2012 Late-Breaking Basic Science Oral Session
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, Möllmann 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 6 hrs and returned to baseline after 10 days. To validate these findings in humans, serial blood samples were taken from 5 patients with hypertrophic cardiomyopathy undergoing transcoronary ablation of septal hypertrophy (TASH). Similar to the swine model, the level of cMyBP-C increased 30 min after TASH (0.25 ± 0.15 ng/ml) and peaked at 4 hrs (0.56 ± 0.27 ng/ml), confirming that cMyBP-C is a promising ultra-early biomarker of MI. Furthermore, cMyBP-C level was determined in patients with acute coronary syndromes (ACS) from the SYNERGY library population and a healthy control group (n=160 and 61, respectively). Seventy-eight percent (125 out of 160) of patients with ACS had detectable cMyBP-C serum levels (2.9 ± 1.2 ng/ml), implicating serum cMyBP-C as a biomarker for ACS. Conclusion: The rapid appearance of proteolyzed cMyBP-C in the circulation post-acute MI in a swine model and in human patients with ACS and post-TASH identify serum cMyBP-C as an ultra-early biomarker of myocardial necrosis.
Author Disclosures: D.W.D. Kuster: None. A. Cardenas-Ospina: None. L. Miller: None. C. Troidl: None. H.M. Nef: None. C. Liebetrau: None. M. Helge: None. K.S. Pieper: None. K.W. Mahaffey: None. N.S. Kleiman: None. B.D. Stuyvers: None. A.J. Marian: None. S. Sadayappan: None.
Key Words: Myocardial infarction; Biomarkers; Myocardial infarction, NSTEMI; Acute coronary syndromes; Contractile proteins
Replacement Of Mybpc3 Mutation By 5’-trans-splicing In A Knock-in Mouse Model: A Step Towards Causal Therapy Of Hypertrophic Cardiomyopathy
Doreen Khajetoorians1, Giulia Mearini1, Elisabeth Kraemer1, Birgit Geertz1, Christina Hornung1, Oliver Mueller2, Thomas Eschenhagen1, Thomas Voit3, Luis Garcia3, Stephanie Lorain3, Lucie Carrier1; 1Univ Med Cntr Hamburg-Eppendorf, Hamburg, Germany, 2Univ Hosp Heidelberg, Heidelberg, Germany, 3Inserm U974, Paris, France
Purpose: Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric septal hypertrophy, diastolic dysfunction, myocardial disarray and lacks curative treatment. It is often caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). Most of the mutations alter mRNA splicing and result in aberrant mRNAs and proteins. In the present study we evaluated the feasibility and efficacy of RNA correction using spliceosome-mediated 5’-trans-splicing to remove the mutation in vitro and in vivo in Mybpc3-targeted knock-in (KI) mice. Methods and Results: KI mice carry a G > A transition in exon 6, which results in low levels of mutant Mybpc3 mRNAs and cMyBP-C proteins. We generated a series of FLAG-tagged pre-trans-splicing molecules (PTM) containing wild-type exons 1-6 with binding domains complementary to intron 6 of Mybpc3. The PTMs were packaged in adeno-associated virus serotype 6 or 9 (AAV6; AAV9) driven by a cardiomyocyte-specific promoter. Cardiac myocytes isolated from neonatal KI mice (NMCMs) were transduced with AAV6 for 7 days and AAV9 was systemically administered in 3-day-old KI mouse for 7 weeks. The efficacy of 5’-trans-splicing was evaluated by RT-PCR, Western Blot and immunofluorescence. The trans-spliced mRNA was amplified with specific primers only in PTM-transduced samples in vitro and in vivo. The correction of the mutation was confirmed by sequencing. By semi-quantitative PCR trans-spliced mRNA was estimated to represent 33% and 0.14% of total Mybpc3 transcripts in vitro and in vivo, respectively. Whereas the trans-spliced cMyBP-C protein was not revealed by Western blot, up to 9% of FLAG-positive striated cardiac myocytes were detected by immunofluoresence and exhibited correct incorporation of trans-spliced cMyBP-C in doublets in the A-band of the sarcomere. Conclusion: These data provide the first proof-of-concept of 5’-trans-splicing to correct cardiac genetic defects in vitro as well as in vivo. Therefore, spliceosome-mediated 5’-trans-splicing may be a promising approach for the treatment of HCM.
Author Disclosures: D. Khajetoorians: None. G. Mearini: None. E. Kraemer: None. B. Geertz: None. C. Hornung: None. O. Mueller: None. T. Eschenhagen: None. T. Voit: None. L. Garcia: None. S. Lorain: None. L. Carrier: None.
Key Words: Hypertrophic cardiomyopathy; Gene therapy
Hematopoietic Id Ablation Contributes to Pathogenesis in the Adult Heart
Corey Chang, Qingshi Zhao, Diego Fraidenraich; Univ of Medicine and Dentistry of New Jersey, Newark, NJ
The Id (inhibitor of DNA binding) genes play a crucial role in cardiovascular development. Id double knockout (Id dKO) embryos lacking both Id1 and Id3 develop multiple cardiac defects reminiscent of the “thin myocardial wall” syndrome and are lethal at mid-gestation. While Id genes are only expressed in non-myocardial layers (epicardium, endocardium, endothelium), the myocardium is affected suggesting a paracrine mechanism of action. Maternal injection of IGF1 (epicardial Id-dependent signal) failed to rescue inner heart defects seen in Id dKO embryos, suggesting that the endocardium/endothelium play an important role in cardiac development. To circumvent embryonic lethality and study the role of Id in the endocardium/endothelium in adult mice, we generated a conditional knockout (Id cKO) using the Tie2Cre/loxP system. These mice are Id3 null with endocardial/endothelial-specific Id1 ablation. Surviving Id cKOs develop dilated fibrotic cardiomyopathy, hematopoietic defects and splenomegaly in adulthood, suggesting that Id loss in Tie2 expressing organs may be responsible. Since Tie2 is also expressed in hematopoietic cells, it is unclear if loss of hematopoietic Id contributes to cardiac pathology in Id cKOs. To address this question, we transplanted WT GFP-labeled bone marrow into lethally irradiated Id cKOs (WT/Id cKOs) to test if a normal hematopoietic system can ameliorate aspects of cardiomyopathy in our model (rescue). We also performed the reverse experiment (Id cKO/WTs) to test if an Id-deficient hematopoietic system negatively impacts a normal heart (dysregulation). Full bone marrow reconstitution occurred. Within the endomyocardium, we found marked acellularity and fibrosis in Id cKOs, decreased fibrosis and improved cellularity in WT/Id cKOs, and emerging interstitial fibrosis and mononuclear invasion in Id cKO/WTs. The marked perivascular fibrosis in Id cKO hearts was reduced in WT/Id cKO hearts. Ejection fraction decreased in Id cKOs, improved in WT/Id cKOs and decreased in Id cKO/WTs (64.4+/-5.7% n=5 WT/Id cKOs; 58.4+/-7.97% n=9 Id cKO/WTs; 56.2+/-13.2% n=56 Id cKOs; 64.3+/-6.8% n=15 WTs). These results suggest that Id levels in bone marrow cells play an important role in the development of dilated, fibrotic cardiomyopathy.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: C. Chang: None. Q. Zhao: None. D. Fraidenraich: None.
Key Words: Cardiovascular development; Adult congenital heart disease; Vascular disease-Cardiomyopathy; Heart development
Detection And Therapy Of Ischemia-reperfusion Injury Using Hydrogen Peroxide-responsive Molecularly Engineered Polymer Nanoparticles
Dongwon Lee1, Soochan Bae2, Donghyun Hong3, On Hwang2, Seunggyu Park2, Joo H Yoon2, Qingen Ke2, Peter M Kang2; 1Chunbuk National Unversity, Jeonju, Korea, Republic of, 2BETH ISRAEL DEACONESS MED CTR, Boston, MA, 3Chunbuk National Univ, Jeonju, Korea, Republic of
The main culprit in the pathogenesis of ischemia/reperfusion (I/R) injury is the overproduction of hydrogen peroxide (H2O2), a causal agent for apoptosis and inflammation that lead to cellular damage and organ dysfunction. In this study, we generated and characterized novel H2O2-responsive nanoparticles formulated from polyoxalate co-polymer containing vanillyl alcohol (PVAX) during I/R injury. PVAX, in the presence of H2O2, degrades completely into three safe compounds, cyclohexanedimethanol, VA and CO2. PVAX effectively scavenges H2O2 in a dose-dependent manner, with the intrinsic anti-oxidant and anti-inflammatory properties in vitro. Using In Vivo Imaging System, we demonstrated that PVAX loaded with rubrene as a fluorophore robustly imaged H2O2 generated during I/R in the hind-limb I/R injury in vivo, demonstrating their potential for bioimaging of H2O2. PVAX nanoparticles injected into the site of hind-limb I/R injury also exerted highly potent anti-inflammatory and anti-apoptotic activities resulting in a significantly less cellular damage compared to the vehicle-treated group. We then examined the effect of PVAX in doxorubicin (DOX)-mediated cardiomyopathy since a major mechanism of DOX-mediated toxicity has been shown to involve increased oxidative stress. We found that i.p. injection of 100 µg of PVAX daily resulted in significant decrease in apoptosis as well as PARP-1 and caspase activation in both heart and liver compared to vehicle treat ed groups. Furthermore, 10 days after DOX administration PVAX treated animals, compared to vehicle treated animals, demonstrated 31% less decrease in body weight, 28% improvement in cardiac function and significantly improved survival rate (Veh= 35% vs PAVX=71%; p<0.05%). Moreover, PVAX administered daily for 7 days demonstrated no histological or functional abnormalities in various organs. Thus, we conclude that PVAX is a novel multifunctional nanoparticle that possesses intrinsic anti-oxidant and anti-inflammatory properties, and has a tremendous potential to be used as a theranostic agent for I/R injury in heart and other organs.
Author Disclosures: D. Lee: None. S. Bae: None. D. Hong: None. O. Hwang: None. S. Park: None. J.H. Yoon: None. Q. Ke: None. P.M. Kang: Research Grant; Significant; Abbott Laboratories.
