. R.T. Peterson: None. D.J. Milan: None.
Key Words: Qt interval, Ion channels, Electrophysiology
Deletion of GSK3-α Worsens Ischemic Injury, Cardiac Remodeling and Contractile Function Following Myocardial Infarction
Hind Lal, Jibin Zhou, Raihana Zaka, Ronald J Vagnozzi, Thomas Jefferson Univ, Philadelphia, PA; James Woodgett, Samuel Lunenfeld Rsch Institute, Toronto, Canada; Erhe Gao, Thomas Force; Thomas Jefferson Univ, Philadelphia, PA
Loss of myocardium following myocardial infarction (MI) is the leading cause of LV dysfunction and heart failure worldwide. However, the molecular pathways regulating remodeling are not well understood. We recently demonstrated that GSK-3α is critical to the heart's response to pressure overload. However the role, if any, of GSK-3α in regulating ischemic injury and post-MI remodeling is not known. To address this, MI was induced in wild-type (WT) vs. GSK-3α(−/−)(KO) mice by LAD ligation and mice were followed with serial echocardiography. Pre-MI, WT and KO hearts had comparable chamber dimensions and ventricular function, but as early as one week post MI, KO animals had a significant increase in end-diastolic (4.44±0.088 vs 3.96±0.1622mm; P<0.05) and end-systolic dimension (3.44 vs 2.57mm; P<0.0001), reflecting accelerated dilative remodeling in KOs. This was associated with marked LV dysfunction as reflected by reduced FS (22.40 vs 34.81; P<0.001). KO mice had significantly increased mortality within the first 10 days post-MI (43% vs 22%; P=0.0418), and post-mortem examination confirmed increased rates of cardiac rupture as the cause of most of the deaths. In the mice that survived the first 10 d, there was no difference in mortality between WT and KO up to the termination of the study (8wks), but LV dilatation and dysfunction remained worse in the KO throughout. Furthermore, markers of hypertrophy, heart failure (BNP and lung weight/body weight) and fibrosis were significantly increased in the KO. Given the increased early deaths due to rupture and significantly worse LV function evident as early as 1 wk post-MI, we examined infarct size at 48h and found it to be significantly increased in the KO. Conclusion: GSK-3α provides a critical ? brake? on both acute ischemic injury and chronic MI-induced LV remodeling and preserves cardiac function. Thus in contrast to our recent report that deletion of GSK-3β is protective against post-MI remodeling, deletion of GSK-3α is detrimental in a similar setting. These striking differences in the isoform-specific effects highlight the need to understand the unique roles of each isoform. More importantly, our studies encourage caution in going forward with agents that inhibit both GSK-3 isoforms nonspecifically.
Author Disclosures: H. Lal: None. J. Zhou: None. R. Zaka: None. R.J. Vagnozzi: None. J. Woodgett: None. E. Gao: None. T. Force: None.
Key Words: Heart failure, Remodeling, Signal transduction
Nrf2 Deficiency Prevents Reductive Stress and Rescues Human Mutant Protein Aggregation Cardiomyopathy (MPAC) in Mice
Gayatri Deepti Khanderao, Divs of Cardiology & Pulomonary, Univ of Utah Health, Salt Lake City, UT; Matthew A Firpo, Dept of Surgery, Univ of Utah Health, Salt Lake City, UT; Curtis D Olsen, Sheldon Litwin, Ivor J Benjamin, Div of Cardiology, Univ of Utah Health, Salt Lake City, UT; Li Wang, Div of Gastroenterology, Univ of Utah Health, Salt Lake City, UT; Rajasekaran Namakkal Soorappan; Rm # 327A, Divs of Cardiology & Pulmonary, Univ of Utah Health, Salt Lake City, UT
Background: We recently discovered that the overexpression of human mutant αB-crystallin (hR120GCryAB) in mouse resulted in protein aggregation cardiomyopathy (PAC) and reductive stress (RS) in the heart tissue (Rajasekaran et.al Cell, 2007). Further, we identified that sustained activation of Nrf2 (nuclear erythroid 2 related factor 2)/ARE signaling as a causal mechanism for RS in the MPAC mice. We hypothesize that abolishing Nrf2 could prevent RS in the R120GTG mice and thereby rescue them from cardiomyopathy and heart failure. Methods: To elucidate loss-of-function mechanisms for Nrf2 on RS and MPAC, we generated (1) NTG/WT, (2) R120GTG/WT, (3) R120GTG/Nrf2± and (4) R120GTG/Nrf2−/− mice by intercrossing R120GTG X Nrf2-KO. Primarily, we determined the rate of survival and ECHO-cardiograph analysis to understand the degree of pathogenesis and cardiac function over time (n=6-10). Next, we analyzed the redox state and protein/mRNA expression for major antioxidants including enzymes that involve in glutathione metabolism in the heart. Results: Disruption of Nrf2 prolonged the survival of MPAC mice along with no signs of cardiac dysfunction (by ECHO). The R120GTG mice with Nrf2 deficiency (either Nrf2± or Nrf2−/−; n=4) had significantly decreased glutathione (but equal to NTG) levels when compared with R120GTG/WT cohorts. Such a decline in GSH resulted in quenching of “reducing power” to prevent “reductive stress” and facilitated redox homeostasis in the myocardium. Interestingly, the intercross mice showed no cardiac hypertrophy even at 12 months while the R120GTG exhibited 90% mortality due to PAC/heart failure. Further, protein and mRNA levels for major antioxidants were found to be normal in the R120GTG/Nrf2± while there was significant down regulation of these parameters in the R120GTG/Nrf2−/− mice when compared to NTG/WT suggesting that Nrf2-independent pathways are inadequate to maintain the antioxidant capacity under stressed conditions. Conclusions: These results demonstrate a critical role for Nrf2 underlying antioxidant potential in the myocardium. Abrogating RS via Nrf2 might be highly relevant to cure the protein aggregation diseases that are coupled with elevated intracellular reducing potential.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: G. Khanderao: None. M.A. Firpo: None. C.D. Olsen: None. S. Litwin: None. I.J. Benjamin: None. L. Wang: None. R. Namakkal soorappan: None.
Key Words: Cardiac hypertrophy, Oxidative stress, Cardiomyopathy
Coordinated Downregulation of MicroRNA-1 And MicroRNA-133 During Myocyte Hypertrophy Is Required For An Increase In Global Gene Expression and Protein Turnover
Danish Sayed, Minzhen He, Zhi Yang, Maha Abdellatif; UMDNJ Newark, Newark, NJ
Cardiac hypertrophy is characterized by an increase in global protein and RNA synthesis that is accompanied by an increase in proteasomal-dependent protein turnover. However, the regulation and coordination of these two functions has not been elucidated. The objective of our study was to investigate the role of microRNA (miR) in this process. MiR-1 and miR-133, which are coexpressed in muscle tissue, are downregulated during skeletal and cardiac muscle hypertrophic growth. Our data reveal a role for miR-1 in regulating global mRNA and protein synthesis by suppressing key molecules that include TATA-binding protein (TBP), general transcription factor IIB (GTFIIB), cyclin-dependent kinase 9 (cdk9), and eukaryotic initiation factor 4E (eIF4E), through highly conserved targeting sites. Indeed, the levels of miR-1 inversely correlate with the expression of these molecules during postnatal cardiac growth and myocyte hypertrophy. Accordingly, supplementing cardiac myocytes undergoing hypertrophic growth with exogenous miR-1 inhibits total mRNA and protein synthesis by 40% and 52%, respectively, preceded by downregulation of miR-1 targets, and vice versa. TBP binds to the TATA box and recruits RNA polymerase II through binding to TFIIB. Thus, via inhibiting the expression of these target proteins, miR-1 should inhibit the association of RNA polymerase II to the genomic DNA. To confirm this possibility, we supplied cardiac myocytes undergoing hypertrophy with exogenous miR-1 or a control adenovirus and performed chromatin immunoprecipitation using RNA polymerase II, followed by library construction and sequencing, and microarray analysis (ChIP-on-ChIP). The results confirmed that miR-1 inhibits the association of RNA polymerase II with a broad spectrum of genes, including house-keeping genes by ∼80%. On the other hand, increasing miR-133 levels inhibited proteasomal-mediated protein degradation and increased ubquitinated proteins in cardiac myocytes equivalent to the effect of the proteasomal inhibitor epoxomicin. Conversely, knockdown of miR-133 reversed the effect epoxomicin. Thus, the coordinated downregulation of miR-1 and miR-133 during hypertrophic muscle growth results in enhancing global gene expression and protein turnover.
