2013 Late-Breaking Basic Science Abstracts
Late-Breaking Basic Science and Featured Research Presentation
Enhanced Myocardial Repair with CardioChimeras
Pearl J Quijada, Jonathan D Cubillo, Claudio Staub, Mark A Sussman; San Diego State Univ, San Diego, CA
Dual stem cell transplantation of c-kit positive cardiac progenitor cells (CPCs) and mesenchymal stem cells (MSCs) after infarction support modest improvements in cardiac function, however there are currently no reports substantiating a single stem cell type supporting both direct and indirect mechanisms of myocardial repair. Therefore we created a CardioChimera, a stem/progenitor cell formed by fusion between CPCs and MSCs, resulting in a unique progeny superior to either individual precursor. CardioChimeras were purified after cell fusion with Hemagglutinating virus of Japan and expanded clonally based on dual expression of fluorescent proteins mCherry and eGFP from CPCs and MSCs respectively. CardioChimeras are mono-nucleated, have comparable growth kinetics to parental CPCs and MSCs and display increased cellular size unrelated to cell cycle arrest and/or senescence. CardioChimeras have increased expression of cardiomyogenic lineage markers cardiac troponin T (2.5-fold), smooth muscle 22 (9.3-fold), and CD31 (10.7-fold) concomitantly associated with decreased c-kit protein expression (50%) relative to parent CPCs. Lineage commitment of CardioChimeras is bolstered by dexamethasone treatment measured by mRNA levels of cardiogenic genes and increases in active mitochondria (2.2-fold) after labeling with MitoTracker. Induction of apoptosis is blunted in cardiomyocytes co-cultured with CardioChimeras compared to co-culturing with CPCs and MSCs alone, or combination of non-fused parent cells. CardioChimeras enhance cardiomyocyte growth similar to parent MSCs owing to an increased propensity to secrete pro-growth factors. Collectively, CardioChimeras represent an adaptable cell therapy combining the beneficial properties of CPCs to undergo cardiac specific commitment as well as MSCs that foster an improved microenvironment with protective paracrine secretion. Clinically, CardioChimeras merge the application of distinct cell types into a single entity for increased engraftment, mitigation of inflammation and blunting the progression of heart failure by promoting myocardial regeneration.
Author Disclosures: P.J. Quijada: None. J.D. Cubillo: None. C. Staub: None. M.A. Sussman: None.
Key Words: Regenerative medicine stem cells; Stem cell therapy; Myocardial infarction; Stem/progenitor cells
Deletion Of P53 In Endothelial Cells Prevents Cardiac Fibrosis And Heart Failure Induced By Pressure Overload In Mice Via Enhanced Angiogenesis
Rajinikanth Gogiraju1, Julia H Steinbrecher1, Stephan E Lehnart1, Matthias Dobbelstein1, Katrin Schaefer2; 1Univ Medicine Goettingen, Goettingen, Germany, 2Univ Medicine Mainz, Mainz, Germany
Cardiac hypertrophy develops as adaptive response of the heart to increased workload, but may progress to heart failure if continued over the long term. An inadequate reduction of cardiac capillaries may be causally involved in the deterioration of cardiac function. The aim of this study was to examine whether prevention of endothelial cell apoptosis, achieved by deletion of tumor suppressor protein p53, improves cardiac remodeling in response to increased afterload and prevents the transition to heart failure. Mice with endothelial cell-specific deletion of p53 (End.p53-KO) were generated by crossing p53flox/flox mice with mice expressing Cre recombinase under control of the tamoxifen-inducible promoter Tie2 (Tie2.ERT.Cre). Cardiac hypertrophy was induced by transverse aortic constriction (TAC). In control mice, cardiac hypertrophy was accompanied by increased p53 expression and elevated levels of apoptosis, as shown by TUNEL and activated caspase-3 staining, whereas reduced apoptotic cell numbers were detected in hearts of End.p53-KO mice. Moreover, End.p53-KO mice exhibited an increased number of CD31-positive, perfused blood vessels. Endothelial p53 deletion also improved reperfusion and angiogenesis in the unilateral hindlimb ischemia model. In vitro analyses confirmed that inhibition of p53, achieved by preincubation of human microvascular endothelial cells with pifithrin-α, promoted endothelial sprouting. In addition to improved cardiac angiogenesis, hearts of End.p53-KO mice exhibited reduced interstitial fibrosis compared to controls and expressed lower levels of genes involved in extracellular matrix remodeling. Echocardiography revealed a less severe reduction in fractional shortening and left ventricular chamber dilatation. Importantly, survival of End.p53-KO mice was significantly improved 20 wks after TAC. Taken together, our findings suggest that accumulation of p53 in endothelial cells contributes to the blood vessel rarefication and fibrosis during cardiac hypertrophy and that endothelial cell survival is essential for the preservation of normal contractile function in response to chronic cardiac overload.
Author Disclosures: R. Gogiraju: None. J.H. Steinbrecher: None. S.E. Lehnart: None. M. Dobbelstein: None. K. Schaefer: None.
Key Words: Angiogenesis; Heart failure; Hypertrophy; Apoptosis; Endothelium
Direct Visualization Of miRNA-22 Biogenesis In Isoproterenol-induced Cardiac Hypertrophy By Bioluminescence Imaging In Vitro And In Vivo
Yingfeng Tu, Lin Wan, Dongliang Zhao, Lihong Bu, Baozhong Shen; Harbin Med Univ, Harbin, China
Evidence from recent studies has shown that miRNAs play key role in cardiac hypertrophy (CH). To measure the expression level of endogenous miRNAs is very conducive to understanding the importance of miRNAs in CH. However, current methods to monitor endogenous miRNAs level, such as northern blotting, qRT-PCR, and miRNA microarrays can not provide real-time information of miRNAs biogenesis in CH in vivo. Here, we constructed a novel miRNA reporter imaging system in which a cytomegalovirus (CMV) promoter driven Gluc gene was fused with three copies of the target sequence of miR-22 before the poly(A) tail to monitor the activity of mature miR-22 repetitively and timely. In the current study we found that with prolongation of isoproterenol (ISO) stimulation in vivo, there was a tendency that the expression level of miR-22 was gradually increased. Accordingly, the bioluminescence imaging analysis revealed that there was a gradually decrease in fluorescence signals of the CMV/Gluc/3хPT_miR-22 under conditions where miRNA-22 was up-regulated by ISO. There was a significant correlation between miRNA-22 and CH by using qRT-PCR and our luciferase reporter system (Figure 1 and 2). However, the firefly luciferase (Fluc) activity of CMV/Fluc, as a positive control for imaging of miR-22 activity, was not affected with ISO treatment (Fig. 1). Furthermore, knockdown of miR-22 by antagomir-22 could reverse the repressed (Gluc) activities (Fig. 2). This preliminary study elucidate the feasibility of using our constructed miRNA reporter imaging system to monitor the location and magnitude of expression level of miRNA-22 in CH in vitro and in vivo.
Author Disclosures: Y. Tu: None. L. Wan: None. D. Zhao: None. L. Bu: None. B. Shen: None.