Key Words: Ischemia reperfusion; Apoptosis Inflammation; New technology; Cardiomyopathy
Communication with Cardiomyocytes in Contact Co-culture Reprograms Mesenchymal Stem Cells for Improved Cardiomyogenesis
Vien Khach Lai, Shujia Jiang, Kelly S Prindle, Khawaja H Haider; Univ of Cincinnati, Cincinnati, OH
Background: Bone marrow mesenchymal stem cells (MSC) can differentiate to morpho-functional cardiomyocytes (CM) when maintained in conductive culture conditions in vitro and post-transplantation in the ischemic heart albeit with an as yet undefined mechanism. We hypothesized that propensity of MSC for reprogramming to adopt cardiac phenotype and their myocardial reparability could be enhanced if the cells were co-cultured with CM in vitro prior to transplantation. Methods and results: Bone marrow cells were isolated from GFP expressing transgenic adult rats and analyzed for MSC specific surface markers by flow cytometry. The cells were co-cultured with PKH-26 labeled rat neonatal CM at 1:3 ratios in a dual chamber dish separated by a semi-permeable membrane or by direct contact co-culture for 0-4 days. Conventional fluorescence microscopy revealed formation of nano-tubular structures between MSC in contact co-culture with CM. Live cell imaging showed transient exchange of cytosolic contents between the two juxtaposed cell types which was indicated by PKH26 red fluorescence dye transfer. Flow cytometry revealed increasing percentage of double positive GFP+ MSC in time-dependent manner (up to 81.08 ± 5.91% of GFP+ MSC received the red dye transferred from CM at day 3 of co-culture). Cardiac specific gene expression including GATA4, Nkx2.5 and MEF2c was elevated in MSC at 3 days after co-culture with CM. These results were substantiated by immunocytochemistry. Transplantation of MSC (co-cultured for 3 days) in a rat heart model of acute coronary artery ligation resulted in significantly reduced infarction sizes (38.2 ± 3.1 vs 25.0 ± 3.8 for control vs co-cultured MSC, p<0.05) and preserved global heart function (left ventricular ejection fraction: 46.0 ± 1.2 vs 56.1 ± 4.0% for control vs co-cultured MSC, p<0.05) after 3 weeks of MSC transplantation compared to that of control. Conclusion: Direct contact with CM (1:3) in co-culture reprograms MSC for enhanced rate of cardiomyogenic differentiation via exchange of their cytosolic contents. These findings provide an insight into the mechanisms which underlie the morpho-functional cardiogenic differentiation of MSC post-transplantation in an infarcted heart.
Author Disclosures: V. Lai: None. S. Jiang: None. K.S. Prindle: None. K.H. Haider: None.
Key Words: Myocardial infarction; Regenerative medicine stem cells; Transplantation; Molecular biology
Telomeric Shortening Induces a Senescent Cardiomyopathy that is Independent from Chronological age
Junghyun Kim, Fumihiro Sanada, Maria Cimini, Christian Arranto, Polina Goihberg, Toru Hosoda, Marcello Rota, Jan Kajstura, Piero Anversa, Annarosa Leri; Brigham and Women’s Hosp, Boston, MA
Telomeric shortening is viewed as the hallmark of organism, organ, and cellular senescence. It remains, however, to be documented whether loss of telomeric DNA in cardiac stem cells (CSCs) and/or cardiomyocytes is causally involved in the manifestations of the aging cardiomyopathy. To address this issue, the heart of mice carrying a deletion of the RNA component of telomerase (Terc−/− mice) was studied at 3-7 months of age. This allowed us to define whether telomere shortening in stem cells and their progeny promotes ventricular dysfunction independently from chronological age. The structural and functional characteristics of the aging cardiomyopathy were defined initially in senescent 30-month-old wild-type mice (WT). By echo-Doppler, MRI, and invasive hemodynamics, deterioration in systolic and diastolic indices of myocardial contractility were detected in these senescent mice. Quantitative parameters obtained by MRI documented the presence of chamber dilation and attenuated ventricular torsion capacity and diastolic strain rate. With respect to young mice, a 50% reduction in telomere length was detected in CSCs and cardiomyocytes isolated from 30 month-old mice. Importantly, 7-month-old Terc−/− mice showed severe ventricular dysfunction comparable to that seen in 30 month-old WT. Telomere length in Terc−/− mouse CSCs and myocytes was ~50-70% shorter than in age-matched WT cells but was comparable to that detected in 30 month-old WT cells. The number of CSCs was 60% lower in Terc−/− than age-matched WT mice, and the fraction of BrdU-positive CSCs decreased 1.4-fold, from 25% to 14%. The absence of Terc led to a 50% reduction in myocyte turnover, which was coupled with myocyte hypertrophy and significant myocyte loss. BrdU and Ki67 labeling were reduced, respectively, by 65% and 60% in Terc-/- myocytes. Old CSCs formed a senescent progeny composed of cardiomyocytes, which carried markedly shortened telomeres, were consistently larger in volume, and showed a severe depression in cell shortening and re-lengthening. Our findings document that telomeric shortening is the critical determinant of CSC aging and reduced myocyte renewal. The accumulation of senescent CSCs and cardiomyocytes leads to pathological cardiac remodeling and heart failure.
Author Disclosures: J. Kim: None. F. Sanada: None. M. Cimini: None. C. Arranto: None. P. Goihberg: None. T. Hosoda: None. M. Rota: None. J. Kajstura: None. P. Anversa: None. A. Leri: None.
Key Words: Stem cells; Aging; Cardiac regeneration
Identification of Novel Non-steroidal Modulators of Vitamin D Receptor with Cardioprotective Property without Hypercalcemic Effect
Ji Yoo Lee1, Santosh A Khedkar1, Sangita Choudhury1, Mohammed Samad1, Dongsheng Zhang1, Ravi I Thadhani2, S A Karumanchi1, Alan C Rigby1, Peter M Kang1; 1BETH ISRAEL DEACONESS MED CTR, Boston, MA, 2Massachusetts General Hosp, Boston, MA
Vitamin D is a multifunctional, steroid hormone responsible for regulating various biological processes. Vitamin D therapy has been shown to prevent cardiac hypertrophy and improve cardiac dysfunction. However, its clinical utility has been limited by hypercalcemia. In this study, we report on a novel vitamin D agonist tool compound discovered using computer aided drug discovery (CADD) that possesses significant anti-hypertrophic activity without hypercalcemia. Using this CADD approach, we screened approximately 4 million unique compounds virtually using chemical features/descriptors from known vitamin D receptor (VDR) agonists that had minimal demonstrable hypercalcemia activity. These hit compounds were vetted using an ensemble structure-based screen of the VDR. The top 174 CADD molecules were evaluated using the GeneBLAzer® Cell-Based VDR Assay to identify those compounds that significantly regulated the transcriptional profile of VDR; we identified 5 compounds that showed considerable activation at 100 µM or lower. The compound possessing the greatest VDR binding activity (known as VDR136) showed a significant concentration-dependent suppression of phenylephrine (PE)-induced cardiac hypertrophy in adult cardiomyocyte culture in vitro and mouse infused with PE via osmotic pump in vivo. In addition, VDR136 significantly suppressed cardiac hypertrophy and progression to heart failure induced by transverse aortic constriction (TAC) as compared to the vehicle treated group as determined by 17% decrease in HW/BW ratio and 41% improvement in fractional shortening. VDR 136 also demonstrated effective suppression secondary hyperparathyroidism in 1α-hydroxylase knockout mice, a model of vitamin D deficiency. Most importantly, we observed no significant hypercalcemia even at supra-physiological concentrations of VDR136. In contrast, calcitriol, a naturally occurring, commercially available vitamin D hormone, caused significant hypercalcemia. Thus, VDR136 represents a novel VDR agonist with significant cardioprotective properties that lack the hypercalcemic effect otherwise common with vitamin D analogs, and may provide a novel therapeutic option for the treatment of cardiac hypertrophy and heart failure.
Author Disclosures: J. Lee: None. S.A. Khedkar: Employment; Significant; Imclone. S. Choudhury: None. M. Samad: None. D. Zhang: None. R.I. Thadhani: None. S.A. Karumanchi: None. A.C. Rigby: Employment; Significant; Imclone. P.M. Kang: Research Grant; Significant; Abbott Laboratories.
Key Words: Heart failure; Hypertrophy; Vitamins; Calcium; Drugs
Sorafenib-induced Cardiotoxicity is Mediated by Inhibition of c-kit+ Cardiac Stem Cells
Catherine A Makarewich, Jason M Duran, Thomas E Sharp, III, Ronald J Vagnozzi, Remus M Berretta, Hajime Kubo, Thomas Force, Steven R Houser; Temple Univ Sch of Medicine, Philadelphia, PA
Introduction: Tyrosine kinase receptor (RTK) inhibitors have been previously shown to be cardiotoxic in cancer patients with comorbidities, but the mechanism of this cardiotoxicity has not been elucidated. Hypothesis: The multi-kinase inhibitor Sorafenib is known to inhibit several RTKs including c-kit, a receptor found on cardiac progenitor cells in the heart and bone marrow. Patients with comorbidities have pre-existing cardiac damage, and administration of Sorafenib inhibits their cardiac stem cell population, thus preventing myocyte turnover and exacerbating cardiac dysfunction. Methods and Results: Male 12 week old C57BL/6 mice were pretreated with 40 mg/kg/d intraperitoneal Sorafenib (n=20) or vehicle (n=17) for 7 days before receiving myocardial infarction (MI) by coronary artery ligation. This dose had no significant effect on cardiac function before MI. By 1 week post-MI, Sorafenib treatment dramatically exacerbated LV dysfunction measured by echocardiography (ejection fraction = 20.6 ± 3.5 v. 30.6 ± 3.1%; p=0.04) and significantly decreased survival (40.0 v. 72.0%; p=0.02) relative to MI controls. C-kit+ bone marrow stem cells (BMC), c-kit+ cardiac-derived stem cells (CDCs) and adult feline left ventricular myocytes (AFLVMs) were isolated and treated with Sorafenib at 1, 5 and 10uM doses for 12-72 hours in vitro. High concentrations (10 uM) of Sorafenib induced complete necrosis in all cell types. Both 1 and 5 uM treatments potently inhibited proliferation of CDCs and BMCs in a dose-dependent manner in vitro. Low dose Sorafenib (1 uM) produced a time-dependent increase in caspase-3 activation in both cultured BMCs and CDCs suggesting induction of apoptosis/necrosis. Importantly, neither the 1 or 5 uM doses of Sorafenib induced caspase-3 activation in AFLVMs, nor did it cause cell death or otherwise affect contractility or alter calcium transients in vitro. Conclusions: These data show that part of the cardiotoxicity associated with RTK inhibitors, such as Sorafenib, is mediated through inhibition of c-kit+ cardiac progenitor pools. This is the first known report of a proposed mechanism of cardiotoxicity based on a possible inhibition in stem cell-activity (repair-based and/or survival signal-based).
Author Disclosures: C.A. Makarewich: None. J.M. Duran: None. T.E. Sharp: None. R.J. Vagnozzi: None. R.M. Berretta: None. H. Kubo: None. T. Force: None. S.R. Houser: None.