Author Disclosures: D. Sayed: None. M. He: None. Z. Yang: None. M. Abdellatif: None.
Key Words: Cardiac hypertrophy, Gene expression, Proteasome
PDE3A: A Component of a Molecular Scaffold That May Integrate Cyclic AMP and SERCA2 Transduction Pathways in Cardiac Muscle
Faiyaz A Khan, Weixing Shen, National Institutes of Health, Bethesda, MD; Judith Krall, Fabrice Vandeput, Univ of Utah, Salt Lake City, UT; Steve Hockman, National Institutes of Health, Bethesda, MD; Eva Degerman, Univ of Lund, Lund, Sweden; Matthew Movsesian, Vincent C Manganiello; National Institutes of Health, Bethesda, MD
Many cardiac functions are regulated by multiple spatially and functionally distinct pools of cAMP. By hydrolyzing cAMP, cyclic nucleotide phosphodiesterases (PDEs) regulate the amplitude, duration, and compartmentation of cAMP-mediated signaling. Although PDE3 inhibitors raise cAMP and produce acute inotropic effects, the detailed mechanisms are unclear. Immunohistochemical (IHC) staining of human myocardium indicates that PDE3A colocalized with desmin, AKAP18 and SERCA2 to sarcomere Z-bands, while PDE3B colocalized with mitochondrial proteins (COX4, ATP synthase, Cyt-C). During sucrose gradient centrifugation of mouse cardiac membranes, PDE3A cofractionates with sarcoplasmic reticulum (SR) Ca+2 -ATPase 2 (SERCA2) and phospholamban (PLB), and IHC staining indicates that PDE3A colocalized with SERCA2 and desmin in mouse heart. In addition, Western blots and LC-MS/MS analysis of PDE3A immunoprecipitates indicates that murine PDE3A coimmunoprecipitated (CO-IP) with SERCA2. Similarly, in solubilized human cardiac microsomes, PDE3A CO-IP with SERCA2 and other signaling molecules thought to be components of an AKAP/SERCA2 macromolecular regulatory complex, including PLB, PKARII, PP2A, and AKAP18, but not AKAP LBC. In human myocardial microsomes, the PKA catalytic subunit (PKAc) phosphorylates PDE3A1 and A2 isoforms, but not PDE3A3, and significantly increases PDE3 catalytic activity. cAMP or PKAc significantly increase Ca2+ uptake into human SR vesicles, and cilostamide, a PDE3-selective inhibitor, potentiates the effect of cAMP on Ca2+ uptake into mice and human SR vesicles. SERCA2 Ca2+ -ATPase activity and Ca2+ uptake were increased in SR vesicles from PDE3A-knockout (KO) mice, compared to WT. In lysates from KO hearts, SERCA2 expression was increased and that of PLB decreased, and phosphorylation of PLB at Ser-16 (pPLB/PLB ratio) was increased. In KO lysates, due to the loss of PDE3A activity, PKA was activated, as evidenced by increased phosphorylation of PKA substrates and PLB. Taken together, these data suggest that, as a component of SERCA2-containing macromolecular complexes in murine and human myocardium, PDE3A regulates a discrete cAMP pool important in regulating contractility by modulating Ca2+ uptake into the SR.
Author Disclosures: F.A. Khan: None. W. Shen: None. J. Krall: None. F. Vandeput: None. S. Hockman: None. E. Degerman: None. M. Movsesian: None. V.C. Manganiello: None.
Key Words: Signal transduction, Muscle, cardiac - see Myocardium, Calcium
Late-Breaking Basic Science Poster Session
Role of MicroRNA-29b (miR 29-b) in the Migration of Induced Pluripotent Stem Cells (iPSC) Derived Cardiac Progenitor Cells (IPS-CPC) in Scar Tissue After Myocardial Infarction>
Wei Huang, Bo Dai, Dongsheng Zhang, Atif Ashraf, Meifeng Xu, Muhammad Ashraf, Yigang Wang; Univ of Cincinnati, Cincinnati, OH
We postulated that manipulation of cardiac collagen deposition by overexpression of miR29b influences the migration of IPS-CPC from a tri-cell patch (cardiomyocytes, endothelial cells, and embryonic fibroblasts) after myocardial infarction (MI). In vitro: Cardiac progenitor cells were generated by transfection of iPSC with lentivirus based cardiac specific tissue promoters in cell patch constitutively expressing a double fusion protein [expressing green fluorescent protein (GFP) and firefly luciferase]. Patch was comprised of IPS-CPC induced by Na+-Ca2+ exchanger (NCX1) and Tie-1 promoters. Nude rats after MI were administered either lentivector-based miRNA29b-1 precursor (miR-29b), anti-miR-29b (Anti-29b), or pSIH1-H1-copGFP (CON) into the hearts. Three days later a cell patch was placed over the infarcted region of the hearts and expression of myocardin-related transcription factor A (MRTF-A), collagen I, and collagen III was measured. Mobilization of IPS-CPC was analyzed by counting the number of GFP+ cells in the infarcted area and by in vivo bioluminescent imaging (BLI). One month after cell patch implantation, echocardiography was performed and hearts were harvested for analysis. The number of GFP+ cells, amount of BLI signals, and heart function were significantly increased and were associated with reduced MRTF-A, collagen I, and collagen III in the rats treated with miR-29b as compared to the rats that received a CON, or anti-29b, p<0.05, (Fig.1). In conclusion, overexpression of miR-29b enhances mobilization of IPS-CPC in the infarcted tissue resulting in heart functional restoration after MI.
Author Disclosures: W. Huang: None. B. Dai: None. D. Zhang: None. A. Ashraf: None. M. Xu: None. M. Ashraf: None. Y. Wang: None.
Key Words: Myocardial infarction, Stem/progenitor cells, Heart failure
S1P Lyase: A Novel Therapeutic Target for Ischemia/Reperfusion Injury of the Heart
Padmavathi Bandhuvula, Children's Hosital Oakland Rsch Institute, Oakland, CA; Norman Honbo, Guan-Ying Wang, Zhu-Qiu Jin, VA Med Cntr, San Francisco, CA; Henrik Fyrst, Cntr for Comparative Medicine, UC Davis, Davis, CA; Meng Zhang, Children's Hosp Oakland Rersearch Institute, Oakland, CA; Alexander D Borowsky, Lisa Dillard, Cntr for Comparative Medicine, UC Davis, Davis, CA; Joel S Karliner, VA Med Cntr, San Francisco, CA; Julie D Saba; Children's Hosp Oakland Rsch Institute, Oakland, CA
Aims: Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes cardiomyocyte survival and contributes to ischemic pre-conditioning. S1P lyase (SPL) is a stress-activated enzyme responsible for irreversible catabolism of S1P. We hypothesized that SPL contributes to oxidative stress by depleting S1P pools available for cardioprotective signaling. Accordingly, our goal was to evaluate the potential efficacy of SPL inhibition as a strategy for reducing cardiac ischemia/reperfusion (I/R) injury. Methods and Results: We measured SPL protein expression and enzyme activity in murine hearts and cardiomyocytes. An ex vivo (Langendorff) model was employed to assess the influence of I/R on cardiac SPL activity and to determine whether genetic disruption of SPL or pretreatment of mice with a nontoxic SPL inhibitor modulates I/R-induced myocardial infarction and hemodynamic recovery. Basal SPL activity was low in wildtype cardiac tissue but was activated in response to 50 min of ischemia (n=5, P < 0.01). Hearts of heterozygous SPL knockout mice exhibited reduced SPL activity, elevated S1P levels, smaller infarct size and increased functional recovery after I/R compared to littermate controls (n=5, P < 0.01). The small molecule tetrahydroxybutylimidazole (THI) is an FDA-approved food additive (caramel coloring # 3) that inhibits SPL. When given overnight at 25 mg/L in the drinking water, THI raised S1P levels and reduced SPL activity (n=5, P < 0.01). THI also reduced infarct size and enhanced hemodynamic recovery in response to 50 min of ischemia and 40 min of reperfusion in ex vivo hearts (n=7, P < .01, Figure). These data correlated with increased levels of Mnk1-, e1F4E- and S6-phosphorylation after I/R, suggesting that SPL inhibition enhances protein translation. Conclusions: These results reveal for the first time that SPL is an ischemia-induced enzyme that is inhibited by a common food additive, and thus is a novel target for preventing cardiac I/R injury.