Key Words: Cardiac hypertrophy; Microrna; Imaging agents; Cardiovascular imaging
Hematopoietic Id Ablation Triggers Endomyocardial Fibrotic and Vascular Defects Within the Murine Heart
Corey Chang, Qingshi Zhao, Diego Fraidenraich; Rutgers New Jersey Med Sch, Newark, NJ
We previously reported that Id (inhibitor of DNA binding protein) deficient mice with endothelial and hematopoietic (Tie2Cre) ablation (Id conditional double knockout mice, Id cKO) survive into adulthood and develop dilated fibrotic cardiomyopathy (DCM) and multiple hematopoietic defects. The Id cKO hearts exhibit perivascular fibrosis and interstitial fibrosis within the endomyocardium along with disruption of the endocardial lining. To determine if hematopoietic Id ablation contributes to cardiac pathology, we conducted a series of bone marrow transplantation studies to control the Id status of the hematopoietic system. We found that Id cKO bone marrow transplantation phenocopies the Id cKO hearts 4 months post-transplantation with a corresponding decrease in ejection fraction in 8 out of 13 WT recipients (Id cKO/WT BMTs). Real-time PCR analysis of Id cKO/WT BMT hearts reveals a 2.1-fold increase (p<0.05) in the expression of thrombospondin-1 (TSP1), which is normally repressed by Id1 compared to WT/WT BMT controls. Connective tissue growth factor (CTGF) - a downstream target of the TSP1/TGFbeta1/Smad pathway - exhibits a 2.3-fold (p<0.05) increase in mRNA expression. Similarly, collagen type III alpha 1 (Col3a1) - a collagen marker typically upregulated in the context of DCM - is upregulated 2.09-fold (p=0.05). No significant changes in Id1 expression was observed although there was a trend toward higher levels in Id cKO/WT BMTs. No significant changes in tumor necrosis factor receptor associated factor 6 (TRAF6) - a member of the non-Smad/CTGF pathway - expression was observed. Insulin growth factor binding protein 3 (IGFbp3), a factor that directly activates the Smad pathway independent of TGFbeta1 signaling, was upregulated 6.45-fold (p<0.05). Treatment of HUVEC cells with Id cKO serum on matrigel shows a 30.7% decrease in the total number of tubes. The results highlight a novel pathway whereby loss of distal Id from bone marrow cells triggers upregulation of TSP1 and local expression of Id in the heart is insufficient to prevent dysregulation of TSP1. The findings suggest that Id ablation in hematopoietic cells triggers dysregulation of vascular and fibrotic pathways in the hearts of Id cKO/WT BMTs, possibly mediated by the TSP1/CTGF pathway.
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: Cardiomyopathy; Heart failure; Fibrosis; Vascular disease; Angiogenesis
Mirna-208a Targeting Purß Gene Regulates The ß-MHC Content In Cardiac Hypertrophy Induced By Exercise Training
Tiago Fernandes, Ursula P Soci, Edilamar M Oliveira; Univ of Sao Paulo, Sao Paulo, Brazil
Aerobic exercise training (ET) results in beneficial adaptations of the heart with a balanced and revertible physiological growth. It counteracts a shift from α-toward β-MHC composition, pathological hypertrophy and cardiac dysfunction in cardiovascular diseases; however, underlying mechanisms remain to be further determined. MiRNA-208a is a cardiac-specific miRNA that regulates the β-MHC content targeting Purβ. We investigated whether ET-induced cardiac hypertrophy (CH) is due to the activation of physiological signaling pathways Akt-mTOR axis-dependent and regulates the miRNA-208a and their target Purβ related with MHC gene switching. Wistar rats (n=30) were assigned to 3 groups: sedentary (S); swimming trained with protocol 1 (T1, moderate-volume training); and protocol 2 (T2, high-volume training). ET promoted CH by left ventricle weight/body weight ratio and cardiomyocytes diameter in T1 compared to S group. These responses were more robust in T2 compared to T1 group. ET increased cardiac p-Aktser473:Akt ratio and p-mTORser2448:mTOR ratio and lowered p-GS3Kβser9:GS3Kβ ratio in both trained groups suggesting Akt-mTOR signaling pathway participate in the physiological CH by ET. Interestingly, ET reduced 62% cardiac miRNA-208a levels analyzed by real-time PCR paralleled with an increase of 40% Purβ levels in T2 compared to S group. Increase in Purβ expression was sufficient to repress β-MHC expression in 70% at the mRNA level accompanied by increased of α- MHC (38%) and lower skeletal α-actin (53%) levels in T2. Cardiac levels of α, β and α/β -MHC, ANF and skeletal α-actin did not change in T1 compared to S. Furthermore, ET improved diastolic function in T2 group by increase of E/A ratio analyzed by echocardiography. LV compliance the increased cardiac mass (13%), normalized β-MHC mRNA and protein levels and improved the loss of diastolic function caused by obesity. These results suggest that the magnitude of CH was dependent of the increase in the volume of ET mediated by Akt-mTOR pathway. Indeed, ET prevent pathological cardiac hypertrophy via β-MHC reduction by regulation of miRNA-208a targeting Purβ, indicating the miRNA-208 in the control of myosin content and muscle performance and as potential therapeutic target for cardiac disorders.
Author Disclosures: T. Fernandes: None. U.P.R. Soci: None. E.M. Oliveira: None.
Key Words: Cardiac hypertrophy; Myosin; Exercise tests; Microrna
Reversal Of Myocardial Fibrosis In A Pre-clinical Model Of Heart Failure By Surgical-mediated Serca2a Gene Overexpression
Michael G Katz1, Elizabeth Brandon-Warner1, Anthony S Fargnoli1, Richard D Williams1, Rachel L Kaplan1, Tracy Walling1, Roger J Hajjar2, Laura Schrum1, Charles R Bridges1; 1Carolinas Healthcare System, Charlotte, NC, 2Mt Sinai Med Cntr, New York, NY
Background: Excessive myocardial fibrosis after ischemia leads to heart failure (HF). Adverse cardiac remodeling is accompanied with the upregulation of transforming growth factor beta signaling system (TGFβ), accumulation of collagen and dysregulation of SERCA2a. Currently fibrosis has no management. Surgical delivery of SERCA2a with “closed-loop” technique can attack molecular mechanisms of HF and is a promising and novel intervention. Methods: HF was induced by ligation of select coronary arteries in sheep resulting in large MI (n=9), or small MI (n=6). Additional sheep (n=6) underwent surgery with multiple recirculating gene delivery of AAV1/SERCA2a four weeks after large MI. MRI for hemodynamic evaluation was performed at baseline, 3 and 12 weeks after MI. Expression of collagen and TGFβ signaling components was assessed in infarct, border and remote zones by qRT PCR. Collagen was determined by electronic microscopy, histochemical and histological analysis. Results: HF (baseline vs. 12 weeks) was confirmed in large MI by a reduction in EF (56±4.2% vs. 32±3.8%, p<0.05), and increased LV end systolic and end diastolic volumes (21±2.7 mL to 55±4.4 mL and 48±1.0 mL to 81±3.8 mL, p<0.05, respectively) and was significantly less pronounced in SERCA2a/large MI and small MI. Active fibrosis, de novo collagen, was evident in infarct, border and remote zones in large MI, while SERCA2a/large MI and small MI showed less fibrosis with a higher ratio of mature to de novo collagen. Expression of TGFβ signaling components was significantly higher in large MI vs. small MI and SERCA2a/large MI (TGFβ1 214.7±65.6, vs. 3.73±1.74, vs. 13.68±2.35; TGFβ receptor 2: 162.02±40.41, vs. 5.08±1.36, vs. 3.93±.0.95; Smad3: 90.40±13.53, vs. 4.96±3.38, vs. 12.05±2.04; collagen 1α1: 98.36±30.51, vs. 5.68±1.80, vs. 1.88±0.63, p<0.05, respectively). Conclusion: The fibrogenic response was elevated significantly in large MI compared to small MI and SERCA2a/large MI sheep. Our data suggest that progression of fibrotic process is significantly different in large MI versus small MI after initial scar formation. Overexpression of SERCA2a via improved calcium regulation significantly impedes progression of fibrosis. This suggests SERCA2a as a novel therapy for HF.
Author Disclosures: M.G. Katz: None. E. Brandon-Warner: None. A.S. Fargnoli: None. R.D. Williams: None. R.L. Kaplan: None. T. Walling: None. R.J. Hajjar: None. L. Schrum: None. C.R. Bridges: None.