Key Words: Heart failure; Cardiomyopathy; Drugs; Myocardial infarction
Cardiac Specific Deletion Of Glycogen Synthase Kinase-3α Attenuates Post Myocardial Infarction-induced Ventricular Remodeling And Preserves Heart Function
Firdos Ahmad1, Hind Lal1, Ronald J Vagnozzi1, Jibin Zhou1, James R Woodgett2, Erhe Gao1, Thomas Force1; 1Sch of Medicine Temple Univ, Philadelphia, PA, 2Samuel Lunenfeld Rsch Institute, Univ of Toronto, Toronto, Canada
Glycogen synthase kinase (GSK)-3α has been reported to regulate cardiac growth, pathologic hypertrophy, mitochondrial integrity and regulation of contractile function. However, it is essential to better define the role of GSK-3α in the heart using appropriate models since previous studies were performed either by using global knock-in or knock-out strategies, complicating interpretation of the data and limiting understanding of true roles in specific tissues. The goal of this study was to define the role of GSK-3α in the heart using a cardiac-specific conditional GSK-3α KO. For these studies, the α-MHC promoter driving MerCreMer was crossed with the GSK-3αfl/fl mouse. Mice (5-6 months of age) of two genotypes: GSK-3αfl/flCre+Tam+ (KO) and littermate controls (GSK-3αfl/flCre-Tam+(WT), were subjected to LAD ligation (MI). Survival was significantly improved post MI in the KO in comparison to the WT (100% vs 75%, p=0.03). At two weeks post MI, chamber dilation and left ventricular (LV) dysfunction were comparable in KO and WT, though the KO had significantly increased LV mass (p=0.02 at 1 wk, p=0.002 at 2 wks vs WT). Importantly, at four weeks post MI, LV chamber dimension was reduced in the KO [LVID;d(mm) 5.16 ± 0.61 vs 5.97 ± 0.82, p=0.002] and LV function was improved (LVEF% 32.03 ± 9.95 vs 22.3 ± 14.6, p=0.02). The diastolic (p=0.01) and systolic (p=0.007) LV volumes in the KO were also significantly decreased. Our observations are distinctly different from the global GSK-3α KO which had increased post MI mortality and marked cardiac dysfunction. These findings demonstrate that cardiac specific deletion of GSK-3α prevents MI-induced cardiac remodeling and preserves heart function. Moreover, the deletion induces cardiac hypertrophy that seems to be physiological and beneficial for cardiac function. Since inhibition of GSK-3β is also beneficial in this setting, a non-selective inhibitor targeting both isoforms could be a viable therapeutic to control post MI remodeling, preserve heart function and ultimately prevent heart failure.
Author Disclosures: F. Ahmad: None. H. Lal: None. R.J. Vagnozzi: None. J. Zhou: None. J.R. Woodgett: None. E. Gao: None. T. Force: Research Grant; Significant; NIH Funding.
Key Words: Heart failure; Myocardial infarction; Ventricular remodeling; Heart function tests
A Clock Gene RORα-mediated Regulation of the Activity of Rho-associated Coiled-coil Protein Kinase 2 (ROCK2) Plays a Key Role in Generating Vascular Intrinsic Circadian Rhythm of Myofilament Ca2+ Sensitivity and Vascular Contractility
Toshiro Saito1, Mayumi Hirano1, Tomomi Ide1, Toshihiro Ichiki1, Noriyuki Koibuchi2, Kenji Sunagawa1, Katsuya Hirano1; 1Grad Sch Med Sci, Kyushu Univ, Fukuoka, Japan, 2Gunma Univ Graduate Sch of Medicine, Maebashi, Japan
The occurrence of cardiovascular events shows diurnal variation with a peak in the morning. The circadian change in vascular contractility is one of the mechanisms for this circadian variation. We hypothesize that the vascular intrinsic clock contributes to generating the circadian rhythm of the contractility. However, its underlying mechanism still remains elusive. We addressed this question by using cultured vascular smooth muscle cells (VSMC) and staggerer mutant mice, which lack functional RORα, and identified RORα and ROCK2 that play a key role in generating circadian rhythm of vascular contractility. In VSMC, the level of myosin light chain (MLC) phosphorylation induced by thrombin or endothelin-1 showed an oscillation of a 25.4-hr cycle length with peaks at 36 and 60 hrs and a nadir at 48 hrs after dexamethasone pulse-triggered synchronization of the circadian clock. Pharmacological inhibition or RNAi knockdown of ROCK2, but not MLC kinase, protein kinase C, or ZIP kinase, abolished the rhythm of MLC phosphorylation. The expression of ROCK2 protein and its activity, as assessed by the phosphorylation of MYPT1, oscillated in phase with MLC phosphorylation. The amount of GTP-bound RhoA did not oscillate. The expression of ROCK2 mRNA and RORα protein oscillated in a circadian manner with a peak 4 hrs earlier than the ROCK2 activity. RORα activated the promoter of the ROCK2 gene in luciferase reporter assay, while RORα knockdown abolished the rhythm of ROCK2 expression. ROCK2 expression also oscillated in a circadian manner with a peak at Zeitgeber time (ZT) 0 and a nadir at ZT12 in the aorta of the control, but not staggerer, mice. In the permeabilized aortic ring preparations, the levels of MLC phosphorylation and contraction induced by GTPγS or thromboxane A2 at ZT0 were higher than those seen at ZT12 in the control, but not staggerer, mice. No diurnal change was seen for the Ca2+-dependent contraction in either mouse. We conclude that RORα-induced circadian oscillation of the ROCK2 activity modulates MLC phosphorylation and plays a key role in generating the circadian rhythm of myofilament Ca2+ sensitivity. This rhythm of vascular contractility may underlie the diurnal variation in the occurrence of cardiovascular events.
Author Disclosures: T. Saito: None. M. Hirano: None. T. Ide: None. T. Ichiki: None. N. Koibuchi: None. K. Sunagawa: None. K. Hirano: None.
Key Words: Smooth muscle; Vascular; Contractility; Signal transduction
Late-Breaking Basic Science Poster Session
Birth Dating of Human Lung Cells by Accelerator Mass Spectrometry
Mario Ricciardi, Ada Pesapane, Francesca Polverino, Christian Arranto, Giorgia Palano, Hilarie Lam, Ana Castano, Alex Matsuda, Marcello Rota, Annarosa Leri, Ivan Rosas, Mark Perrella, Bruce A Buchholz, Jan Kajstura, Joseph Loscalzo, Piero Anversa; Brigham and Women’s Hosp, Boston, MA
The respiratory units of the distal airway in the human lung are composed of the alveoli and the adjacent capillary structures. Alveoli are defined by type 1 and type 2 epithelial cells, while endothelial cells form a dense capillary network between alveoli. Fibroblasts are distributed at the interface between the alveolar epithelium and endothelial cells (ECs). There is general consensus that type 1 and type 2 pneumocytes, and ECs have limited renewal capacity physiologically, although the degree of regeneration of these cells has never been carefully studied. Similarly, the effects of idiopathic pulmonary fibrosis (IPF) on cell formation are currently unknown. To address these issues, the turnover rate of these lung cell categories was measured in normal human lungs not used for transplantation and in explanted lungs with IPF. For this purpose, lung cells were exposed during isolation by enzymatic digestion to 3H-thymidine for 1 hour to label cycling cells. Alveolar epithelial cells, ECs, and fibroblasts were separated by FACS sorting and the DNA was extracted. The level of 3H-thymidine was determined by Accelerator Mass Spectrometry (AMS); each measurement was corrected for potential contamination from the other cell classes. Based on this approach, the yearly turnover of fibroblasts, ECs, type 1 and type 2 pneumocytes was 22%, 14%, 13%, and 2%, respectively. Corresponding values with IPF were 961%, 28%, 22%, and 4%, documenting that the components of the respiratory units were markedly affected by IPF and the response of the fibroblasts was striking. In controls, the average age of fibroblasts, ECs, type 1 and type 2 pneumocytes was, respectively, 5, 7, 8, and 12 years, while organ age was 56 years. In IPF, the average age of fibroblasts, ECs, type 1 and type 2 pneumocytes was, respectively, 0.2, 4, 5, and 9 years, while organ age was 69 years. As a consequence, all cell categories were significantly younger with IPF. Thus, the cellular compartments constituting the gas exchange units in the adult normal and severely diseased human lung are replaced several times during the course of life, and this growth response is potentiated in IPF. In conclusion, the human lung is a highly dynamic organ with an unexpected regenerative potential.
Author Disclosures: M. Ricciardi: None. A. Pesapane: None. F. Polverino: None. C. Arranto: None. G. Palano: None. H. Lam: None. A. Castano: None. A. Matsuda: None. M. Rota: None. A. Leri: None. I. Rosas: None. M. Perrella: None. B.A. Buchholz: None. J. Kajstura: None. J. Loscalzo: None. P. Anversa: None.
Key Words: Pulmonary
Differential Roles of Cardiac Fibroblast Glycogen Synthase Kinase-3 Isoforms in Myocardial Infarction Induced Ventricular Remodeling and Heart Failure
Hind Lal1, Firdos Ahmad1, Ronald Vagnozzi1, Jibin Zhou1, Mahek Shah1, James Woodgett2, Erhe Gao1, Thomas Force1; 1Temple Univ, Philadelphia, PA, 2Samuel Lunenfeld Rsch Institute, Toronto, Canada
Most cardiac diseases are associated with myocardial fibrosis, accompanied by excess deposition of extracellular matrix (ECM) and fibroblast activation. Fibroblasts are the principal producers of ECM and contribute significantly to myocardial fibrosis. However, fibroblasts are considered to play a secondary role in cardiac hypertrophy, remodeling and heart failure. Herein, we evaluated the isoform specific role of cardiac fibroblast GSK-3α (CF-GSK-3α) and CF-GSK-3β in the regulation of myocardial infarction (MI)-induced remodeling and heart failure. We achieved cardiac fibroblast-specific deletion of GSK-3α or GSK-3β by employing Cre recombinase driven by a 3.9-kb mouse Postn promoter in GSK-3αfl/fl and GSK-3βfl/fl mice. These mice, CF-GSK3-α-KO and CF-GSK-3βKO were subjected to permanent occlusion of LAD (MI) at the age of 8 wks. Mice were followed with serial echocardiography. Pre-MI, hearts of WT and KO mice had comparable chamber dimensions and ventricular function, but as early as two weeks post MI, CF-GSK3β-KO animals had a significant increase in end-diastolic (4.23 ± 0.14 vs 4.83 ± 0.26mm; P=0.035) and end-systolic dimension (2.96 ± 0.16 vs 3.86 ± 0.33mm; P=0.032). This was associated with marked LV dysfunction as reflected by reduced EF (57.36 ± 3.42 vs 40.71 ± 5.60; P=0.025) Both HW/TL (0.84 ± 0.03 vs 1.11 ± 0.07; P=0.009) and LW/TL (0.98 ± 0.02 vs 1.26 ± 0.12; P=0.06) were elevated in CF-GSK-3βKO mice. Post-MI mortality was also increased in CF-GSK-3βKO mice (57.1% vs. 29.4% P=0.01). In distinct contrast to CF-GSK-3βKO with exaggerated remodeling vs. WT, MI-induced chamber dilation and LV dysfunction were reduced in CF-GSK3α-KO compared to WT (EF (35.75 ± 3.40 vs 47.96 ± 4.06; P=0.0291) at 8 weeks post MI. These finding suggest that deletion of GSK-3α in cardiac fibroblasts prevents MI-induced myocardial remodeling and dysfunction. Thus cardiac fibroblast GSK3α and GSK-3β exhibit opposite effects on MI-induced cardiac remodeling and dysfunction. Furthermore, these data are, to our knowledge, the first to establish the driving role of cardiac fibroblasts in MI-induced remodeling.