Author Disclosures: P. Bandhuvula: None. N. Honbo: None. G. Wang: None. Z. Jin: None. H. Fyrst: None. M. Zhang: None. A.D. Borowsky: None. L. Dillard: None. J.S. Karliner: None. J.D. Saba: None.
Key Words: Cardioprotection, Enzyme inhibitors, Ischemia reperfusion
Impaired Lymphocyte Homeostasis and Autoimmune Disorder in Mice Deficient in HDL Receptor SR-BI
Hong Feng, Ling Guo, Dan Wang, Alan Daugherty, University of Kentucky, Lexington, KY; Haiqing Gao, Sandong Univ, Jinan, China; Guihua Hou, Shandong Univ, Jinan, China; Xiang-An Li; University of Kentucky, Lexington, KY
Scavenger receptor BI (SR-BI) is a well-established HDL receptor. Recent studies reveal that SR-BI modulates inflammatory response in macrophage and protects against septic death in mice. In spite of this established role in regulating innate immunity, the function of SR-BI in adaptive immunity remains unknown. In this study we report that SR-BI plays pivotal roles in lymphocyte homeostasis and autoimmunity. Mice deficient in SR-BI display splenomegaly with a 60% increase in B lymphocytes and significant decrease in T-to-B ratio. Furthermore, both T and B cells exhibit an overall hyperactivation status, characterized by the accumulation of activated CD19+CD69high B cells and CD4+CD44high T cells in SR-BI null mice spleen. To explore the underlying mechanisms, we elucidated the effects of SR-BI on lymphocyte development and no difference was observed in thymocyte development in thymus or in B cell development in bone marrow or in spleen between SR-BI null and wild type littermates. Considering that mice deficient in SR-BI have impaired HDL metabolism as shown by 2-fold increase in HDL-cholesterol levels and abnormally large HDL particles, we tested our hypothesis that the abnormal HDL acts as “in situ” stimulator of lymphocyte activation and proliferation. Elevation in abnormal HDL by high fat diet treatment markedly exacerbated lymphocyte expansion and activation phenotype, and remarkably, caused a 2-fold increase in B cell proliferation in vivo revealed by BrdU incorporation in SR-BI null mice. Oppositely, reduction of the abnormal HDL levels by Probucol treatment relieved the lymphocyte expansion and activation phenotype. Importantly, given the progressively developing hyperactivation status of lymphocytes, SR-BI null mice developed systemic autoimmune disorders with aging, characterized by elevation of serum auto-antibodies against dsDNA, ssDNA and histone, and the deposition of IgG and C3 in kidney glomeruli. In conclusion, our findings reveal a previously unrecognized role of SR-BI in modulating lymphocyte homeostasis and autoimmunity, and demonstrate that abnormal HDL due to defect in SR-BI acts as a stimulator of lymphocyte activation and proliferation. Our findings provide an insight into the link between HDL and adaptive immunity.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: H. Feng: None. L. Guo: None. D. Wang: None. A. Daugherty: None. H. Gao: None. G. Hou: None. X. Li: Research Grant; Significant; R01GM085231.
Key Words: HDL, Immune system, Receptors
Plasminogen Regulates SDF-1/CXCR4-mediated Hematopoietic Stem Cell Mobilization by Activation of Matrix Metalloproteinase-9
Yanqing Gong, Jane Hoover-Plow; Cleveland Clinic Foundation, Cleveland, OH
Ischemic heart diseases including myocardial infarction (MI) are the leading causes of death in the US. Use of hematopoietic progenitor and stem cells (HPSC) to improve recovery of the injured heart after MI is an important emerging therapeutic strategy. Granulocyte colony-stimulating factor (G-CSF) is a widely-used therapeutic agent for HPSC bone marrow (BM)mobilization, however, poor mobilization is observed in 20% patients and healthy donors. Therefore, a better understanding of the underlying mechanisms regulating HPSC mobilization may offer novel approaches for stem cell therapy. Our data show that plasminogen (Plg) deficiency (Plg−/−) significantly decreases HPSC mobilization from BM to the circulation induced by G-CSF indicating that Plg is required for HPSC mobilization. After G-CSF treatment, there was no difference in proMMP-9 between Plg+/+ and Plg−/−mice, but actMMP-9 was higher in Plg+/+ mice than Plg−/− mice. Reconstitution of actMMP-9, but not proMMP-9, rescued HPSC mobilization in Plg−/− mice. Thus, Plg-dependent HPSC requires MMP-9 activation. SDF-1 is a key chemoattractant for HPSC mobilization from BM, and G-CSF decreases SDF-1 in BM of Plg+/+mice creating a gradient of SDF-1 between BM and blood. Our data show that Plg deficiency abolishes the gradient of SDF-1. Furthermore, the expression of CXCR-4, a major receptor of SDF-1 on HPSC, was significantly increased in Plg+/+ mice but not in Plg−/− mice. These data suggested that Plg could decrease BM SDF-1 and increase CXCR-4 expression and, therefore favor the recruitment of HPSC from BM to the peripheral blood, where SDF-1 concentration is higher. Moreover, MMP-9 deficiency and reconstitution of MMP-9 activity in Plg−/−mice selectively reversed CXCR4 expression on HPSC but not SDF-1 level, indicating distinct and novel mechanism by which Plg regulates HPSC mobilization through SDF-1/CXCR-4. In addition, our data show that Plg deficiency inhibits G-CSF-induced neovascularization after LAD ligation induced MI in the infarcted area, a key step of cardiac repair. Taken together, these data suggest that Plg may enhance HPSC recruitment by regulating both MMP-9-mediated CXCR-4 expression and BM SDF-1 function. Those pathway cascades may also contribute to cardiac repair after MI.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: Y. Gong: None. J. Hoover-Plow: None.