Key Words: Gene therapy; Fibrosis; Heart failure; Gene transfer
Paroxetine-mediated GRK2 Inhibition Improves Cardiovascular Outcomes Post Myocardial Infarction
Sarah M Schumacher-Bass1, Erhe Gao1, Kurt Chuprun1, John J Tesmer2, Walter J Koch1; 1Temple Univ, Philadelphia, PA, 2Univ of Michigan, Ann Arbor, MI
During heart failure (HF), cardiac levels and activity of the G protein-coupled receptor (GPCR) kinase (GRK) GRK2 are elevated, increasing phosphorylation, desensitization and down-regulation of β-adrenergic receptors (βARs) and other cardiac GPCRs. Increased GRK2 has been shown to participate in adverse remodeling and contractile dysfunction during HF, while GRK2 inhibition via a carboxyl-terminal peptide, βARKct, enhances heart function and can prevent and reverse HF. Recently, a collaborative study, Thal et al, discovered that the selective serotonin reuptake inhibitor paroxetine could inhibit GRK2 with selectivity over other GRK subfamilies. Paroxetine inhibited GRK2 target phosphorylation in vitro and significantly potentiated the isoproterenol-mediated increase in contractility in vitro and in vivo, with no effect beyond βARKct, supporting an inhibition of GRK2 in myocytes. In this study, wild-type mice underwent myocardial infarction (MI) or sham surgery and were allowed 2 weeks for infarct development before a 4 weeks treatment with vehicle (v), paroxetine (p), or fluoxetine via subcutaneous mini-osmotic pumps. Echocardiography was used to monitor cardiovascular function at baseline and every 2 weeks, followed by terminal hemodynamics. Heart weight normalized to tibia length was significantly reduced in paroxetine treated post-MI mice compared to vehicle-treated controls (sham v 6.65, p 6.63; MI v 13.27, p 10.51±0.68, p<0.01). All MI subjects exhibited similar cardiac function at baseline and 2 weeks post MI, demonstrating a decrease in EF from 68 to ~35%. In contrast to the continuing degradation of function in the vehicle-treated group, paroxetine treatment led to significant improvement (EF 46±4.5%, FS 23±2.5%, p<0.05). Hemodynamics revealed no difference in the isoproterenol-mediated chronotropy between groups. Importantly, the lack of isoproterenol-mediated inotropy in vehicle-treated mice was restored by paroxetine, but not fluoxetine, treatment. Together, these data demonstrate that paroxetine-mediated inhibition of GRK2 improves cardiac function post MI. The ability to deliver a small molecular inhibitor that would temper GRK2 activity ubiquitously presents an intriguing avenue for treatment of human HF.
Author Disclosures: S.M. Schumacher-Bass: None. E. Gao: None. K. Chuprun: None. J.J. Tesmer: None. W.J. Koch: None.
Key Words: Heart failure; Cardiovascular therapeutics; Myocardial infarction
Fulminant Lethal Cardiomyopathy Reveals Essential Roles of Organelle Fission/Fusion in Cardiomyocyte Mitochondrial Regeneration
Moshi Song1, Yun Chen1, Stephan Frank2, Luca Scorrano3, Gerald W Dorn II1; 1Washington Univ Sch of Medicine, Saint Louis, MO, 2Basel Univ, Basel, Switzerland, 3Venetian Institute for Molecular Medicine, Padua, Italy
Cardiomyocytes show little evidence of mitochondrial remodeling, but protein mediators of fission (Drp1) and fusion (Mfn 1 and 2) are highly expressed suggesting non-canonical functions. Previously, interrupting mito fusion (combined ablation of Mfn1 and Mfn2) evoked dilated cardiomyopathy with organelle fragmentation. Here, we interrogated both fission and fusion to define relationships with mitochondrial clearance and renewal. Postnatal overexpression of Drp1 and Mfn2 was tolerated, whereas postnatal Drp1 and Mfn1/Mfn2 ablation (myh6-Cre) was lethal by 6 weeks. To study adult hearts we conditionally ablated (MER-Cre-MER) Drp1 and Mfn1/Mfn2. Rapid cardiomyopathic dilatation was evoked by both fission- and fusion-deficiency (LV EDD 132% and 129% of ctrl, LV FS 31% and 58% of ctrl, p<0.001), but Drp1 null hearts showed massive mito enlargement by TEM and flow cytometry whereas mito fragmentation occurred in Mfn1/Mfn2 null hearts. Strikingly, mitochondria were depleted after Drp1 ablation, but accumulated after Mfn1/Mfn2 ablation. Mito respiration decreased and ROS production increased to a much greater extent in Drp1 null than Mfn1/Mfn2 null hearts, linking organelle dysfunction mostly strongly to defective fission. Parkin-mediated mitophagy was interrupted in Mfn1/Mfn2 null hearts, but increased after Drp1 ablation as measured by mito ubiquitination, Parkin translocation and p62 accumulation. Likewise, histological cardiomyocyte drop-out and apoptosis increased after Drp1 ablation, but not after Mfn1/Mfn2 ablation. These results reveal distinct roles for mito fission and fusion in the heart: Damaged mitochondria undergo fission, producing one healthy daughter organelle (that undergoes regeneration by fusing with other healthy ones) and one damaged daughter targeted for mitophagic elimination. Lack of Drp1 precludes fission-mediated segregation of functional and dysfunctional components into daughters, preventing repair of ROS-producing parent organelles and provoking generalized mitophagic depletion of mitochondria. In contrast, absence of Mfn1/Mfn2 prevents re-incorporation of healthy daughter organelles into the cell pool while also interrupting Parkin-mediated mitophagic elimination of damaged daughters.
Author Disclosures: M. Song: None. Y. Chen: None. S. Frank: None. L. Scorrano: None. G.W. Dorn: None.
Key Words: Cardiomyopathy; Mitochondria; Gene expression; Molecular biology
Biological Pacemaker Created By Percutaneous TBX18 Gene Transfer In A Porcine Model Of Complete Heart Block
Yu-Feng Hu, James Dawkins, Hee Cheol Cho, Eduardo Marbán, Eugenio Cingolani; Cedars-Sinai Heart Institute, Los Angeles, CA
Background: Re-expression of an embryonic transcription factor, TBX18, converts ordinary cardiomyocytes to pacemaker cells (Kapoor et al, Nat Biotech 2013). Gene- and cell-based biological pacemakers (BioP) have been demonstrated successfully in animal models of heart block, but transcriptional reprogramming has never been tested in a realistic translational model. We tested whether TBX18 gene transfer could create BioP activity in vivoin a porcine model of complete heart block, with focal percutaneous transgene delivery. Methods: Adenoviral vectors expressing either TBX18 or GFP as a control were delivered into the high right ventricular septum via venous catheters (NOGA™ Myostar). Animals were followed continuously for 14 days by implanted telemetry and serial pacemaker interrogations. Physical activity was measured by a built-in accelerometer. Electro-anatomic activation maps and electrophysiological studies (EPS) were conducted 2 weeks after gene delivery. Results: BioP activity was evident in TBX18-transduced animals starting at day 3 and persisted during the follow-up period. Mean heart rate (HR) at day 7 was higher in TBX18-transduced animals compared to GFP controls (HR=80±2 vs. 65±4bpm, p<0.05). Daytime HR (89±6 vs. 70±6bpm, p<0.05), HR during maximal activity (91±8 vs. 70±6bpm, p<0.05), and the increment of 11.2% in mean daily activity were all greater in the TBX18-transduced group. HR variability showed different low frequency/high frequency ratios between the two groups (5.7±2.6 vs. 1.1±0.2, p<0.05). Activation mapping and 12-lead ECG morphology localized the site of initiation to the TBX18injection site. No significant differences in QT dispersion or APD90dispersion by monophasic action potential were seen, nor were inducible arrhythmias observed after programmed stimulation in either group. Cardiac enzymes, liver function, renal function, and systemic inflammatory markers remained unchanged in both groups during follow-up. Conclusions: This proof-of-concept study shows that TBX18injection can create stable BioP activity for at least 14 days. The minimally invasive delivery method, combined with favorable local/systemic safety profiles, bring this approach closer to clinical translation.
Author Disclosures: Y. Hu: None. J. Dawkins: None. H. Cho: None. E. Marbán: None. E. Cingolani: None.