Author Disclosures: H. Lal: None. F. Ahmad: None. R. Vagnozzi: None. J. Zhou: None. M. Shah: None. J. Woodgett: None. E. Gao: None. T. Force: Research Grant; Significant; NIH funded.
Key Words: Myocardial infarction; Fibrosis; Heart failure; Remodeling
Modeling Cardiovascular Disease in Supravalvular Aortic Stenosis and Williams-Beuren Syndrome Using Human Induced Pluripotent Stem Cells
Yibing Qyang; Yale Univ, New Haven, CT
Supravalvular aortic stenosis (SVAS) and Williams-Beuren syndrome (WBS) are genetic disorders of elastin (ELN) and are characterized by hyper-proliferation of vascular smooth muscle cells (SMCs) that can lead to blockage of the arterial vessels and sudden cardiac death. While SVAS is typically caused by heterozygous, loss-of-function mutations in the ELN gene, WBS is a microdeletion disorder resulting from heterozygous loss of 1.5-1.8 Mb pairs of DNA from chromosome 7 including the ELN gene. Although WBS patients display a complex phenotype including neurobehavioral defects, the cardiovascular lesions in patients with SVAS and WBS are virtually identical and manifested as hyper-proliferation of vascular SMCs. The use of animal models and primary cell culture to study SVAS and WBS has been very informative. However, Eln+/- and WBS haploinsufficiency mice do not develop human-like aortic occlusive defects, and human tissue accessibility is limited. It is therefore of great importance to establish a self-renewable human model for studying SVAS and WBS. Human induced pluripotent stem cells (hiPSCs) can be derived from a person’s own somatic cells by forced gene expression and self perpetuate. We recently established hiPSC lines from SVAS and WBS patients. SVAS and WBS iPSC-derived SMCs (iPSC-SMCs) had reduced ELN expression, decreased smooth muscle alpha actin (SM α-actin) filament bundles, and increased proliferation and migration, compared to control iPSC-SMCs. Recombinant ELN or enhancement of small GTPase RhoA signaling rescued actin filament bundle formation and inhibited hyper-proliferation. Several candidate small molecules including vinblastine have been tested to see whether rescue of actin filament bundle formation in SVAS or WBS iPSC-SMCs leads to inhibition of hyper-proliferation. Our results showed that low dose of vinblastine rescued actin filament bundle formation, inhibited hyper-proliferation, and decreased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in SVAS iPSC-SMCs without effect on cellular apoptosis. Thus, SVAS and WBS iPSC-SMCs recapitulate key cardiovascular pathological features of patients and may provide a promising strategy to study disease mechanisms and to develop novel therapies.
Author Disclosures: Y. Qyang: None.
Key Words: Stem cell biology; Smooth muscle regulation
MiR-24 Up-regulation Underlies Excitation-contraction Uncoupling in Heart Failure
Ming Xu1, Hao-Di Wu1, Rong-Chang Li1, Meng Wang2, Jin Tao1, Su-Fang Li1, Yun-Bo Guo1, Shao-Ting Lai1, Guan-Zheng Luo2, Yan Bai1, Wei Gao1, You-Yi Zhang1, Xiu-Jie Wang2, Shi-Qiang Wang1; 1Peking Univ, Beijing, China, 2Institute of Genetics and Developmental Biology, Beijing, China
Chronic heart failure is a complex clinical syndrome with impaired myocardial contractility. Failing cardiomyocytes exhibit decreased efficiency of Excitation-contraction (E-C) coupling. The down-regulation of junctophilin-2 (JP2), a structural protein anchoring the sarcoplasmic reticulum (SR) to T-tubules (TTs), has been identified as a major mechanism underlying the defective E-C coupling. However, the regulatory mechanism of JP2 remains unknown. Here we report that miR-24, a microRNA up-regulated in heart failure, is an immediate upstream suppressor of JP2. Bioinformatic analysis predicted two potential binding sites of miR-24 in the 3’-untranslated regions of JP2 mRNA. Luciferase assays confirmed that miR-24 suppressed JP2 expression by binding to either of these sites. In the aortic stenosis model, miR-24 was up-regulated in failing cardiomyocytes. Adenovirus-directed over-expression of miR-24 in cardiomyocytes decreased JP2 expression and reduced Ca2+ transient amplitude and E-C coupling gain. In vivo silencing of miR-24 by a specific antagomir in an aorta-constricted mouse model effectively protected cardiomyocytes from structural and functional E-C uncoupling, and prevented the mouse from developing heart failure. As miR-24 is a member of the miR-23a~27a~24-2 cluster regulated by calcineurin-NFATc3 signaling, this finding mechanistically links upstream heart failure signaling to the ultimate defect in cardiomyocyte contractility, and suggests novel therapeutic strategies against heart failure.
Author Disclosures: M. Xu: None. H. Wu: None. R. Li: None. M. Wang: None. J. Tao: None. S. Li: None. Y. Guo: None. S. Lai: None. G. Luo: None. Y. Bai: None. W. Gao: None. Y. Zhang: None. X. Wang: None. S. Wang: None.
Key Words: Heart failure; Excitation-contraction coupling (ECC); Calcium; Microrna
Antifibrotic Effect Of Transplanted Cardiospheres Mediated By Dynamic Activation Of The Matrix Proteolytic Cascade In Acute Myocardial Infarction
ELENI TSELIOU1, Sara Pollan1, John Terrovitis2, Baiming Sun1, Linda Marbán1, Eduardo Marbán1; 1CEDARS SINAI HEART INSTITUTE, Los Angeles, CA, 2Third Dept of Cardiology, Univ of Athens, Athens, Greece
Background: Intramyocardial cardiosphere transplantation in the infarcted myocardium enhances tissue viability and improves left ventricular (LV) function in both small and large animal models. However, the underlying biological mechanisms by which cardiospheres exert their beneficial effects have not yet been fully elucidated. Objective: We tested the hypothesis that injected cardiospheres would alter the proteolytic pathway post myocardial infarction (MI) and thereby attenuate adverse remodeling. Methods: MI was induced in Wistar Kyoto rats, followed by peri-infarct injections of 2x106 syngeneic cardiospheres (n=25). Animals injected with vehicle only served as controls (n=20). Cocultures were performed to test potential direct interaction between fibroblasts and cardiospheres. Results: In vitro, cardiospheres stimulated fibroblasts’ excretion of gelatinases MMP2 and MMP9 as early as 24 hours in coculture. In vivo, a differential MMP/TIMP profile was evident in the peri-infarct region 7 days post MI and treatment: MMP2 and MMP13 were upregulated in the cardiosphere-treated myocardium compared to control (p<0.05 treated vs control), where increased MMP9 was detected. Plasma levels of ICTP, an index of collagen degradation, were increased by day 7 post cardiosphere transplantation (p<0.01 treated vs control). In addition, collagen synthesis, evaluated by hydroxyproline assay, was decreased (p<0.01 treated vs control). Picrosirius red tissue staining for regional collagen deposition showed less fibrosis in the treated compared to the control group at day 21. These changes in the cardiosphere-transplanted post-MI myocardium were associated with significant improvements in LV morphological and functional parameters and increased capillary and vessel formation. Conclusion: Cardiospheres intramyocardially-injected in the infarcted myocardium alter the MMP pathway to favor a collagenolytic and antifibrotic profile. These findings begin to reveal how cardiospheres attenuate post MI adverse remodeling, a principle central to their beneficial actions in ischemic heart disease.
Author Disclosures: E. Tseliou: None. S. Pollan: None. J. Terrovitis: None. B. Sun: None. L. Marbán: Employment; Significant; Capricor Inc. Ownership Interest; Significant; Capricor Inc. E. Marbán: Ownership Interest; Significant; Capricor Inc.
Key Words: Stem cell therapy; Stem cell biology; Myocardial infarction, STEMI; Remodeling; Extracellular matrix
Global RNA Splicing and Regulation in Fetal and Diseased Hearts
CHEN GAO, Vincent Ren, Jae-Hyung Lee, Xinshu(Grace) Xiao, Jau-nian Chen, Yibin Wang; UCLA, Los Angeles, CA
Background: The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is also implicated in many human diseases including cancer. However, little knowledge is available about the scope of alternative splicing at whole genome level during cardiac development and diseases and even less about the mechanisms underlying the regulation of mRNA splicing in response to pathological injury in heart. Methods and Results: In our current study, we have identified global alternative splicing changes associated with both development and pathological remodeling in mouse heart using deep RNA-Sequencing. The alternative RNA splicing events observed in failing hearts resemble the profile observed in fetal hearts, suggesting a fetal-like RNA splicing program induced in diseased hearts. We subsequently examined the expression profiles of RNA splicing regulators in neonatal, normal adult, and failing adult mouse hearts, and identified Fox1 as a significantly induced regulator during cardiac development in both mouse and zebrafish, and down-regulated in both mouse and human failing hearts. Fox1 knock-down mediated by morpholino in zebrafish embryos led to lethal phenotype associated with impaired cardiomyocytes maturation and differentiation. Interestingly, this phenotype could be rescued by re-expressing both zebrafish and mouse Fox1 gene. Finally, using bioinformatic analysis, a significant number of the alternatively spliced exons identified in the failing heart harbor a conserved Fox1 binding motif in flanking introns, suggesting that Fox1 may serve as a conserved and major regulator for alternative RNA splicing during cardiac development and diseases. Conclusion: Our study provided the first comprehensive analysis of mRNA splicing regulation in heart during post-natal development and heart failure, and identified Fox1 as a potential key regulator for RNA splicing in heart. This study expands our current understanding to the complexity of cardiac transcriptome in both development and diseases, and reveals the functional importance of RNA-splicing regulation in heart.