Key Words: Stem/progenitor cells, Myocardial infarction, Chemokines
Opposite Effects of Genetic Deletion of the IL-1 Receptor and IL-1 Receptor Antagonist in an Experimental Mouse Model of Pulmonary Hypertension
Allan Lawrie, Abdul G Hameed, Janet Chamberlain, Nadine A Arnold, David C Crossman, Sheila E Francis; Univ of Sheffield, Sheffield, United Kingdom
Pulmonary arterial hypertension (PAH) is a life threatening condition with high morbidity and mortality. Inflammatory mechanisms are proposed to play a significant role in disease progression, particularly PAH associated with other diseases e.g. systemic sclerosis. Previous studies have described PAH in fat fed Apolipoprotein E knock out mice (ApoE−/−). Our group has previously reported that ApoE−/− mice with interleukin-1 receptor deletion (IL-1R1−/−) have reduced diet-induced atherosclerosis, and lower systemic blood pressure compared to ApoE−/− mice on the same diet. We subsequently hypothesized that ApoE−/−/IL-1R1−/− double null mice would exhibit a reduced PAH phenotype compared to ApoE−/−. We reported at the AHA Scientific Session last year that, surprisingly, after 8 weeks on a high fat (Paigen) diet ApoE−/−/IL-1R1−/− mice exhibited significantly higher RVSP (mean 79 mmHg vs 50 mmHg, n=7 p<0.01) and more severe pulmonary vascular muscularisation than ApoE−/−s. Further investigation has revealed expression of a putative alternatively primed IL-1R1 transcript expressed within the lungs (but not aorta) of the ApoE−/−/IL-1R1−/− double null mice. To assess the functionality of this putative IL-1 receptor form, we treated both ApoE−/− and ApoE−/−/IL-1R1−/− mice with Interleukin 1 receptor antagonist (IL-1Ra) or placebo for 4 wks via a subcutaneous osmotic mini pump, following an initial 4 weeks on diet. Echocardiography and cardiac catheterization was performed at 8 weeks prior to collecting lung tissue and serum. ApoE−/− and ApoE−/−/IL-1R1−/− mice treated with IL-1Ra had significantly reduced RVSP (23 and 33 mmHg respectively) and significantly reduced pulmonary vascular remodeling (55% and 66% respectively.) These data suggest that IL-1Ra may have beneficial effects in treating PAH and that alternative IL-1 receptor signaling in the lung may be important in driving PAH pathogenesis.
Author Disclosures: A. Lawrie: None. A.G. Hameed: None. J. Chamberlain: None. N.A. Arnold: None. D.C. Crossman: None. S.E. Francis: None.
Key Words: Pulmonary hypertension, Transgenic models, Inflammation
Epigenetic Control of Cardiac Growth by the Histone Methyltransferase Ezh2
Paul Delgado-Olguin, Gladstone Institutes, San Francisco, CA; Alexander Tarakhovsky, The Rockefeller Univ, New York, NY; Benoit G Bruneau; Gladstone Institutes, San Francisco, CA
Heart homeostasis is controlled by cardiac-specific gene transcription. Much less is known on the simultaneous gene repression that must occur to allow execution of specialized functions during the heart's lifetime. Therefore, stable repression of non-cardiac transcriptional programs is likely essential for cardiac maintenance. Stable gene repression is established by tri-methylation of the lysine 27 of histone H3 (H3k27me3) via Enhancer of zeste homolog 2 (Ezh2). However, whether Ezh2 functions in the mammalian heart or in heart disease is not known. Methods: Ezh2 was inactivated in the mouse second heart field by Mef2c-Cre driven recombination. In vivo, cellular and molecular approaches were used to identify key Ezh2 targets whose epigenetic repression is essential for cardiac maintenance. Results: Ezh2 inactivation led to cardiac hypertrophy, fibrosis and fetal gene reactivation in adult mice. Ezh2-deficient hearts developed hyperfibrosis upon hypertrophic stimuli, suggesting that Ezh2 represses a pro-fibrosis program in the heart. Indeed, adult Ezh2-deficient hearts over-expressed fibrosis markers (Vimentin, collagens) and the pro-fibrosis factors periostin (Postn), connective tissue growth factor (Ctgf) and Tgfβ3. Mutations upregulating Tgfβ3 are present in arrhythmogenic right ventricular dysplasia (ARVD), characterized by adherens junction remodeling. Indeed, Ezh2-deficient cardiomyocytes remodeled adherens junctions, as shown by induction of E-cadherin and redistribution of β-catenin, modeling some aspects of ARVD. We uncovered key Ezh2 targets acting upstream Tgfβ3 in the heart. The homeodomain transcription factor Six1, which activates Tgfβ3 signaling, and its coactivator Eya1, were upregulated in Ezh2-deficient cardiomyocytes. Indeed, the Six1 promoter had decreased H3K27me3 and increased PolII and H3 acethylation in Ezh2-deficient hearts. Furthermore, Six1 and Eya1 induced cardiac hypertrophy in cardiomyocytes and coactivated Nppa expression. Conclusion: Ezh2 is essential for heart maintenance, as it epigenetically represses key transcription factors like Six1 and Eya1, which, upon derepression, activate Tgfβ3, a pro-fibrosis program and desmosome remodeling, contributing to cardiac dysfunction.
Author Disclosures: P. Delgado-Olguin: None. A. Tarakhovsky: None. B.G. Bruneau: None.
Key Words: Gene expression, Cardiac hypertrophy, Fibrosis
MicroRNA-21 Mediates Cardioprotection with PKGI-α Over-expression through Upregulation of Hydrogen Sulfide
Fadi N Salloum, Nicholas N Hoke, Anindita Das, Gregory R Sturz, Christopher S Thomas, Rakesh C Kukreja; Virginia Commonwealth Univ, Richmond, VA
Background: Activation of cyclic GMP-dependent protein kinase (PKG) with pharmacologic agents or its overexpression protect against ischemia/reperfusion injury in the heart and in cardiomyocytes. Moreover, Akt phosphorylation and hydrogen sulfide (H2S) generation were involved in the protective effect. Since Akt positively regulates microRNA (miR)-21, we hypothesized that PKG-driven H2S generation is mediated by miR-21. Methods and Results: Ventricular myocytes were isolated from adult male Wistar rats, infected with adenoviral vector encoding either PKGI-α or mutant (K390A) overnight, with/without adenoviral vector encoding miR-21 eraser, and exposed to 90 min of simulated ischemia. Myocyte necrosis and apoptosis were determined after 1 h or 18 h of reoxygenation using trypan blue exclusion and LDH release or TUNEL assay, respectively. Cardiomyocyte H2S concentration was measured using a H2S sensor connected to a single channel analyzer. miR-microarray revealed a clear trend in miR-21 upregulation with PKGI-α as compared with control or K390A infected cells. Moreover, the miR-21 eraser abolished the protection exerted by PKGI-α as evidenced by an increase in necrosis (33±2%, Fig. 1A), LDH release (% of control) (281±22) and apoptosis (20±2%, Fig. 1B) compared to PKGI-α infected cells (necrosis: 18±1%, n=4, P<0.001; LDH: 156±12, n=4, P<0.05; apoptosis: 13±1%, n=4, P<0.05). Furthermore, cardiomyocyte H2S concentration (μM/mg protein) was increased with PKGI-α (2.3±0.1) as compared with control (0.8±0.1) or K390A infected cells (0.9±0.3). Interestingly, miR-21 eraser blocked the increase in H2S levels with PKGI-α (0.7±0.3, Fig. 1C). Conclusion: This study provides first evidence that over-expression of PKG1-α in primary adult cardiomyocytes enhances H2S generation through miR-21-dependent pathway, which leads to attenuation of necrosis and apoptosis following simulated ischemia and reoxygenation.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: F.N. Salloum: None. N.N. Hoke: None. A. Das: None. G.R. Sturz: None. C.S. Thomas: None. R.C. Kukreja: None.
Key Words: Ischemia reperfusion, Microrna, Gene transfer
Optically Activated Light-Sensitive Channels Can Pace Cardiac Tissue and Generate Propagating Cardiac Impulses
Zhiheng Jia, Zhongju Lu, Harold Bien, Huilin Liu, Barbara Rosati, Ira Cohen, Emilia Entcheva; Stony Brook Univ, Stony Brook, NY
After the recent cloning of light-sensitive ion channels, e.g. ChannelRhodopsin2 (ChR2), and their expression in mammalian cells, a new field - optogenetics emerged in neuroscience, allowing precise perturbations of neural circuits by light in vitro and in vivo. Thus far, functionality of optogenetic tools has not been demonstrated outside neuroscience. Considering common excitable tissue properties, we hypothesized that optical control can be extended to cardiac muscle, where precise spatiotemporal pacing can initiate or stop waves of excitation and contraction. We combine here for the first time optical pacing and optical mapping of the induced waves in a multicellular setting (cell culture). In addition to direct expression of ChR2 in myocytes, we demonstrate a robust cell delivery approach, where non-excitable cells carry exogenous light-sensitive ion channel(s), providing inward current, and when electrically coupled to the host cardiomyocytes (CM), produce optically-excitable tissue. A stable ChR2 HEK cell line expressing Cx43 was developed and used as a cell delivery platform into cardiomyocytes in vitro as a proof or principle. We combined optical excitation and ultra-high resolution large-scale optical mapping to capture light-triggered electrical waves and muscle contractions. We found optically-paced cardiac impulses to be indistinguishable from electrically-triggered waves (calcium transient duration, conduction velocity, contractility), Figure 1. In conclusion, our results demonstrate feasibility to control excitation and contraction in cardiac (and other) muscle by light. The cell delivery approach can be extended to stem cell therapy for in vivo applications. Light stimulation provides superior spatiotemporal control, remote access, is a cost and energy-efficient solution and can be an important tool in basic research on control of cardiac arrhythmias, as well as in development of light-driven pacemakers and muscle actuators.