Key Words: Arrhythmias, treatment of; Gene therapy; Pacemakers
A Novel Non-canonical Role Of Cx43 In The Heart: Ensuring The Arrival Of Nav1.5 To The Intercalated Disc.
Esperanza Agullo Pascual1, Xianming Lin1, Anna Pfenniger1, Indra Lübkemeier2, Klaus Willecke2, Eli Rothenberg1, Mario Delmar1; 1New York Univ - Sch of Medicine, New York, NY, 2Univ of Bonn, Bonn, Germany
Background and objective: In its classical description, the function of connexins is to form gap junctions. While this description stands, recent studies show that connexin43 (Cx43) is not only a pore-forming gap junction protein. In fact, Cx43 is a part of a protein interacting network (the connexome), likely to regulate other functions in a gap junction-independent manner. Recently, it was reported that loss of the last five C-terminal amino acids of Cx43 (Cx43D378stop) leads to lethal ventricular arrhythmias in mice. Interestingly, the mutated Cx43 protein localized at the intercalated disc (ID), and gap junction channels showed normal properties. However, whole-cell sodium current (INa) density was decreased. Here, we explored the mechanisms relating Cx43 to INa in ventricular myocytes. Methods and Results: Functional assays: Adult myocytes from Cx43D378stop and Cre-negative (control) mice were tested by cell-attached macropatch, scanning ion conductance microscopy and super-resolution scanning patch clamp. We observed that Cx43D378stop caused a selective reduction in the number of functional sodium channels exclusively at the ID pool. Super-resolution fluorescence microscopy: Two-color direct stochastic optical reconstruction microscopy (20 nm resolution) showed Nav1.5 clusters in close proximity (or overlapping) with N-cadherin plaques. The distance between NaV1.5 clusters and the cell end increased from 57.2±12nm, n=365 in control to 111.7±11nm, n=446 in Cx43D378stop myocytes (p<0.001), indicating that mutation Cx43D378stop reduced NaV1.5 surface expression. This coincided with separation of the microtubule plus-end protein EB1 from the N-cadherin-rich cell end, from 23.7±31.9nm, n=665 in control, to 123.5±13.5nm, n=502 in Cx43D378stop cells (p<0.05). The distance to the stabilized end of the microtubule (glu-tubulin) also increased from 83.7±16.5nm, n=681 to 179±22.5nm, n=476 (p<0.05). Conclusions: Functional surface expression of NaV1.5 at the ID depends on preservation of the Cx43 C-end. We propose that Cx43 is necessary for proper arrival of the microtubule plus-end to the ID and delivery of its cargo, including NaV1.5. These gap junction-independent functions of Cx43 are vital to proper electrical function.
Author Disclosures: E. Agullo Pascual: None. X. Lin: None. A. Pfenniger: None. I. Lübkemeier: None. K. Willecke: None. E. Rothenberg: None. M. Delmar: None.
Key Words: Sodium channel; Connexin
Late-Breaking Basic Science Posters
Enhanced Myocardial Repair with CardioClusters
Megan M Monsanto, Sadia Mohsin, Kristina Fisher, Mark Sussman; San Diego State Univ, San Diego, CA
Existing approaches to modify stem cells for myocardial regeneration desperately need an innovative, novel, and creative solution that builds upon existing knowledge and will raise efficacy of repair to a new level. Although this deficiency has been attacked through combinatorial stem cell delivery and formation of cardiospheres, there is no evidence that these stem cell injections provide for direct cellular cross talk to promote stem cell survival and proliferation. Therefore, we created a CardioCluster, a three-dimensional (3-D) microenvironment consisting of three defined cell populations from the human heart: c-kit+ cardiac progenitor cells (CPCs), CD90+/CD105+ mesenchymal stem cells (MSCs) and CD133+ endothelial progenitor cells (EPCs). The size of the CardioCluster can be controlled by the quantity of cells used to create the cluster, allowing them to be infused into the heart without being reduced to single cell suspensions as is the case for cardiosphere-derived cells where the structural and cell-cell contact information is lost when delivered. Unlike cardiospheres, these cardiac cells are combined into a rationally designed cluster with MSCs and CPCs in the central core and EPC forming the outer layer. EPC play a vital role in forming neovasculature that will connect the CardioClusters to living heart tissue not damaged by ischemia and allow for revascularization of the damaged myocardium. We have found that EPC have increased resistance to apoptotic stress and would therefore be an ideal cell type for the exterior of our CardioCluster. In vitro we have shown that EPC are better able to form tubular networks on matrigel-coated plates compared to either CPC or MSC. MSCs reinforce the 3-D structure by releasing growth factors that attract and maintain cells within the cluster. Clinically, CardioClusters broaden the application of cell types into a single structure to increase engraftment, mitigate inflammation and prevent the progression of heart failure.
Author Disclosures: M.M. Monsanto: None. S. Mohsin: None. K. Fisher: None. M. Sussman: None.
Key Words: Angiogenesis; Regenerative medicine stem cells
Differential Regulation of Senescence and Lineage Commitment by Prolyl Isomerase Pin1 in Cardiac Progenitor Cells
Haruhiro Toko, Nirmala Hariharan, Mathias Konstandin, Lucia Ormachea, Balaji Sundararaman, Eri Joyo, Anya Y Joyo, Brett Collins, Natalie A Gude, Shabana Din, Sadia Mohsin, Michael McGregor, Mark A Sussman; San Diego State Univ, San Diego, CA
Autologous c-kit+ cardiac progenitor cells (CPCs) are currently used in the clinic to treat heart disease. CPC-based regeneration may be further augmented by better understanding molecular mechanisms of endogenous cardiac repair and enhancement of pro-survival signaling pathways that antagonize senescence while also increasing lineage commitment. The prolyl-isomerase Pin1 regulates signaling cascades by modulating protein folding, activity and stability of phosphoproteins. In this study, we examine the heretofore unexplored role of Pin1 in CPCs. Pin1 colocalizes with c-kit in mice hearts with myocardial infarction (MI) where CPCs are detected. Pin1 expression is associated with proliferating CPCs in vitro with a decline in Pin1 protein seen in conditions where proliferation is limited, such as serum starvation (-40%) and treatment with dexamethasone (Dex;-60%, p<0.05), a non-specific inducer of CPC differentiation. Pin1 is required for cell cycle progression and loss of Pin1 attenuates proliferation (-38%, p<0.05), causes G1/S phase arrest (p<0.05) concomitantly associated with decreased expression of Cyclin D (-30%, p<0.05) and Cyclin B (-55%, p<0.05) and increased expression of cell cycle inhibitors p53 (4.8 fold, p<0.05) and Retinoblastoma (Rb; 1.7 fold) in CPCs. Deletion of Pin1 increases cellular senescence (3 fold, p<0.001) but decreases lineage commitment (p<0.05) and cell death (p<0.001) of CPCs. Endogenous CPC expansion in ischemic hearts is Pin1 dependent as Pin1 knockout mice have fewer cycling CPCs (-42%, p<0.05) after MI. Interestingly, Pin1 overexpression also impairs proliferation (-52%, p<0.05) and causes G2/M phase cell cycle arrest (p<0.05) with concurrent downregulation of Cyclin B (-25%), p53 and Rb (-30% both, p<0.01). Additionally, Pin1 overexpression inhibits replicative senescence (-54%), increases Dex-induced lineage commitment (p<0.05) and inhibits cell death of CPCs (p<0.05), indicating that the cell cycle arrest caused by Pin1 overexpression is a consequence of lineage commitment and not senescence or cell death. In conclusion, Pin1 has pleiotropic roles in CPCs and may be a molecular target to promote survival, enhance repair, improve lineage commitment and antagonize senescence.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: H. Toko: None. N. Hariharan: None. M. Konstandin: None. L. Ormachea: None. B. Sundararaman: None. E. Joyo: None. A.Y. Joyo: None. B. Collins: None. N.A. Gude: None. S. Din: None. S. Mohsin: None. M. McGregor: None. M.A. Sussman: None.