Author Disclosures: C. Gao: None. V. Ren: None. J. Lee: None. X. Xiao: None. J. Chen: None. Y. Wang: None.
Key Words: Heart failure; Genomics; Cardiac development; Molecular biology
Treatment with Cell Lysate from Genetically Modified Stem Cells Containing Pre-formed Trophic Factors Effectively Rescues the Infarcted Heart
Vien Khach Lai, Kelly S Prindle, Brian Sidow, Shujia Jiang, Khawaja H Haider; Univ of Cincinnati, Cincinnati, OH
Overview: A plethora of bioactive molecules released from mesenchymal stem cells (MSC) has shown their paracrine activity and contributed to preserved infarcted heart function. Here, we demonstrated the therapeutic benefits of cell lysate containing pre-formed bioactive molecules from MSC genetically modulated to co-overexpress Akt and angiopoietin-1 (Ang-1). Methods and Results: Rat MSC were genetically modified for simultaneous overexpression of Akt and Ang-1 genes (AAMSC) using our optimized protocol. MSC transduced with adenoviral vector without therapeutic gene (EmpMSC) were used as controls. After 48 hours of respective treatments, protein lysates were isolated by freeze-thaw cycles. Real-time PCR based array data showed that beside Akt and angiopoietin-1, AAMSC had 25 growth factor genes including VEGF, BMP, interleukin, HGF and epiregulin with>2 fold upregulation as compared to EmpMSC. AALysate was cytoprotective for cardiomyocytes and endothelial cells and promoted endothelial cell migration and tube formation in vitro. Treatment of neonatal cardiomyocytes with AAlysate enhanced their proliferation (22.7 ± 1.8% vs 9.4 ± 1.3% for AAlysate vs Emplysate). Treatment of the infarcted heart with AAlysate (n=12 animals/group) significantly reduced cardiomyocyte apoptosis, increased cardiomyocyte DNA synthesis (Ki67+ cardiomyocytes/ histological section 2.1 ± 0.4 vs 0.7 ± 0.2 for AAlysate vs Emplysate) and increased blood vessel density (29.4 ± 0.5 vs 16.1 ± 0.3 for AAlysate vs Emplysate). AAlysate treated infarcted hearts also showed enhanced mobilization of bone marrow stem cells into the infarcted heart. These molecular and cellular events led to attenuated infarct size (27.6 ± 4.0% vs 37.2 ± 1.1% for AAlysate vs Emplysate) and preserved left ventricular function in AAlysate treated animal hearts. Conclusion: Treatment with cell lysate containing pre-formed trophic factors effectively rescued the infarcted heart by intricate mechanisms involving reduced myocardial apoptosis, enhanced cardiomyocyte proliferation, higher mobilization and homing of endogenous stem cells and improved angiogenesis. Our noninvasive approach will facilitate future stem cell therapy for heart failure in the clinical perspective.
Author Disclosures: V. Lai: None. K.S. Prindle: None. B. Sidow: None. S. Jiang: None. K.H. Haider: None.
Key Words: Stem cells; Cardiac regeneration; Angiogenesis; Proteins; Apoptosis
A Novel Paradigm Of MicroRNA Regulated B Cell Functions On Insulin Resistance: Mir-150 Regulates Insulin Sensitivity Through Controlling Antibody Production
Wei Ying, Cong Meng, Sara Mashoof, Guoqing Zhuang, Patali Cheruku, Lei Shi, Fuller Bazer, Guoyao Wu, Stephen Safe, Michael Cristiello, Beiyan Zhou; Texas A&M Univ, College Station, TX
Low degree adipose tissue inflammation induced by chronic nutrient excess is a causal factor for insulin resistance, which is the hallmark of type II diabetes and a major contributor to cardiovascular diseases associated with obesity. Extensive efforts have been contributed to define the roles of myeloid cells and T cells in this picture. However, how B cells are involved in this context and how their function is regulated, especially by microRNAs, are poorly understood. Using a unique knockout mouse model, we discovered a novel mechanism that miR-150 exerts profound effects on chronic nutrient overload induced systemic insulin resistance by controlling B cell functions, specifically antibody production. Our previous study demonstrated that miR-150 is a key regulator for hematopoietic lineage development, especially for the formation for B lymphocytes. High-fat diet (HFD) feeding exacerbated the development of systemic insulin resistance and glucose intolerance in miR-150KO mice compared to wild type control mice. No significant differences were observed between miR-150 and control groups in the aspects of HFD induced body weight gain, food intake, and adiposity. miR-150 deficiency did not alter macrophage activation, which has been previously demonstrated as the major contributor to diet induced adipose tissue inflammation and insulin resistance. Interestingly, miR-150KO mice exhibited an increased adipose tissue B cell infiltration, adipose tissue inflammatory cytokine production, and liver steatosis, which is accompanied by dramatically enhanced circulating antibody production that can directly contribute to the development of insulin resistance. Four genes are identified as bona fide miR-150 target genes, including ELK1, CBL, EGRs, and ETF1, which are important components of multiple metabolic related signaling pathways. In summary, our study demonstrated that miR-150 is a novel and potent regulator for obesity induced systemic insulin resistance through controlling antibody productive B cells. Thus, it provides a novel paradigm of microRNA regulation in the context of insulin resistance, and opens a new direction for treating insulin-resistance related diseases by generating specific antibodies.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: W. Ying: None. C. Meng: None. S. Mashoof: None. G. Zhuang: None. P. Cheruku: None. L. Shi: None. F. Bazer: None. G. Wu: None. S. Safe: None. M. Cristiello: None. B. Zhou: None.
Key Words: Insulin resistance; MicrornaImmune system; Obesity; Cardiovascular disease
Newly Identified Pro-inflammatory Mechanism of Calcification via Formation of a S100A9-Annexin5 Membrane Complex
Sophie E New1, Claudia Goettsch1, Masanori Aikawa2, Julio Marchini2, Manabu Shibasaki1, Katsumi Yabusaki1, Peter Libby2, Catherine Shanahan3, Kevin Croce4, Elena Aikawa4; 1Brigham and Women’s Hosp, Boston, MA, 2Brigham and Women’s Hosp, Harvard Med Sch, Boston, MA, 3King’s College London, London, United Kingdom, 4Brigham and Women’s Hosp, Harvard Med Sch, Boston, MA
Atherosclerotic plaques containing “spotty” calcifications consisting of calcified vesicles _ precursors to microcalcification _ have increased local stress, which may promote microfractures and fatal rupture. Chronic renal disease (CRD) and mineral imbalance accelerates calcification and the release of matrix vesicles (MV). Using molecular imaging, we previously linked early calcification with inflammation determined as macrophage accumulation. Here, we hypothesized an inflammation-dependent mechanism of calcification via macrophage-released microcalcification-generating MV _ in addition to the commonly accepted pathway of osteogenic differentiation of vascular smooth muscle cells. In human carotid plaques, macrophages associated with calcified vesicular structures (n=127; p<0.0001), while a hydroxyapatite-binding molecular imaging agent visualized calcifying MV. Immunogold labeling showed release of CD68-positive MV containing S100A9 and Annexin5 (Anx5) (Fig. A and B). Under CRD conditions, including levels of calcium and phosphate (Ca/P) comparable to those observed in CRD patients, macrophages released MV with high calcification (p<0.01) and aggregation potential (n=3; Fig. C), which expressed exosomal markers (TSG101 and CD9). Ca/P-stimulated mouse macrophages (RAW 267.4) have increased pro-inflammatory (M1) markers (n=3, *p<0.01; Fig. D). Silencing S100A9 in vitro and genetic deficiency in S100A9-/- mice reduced MV calcification, while stimulation with S100A9 increased their calcification potential. Externalization of phosphatidylserine (PS; Fig. E, green), after Ca/P stimulation and interaction of S100A9 and Anx5, indicated that an Anx5-S100A9 membrane complex facilitates hydroxyapatite nucleation within the macrophage-derived MV membrane. Our results support the novel concept that macrophages release calcifying MV, enriched in S100A9-Anx5 complex, which accelerate microcalcification formation in CRD.
Author Disclosures: S.E. New: None. C. Goettsch: None. M. Aikawa: None. J. Marchini: None. M. Shibasaki: Employment; Significant; Kowa Company, Ltd. K. Yabusaki: Employment; Significant; Kowa Company, Ltd. P. Libby: None. C. Shanahan: None. K. Croce: None. E. Aikawa: None.
Key Words: Calcification; Inflammation; Arteriosclerosis; Vascular
Inhibition of PAR-4 Prevents Myocyte Apoptosis but Impairs Cardiac Healing and Function after Myocardial Ischemia
Mikhail A Kolpakov, Bahman Hooshdaran, Khadija Rafiq, Liudmila Vlasenko, Rachid Seqqat, Steven R Houser, Satya P Kunapuli, Abdelkarim Sabri; Temple Univ, Philadelphia, PA
Background: Protease-activated receptors-4 (PAR-4) is a low affinity thrombin receptor with less understood function. PAR-4 is involved in platelet activation, but its role in inflammation is controversial. Specific functions of PAR-4 in myocyte growth and cardiac function have not yet been described. In this study, we elucidated the contribution of PAR-4 as a potential mediator of remodeling in human ischemic cardiomyopathy and following myocardial ischemia in mice. Methods and Results: PAR-4 expression was significantly up-regulated in human hearts with ischemic cardiomyopathy compared to non-failure heart controls and in mice after myocardial ischemia. Immunofluorescence microscopy showed increased PAR-4 expression in cardiomyocytes and coronary smooth muscle cells, but not in fibroblasts or endothelial cells. PAR-4 knockout (KO) mice showed less myocyte apoptosis, reduced infarct size and improved cardiac function in response to ischemia/reperfusion injury. Concomitant with these findings in vivo, adenoviral expression of wild type PAR-4 in cardiac myocytes significantly enhanced myocyte apoptosis in response to high concentrations of thrombin or PAR-4 agonists through JNK activation. In contrast inhibition of PAR-4 expression by shRNA significantly reduced myocyte apoptosis induced by thrombin or PAR-4 agonists. Surprisingly, PAR-4 KO mice have significantly lower survival rate after chronic myocardial infarction compared to WT mice (46 vs. 75%, p<0.05). Pathological evaluation of hearts from PAR-4 KO mice demonstrated a greater infarct size, delayed infiltration of inflammatory cells and impaired healing, along with greater left ventricle dilatation and decreased contractility compared to WT mice at 7 and 30 days post infarct. Conclusions: These studies show that PAR-4 is a positive regulator of myocyte apoptosis. However, PAR-4 deletion impaired myocardial healing and results in adverse cardiac remodeling and function after chronic myocardial infarction. These results indicate that the use of PAR-4 antagonists should be limited to acute phases after ischemic insults to preserve myocyte loss and should be avoided as long term therapy against the progression to heart failure.