Author Disclosures: Z. Jia: None. Z. Lu: None. H. Bien: None. H. Liu: None. B. Rosati: None. I. Cohen: None. E. Entcheva: None.
Key Words: Pacing, Cells, Cardiac mapping
Antibody-Sortagging: A Universal Approach Towards Targeted Molecular Imaging and Cell Homing in Cardiovascular Disease
Hang T Ta, Sandeep Prabhu, Ephraem Leitner, Fu Jia, Katherine Putnam, Nicole Bassler, Karlheinz Peter, Christoph E Hagemeyer; Baker IDI Heart and Diabetes Insitute, Melbourne, Australia
Targeting of contrast agents to unstable atherosclerotic plaques offers the potential to identify such plaques before rupture, allowing suitable interventions and thus avoiding myocardial infarction and death. Similarly, homing of stem cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen binding activity and functionality of the targeted biological component (e.g. stem cells). Here we report a novel, gentle, robust, highly reproducible, and site-specific coupling method utilizing the Staphylococcus aureus sortase A enzyme to conjugate a single-chain antibody (scFv), anti-GPIIb/IIIa-scFv, to nanoparticles and cells for molecular imaging and stem cell homing in cardiovascular disease. This scFv specifically binds to activated platelets, which play a pivotal role in atherosclerosis, thrombosis and inflammation. The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to magnetic particles of iron oxide (powerful contrast agents for magnetic resonance imaging), and to model CHO cells. The conjugation efficiency was between 50-70% and the bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled CHO cells and nanoparticles to activated platelets was strong and specific as demonstrated in in-vitro static adhesion assays, in a flow chamber system under shear stress and in mouse intravital microscopy. In conclusion, this unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy as well as targeted molecular imaging in cardiovascular, inflammatory diseases and beyond.
Author Disclosures: H.T. Ta: Employment; Significant; Baker IDI. Research Grant; Significant; National Heart Foundation. S. Prabhu: Employment; Significant; Baker IDI. E. Leitner: ; Baker IDI. F. Jia: ; Baker IDI. K. Putnam: ; Baker IDI. N. Bassler: ; Baker IDI. K. Peter: ; Baker IDI. Research Grant; Significant; NHMRC. C.E. Hagemeyer: Employment; Significant; Baker IDI. Research Grant; Significant; NHMRC. Other Research Support; Significant; National Heart Foundation.
Key Words: Molecular, Cardiovascular imaging, Regenerative medicine stem cells
Identification of a Novel Loss-of Function Calcium Channel Gene Mutation in Short QT Syndrome
Christian Templin, Jelena-Rima Ghadri, Univ Hosp Zurich, Zurich, Switzerland; Jean-Sebastien Rougier, Univ of Bern, Bern, Switzerland; Alessandra Baumer, Vladimir Kaplan, Univ Hosp Zurich, Zurich, Switzerland; Heinrich Sticht, Friedrich-Alexander-Univ Erlangen-Nuernberg, Erlangen, Germany; Anita Rauch, Thomas F Lüscher, Firat Duru, Univ Hosp Zurich, Zurich, Switzerland; Hugues Abriel; Univ of Bern, Bern, Switzerland
Background: Short QT Syndrome (SQTS) is a genetically determined ion-channel disorder, which may cause malignant tachyarrhythmias and sudden cardiac death (SCD). Thus far, mutations in 5 different genes encoding potassium and calcium channel subunits have been reported. We present, for the first time, a novel loss-of-function mutation coding for a L-type calcium channel subunit in a 17-year old female patient who survived an episode of SCD. Methods and Results: The ECG revealed a QT interval of 317 ms (QTc 329 ms) with tall, narrow, and symmetrical T-waves. Invasive electrophysiologic testing showed short ventricular refractory periods and increased vulnerability to induce ventricular fibrillation. DNA screening of the patient identified no mutation in previously known SQTS genes, however a new variant at a heterozygous state was identified in the CACNA2D1 gene (nucleotide c.2264G>C; amino acid p.Ser755Thr), coding for the Cavα2δ-1 subunit of the L-type calcium channel. This gene variant was not present in the sequenced 404 chromosomes of an ethnically matched control population. The pathogenic role of the p.Ser755Thr variant of the CACNA2D1 gene was analyzed by using co-expression of the two other L-type calcium channel subunits, Cav1.2α1 and Cavβ2b, in HEK-293 cells. Barium currents (IBa) were recorded in these cells under voltage-clamp conditions using the whole-cell configuration. Co-expression of the p.Ser755Thr Cavα2δ-1 subunit strongly reduced the IBa by more than 70% when compared to the co-expression of the wild-type (WT) variant. Small positive shifts of the inactivation and activation curves were observed when the mutant α2δ-subunit was expressed. Protein expression of the three subunits was verified by performing Western blots of total lysates of HEK-293 cells. The p.Ser755Thr variant of the Cavα2δ-1 subunit was expressed at a similar level compared to the WT subunit. This variant did not modify the expression of the pore-forming subunit of the L-type Ca channel, Cav1.2α1, suggesting a deficient trafficking of the L-type channel towards the cell membrane. Conclusions: In the present study, we report the first pathogenic mutation in the CACNA2D1 gene in humans, which causes a new variant of SQTS.
Author Disclosures: C. Templin: None. J. Ghadri: None. J. Rougier: None. A. Baumer: None. V. Kaplan: None. H. Sticht: None. A. Rauch: None. T.F. Lüscher: None. F. Duru: None. H. Abriel: None.
Key Words: Sudden cardiac death, Ventricular arrhythmia, Gene mutations
Synoviolin is a Novel Central Regulator of Endoplasmic Stress Response and Protein Quality Control in the Heart
Shirin Doroudgar, Christopher C Glembotski; San Diego State Univ, San Diego, CA
Recently the endoplasmic reticulum stress response (ERSR) has been shown to be critical for minimizing ischemic damage in the heart. We now identify synoviolin1 (Syvn1; Hrd1) as a novel global regulator of the ERSR. Accordingly, Syvn1 may be an important modulator of ischemic damage in the heart. Ischemia results in misfolding of proteins synthesized in the ER, activating the ERSR, which can be cytoprotective or death-promoting. In the heart, ERSR-mediated protection is afforded through the activation of ATF6, a nodal transcription factor of the ERSR that we have shown to be activated by ischemia. Activated ATF6 up-regulates proteins that protect from cell death during ischemia. Here we show that the E3 ubiquitin ligase, Syvn1, is up-regulated by ATF6 in the adult mouse heart, in vivo, and is a central regulator of ER stress that has never been studied in the heart. Using neonatal rat ventricular cardiomyocytes (NRVCMs), we demonstrate, for the first time, that Syvn1 is expressed in cardiac cells, and that it is up-regulated by ER stress. Knockdown of Syvn1 in NRVCMs using Syvn1-targeted siRNA decreases activation of the ERSR, as measured by the expression of nodal transcription factors AFT6 and XBP1, and downstream targets, indicating that Syvn1 is a global regulator of the ERSR. Furthermore, knockdown of Syvn1 increases cell death during ER stress, demonstrating critical roles for endogenous Syvn1 in ER stress-mediated protection during ischemic stress. It is not yet known whether protein quality control machinery in the ER responds to protein misfolding in other cellular compartments. However, since in addition to degrading misfolded proteins in the ER, as an E3 ligase, Syvn1 is involved in posttranslational, proteasome-mediated degradation of key regulatory cytosolic proteins that are not misfolded, it is probable that Syvn1 is a cornerstone of protein quality control in both ER and cytosolic compartments.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: S. Doroudgar: None. C.C. Glembotski: None.