Key Words: Stem/progenitor cells; Signal transduction; Molecular biology; Stem cell biology
Development Of Recipient-matched Engineered Heart Tissue Using 3d Printing
Yalin Yildirim1, Simon Pecha1, Hiroshi Naito2, Bijoy Karikkineth2, Wolfram H Zimmermann3, Hermann Reichenspurner1, Thomas Eschenhagen2; 1Univ Heart Cntr Hamburg, Hamburg, Germany, 2Dept of Experimental Pharmacology and Toxicology Hamburg, Hamburg, Germany, 3Institute of Pharmacology, Univ Med Cntr Göttingen, Göttingen, Germany
Background: Engineered pouch-like heart muscle constructs can be applied as heart-embracing cardiac grafts in vivo and might be used as biological ventricular assist devices (BioVAD®). Implantation studies showed micro-gaps between the graft and native heart due to a shape mismatch. The incoherence can lead to electrical coupling problems and loss of support force. Here we developed a procedure to create BioVADs individually matched to the recipient heart. Methods and Results: In group A, small animal magnetic resonance imaging (MRI) heart scan was performed in female wistar rats (n=10). 3D reconstruction of enddiastolic MRI data was performed and used to create a three-dimensional model of the heart by 3D printing. We used the recipient-matched 3D heart model and grew BioVADs from neonatal rat heart cells, fibrinogen and serum-containing culture medium. In group B, instead of the 3D heart model, a spheric glas spacer (diameter 10mm) was used to construct pouch like BioVADs. The engineered tissue beat spontaneously and showed contractile properties of native heart muscle including positive inotropic responses to calcium and isoprenaline. In group A recipient heart matched BioVADs were implanted into the corresponding healthy rats (n=10), while in group B the spheric BioVADs were implanted (n=10). 14 days after implantation hearts were explanted for histology. Histological analysis of recipient-matched BioVADs showed excellent coverage of the epicardial heart surface, while spheric BioVADs showed high number of micro and macro gaps. Furthermore direct alignment of the BioVADs and recipients hearts was measured in cross-sectioned histological compounds. In group B, a statistically higher number of areas with loss of contact between BioVADs and recipient hearts was seen compared to group A (67% vs 38%; p<0.001) Conclusion: We developed a novel casting technology using small animal MRI and 3D printing to generate artificial cardiac tissue that is perfectly matched to the recipient heart and exhibits structural and functional properties of native myocardium.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: Y. Yildirim: None. S. Pecha: None. H. Naito: None. B. Karikkineth: None. W.H. Zimmermann: None. H. Reichenspurner: None. T. Eschenhagen: None.
Key Words: Tissue engineering; Cardiac regeneration
Targeted Injection Of A Truncated Form Of Tissue Inhibitor of Metalloproteinase Alters Post-MI Remodeling; Evidence for Different Functional Domains
David C Lobb1, Christina B Logdon1, Heather Doviak1, Jason W O'Neill2, James A Shuman1, Craig P Novack1, Kia N Zellars1, Sara Pettaway1, Aarif Khakoo2, TaeWeon Lee2, Francis G Spinale1; 1Univ of South Carolina Sch of Medicine, WJB Dorn VA, Columbia, SC, 2Amgen Inc, San Francisco, CA
Background: Infarct expansion occurs after myocardial infarction (MI), leading to adverse left ventricular (LV) remodeling and failure due to an imbalance between MMPs and endogenous inhibitors (TIMPs). Clinical studies identified reduced levels of TIMP-3 were associated with adverse remodeling, and transgenic studies established a cause-effect relation of TIMP-3 to post-MI remodeling. We demonstrated that slow release of a recombinant, full length TIMP-3 (F-TIMP-3) within the MI region improved indices of LV remodeling, such as LV ejection fraction (LVEF) and end-diastolic volume (LVEDV). However, the biological effects of TIMP-3 are diverse and may depend upon specific sequence domains within the TIMP-3 molecule. The goal of this completed study was to compare effects of F-TIMP-3 to a truncated form encompassing the N-domain (N-TIMP-3) on post-MI remodeling. Methods/Results: MI was induced in adult pigs, randomized to receive MI injections of identical amounts (30 mg) of F-TIMP-3 (n=8), N-TIMP-3 (n=9), or saline injection (MI-only, n=11). At 14 days post-MI, LVEF fell in all post-MI groups but remained higher in the TIMP-3 injection groups. LVEDV doubled in the MI only group and was significantly lower in the TIMP-3 groups. Pulmonary capillary wedge pressure increased by approximately 3-fold in the MI only group but was reduced in the TIMP-3 groups. MMP-2 and IL-8 mRNA (RT-PCR) levels were increased by over 10-fold in the MI only and N-TIMP-3 groups (p<0.05) but fell from these values with F-TIMP-3 (p<0.05). Conclusion: The significance is 2-fold. A truncated form of TIMP-3 is sufficient to favorably alter the course of post-MI remodeling. Second, the functional and differential relevance of TIMP-3 domains has been established in-vivo since the TIMP-3 constructs demonstrated different MMP/cytokine expression profiles. These studies identify a unique and specific therapeutic strategy to alter the course of LV remodeling and dysfunction following MI.⇓
Author Disclosures: D.C. Lobb: None. C.B. Logdon: None. H. Doviak: None. J.W. O'Neill: Employment; Modest; Amgen. J.A. Shuman: None. C.P. Novack: None. K.N. Zellars: None. S. Pettaway: None. A. Khakoo: Employment; Modest; Amgen. T. Lee: Employment; Modest; Amgen. F.G. Spinale: Research Grant; Modest; NIH. Consultant/Advisory Board; Modest; Boston Scientific, Amgen Incorporated.
Key Words: Extracellular matrix; Fibrosis; Myocardial infarction; Pharmacology; Proteolytic enzymes
Evidence for a shared genetic determination of Ischemic Stroke And Coronary Artery Disease - a genome-wide analysis
Martin Dichgans1, Rainer Malik1, Inke R König2, Jonathan Rosand3, Robert Clarke4, Solveig Gretarsdottir5, Braxton D Mitchell6, Jeanette Erdmann7, Sekar Kathiresan8, Ruth McPherson9, Cathie Sudlow10, Muredach P Reilly11, John R Thompson12, Pankaj Sharma13, John C Chambers14, Hugh Watkins15, Peter M Rothwell16, Robert Roberts17, Hugh S Markus18, Nilesh J Samani19, Martin Farrall20, Heribert Schunkert21; 1Institute for Stroke and Dementia Rsch, Klinikum der Universität München, Ludwig- Maximilians-Universität, Munich, Germany, 2Institut für Medizinische Biometrie und Statistik, Universität zu Lübeck, Lübeck, Germany; Universitätsklinikum Schleswig-Holstein, Campus Lübeck, Lübeck, Germany, 3Dept of Neurology and Cntr for Human Genetic Rsch, Massachusetts General Hosp, Boston, MA, 4Clinical Trial Service Unit and Epidemiological Studies Unit, Univ of Oxford, Oxford, United Kingdom, 5deCODE Genetics, Reykjavik, Iceland, 6Univ of Maryland Sch of Medicine, Dept of Medicine, Maryland, MD, 7Institut für integrative und experimentelle Genomik, Universität zu Lübeck, Lübeck, Germany, 8Cntr for Human Genetic Rsch, Massachusetts General Hosp, Boston, MA, 9Div of Cardiology, Univ of Ottawa Heart Institute, Ottawa, Canada, 10Div of Clinical Neurosciences, Univ of Edinburgh, Edinburgh, United Kingdom, 11Univ of Pennsylvania Sch of Medicine, Philadelphia, PA, 12Leicester NIHR Biomedical Rsch Unit in Cardiovascular Disease, Glenfield Hosp, Leicester, United Kingdom, 13Imperial College London & Hammersmith Hosps, London, United Kingdom, 14Imperial College Sch of Medicine, London, United Kingdom, 15Cardiology Div Dept of Medicine, Brigham and Women's Hosp, Harvard Med Sch Boston, Boston, MA, 16Stroke Prevention Rsch Unit, Univ Dept of Clinical Neurology, Radcliffe Infirmary, Oxford, United Kingdom, 17The John & Jennifer Ruddy Canadian Cardiovascular Genetics Cntr, Univ of Ottawa, Ottawa, Canada, 18Dept of Neurology, King’s College Sch of Medicine and Dentistry, London, United Kingdom, 19Dept of Cardiovascular Sciences, Univ of Leicester, Glenfield Hosp, Leicester, United Kingdom, 20Wellcome Trust Cntr for Human Genetics, Univ of Oxford, Oxford, United Kingdom, 21Dept of Cardiology, German Heart Cntr Munich, Munich, Germany
Background: Ischemic stroke (IS) and coronary artery disease (CAD) share several risk factors and each have a substantial heritability. We conducted a genomewide analysis to evaluate the extent of shared genetic determination of the two diseases.