Author Disclosures: M.A. Kolpakov: None. B. Hooshdaran: None. K. Rafiq: None. L. Vlasenko: None. R. Seqqat: None. S.R. Houser: None. S.P. Kunapuli: None. A. Sabri: None.
Key Words: Proteolytic enzymes; Ischemic heart disease; Thrombin; Inflammation; Ventricular remodeling
Endogenous Myocyte Regeneration and Diabetic Cardiomyopathy
Alex Matsuda, Barbara Ogórek, Maria-Virginia Caballero, Ewa Wybieralska, Yingnan Bai, Weining Bian, Donato Cappetta, Sergio Signore, Piero Anversa, Marcello Rota, Annarosa Leri, Jan Kajstura; Brigham and Women’s Hosp, Boston, MA
The recognition that the mammalian heart contains a pool of resident c-kit-positive cardiac stem cells (CSCs) that regulate cell turnover and tissue repair raises questions concerning their role in the etiology of the diabetic myopathy. The objective of the current study was to determine whether the negative effects of diabetes on the adult heart are dictated by defects in CSC growth and lineage commitment. Type I insulin-dependent diabetes mellitus was induced in mice by streptozotocin administration. The kinetics of CSCs and cardiomyocytes was measured 3, 7, 10, 20, and 30 days after the onset of diabetes, by implementing a hierarchically structured cell system, which allows the quantitative analysis of the rate of cell turnover. The multiple variables required to apply this mathematical model were measured. They included first the number of CSCs, LCCs (lineage committed cells: myocyte progenitors-precursors), transit amplifying myocytes, and post-mitotic myocytes in the left ventricular (LV) myocardium. Additionally, the fraction of cycling CSCs and the length of their cell cycle were measured, together with the number of cardiomyocytes dying by apoptosis and necrosis. The diabetic heart was characterized by a severe time-dependent loss in LV post-mitotic myocytes, dictated primarily by a defect in cell renewal. Physiologically, 20% of myocytes were replaced per month in the adult mouse heart through activation, cell cycle reentry, and differentiation of CSCs. Surprisingly, a value of 6%, 1.4%, 0.5%, 0.07%, and 0.05% was found at 3, 7, 10, 20 and 30 days after the induction of diabetes, respectively. Typically, the myocardium showed a progressive increase in old cardiomyocytes expressing the senescence-associated protein p16INK4a and p53. Importantly, the severe impairment in myocyte regeneration was coupled with an increase in LV end-diastolic pressure, and a decrease in LV systolic pressure, LV developed pressure, positive and negative dP/dt. Collectively, our data indicate that the diabetic myopathy has to be viewed as a stem cell disease in which the alterations in the other cardiac cell classes are all secondary events, resulting from defects in replication and lineage specification of the controlling cell, i.e., the CSC.
Author Disclosures: A. Matsuda: None. B. Ogórek: None. M. Caballero: None. E. Wybieralska: None. Y. Bai: None. W. Bian: None. D. Cappetta: None. S. Signore: None. P. Anversa: None. M. Rota: None. A. Leri: None. J. Kajstura: None.
Key Words: Stem cells
Cardiotrophin-1: A New Key Molecule In Vascular Fibrosis, Arterial Stiffness And Senescence
Natalia Lopez-Andres1, Laurent Calvier1, Renaud Fay2, Carlos Labat1, Javier Diez3, Athanase Benetos1, Faiez Zannad2, Patrick Lacolley1, Patrick Rossignol2; 1INSERM U 961, Vandoeuvre-les-Nancy, France, 2Cntr d’Investigation Clinique Plurithématique Pierre Drouin -INSERM CHU de Nancy, Vandoeuvre-les-Nancy, France, 3Cntr for Applied Med Rsch, Pamplona, Spain
Background: Cardiotrophin-1 (CT-1), a cytokine belonging to the interleukin-6 family, is increased in hypertension and in heart failure. We hypothesized that CT-1 excess promotes vascular fibrosis and dysfunction, whereas CT-1 deficiency influences lifespan by decreasing arterial stiffness. Methods: CT-1 (20 µg/Kg/day) or vehicle were administrated to Wistar rats for 6 weeks, and 29 month-old WT and CT-1-null mice were used. Vascular function was analysed in vivo using echotracking device and ex vivo employing a scanning acoustic microscopy. Vascular histomorphology, senescence, metabolic, inflammatory and oxidative stress parameters were measured by immunohistochemistry, RT-PCR, Western Blot and ELISA. Results: CT-1 treatment did not modify blood pressure levels. In CT-1-treated rats, the circumferential wall stress-incremental elastic modulus curve was shifted leftward and the acoustic speed of sound in the aorta was augmented, indicating indicating increased arterial stiffness. Vascular media thickness, collagen and fibronectin content were increased by CT-1 treatment. The wall stress-incremental elastic modulus curve of old CT-1-null mice was shifted rightward as compared to WT, indicating decreased arterial stiffness. Media thickness and wall fibrosis were lower in CT-1-null mice. CT-1-null mice showed decreased levels of inflammatory, apoptotic and senescence pathways, whereas telomere-linked proteins, DNA repair proteins and antioxidant enzyme activities were increased. CT-1-null mice displayed a 5-month increased median longevity compared with WT. Conclusion: CT-1 is a new potent vascular fibrotic agent able to induce arterial stiffness independently from blood pressure. CT-1 absence is associated with decreased arterial fibrosis, stiffness and senescence and increased longevity in mice likely through downregulating apoptotic, senescence and inflammatory pathways. CT-1 may be a major regulator of arterial stiffness with a major impact on the aging process.
Author Disclosures: N. Lopez-Andres: None. L. Calvier: None. R. Fay: None. C. Labat: None. J. Diez: None. A. Benetos: None. F. Zannad: None. P. Lacolley: None. P. Rossignol: None.
Key Words: Aging; Arteries; Biomarkers; Chemokines; Carotid arteries
β-arrestin1 Stimulates the Processing of a Subset of MicroRNAs
Yongchao Wang1, Corey L Neal1, Weili Zou2, Lan Mao2, Barbara Williams2, Il-man Kim1; 1Georgia Health Sciences Univ, Augusta, GA, 2Duke Univ Med Cntr, Durham, NC
MicroRNAs (miRs) are small, non-coding RNAs that modulate diverse biological functions through the repression of target genes. While several signaling pathways have been shown to regulate miR biogenesis, the seven transmembrane receptor (7TMR; also known as G protein-coupled receptor)-mediated signaling pathways that control miR biogenesis are only beginning to be unveiled. Originally identified as mediators of 7TMR desensitization, β-arrestins are now recognized as adaptor proteins that transduce signals to multiple effector pathways, resulting in unique downstream responses such as promotion of cardiomyocyte survival. Based on a published β-arrestin interactome, we tested the hypothesis that β-arrestins play a role in the regulation of miR biogenesis. Using both HEK 293 cells overexpressing β1-adrenergic receptors (β1ARs) and in vivo mouse hearts, we examined whether the β-blocker carvedilol (Carv), which activates β-arrestin-mediated signaling, can regulate miR expression. Up-regulation of human miR-190 was induced by Carv and was prevented by using siRNA directed against β-arrestin1. The Carv effect was also blocked by the β1AR antagonist metoprolol, indicating a receptor-mediated mechanism of action. Interestingly, while Carv did not increase the expression of human primary miR-190 transcript, it did increase the expression of the premature form of miR-190. Similarly, Carv activated the expression of five premature or mature miRs from mouse hearts in a β-arrestin1-dependent manner. Mechanistically, we demonstrated that β-arrestin1 controls miR processing by forming a nuclear complex with hnRNPA1 and Drosha, which are two components of the Drosha microprocessor complex. Lastly, HL-1 cardiomyocyte studies indicated that β-arrestin1-regulated miRs function as cardioprotective miRs by repressing apoptotic or anti-proliferative genes. In conclusion, we identify a novel mechanism by which β1AR-mediated β-arrestin1 signaling pathways regulate miR processing and discover new and potentially important miR-target pairs that are required for cardioprotective signaling.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: Y. Wang: None. C.L. Neal: None. W. Zou: None. L. Mao: None. B. Williams: None. I. Kim: None.
Key Words: Microrna; Signal transduction; Beta-blocker; Cardioprotective drugs; Heart failure
A Novel Bradykinin-2 Receptor Agonist Antibody that Improves Diabetic and Cardiovascular Endpoints
Mark S Williams, Matthew Charles; DiaMedica, Winnipeg, Canada
Background: The kallikrein-kinin system (KKS) has been previously linked to glucose homeostasis via increasing insulin sensitivity, vasodilation and more recently decreasing hepatic gluconeogenesis. The Bradykinin 2 receptor (BK2R) is a widely expressed G protein coupled receptor that is associated with these beneficial effects, and herein we report a novel agonist monoclonal antibody (mAb) to the BK2R with beneficial anti-diabetic and cardiovascular properties. Methods: In vivo – male ZDF fa/fa rats (11 weeks of age, n=8 per arm) were treated twice weekly SC with the BK2R antibody (0.2, 0.04, 0.008 mgs/kg) for three weeks. In vitro – an insulin-stimulated in vitro radio-labeled glucose uptake assay using isolated rat adipocytes was used to analyze insulin sensitization activity of the antibody (1 mg/mL) after a 1 hour incubation with a humanized mAb. Results: In vivo – Overall there dose dependent reductions in various markers including HbA1c (6.2 vs. 8.8% control, p=0.0103), a reduced increase in FBG (33.1 vs. 110 mg/dL, p=0.0058), and a reduction in total cholesterol (-12.1 vs. + 28 mg/dL, p=0.0156) after 21 days. Treatment also prevented the development of hypertension (unchanged vs. control where BP increased 25 mm Hg, p=0.0004) in addition to a reduction in heart rate (~60 bpm, p=0.001). The mAb treatment saw no effect on weight gain or food intake compared to controls. The BK2R antibody outperformed Sitaglitpin (10 mg/kg po daily) and Exenatide (1 ug/rat BID) controls on all measures except lipid reduction, where the BK2R mAb had no discernible effect. In an OGTT at day 21 the mAb reduced glucose iAUC compared to the negative control (2495 vs. 3773, p<0.0001) and exhibited a clear secretagogue effect. In vitro – A glucose uptake assay demonstrated insulin stimulated glucose uptake activity (up to 57%, p=0.01) following incubation with a humanized version of the mAb suggesting an additional insulin sensitization MOA. Conclusion: A novel agonist GPCR antibody to the BK2R with insulin secretagogue and insulin-sensitizing activity has been reported with beneficial anti-diabetic and CV effects.