Key Words: Cardioprotection, Stress, Ischemic heart disease
In vivo Differentiation of Epigenetically Reprogrammed Endothelial Progenitor Cells into Cardiomyocytes Enhances Functional and Anatomical Post-infarct Myocardial Repair
Melissa Thal, Prasanna Krishnamurthy, Erin Lambers, Eneda Hoxha, Suresh Verma, Douglas Losordo, Raj Kishore; Northwestern Univ, Chicago, IL
Background: Currently, bone marrow derived endothelial progenitor cells (BM-EPCs) are being used clinically to improve vascularization in patients with ischemic heart disease. While it is generally accepted that EPCs participate in vascular repair of the ischemic myocardium, there exists no convincing evidence that these cells are capable of trans-differentiating into functional cardiomyocytes (CMC) Since ischemic heart disease leads to substantial loss of CMC, cardiomyogenic plasticity of an existing autologous cell therapy is of obvious import. EPCs and CMC both differentiate from a common mesodermal progenitor cell however; during specific lineage differentiation to EC phenotype CMC specific genes are epigenetically silenced. We hypothesized that reprogramming of EPC using small molecules targeting key epigenetic repressive marks may recapitulate cardiomyogenic potential in EPCs. Method and Results: Mouse Lin-Sca1+CD31+ BM cells (EPCs) were sorted and treated with inhibitors of DNA methyltransferases (5-Azacytidine), histone deacetylases (valproic acid) and G9a histone methyltransferase. Forty eight hour treatment led to the reactivation of pluripotency associated and CMC specific mRNA expression while EC specific genes were significantly up-regulated. When cultured under appropriate differentiation conditions, reprogrammed EPCs showed efficient differentiation into CMC and vascular smooth muscle cells. Epigenetically, treatment of EPCs showed significant demethylation of Nkx2.5 promoter and de-methylation of Histone 3 lysine 9 (H3-K9). Intra-myocardial transplantation of sex-mismatched male reprogrammed eGFP-EPCs in an acute myocardial infarction mouse model (female) showed significant improvement in LV functions compared to control EPCs and this was histologically supported by the de novo CMC differentiation (GFP+sarcomeric actinin double positive as well as GFP+Y chromosome +SA positive cells) as well as increased capillary density and significantly reduced fibrosis. Conclusions: Taken together, our results suggest that epigenetically reprogrammed EPCs display a more plastic phenotype and improve post-infarct cardiac repair by both neo-cardiomyogenesis and neovascularization.
Author Disclosures: M. Thal: None. P. Krishnamurthy: None. E. Lambers: None. E. Hoxha: None. S. Verma: None. D. Losordo: None. R. Kishore: None.
Cardiac regeneration, Endothelial progenitor cell, Stem/progenitor cells,
Increases in Sodium Permeability Trigger the Activation of MEF2/NFAT and BNP/MHC Gene Expression in Cardiac Myocytes via the Salt-Inducible Kinase 1 Network
Popov Sergej, Kyriaki Venetsanou, Pedro J Chedrese, Karolinska Institutet, Stockholm, Sweden; Hiroshi Takemori, National Institute for Biomedical Innovations, Osaka, Japan; Naoki Mochizuki, National Cerebral and Cardiovascular Cntr, Osaka, Japan; Alejandro M Bertorello; Karolinska Institutet, Stockholm, Sweden
Cardiac hypertrophy (CH) generally occurs as the result of a sustained mechanical stress, due to elevated systemic arterial blood pressure (BP). In animal models however, elevated salt intake associates with CH without significant increases in BP. Thus, we hypothesize that CH may not be entirely related to a mechanical stress but also to other factors associated with elevated BP such as abnormal sodium homeostasis. To test this hypothesis, we examined the effect of increases intracellular in sodium ([Na+]i) (5-10 mM) on transcription factors (TF) and genes associated with CH by incubating HL-1cells (a line of cardiac origin) with 3 μM monensin. Elevations in [Na+]i induced a large increase of brain natriuretic peptide gene expression (11 fold, n=6 at 12 hs), and a moderate increase in structural genes such as myosin heavy chain (% increase 160±20, n=5 at 12 hs) (mRNA expression levels by Taqman and also luciferase-tagged reporters). Furthermore, the TF MEF2 (ANOVA P=0.008) and NFAT (ANOVA P=0.003) also increased in a time dependent manner. Elevation of [Na+]i is associated with activation of salt-inducible kinase 1 (SIK1, or snflk-1), an essential kinase for cardiac development. In HL-1 cells, elevations in [Na+]i increased SIK1 activity, revealed by an intramolecular Förster resonance energy transfer (FRET)-based sensor showing the structural alteration of SIK-dependent phosphorylation of T182 residue. [Na+]i not capable to increase MEF2/NFAT activity or gene expression in cells expressing two SIK1 mutants (lacking kinase activity or the CaMK1 phosphorylation motif) when compared to cells expressing the wild type SIK1. SIK1-dependent activation of MEF2 involved phosphorylation of HDAC5, because its activation was abolished in cells expressing HDAC5 lacking the SIK1 phosphorylation domain (S255A). The mechanisms by which increases in [Na+]i activate SIK1 and TF are initiated by a parallel increase in intracellular calcium via the reverse Na+/Ca2+ exchanger, activation of CaMK1 and phosphorylation of SIK1 (T322). In conclusion, sustained and moderate increases in myocyte sodium permeability may directly influence myocardial growth by controlling transcription activation and gene expression throughout the activation of the SIK1 network.
Author Disclosures: P. Sergej: None. K. Venetsanou: None. P.J. Chedrese: None. H. Takemori: None. N. Mochizuki: None. A.M. Bertorello: None.
Key Words: Cardiac hypertrophy, Hypertension, Sodium
Novel Cell Surface Markers to Identify and Isolate Endogenous or Induced Pluripotent Stem Cell-Derived Human Cardiovascular Progenitor Cells
Ali Nsair, Katja Schenke-Layland, Ben Van Handel, Hanna Mikolla, Robb MacLellan; UCLA, Los Angeles, CA
Introduction: Given their robust capacity for cardiovascular differentiation, the recently described Isl-1 positive cardiovascular progenitor cell (CPC) would be of great benefit for cardiac cell therapy. However, at present, surface markers that characterize early human CPCs are unknown. Surface markers that identify mouse CPCs (Kdr, c-Kit) are less specific for identifying human CPCs and intracellular markers (Isl-1, Nkx2.5) would require genetic modification of the cells and complicate their clinical use. Purpose of study: We sought to develop markers capable of identifying endogenous human CPCs and determine culture conditions that support their expansion in vivo. A secondary goal was to utilize these markers to isolate and characterize multipotent CPCs from human induced pluripotent stem cells (iPSC) for potential use in regenerative therapies. Results: To determine more specific surface markers we performed microarray analysis of Isl-1+/Kdr+ CPCs isolated from mouse embryonic stem cells and identified eight potential candidate cell surface markers. We utilized FACs and immunofluorescence on fetal human hearts to determine the sensitivity and specificity of these markers alone and in combination to identify Isl-1+ CPCs. The combination of Flt1 and Flt4 best identified the Isl-1+ CPCs in human hearts. Isolated endogenous and human iPSC-derived Flt1+/ Flt4+ cells expressed CPC-specific genes including Nkx2.5, Sox18, Mef2C and c-Myc, but no markers of differentiated cardiovascular cells. When endogenous and human iPSC-derived Flt1+/ Flt4+ cells were plated on matrigel-coated plates and exposed to cardiac, smooth muscle and endothelial-specific differentiation media, spontaneously beating colonies were seen after 21 days. Immunocytochemical staining of theses colonies identified cardiomyoctyes expressing Troponin C and sarcomeric actin, smooth muscle cells expressing alpha smooth muscle actin and myosin, h-caldesmon, and endothelial cells expressing CD31, vWF and eNOS. Conclusion: Cell surface markers Flt1+/ Flt4+ can be used to reliably identify and isolate CPCs. Flt1+/ Flt4+ CPCs from human iPS differentiate into all three cell types of the cardiovascular lineage making them potential candidates for clinical cell therapy.