Methods and Results: Genome-wide association data of ~2.5 million genetic variants from 12389 individuals with IS, 33398 individuals with CAD, and more than 110000 controls were obtained from the METASTROKE, CARDIoGRAM, and C4D consortia. We analyzed common variants reaching nominal threshold of significance (p<0.01) for CAD for their association with IS and specific overlap across phenotypes for variants that reached genome-wide significance for CAD and for variants that reached a high (p<1x10-5) threshold of significance for IS. We conducted a joint meta-analysis on the combined phenotype of IS or CAD to identify additional variants that affect their risk. Corresponding analyses were performed restricted to the 2167 individuals with ischemic large artery stroke (LAS) subtype. Common genetic variants associated with CAD at p<0.01 were associated with a significant excess risk for IS (pIS/CAD=9x10-94) and for LAS (pLAS/CAD=10-185) and vice versa (pCAD/IS=10-236 pCAD/LAS=2x10-11). Among the 42 genome-wide significant loci for CAD, three and five loci were significantly associated with IS and LAS (p<0.0012). In a meta-analysis 15 adjusted loci passed genomewide significance (p<5x10-8) for the combined phenotype of IS or CAD and 17 loci passed genomewide significance for the combined phenotype of LAS or CAD. Since these loci had prior evidence for genome-wide significance for CAD we analyzed respective signals for IS and LAS and found evidence for association at chr12q24/SH2B3 (pIS=1.62x10-07), ABO (pIS=2.6x10-4), HDAC9 (pLAS=2.32x10-12), 9p21 (pLAS =3.70x10-6),RAI1-PEMT-RASD1 (pLAS=2.69x10-5),EDNRA (pLAS=7.29x10-4) and CYP17A1-CNNM2-NT5C2 (pLAS=4.9x10-4).
Conclusions: Our results demonstrate substantial overlap in the genetic risk between ischemic stroke and coronary artery disease and, in particular between the large artery stroke subtype and coronary artery disease. This shared genetic architecture may partly explain the clustering of different vascular diseases within families.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: M. Dichgans: None. R. Malik: None. I.R. König: None. J. Rosand: None. R. Clarke: None. S. Gretarsdottir: None. B.D. Mitchell: None. J. Erdmann: None. S. Kathiresan: None. R. McPherson: None. C. Sudlow: None. M.P. Reilly: None. J.R. Thompson: None. P. Sharma: None. J.C. Chambers: None. H. Watkins: None. P.M. Rothwell: None. R. Roberts: None. H.S. Markus: None. N.J. Samani: None. M. Farrall: None. H. Schunkert: None.
Key Words: Genome-wide association studies (GWAS); Coronary artery disease; Stroke; Risk factors; Genetics
Aggravation Of Mitophagic Cardiomyopathy By Highly Expressed Mitochondrial Catalase Reveals The Essential Function Of Mitochondrial-generated Ros In Mitophagy
Yun Chen1, Moshi Song1, Peter S Rabinovitch2, Gerald W Dorn II1; 1Washington Univ in St. Louis, saint louis, MO, 2Dept of Pathology, Univ of Washington, Seattle, WA
Reactive oxygen species (ROS) generated by damaged mitochondria are cytotoxic and strongly implicated in organ and organism senescence; ROS suppression by high-level (100x normal) transgenic expression of mito-targeted catalase (mCAT-100x) extended life by 5½ months in mice and protected against stress-mediated cardiomyopathies. Accordingly, we hypothesized that cardiac mCAT expression would prevent cardiomyopathy evoked by interruption of cardiac mitophagy and resulting accumulation of dysfunctional mitochondria. Mfn2 is the mitochondrial receptor for Parkin and essential for mitophagic elimination of damaged/senescent organelles; cardiac-specific Mfn2 ablation induces a progressive mitophagic cardiomyopathy over 30 weeks. We bred 100x and moderate (~10x) expressing mCAT transgenes onto cardiac Mfn2 null mice and analyzed effects on mito ROS production, respiratory function, and clearance and integrity of mitophagic signaling, and cardiac function. Compared to control mice, cardiac Mfn2 ablation depressed LVFS (26%), increased heart/body weight (44%), increased mito size by TEM and flow cytometry (33%), partially dissipated mito inner membrane potential (αψm), and decreased maximal mito respiration (22%). mCAT-100x alone reduced cardiac mito H2O2 production without adversely affecting any other measured parameter. mCAT-100x also suppressed mito H2O2 generation by Mfn2 null hearts, but markedly exacerbated the cardiomyopathy at 30 wks: Compared to controls, LVFS decreased by 43%, Ht/b wt increased by 127%, mito size increased by 55%, αψm decreased by 25%, and respiration diminished by 37%. By contrast, mCAT-10x protected against cardiomyopathy in cardiac Mfn2 null mice. Aggravation, rather than rescue, of mitophagic cardiomyopathy by highly expressed mito-targeted CAT reveals an essential function of mito-derived H2O2 for normal mitophagic quality control in hearts. Conventional wisdom holds that loss of mito αψm is the signal for activating the PINK1-Mfn2-Parkin pathway leading to mitophagy, but dissociation of αψm and mito-derived H2O2 herein identifies the critical signal as mito-generated ROS, over-suppression of which constitutes a second “hit” to mitophagy in Mfn2-deficient hearts.
Author Disclosures: Y. Chen: None. M. Song: None. P.S. Rabinovitch: None. G.W. Dorn: None.
Key Words: Oxidative stress; Mitochondria; Autophagy; Cardiovascular disease
Nanotechnological Coating Of Prosthetic Grafts Reduces Bacterial Growth
Yalin Yildirim1, Simon Pecha1, Tobias Deuse1, Thomas Eschenhagen2, Hermann Reichenspurner1; 1Univ Heart Cntr Hamburg, Hamburg, Germany, 2Dept of Experimental Pharmacology and Toxicology Hamburg, Hamburg, Germany
Objectives: The growing number of implanted prosthetic material may lead to divers problems, including bacterial infections. Prosthetic infections may cause serious infection leading to high mortality and morbidity. We produced superhydrophobic vascular grafts that showed resistance to bacterial growth. Methods: Vascular grafts were silicon dioxide (SiO2) coated and developed a nano-scale roughness with all properties of superhydrophobic surfaces. Surface properties were investigated by water contact angle and water roll-off angel (tilted-drop measurement) analysis. Vascular grafts (n = 20) with a water contact angel >140° were incubated with a bacteria solution (Escherichia coli, Staphylococcus aureus) for 24 hours. A control group (n = 20) of uncoated vascular grafts were processed the same way. Bacterial growth was investigated by counting colony-forming units. A mock circulation was created, using a Heartware ventricular assist device pump with a nanotechnologically coated- or uncoated outflow grafts. Results: All coated vascular grafts showed a water contact angel >140° (mean 153°± 5.6) and a mean water roll-off angel of 15 ± 4.5. Grafts with nano-scale roughness showed a 86% reduction in colony-forming units compared to the control group. Microscopic analysis of fluorescence labeled bacteria showed 98% reduction of bacterial growth on superhydrophobic coated vascular grafts. Human heparinized blood with above mentioned bacteria were used for mock circulation experiments. Analysis of outflow grafts by laser scanning microscopy and counting of colony forming units showed 81% and 76% reduction of bacterial growth, respectively. Conclusion: Bacterial growth on vascular grafts can be markedly reduced by superhydrophobic coating in vitro. In vivo experiments have to be done to proof biocompatibility and confirm the protection against bacterial growth.⇓
Author Disclosures: Y. Yildirim: None. S. Pecha: None. T. Deuse: None. T. Eschenhagen: None. H. Reichenspurner: None.