Author Disclosures: M.S. Williams: Employment; Significant; DiaMedica Inc. M. Charles: Employment; Significant; DiaMedica Inc.
Key Words: Monoclonal antibodies; Type 2 Diabetes; Insulin resistance; Cardiovascular disease
A Novel Protein Phosphatase Exacerbates Myocytes Death and Represses Cardiac Contractility under Stress
Toru Akaike, Gang Lu, Yibin Wang, Hongmei Ruan; UCLA, Los Angeles, CA
Regulation of sarcoplasmic reticulum (SR) calcium-ATPase (SERCA) activity is necessary for calcium homeostasis in cardiomyocytes, has a major impact on cellular viability and cardiac contractility. The key regulators for SERCA activity include protein kinases like cAMP dependent protein kinase A and calcium/calmodulin dependent protein kinase II, and protein phosphatase like protein phosphatase 1. In this report, we have discovered that protein phosphatase 2Ce (PP2Ce) is a novel serine/threonine protein phosphatase specifically targeted to SR membrane in cardiomyocytes. Here we assessed the hypothesis that PP2Ce is an important player in calcium cycling and cardiac remodeling in heart. PP2Ce was detected to interact with phosphlamban in heart. Expression of PP2Ce blunted β-adrenergic stimulated increase of phospholamban phosphorylation without affecting phosphorylation of ryanodine recepter or troponin I in cultured neonatal rat ventricular myocytes. Recombinant PP2Ce protein specifically dephosphorylated β-adrenergic stimulated increase of phospholamban phosphorylation at thereonin 17 site in heart. PP2Ce expression was significantly increased in patients with dilated cardiomyopathy compared with control patients, was significantly increased under stimulation of hydrogen peroxide and β-adrenergic in cultured neonatal rat ventricular myocytes. PP2Ce expression reduced β-adrenergic stimulated intracellular calcium transient in isolated adult rabbit ventricular myocytes, and promoted hydrogen peroxide and ionomycin induced cell death in cultured neonatal rat ventricular myocytes. Hydrogen peroxide induced Akt dephosphorylation and p38 phosphorylation in cultured PP2Ce expressing neonatal rat ventricular myocytes. Transgenic mice with cardiac specific expression of PP2Ce showed no significant basal phenotype. However, in isolated perfusion heart preparation, we observed significantly larger infarct sizes and more impaired functional recovery following global ischemia/reperfusion injury in the transgenic hearts comparing to wild type controls. Therefore, PP2Ce is a novel component of SR calcium regulatory network that has a potentially important role in cell death regulation and cardiac contractility.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: T. Akaike: None. G. Lu: None. Y. Wang: None. H. Ruan: None.
Key Words: Phosphatases; Phospholamban; Calcium
Regulation Of Sirt3 And Autophagy By Bone Marrow Cells Therapy Improves Cardiac Repair In Post-myocardial Infarction Mice
Jian-Xiong Chen; Univ of Mississippi Med Cntr, Jackson, MS
Bone marrow-derived cells (BMCs) therapy promotes cardiac repair and improves functional recovery of post myocardial infarction (MI); however, the molecular mechanisms are not yet completely understood. Sirtuin-3 (Sirt3) is key regulator of mitochondria reactive oxygen species (ROS) formation and autophagy, and modulates the pathophysiology of cardiac hypertrophy and aging-related cardiac hypertrophy. The present study investigates whether BMCs cell therapy affects cardiac Sirt3 and autophagy thereby mediating repair and functional recovery post-MI. We demonstrated that intramyocardial delivery of BMCs in infarcted mice increases the expression of cardiac Sirt3 and significantly attenuates ischemia-induced NADPH oxidase subunits p47phox and gp91phox expression followed by a significant reduction of myocardial ROS formation in post-MI mice. Treatment with BMCs also led to significant increases in autophagy beclin-1 and LC3-II gene expression. Mice receiving BMCs treatment demonstrated upregulation of VEGF expression and dramatically increased Akt and eNOS phosphorylation in the ischemic hearts. This was accompanied by a significant reduction of myocardial apoptosis. Further, treatment with BMCs promoted myocardial angiogenesis, attenuated cardiac fibrosis and hypertrophy together with a significant improvement of cardiac function at 28 days post-MI mice. Knockout of Sirt3 in the BM significantly attenuated the protective effects of BMCs. Our data demonstrate that the regulation of cardiac Sirt3 and autophagy by transplanted BMCs may contribute to the protective effects of cell therapy.
Author Disclosures: J. Chen: None.
Key Words: Progenitor cell; Cardioprotection; Angiogenesis; Stem cell therapy; Myocardial infarction
Ablation of p110α PI3-Kinase Activity in Vascular Smooth Muscle Cells Prevents Vascular Remodeling and Hypoxia-Induced Pulmonary Hypertension in Mice
Eva M Berghausen1, Wiebke Janssen2, Marius Vantler1, Miguel A Alcazar1, Henrik ten Freyhaus1, Soni Pullamsetti2, Jean J Zhao3, Ralph T Schermuly1, Stephan Rosenkranz1; 1Univ of Colonge, 50931 Köln, Germany, 2Univ of Giessen, 35392 Giessen, Germany, 3Dana-Farber Cancer Institute, Boston, MA
Introduction: Growth factor (GF) induced proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) are a hallmark of vascular remodeling in pulmonary arterial hypertension (PAH). PI 3-kinase (PI3K) is a crucial downstream mediator of GF signaling. Especially, the p110α isoform seems to have a critical function. Therefore, we investigated the relevance of p110α signaling in pulmonary hypertension (PH) by both a genetic and a pharmacological approach. Methods: Smooth muscle (SM)-specific p110α deficient mice and mice treated with the p110α-specific inhibitor PIK75 (50 mg/kg daily; i.p. during hypoxia) were subjected to chronic hypoxia (21 days). Right ventricular (RV) systolic pressure was determined via pressure-catheter measurement, RV hypertrophy as RV/LV+septum weight ratio. Vascular remodeling was determined by morphometric analysis of vascular muscularization and medial thickness of lung tissue sections. The function of p110α in vitro was investigated using either PIK75 treated human PASMCs or p110α deficient mouse PASMCs. Starved cells were stimulated with diverse GFs. Chemotaxis was quantified using a modified Boyden-chamber. Proliferation was detected via BrdU incorporation. Activation of AKT (Phospho-Thr308) was evaluated by Western blotting. Results: Significantly elevated AKT activation in lung tissue from PAH patients and mice suffering hypoxia induced PH, indicate increased PI3K signaling in PH (n≥6, p<0,05). Consequently, PIK75 treatment or SM-specific p110α deficiency led to significantly lower RV systolic pressure and RV hypertrophy in mice exposed to hypoxia compared to their corresponding controls (n≥7, p<0,05). In both approaches, ablation of p110α activity significantly diminished muscularization and medial wall thickness of small pulmonary arteries compared to controls assigning SM-p110α a key function in hypoxia-induced vascular remodeling. Consistently, GF-induced proliferation and chemotaxis were both inhibited in human PASMCs by PIK75 (IC50: 300nM) and abrogated in p110α deficient mouse PASMCs. Conclusion: These results indicate a crucial function of p110α in hypoxia-induced PH. Therefore, inhibition of p110α is a promising strategy for the treatment of PAH.
Author Disclosures: E.M. Berghausen: None. W. Janssen: None. M. Vantler: None. M.A. Alcazar: None. H. ten Freyhaus: None. S. Pullamsetti: None. J.J. Zhao: None. R.T. Schermuly: None. S. Rosenkranz: None.
Key Words: Pulmonary hypertension; Cell signaling; Hypoxia; Remodeling; Vascular
Is Myocardin a limiting factor for cardiac programming?
Elisa Belian, Michela Noseda, Thomas Leja, Mutsuo Harada, Richard T Lumbers, Marta S Abreu Paiva, Nick East, Robert Sampson, Michael D Schneider; Imperial College London, London, United Kingdom
Recent reports have shown that non-myocytes may be reprogrammed into cardiomyocytes using three cardiac transcription factors (cTFs), Gata4 (G), Mef2c (M) and Tbx5 (T). Here, we sought to identify potentially sensitized substrates for reprogramming strategies and test the effect of the co-activator Myocardin. Adult mouse Sca-1+ cardiac progenitor cells (CPCs) that express many endogenous cTFs, were used in parallel with adult mouse tail tip fibroblasts (TTFs). Exogenous cTFs were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput QRT-PCR was used to monitor 29 cardiovascular lineage markers two weeks post-induction. Co-expressing GMT induced more than half the analysed cardiac transcripts in both CPCs and TTFs, with no significant difference between the cell types under the conditions tested. However, in neither cell background was protein detected for the induced sarcomeric genes (Actc1, Myh6, Myl2) or calcium regulators (Ryr2, Pln). Myocardin, which is a transcriptional co-activator for Srf, Gata4 and Tbx5, is not expressed in cardiac progenitor cells. We therefore speculated that Myocardin was a limiting factor in reprogramming. Adding the cardiac-enriched Myocardin isoform B (Myo) to GMT activated an increased number of genes in CPCs and TTFs with 60% and 94% overlap, respectively, with the genes induced by GMT alone. Importantly, MyoGMT triggered detectable protein expression for the targets examined (Actc1, Myh6, Nppa) in CPCs and to a lesser extent in TTFs. Alone, Myocardin was sufficient to induce 70% of the cardiac markers obtained with MyoGMT in CPCs but only 27% of those in TTFs. In summary: (1) GMT induced cardiac gene expression in CPCs and TTFs, but not cardiac protein expression under the conditions tested. (2) Complementing GMT with Myocardin induced additional cardiac transcripts and also cardiac protein expression, indicating a more complete cardiac differentiation program by this combination. (3) Myocardin alone induces a more efficient cardiac program in CPCs than in fibroblasts, suggesting that the presence of endogenous cTFs in CPCs predisposes them to cardiac differentiation and confirming the role of Myocardin as a critical cofactor.
Author Disclosures: E. Belian: None. M. Noseda: None. T. Leja: None. M. Harada: None. R.T. Lumbers: None. M.S. Abreu Paiva: None. N. East: None. R. Sampson: None. M.D. Schneider: None.