Author Disclosures: A. Nsair: None. K. Schenke-Layland: None. B. Van Handel: None. H. Mikolla: None. R. MacLellan: None.
Key Words: Cardiac regeneration, Stem cell therapy, Progenitor cell
Specific Interaction with Tyrosine 308 on β2-Adrenoceptor Promotes Preferential Gs Coupling
Anthony Yiu-Ho Woo, NIA NIH, Baltimore, MD; Krzysztof Jóźwiak, Anita Plazinska, Michal Kolinski, Med Univ of Lublin, Lublin, Poland; Joseph A Kozocas, Larry Toll, Mary J Tanga, SRI International, Menlo Park, CA; Valentina Zernetkina, Rajib Paul, Michel Bernier, Irving W Wainer, NIA NIH, Baltimore, MD; Rui-Ping Xiao; Peking Univ, Beijing, China
There are two clinically important subtypes of β-adrenoceptors (βARs), β1AR and β2AR. We have previously proposed a novel therapeutic strategy for heart failure by combination of β1AR blocker and β2AR agonists. Unlike the Gs-coupled β1AR, β2AR couples dually to Gs and Gi proteins. Our previous studies in cardiomyocytes have demonstrated that while most β2AR agonists promote dual Gs and Gi coupling, fenoterol and methoxyfenoterol stereoisomers induce differential Gs/Gi coupling with the R,R-isomers being Gs-selective and the S,R-isomers dually Gs/Gi-activating. The data demonstrate that the two stereoisomers stabilize distinct active conformations of β2AR. As revealed by comparative molecular field analysis of fenoterol derivatives, hydrogen bond formation with tyrosine residue Y308 confers the agonist high selectivity to β2AR. Moreover, stereochemistry is an important factor affecting both receptor binding affinity and functional selectivity of fenoterol derivatives. Thus we hypothesized that the preferential Gs-activating effect of the R,R-isomers depends on hydrogen bond formation with the p-oxygen moiety of Y308 and no π-π interaction with the cassette of aromatic residues nearby. Consistent with model prediction and the binding data, cardiomyocyte contractile response to R,R-4-methoxy-1-naphthylfenoterol was sensitized by the Gi disruptor, pertussis toxin (PTX) (EC50=16±7 nM, EC50PTX=3±2 nM; p<0.05), albeit the agonist's high potency. PTX-sensitive responses were also observed for the R,R-1-naphthyl (EC50=59±16 nM, EC50PTX=12±5 nM; p<0.05) and R,R-phenyl (EC50=607±122 nM) derivatives. In contrast, the positive inotropic effect of R,R-aminofenoterol was PTX-insensitive (EC50=176±78 nM, EC50PTX=145±64 nM; p=0.64), akin to the case of R,R-fenoterol. Also, R,R-aminofenoterol-stimulated activation of extracellular signal-regulated kinase 1/2 is PTX-insensitive in HEK293 cells stably expressing wild-type β2AR but PTX-sensitive in HEK293 cells expressing the β2AR-Y308A mutant. Thus we conclude that specific interaction between certain R,R-isomers and Y308 stabilizes the β2AR in a conformation favoring selective Gs coupling, a criteria in the identification of novel therapeutic agents for heart failure.
Author Disclosures: A. Woo: None. K. Jóźwiak: None. A. Plazinska: None. M. Kolinski: None. J.A. Kozocas: None. L. Toll: None. M.J. Tanga: None. V. Zernetkina: None. R. Paul: None. M. Bernier: None. I.W. Wainer: None. R. Xiao: None.
Key Words: Beta-adrenergic receptor agonists, Myocardial contraction, Pharmacology
Nerve Growth Factor Gene Therapy via Either Intra-Myocardial or Systemic Delivery of Adeno-associated Viral Vectors Prevents Diabetic Cardiomyopathy in Mice
Marco Meloni, Andrea Caporali, Bristol Heart Institute, Bristol, United Kingdom; Lorena Zentilin, Mauro Giacca, ICGEB, Trieste, Italy; Costanza Emanueli; Bristol Heart Institute, Bristol, United Kingdom
Diabetes mellitus causes cardiac dysfunction and heart failure. Nerve growth factor (NGF), which exerts cardiovascular protective and regenerative effects, is downregulated in the diabetic heart. Gene transfer (GT) with adeno-associated viruses (AAVs) is capable of long lasting transgene expression. Moreover, AAV-serotype 9 (AAV-9) shows a preferential trophism for myocytes. Here, we investigated whether intramyocardial or systemic AAV-mediated NGF GT could prevent cardiomyopathy in a mouse model of diabetes. Two weeks after type-1 diabetes induction by streptozotocin (40 mg/Kg/day IP for 5 days), diabetic CD1 male mice were randomized (n=12 mice per group) to receive human NGF or β-Gal control via either 4 injections of an AAV-2 vector (total dose: 1x1011 viral particles) in the left ventricle (LV) wall or a single injection of an AAV-9 vector (1.5x1013 viral particles) in the tail vein. Age-matched CD1 male mice received intra-myocardial AAV-2-β-Gal or intra-venous AAV-9-β-Gal to serve as non-diabetic controls. In separate mice, successful LV GT at 2 weeks after AAV-2-βGal and AAV-9-β-Gal was confirmed by X-Gal staining and β-Gal activity assay, respectively. In comparison with non-injected mice, intra-venous AAV-9-β-Gal enhanced β-Gal activity in limb muscles, but not in other studied organs, including liver and kidneys. High concentrations of human NGF were detected (ELISA) in plasma at 2 and 12 weeks after GT of NGF, but not of β-Gal. As presented in Table 1, echocardiography showed a progressive deterioration of cardiac function and LV chamber dilatation in β-Gal-treated diabetic mice. By contrast, NGF-treated diabetic mice were protected. Moreover, diabetes caused microvascular rarefaction in the LV myocardium, which was prevented by NGF. Our results provide the first evidence that AAV-mediated intra-myocardial or systemic NGF GT can prevent diabetes-induced cardiac dysfunction and suggest the therapeutic potential of this gene therapy approach.
Author Disclosures: M. Meloni: None. A. Caporali: None. L. Zentilin: None. M. Giacca: None. C. Emanueli: None.