Key Words: New technology; Inflammation
Sorafenib Decreases Survival after Myocardial Infarction by Enhancing Cell Death Of Cardiac Myocytes and ckit+ Stem Cells
Jason M Duran, Catherine A Makarewich, Polina Gross, Sharmeen Husain, Danielle Trappanese, Jonathan A Dunn, Timothy Starosta, Hind Lal, Thomas E Sharp, Ronald J Vagnozzi, Remus M Berretta, Mary F Barbe, Hajime Kubo, Thomas Force, Steven R Houser; Temple Univsersity Sch of Medicine, Philadelphia, PA
Introduction: Sorafenib is an effective treatment for liver and renal cancers, but it can be cardiotoxic in patients with preexisting cardiac comorbidities through unknown mechanisms. Hypothesis: Sorafenib reduces the ability of the heart to respond to disease stressors by predisposing myocytes and c-kit+ stem cells in the heart and bone to premature death via necrosis or apoptosis, thus enhancing disease related injury. Methods and Results: Mice were treated with 30 mg/kg/d intraperitoneal sorafenib (n=40) or vehicle (n=23) for 3 weeks. After 1 week of treatment, myocardial infarction (MI) was induced by coronary artery ligation, and minipumps containing BrdU were implanted to label mitotic cells. Sorafenib treatment markedly decreased 2-week survival post-MI relative to vehicle-treated controls (40.4% v. 11.3%, p<0.05). Echocardiography at 1 and 2 weeks post-MI detected no differences in cardiac function between groups, but sorafenib treated hearts had significantly smaller diastolic and systolic volumes and reduced heart weights. Sorafenib induced complete necrotic death of isolated feline left ventricular myocytes in vitro at high doses (>10 uM), but lower doses (<5 uM) did not induce myocyte death or affect inotropy (fractional shortening or calcium transients). Masson’s trichrome-stained tissue sections showed decreased numbers of myocytes per visual field with increased myocyte cross-sectional area in sorafenib treated animals. Sorafenib inhibited in vitro proliferation of cardiac- and bone-derived c-kit+ stem cells and showed activation of caspase-3 in a dose-dependent fashion. At 1 week post-MI fewer BrdU-labeled myocyte nuclei were detected at the infarct border zone in sorafenib-treated hearts than in vehicle-treated controls (5.7% v. 0.9%, p<0.05). Sorafenib had no effect on infarct size, fibrosis, or blood vessels. Conclusion: Sorafenib cardiotoxicity results from myocyte necrosis and cell loss, and not from any direct effect on myocyte function. Surviving myocytes undergo pathological hypertrophy. Inhibition of c-kit+ stem cells further exacerbates damage by decreasing myocyte turnover. In the setting of MI injury, which also causes large-scale cell loss, sorafenib cardiotoxicity dramatically increases mortality.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: J.M. Duran: None. C.A. Makarewich: None. P. Gross: None. S. Husain: None. D. Trappanese: None. J.A. Dunn: None. T. Starosta: None. H. Lal: None. T.E. Sharp: None. R.J. Vagnozzi: None. R.M. Berretta: None. M.F. Barbe: None. H. Kubo: None. T. Force: None. S.R. Houser: None.
Key Words: Pharmacology; Myocardial infarction; Hypertrophy; Apoptosis; Stem cells
Rare Variation in Sarcomeric Genes Accompanies MYH7 Hypertrophic Cardiomyopathy
Sebastian J Caliri1, Frederick E Dewey1, Megan E Grove1, James Priest1, Suyash Shringarpure1, Cuiping Pan1, Somalee Datta1, Megan J Puckelwartz2, Jessica R Golbus2, Michael Snyder1, Carlos D Bustamante1, Sharlene Day3, Elizabeth McNally2, Thomas Cappola4, Gerald Dorn5, Euan A Ashley1; 1Stanford Univ, Stanford, CA, 2Univ of Chicago, Chicago, IL, 3Univ of Michigan, Ann Arbor, MI, 4Univ of Pennsylvania, Philadelphia, PA, 5Washington Univ in St. Louis, St. Louis, MO
Background: Deleterious variation in genes of the cardiac sarcomere explains many cases of hypertrophic cardiomyopathy (HCM) but variable penetrance and phenotypic heterogeneity suggests the possibility of genetic modifiers. To interrogate the genetic architecture of MYH7 HCM, we investigated the total burden of rare variation in sarcomeric genes using whole genome sequencing. Methods and Results: We performed whole genome (40x coverage) or whole exome (54x) sequencing (Illumina, Inc) in 96 individuals, 47 HCM patients with known pathogenic mutations in MYH7 and 49 individuals without clinical evidence of HCM. Sequence reads were processed using the Burrows Wheeler Aligner, the Genome Analysis Toolkit, and Real Time Genomics variant analysis. Variants were annotated using the Stanford genome analysis pipeline. The primary variant was identified in each HCM patient. Other variants were filtered to include only those rare (<5%), non-synonymous variants within exonic or splicing regions of cardiac sarcomere genes (MYBPC3, MYL2, MYL3, MYH7, ACTC1, TPM1, TNNI3, and TNNT2), yielding 35 variants in HCM cases, and 20 in controls. Interrogation of 1,092 genome sequences (86x) from apparently healthy individuals participating in the 1,000 Genomes Project revealed 202 variants. Outside of the primary genetic variant in MYH7, HCM patients were thus found to harbor an average of 0.745±0.988 additional rare, potentially deleterious mutations in sarcomeric genes with 51% lying outside MYH7, in either MYBPC3or TNNT2. Controls sequenced locally and participants in the 1,000 Genomes Project each had fewer variants per individual: 0.408±0.762 (p=0.0641) and 0.185±0.43 (p=2.046x10-15). Conclusions: Rare variation in sarcomeric genes is greater in HCM patients than controls. A majority of this variation lies outside of MYH7. These results support an oligogenic contribution to the genetic architecture of MYH7 HCM, which carries important implications for predictive genetic testing and cardiac screening recommendations for at-risk family members.
Author Disclosures: S.J. Caliri: None. F.E. Dewey: None. M.E. Grove: None. J. Priest: None. S. Shringarpure: None. C. Pan: None. S. Datta: None. M.J. Puckelwartz: None. J.R. Golbus: None. M. Snyder: None. C.D. Bustamante: None. S. Day: None. E. McNally: None. T. Cappola: None. G. Dorn: None. E.A. Ashley: None.