Key Words: Stem/progenitor cells; Cardiac regeneration; Gene expression; Myocardin; Stem cell therapy
Integrin β1 Signals Through Extracellular Signal-regulated Kinase To Promote The Survival Of Adipose Tissue-derived Stem Cells In The Infarcted Hearts
Zeljko Bosnjak, Yasheng Yan, Xiaowen Bai; Med College of Wisconsin, Milwaukee, WI
Adipose tissue-derived stem cells (ASCs) are a promising cell source for myocardial regeneration. However, reported improvement of cardiac function has been modest, partly due to low survival rate of injected ASCs in hostile ischemic hearts, with the underlying mechanisms remaining largely unknown. Integrin β1 belongs to a family of receptors for many extracellular matrix proteins. This study was to investigate the roles of integrin β1 in mouse ASC survival using both a murine model of myocardial infarction and an in vitro cell culture. Integrin β1 expression in ASCs was either eliminated via lentivirus carrying shRNA (ASCintegrin β1-) or overexpressed by lentiviral infection (ASCintegrin β1+). For in vivo study, BALB/c mice (n=3/group) were subjected to a permanent occlusion of left anterior descending coronary artery. Luciferase-labeled control ASC (ASCcontrol), ASCintegrin β1+, or ASCintegrin β1- were injected into the border zone of hearts. Injected ASCs in live mice was imaged using Bioluminescent Imaging System. For in vitro study, ASCs were treated with 10 mM H2O2 and/or extracellular signal-regulated kinase (ERK) inhibitor PD98059 for 2 h. Cell viability was measured by lactate dehydrogenase (LDH) release from cells with damaged membrane. The in vivo results showed that injected ASCs survived in infarcted hearts for 7 days. There were more ASCs detected in ASCintegrin β1+-injected hearts, while less ASCs in ASCintegrin β1--injected hearts compared to ASCcontrol-injected hearts, indicating that integrin β1 is involved in survival of injected ASCs in ischemic hearts, and overexpression of integrin β1 enhances ASCs to resist hostile myocardial environment. The following in vitro data confirmed our in vivo findings. Upon H2O2 exposure, overexpression of integrin β1 in ASCs significantly decreased LDH release compared with other groups, while integrin β1 knockout increased LDH release from ASCs. In addition, PD98059 significantly inhibited LDH release from ASCcontrol but not from ASCintegrin β1-, suggesting that ERK pathway participates in integrin β1-mediated ASC survival. This finding may help to guide the design of novel therapies for improving ASC-mediated repair capacity by increasing the ability of ASCs to persist in ischemic hearts.
Author Disclosures: Z. Bosnjak: None. Y. Yan: None. X. Bai: None.
Key Words: Stem/progenitor cells
Sarcolemmal KATP Channelopathy in Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells of Diabetic Patients
Scott G Canfield, Chika Kikuchi, Zeljko J Bosnjak, Anna Stadnicka; Med College of Wisconsin, Milwaukee, WI
Background: Cardiovascular complications are one of the major causes of death in patients with metabolic disorders such as diabetes mellitus. Cardiac sarcolemmal KATP (sarcKATP) channels function as metabolic sensors that link energy metabolism to membrane excitability and are required for adaptation of the myocardium to physiological and pathological stress. To investigate whether the functional sarcKATP channel is expressed in patient-specific cells, we derived cardiomyocytes from induced pluripotent stem cells reprogrammed from fibroblasts of non-diabetic (N-iPSC-CMs), type 1 (T1DM-iPSC-CMs), and type 2 diabetic (T2DM-iPSC-CMs) patients. Methods and Results: Efficient cardiac differentiation was confirmed with cardiac-specific immunostaining and live-labeling of cardiomyocytes with GFP under transcriptional control of cardiac promoter myosin light chain-2v. Immunohistochemistry showed that expression and co-localization of the cardiac sarcKATP channel subunits Kir6.2 and SUR2A were higher in N-iPSC-CMs compared to T1DM-iPSC-CMs and T2DM-iPSC-CMs. Single channel recordings in the inside-out patch clamp configuration demonstrated the presence of functional sarcKATP channels in all groups. These channels were reversibly blocked by 10 µM glibenclamide and 2 mM ATP. Single sarcKATP channel kinetics were similar in N-iPSC-CMs and T1DM-iPSC-CMs with a typical single channel current amplitude of 2.17 ± 0.03 pA and unitary conductance 54 ± 0.68 pS. However, T2DM-iPSC-CMs channels exhibited fast flickering between the open and closed state resulting in significantly lower channel amplitude (1.53 ± 0.01 pA) and conductance (38 ± 0.2 pS). Conclusions: Our study shows for the first time that iPSC-CMs differentiated from non-diabetic, type 1 and type 2 diabetic patients express functional sarcKATP channels confirming a cardiac phenotype. In addition, significant differences were observed in the sarcKATP channel kinetics between T2DM-iPSC-CMs and N-iPSC-CMs. Because iPSC-derived cardiomyocytes may recapitulate the channelopathies diagnosed in cardiac patients, it is likely that differences in channel kinetics contribute to compromised cardiac preconditioning in type 2 diabetic patients.
Author Disclosures: S.G. Canfield: None. C. Kikuchi: None. Z.J. Bosnjak: None. A. Stadnicka: None.
Key Words: Stem cells; Potassium channel; Type 1 Diabetes; Type 2 Diabetes; Cardioprotection
Notch Inhibition Prevents the Progression of Small Abdominal Aortic Aneurysms in AngII-Induced Mouse Model
Chetan P Hans, Sara Koenig, Nianyuan Huang, Vidu Garg; Nationwide Children’s Hosiptal, Columbus, OH
Progression of abdominal aortic aneurysm (AAA) is characterized by activation of the inflammatory pathway and an imbalance between the synthesis and degradation of aortic wall structural components including elastin and collagen. Our previous studies have demonstrated that loss of Notch reduces the incidence of AAA in the angiotensin II (AngII) mouse model by preventing the influx of inflammatory macrophages. The present study was performed to determine if pharmacological inhibition of Notch (DAPT) prevents the progression of active AAA in an AngII-induced mouse model. Active aneurysm was introduced in Apoe−/− mice (n=36) by administering AngII. DAPT (10mg/kg; 3 times a week) was injected in these mice starting either 3 days (Group II; n=12) or 8 days (Group III; n=12) after AngII infusion. Group I (n=12) received vehicle (10% alcohol in corn oil) only. DAPT treatment significantly reduced luminal expansion of the abdominal aorta in both the groups as compared to vehicle-treated Apoe−/− mice (p<0.05) as detected by echocardiography. DAPT treatment also prevented characteristic aneurysmal traits of elastin fragmentation and aortic remodeling in both the groups as detected by histology. Marginal increase in macrophage content (CD-68) was observed in Group III compared to Group II, but was significantly less than Group I. However, the increased macrophage content in Group III was not associated with increased monocyte chemotactic protein-1 suggesting that DAPT treatment prevents continuous influx of macrophages. In vitro data suggest that Notch1 inhibition promotes the M2 polarization of macrophages. Interestingly, increased expression of newly synthesized tropoelastin and collagen staining was detected in Group III suggesting that the vascular injury was repaired in the absence of recruitment of macrophages. Expressions of MMP2 and MMP9 were also decreased in the aorta of Group II and Group III compared to Group I (vehicle) as determined by quantitative real-time PCR and immunohistochemistry. The present study demonstrates that Notch inhibition stabilizes the progression of AAA and is associated with increased elastin and collagen regeneration and decreased MMP activity suggesting the potential use of Notch inhibitors in the treatment of AAA.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: C.P. Hans: Research Grant; Significant; AHA Scientific Development Grant. S. Koenig: None. N. Huang: None. V. Garg: None.
Key Words: Abdominal aortic aneurysm; Angiotensin II; Collagen; Inflammation; Extracellular matrix
TNF/TNFR1 Signaling Inhibits Cardiomyogenic Differentiation Of Cardiac Stem Cells And Promotes A Neuroadrenergic Phenotype
Tariq Hamid1, Yuanyuan Ma1, Mohamed Ameen Ismahil1, Qianghong Li2, Steven P Jones2, Aruni Bhatnagar2, Roberto Bolli2, Sumanth D Prabhu3; 1Univ of Alabama at Birmingham, Birmingham, AL, 2Univ of Louisville, Louisville, KY, 3Univ of Alabama at Birmingham and Birmingham VMAC, Birmingham, AL
Heart failure (HF) progression in humans occurs despite augmented tissue resident cardiac stem cells (CSCs), suggesting that the HF microenvironment opposes endogenous cardiac repair. Tumor necrosis factor-α (TNF) levels are increased in HF; its effects are receptor (R) specific, such that TNFR1 promotes whereas TNFR2 alleviates pathological remodeling. We hypothesized that TNF modulates the CSC differentiation and function in an analogous manner. c-Kit sorted, Lin-/Sca1+ CSCs were isolated from wild type (WT) and TNFR1-/- mice. WT CSCs constitutively expressed TNFR1 and exhibited intact downstream TNF signaling. CSCs were then differentiated in the presence and absence of TNF (20 ng/mL) using 5-azacytidine and TGF-β. Differentiated WT CSCs developed striated morphology and increased abundance of multinucleated cells. In contrast, WT CSCs exposed to TNF acquired neuronal morphology with axonal-like projections. However, TNF-exposed TNFR1-/- CSCs appeared morphologically similar to WT CSCs. Cardiac specific gene expression (Mef2c, Nkx2.5 β-MHC) increased (p<0.05) in differentiated WT CSCs in absence of TNF, but was either inhibited (Mef2c) or decreased (Nkx2.5 and β-MHC) with TNF treatment. TNF-exposed WT CSCs also exhibited increased (p<0.05) expression of the neuroadrenergic marker tyrosine hydroxylase (TH). Also, culture supernatants from TNF-exposed WT CSCs revealed 3-4 fold increased abundance of epinephrine and norepinephrine. These changes were significantly attenuated in TNFR1-/- CSCs. Intramyocardial injection of CSCs in mice following reperfused infarction indicated increased abundance of TH staining in hearts (border zone and scar) injected with WT GFP+ CSCs as compared to vehicle-injected hearts. TH staining, however, was significantly diminished, and cardiac function significantly improved in TNFR1-/- CSC-injected hearts. Conclusion: TNF/TNFR1 signaling inhibits cardiomyogenic differentiation of CSCs and promotes a neuroadrenergic phenotype that can serve as a local source of catecholamines. Modulation of TNFR-specific signaling in CSCs may be a novel approach for augmenting endogenous cardiac repair and reducing adrenergic activation in HF, and may also enhance the efficacy of exogenous stem cell therapy.
Author Disclosures: T. Hamid: None. Y. Ma: None. M. Ismahil: None. Q. Li: None. S.P. Jones: None. A. Bhatnagar: None. R. Bolli: None. S.D. Prabhu: None.
Key Words: Stem/progenitor cells; Stem cell therapy; Inflammation
- © 2012 American Heart Association, Inc.