Key Words: Gene therapy, Growth factors, Cardiomyopathy
Identification of Nitric Oxide-dependent miRNAs Determining Chromatin Changes During Mouse Embryonic Stem Cells Differentiation
Jessica Rosati, Inst Dermopatico Dell Immacola, Roma, Italy; Francesco Spallotta, Istituto Cardiologico Monzino, Milano, Italy; Barbara Illi, Casa Sollievo Sofferenza- Istituto Mendel, Roma, Italy; Maurizio C Capogrossi, Carlo Gaetano; Inst Dermopatico Dell Immacola, Roma, Italy
Introduction: MicroRNAs (miRNA) are a class of small, non-coding RNA molecules recently emerged as important regulators of gene function through their effects at post-transcriptional level. No or little information, however, is available about the effect of miRNAs on chromatin structure during ES differentiation. Methods & Results: A miRNA profiling was performed in mouse ES (mES) cultured with or without leukemia inhibitory factor (LIF) and in the presence or absence of the NO donor DETANO which elicits an early expression of mesoendodermic/vascular differentiation markers. In this condition, the presence of NO determined a rapid expression of miRNA-200 family members. Real-time PCR analysis confirmed, in fact, that miRNA-200a, −200b, −200c, −429 were up-regulated about 5 fold on average above control level. Direct over-expression of miRNA-200a, −200b, −200c and −429 alone or in combination had an important effect on chromatin structure. Specifically, the global acetylation of histone H3 lysine K9 (H3K9Ac) was significantly increased in mES as well as NIH-3T3 cells paralleled by a significant upregulation of global histone acetylase activity (HAT). The expression of miRNA-200 family members also had a positive effect on histone H4 Lysine 20 tri-methylation (H4K20me3) which is a modification known to be associated to differentiation, aging and repressed chromatin formation. Mechanistically, the miRNA-200 family is known to repress the Smad interacting protein Sip1/ZEB2. Consistently, the direct knockdown of SIP1/ZEB2 by shRNA interference elicited a similar effect on chromatin structure thus suggesting this factor as an important effector of the NO- and miRNA-200-dependent chromatin remodeling. Conclusion: Taken altogether our data identifies for the first time the miRNA-200 family as responsible of adaptive chromatin changes during the NO-dependent process of mES differentiation and suggest SIP1/ZEB2 as an important effector of this process.
Author Disclosures: J. Rosati: None. F. Spallotta: None. B. Illi: None. M.C. Capogrossi: None. C. Gaetano: None.
Key Words: Stem cells, Nitric oxide, Microrna
Inhibition of Plasminogen Activator Inhibitor-1 (PAI-1) Corrects Diabetic Endothelial Progenitor Cell (EPC) Dysfunction in vitro and in vivo
Ashay D Bhatwadekar, Sugata Hazra, Yagna P.R. Jarajapu, Li Liu, Sergio Caballero, Univ of Florida, Gainesville, FL; Valerie Stepps, Beta Stem Therapeutics Inc, San Francisco, CA; Michael E Boulton, Carl J Pepine, Univ of Florida, Gainesville, FL; Paul. J Higgins, Pharmacology, Albany Med College, NY; Stephen H Bartelmez, Beta Stem Therapeutics Inc, San Francisco, CA; Maria B Grant; Univ of Florida, Gainesville, FL
The dysfunction of diabetic CD34+ cells limits their utility in autologous cell therapy for vascular complications. Previously, we showed that transient inhibition of transforming growth factor-beta 1 (TGF-β1) enhances vascular reparative function of diabetic CD34+ cells. Expression of PAI-1, the major gene product of TGF-β1 activation, is increased by high glucose and insulin exposure in endothelial cells and serum of diabetics. We asked whether the beneficial effects of TGF-β1 blockade on CD34+ cells function are mediated by inhibition of PAI-1 and whether blocking of PAI-1 could correct diabetes associated dysfunction. Plasma determinations of PAI-1 and TGF-β1 were compared in type 2 diabetic (n=17) and type 1 (n=7) diabetic patients with micro and macro-vascular disease. CD34+ cells from these individuals were analyzed for cell survival, proliferation, cell cycle analysis and migration. The effect of TGF-β1 phosphorodiamidate morpholino oligomers (PMO) treatment on PAI-1 levels was also determined. In CD34+ cells, PAI-1 was blocked using either lentivirus expressing PAI-1 shRNA or PAI-1 siRNA. In vivo homing ability of PAI-1 inhibited CD34+ cells was assessed using ischemia/reperfusion (I/R) injury model. Plasma PAI-1 level was markedly increased in type 2 diabetic patients compared to type 1 (p <0.05) and directly correlated with TGF-β1 plasma levels (r= 0.44). TGF-β1 PMO treatment resulted in a reduction of PAI-1 mRNA expression (p=0.0018 in diabetic, p=0.05 in non-diabetic). PAI-1 blockade promoted CD34+ cell proliferation in vitro and bypassed the inhibitory effect of TGF-β1 on cell survival (p<0.001). PAI-1 blockade resulted in advancement of cells through G0 to G1 and allowed cell survival in the absence of growth factors (p<0.05). PAI-1 blockade enhanced the migration of these cells in response to SDF-1α (p<0.01) and improved the in vivo re-endothelialization by CD34+ cells in the I/R model. Our results show that the cytostatic activity of TGF-β1 in CD34+ cells is mediated through PAI-1. Blocking PAI-1 corrects key functional defects in diabetic CD34+ cells. This approach will offer a promising novel therapeutic strategy to allow autologous cells therapy in diabetics with severe vascular dysfunction.
Author Disclosures: A.D. Bhatwadekar: Employment; Significant; University of Florida. S. Hazra: None. Y. P.R. Jarajapu: University of Florida. L. Liu: University of Florida. S. Caballero: University of Florida. V. Stepps: Beta Stem Therapeutics. M.E. Boulton: University of Florida. Research Grant; Significant; NIH research grant. C.J. Pepine: Employment; Significant; University of Florida. P.J. Higgins: Albany Medical College. S.H. Bartelmez: Beta Stem Therapeutics. Research Grant; Significant; NIH research grant. M.B. Grant: Employment; Significant; University of Florida. Research Grant; Significant; NIH research grant.
Key Words: Endothelial progenitor cell, Type 2 Diabetes, Angiogenesis
Density of Collagen in the Infarcted Myocardium Determines Engraftment and Angiomyogenesis by Induced Pluripotent Stem Cells (iPSC)
Bo Dai, Wei Huang, Dongsheng Zhang, Univ of Cincinnati, Cincinnati, OH; Mei Hua Gao, Univ. of California, San Diego, CA; Christian Paul, Atif Ashraf, Ronald Millard, Univ of Cincinnati, Cincinnati, OH; Kirk Hammond, Univ. of California, San Diego, CA; Donald Menick, Gazes Cardiac Rsch Institute, Charleton, SC; Meifeng Xu, Muhammad Ashraf, Yigang Wang; Univ of Cincinnati, Cincinnati, OH
We hypothesized that amount of collagen deposition determines the engraftment of iPSC from a patch applied to infarcted area after myocardial infarction (MI). Transgenic mouse with overexpression of adenylyl cyclases VI (AC6) in which collagen synthesis is decreased upon ischemia was used. In vitro: iPSC were transduced with plv-IRES-ZsGreen-firefly luciferase lentivirus vectors bearing a cardiac sodium-calcium exchange promoter (iPSNCX1) for generation of cardiac progenitors in the patch. Expression of collagen I and collagen III was measured in heart tissues of both AC6 and wild-type (WT) mice. In vivo: iPSNCX1 patch (1×107 cells) was placed over the entire infarcted area at 7 days after MI in AC6 and WT mice. Engraftment efficiency of progenitor cells was assessed using a combination of in vivo bioluminescent imaging (BLI) and postmortem ex-vivo analysis. Echocardiography was performed and hearts were harvested for analysis at 4 weeks after cell patch. iPSNCX1patch hearts showed significantly higher BLI together with differentiation of cardiomyocytes (31%±1.68) from baseline (7%±0.06). Heart function was progressively improved from week 2 to week 4 and was associated with reduced left ventricular fibrosis, collagen I, and collagen III in AC6 mice as compared to WT mice (Fig.1). Myocyte progenitors derived from iPSC showed significantly higher engraftment and angiomyogenesis in AC6 mice with sparsely distributed connective tissues suggesting that density of collagen determines the penetration and engraftment of iPSC in the infarcted myocardium.
Author Disclosures: B. Dai: None. W. Huang: None. D. Zhang: None. M. Gao: None. C. Paul: None. A. Ashraf: None. R. Millard: None. K. Hammond: None. D. Menick: None. M. Xu: None. M. Ashraf: None. Y. Wang: None.
Key Words: Stem/progenitor cells, Myocardial infarction, Gene expression
- © 2010 American Heart Association, Inc.