Key Words: Hypertrophic cardiomyopathy; Myosin; Genomics; Gene mutations
Cardiac Myosin Binding Protein-C Phosphorylation: a Potential Mechanism for the Treatment of Heart Failure with Preserved Ejection Fraction
Paola C Rosas1, Yang Liu1, Mohamed Abdalla1, Candice Thomas1, Kidwell David1, Rajesh Kumar1, Kenneth Baker1, Patricia Powers2, Richard Moss2, Carl Tong1; 1Texas A&M, Temple, TX, 2Univ of Wisconsin, Madison, WI
Heart failure with preserved ejection fraction (HFpEF), which accounts for ~50% of all cases of heart failure, lacks effective treatment. Cardiac myosin binding protein-C (MyBPC3), a thick filament-associated protein, releases its inhibition on myosin-actin cross-bridge cycling when phosphorylated. MyBPC3 phosphorylation is decreased in heart failure. Thus, we hypothesize that phosphorylated MyBPC3 enhances lusitropy to mediate normal diastolic function. We test this idea by comparing mouse models of phosphorylation deficient MyBPC3(S273A, S282A, S302A)-MyBPC3(t3SA), phosphorylation mimetic MyBPC3(S273D, S282D, S302D)-MyBPC3(t3SD), and WT-control MyBPC3(tWT). MyBPC3(t3SA) hearts exhibit diastolic dysfunction but MyBPC3(t3SD) hearts exhibit enhanced relaxation by echocardiographic mitral blood inflow Doppler to tissue Doppler of myocardial relaxation ratios (E/Ea). We assess the underlying mechanism by simultaneously measuring force and intracellular calcium [Ca2+]i on intact papillary muscles with increasing pacing frequency 1-2.5 Hz. We use peak relaxation rate (-dF/dt)min to peak force generation rate (+dF/dt)maxratio (dFR) to compare lusitropy. MyBPC3(t3SD) muscles show increased dFR, signifying enhanced relaxation. Increasing pacing frequency increased dFR (i.e, accelerated relaxation) in MyBPC3(tWT) and MyBPC3(t3SD) but not in MyBPC3(t3SA) muscles. We use single negative exponential to calculate [Ca2+]Idecay rate constant kCa. All models show similar kCa; therefore, relaxation enhancement is attributable to cross-bridge cycling but not differences in calcium handling. MyBPC3(t3SD) hearts show enhanced (-dP/dt)min/(+dP/dt)maxby direct pressure measurements, corroborating papillary muscle findings. Thus, we conclude that the loss of MyBPC3 phosphorylation retards relaxation; whereas, phosphorylated MyBPC3 enhances relaxation, constituting a potential mechanism of treatment for HFpEF.⇓
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: P.C. Rosas: None. Y. Liu: None. M. Abdalla: None. C. Thomas: None. K. David: None. R. Kumar: None. K. Baker: None. P. Powers: None. R. Moss: None. C. Tong: None.
Key Words: Heart failure; Contractile proteins; Diastolic function
Parkin Moderates Hypertrophy and Heart Failure Following Cardiac Pressure Overload
Eileen R Gonzalez, Dieter A Kubli, Nicole H Purcell, Åsa B Gustafsson; Univ of California, San Diego, San Diego, CA
The E3 ubiquitin ligase Parkin has been identified as an important regulator of mitochondrial autophagy (mitophagy) by selectively marking damaged mitochondria for removal. We previously found that Parkin plays an important role in clearing dysfunctional mitochondria after myocardial infarction (MI), and mice deficient in Parkin (Parkin-/-) have increased injury and mortality after MI. However, it is currently unknown if Parkin also plays a role in adapting to hemodynamic stress. Here, we investigated the functional importance of Parkin in pathological cardiac hypertrophy. Wild type (WT), Parkin-/-, and cardiac-specific Parkin transgenic (Parkin-TG) mice were subjected to trans-aortic constriction (TAC). Compared to WT mice, Parkin-/- mice failed to develop cardiac hypertrophy in response to TAC. While WT mice showed increased heart weight/tibia length, myocyte size, and upregulation of hypertrophy markers ANF, skeletal muscle actin, and β-myosin heavy chain (MHC-β) two weeks post-TAC, Parkin-/- mice did not. Parkin-/- hearts did have a blunted hypertrophic response, but cardiac function was still preserved. In contrast, Parkin-TG mice had an exacerbated hypertrophy response compared to WT mice. Parkin-TG mice had increased expression of MHC-β, significantly lower %FS (14.3% ± 2.7% for Parkin-TG vs. 23.1% ± 2.6% for WT) and %EF (30.2% ± 5.5% for Parkin-TG vs. 46.7% ± 4.5% for WT) than WT, and had developed pulmonary edema at this time, indicative of accelerated progression to heart failure. These data suggest that overexpression of Parkin may lead to excessive clearance of mitochondria via mitophagy and increased susceptibility to heart failure in response to hemodynamic stress. However, lack of Parkin results in a failure to activate hypertrophy in response to hemodynamic stress, suggesting that Parkin plays a critical role in the hypertrophy response. This study also demonstrates that the functional role of Parkin differs depending on the stress stimulus; Parkin may have a novel role in regulating hypertrophy via pathways distinct from its canonical role in mitophagy. Although enhancing Parkin-mediated mitophagy has therapeutic potential, our results indicate that this pathway can be detrimental to heart function if it is over-activated.
Author Disclosures: E.R. Gonzalez: None. D.A. Kubli: None. N.H. Purcell: None. Å.B. Gustafsson: None.
Key Words: Cardiac hypertrophy; Autophagy; Mitochondria; Heart failure
Missense Mutations In Plakophilin-2 Can Lead To Brugada Syndrome Phenotype By Decreasing Sodium Current And Nav1.5 Membrane Localization
Marina Cerrone1, Xianming Lin1, Mingliang Zhang1, Esperanza Agullo-Pascual1, Anna Pfenniger1, Halina Chkourko Gusky1, Valeria Novelli2, Changsung Kim3, Tiara Tirasawadichai4, Daniel P Judge5, Eli Rothenberg1, Huei-Sheng Vincent Chen4, Carlo Napolitano2, Silvia G Priori2, Mario Delmar1; 1NYU Sch of Medicine, New York, NY, 2Fondazione S. Maugeri, Pavia, Italy, 3Sanford-Burnham Med Rsch Institute, La Jolla, CA, 4Sanford-Burnham Med research Institute, la Jolla, CA, 5Johns Hopkins Univ Sch of Medicine, Baltimore, MD
Background: Brugada syndrome (BrS) is associated with loss of sodium channel function. Previous studies showed features consistent with sodium current (INa) deficit in patients carrying desmosomal mutations, diagnosed with arrhythmogenic cardiomyopathy (AC; or arrhythmogenic right ventricular cardiomyopathy, ARVC). Experimental models showed correlation between loss of expression of desmosomal protein plakophilin-2 (PKP2), and reduced INa. We hypothesized that PKP2 variants that reduce INa could yield a BrS phenotype, even without cardiomyopathic features of AC. Methods and Results: We searched for PKP2 variants in genomic DNA of 200 patients with BrS diagnosis, no signs of AC, and no mutations in BrS-related genes SCN5A, CACNa1c, GPD1L and MOG1. We identified 5 cases of single amino acid substitutions. One (Q62K) was previously described in AC patients as variant of unknown significance; 4 were unreported. In a family with multiple cases of syncope and/or suspect ECG, novel variant R635Q co-segregated with the phenotype in all affected relatives and was absent in the non affected ones. Mutations were tested in HL-1-derived cells endogenously expressing NaV1.5 but made deficient in PKP2 (PKP2-KD). Loss of PKP2 caused decreased INa and NaV1.5 at site of cell contact. These deficits were restored by transfection of wild-type PKP2 (PKP2-WT), but not of BrS-related PKP2 mutants. Similar results were obtained when cells were co-transfected with PKP2-WT and the BrS-related PKP2 variants, to mimic heterozygosity. Human induced pluripotent stem cell cardiomyocytes (hIPSC-CMs) from a patient with PKP2 deficit showed drastically reduced INa. The deficit was restored by transfection of WT, but not BrS-related PKP2 variant R635Q. Super-resolution microscopy in murine PKP2-deficient cardiomyocytes related INa deficiency to reduced number of channels at the intercalated disc, and increased separation of microtubules from the cell-end. Conclusions: This is the first systematic retrospective analysis of a patient group to define the co-existence of sodium channelopathy and genetic PKP2 variations. PKP2 mutations may be a molecular substrate leading to the diagnosis of BrS.
Author Disclosures: M. Cerrone: None. X. Lin: None. M. Zhang: None. E. Agullo-Pascual: None. A. Pfenniger: None. H. Chkourko Gusky: None. V. Novelli: None. C. Kim: None. T. Tirasawadichai: None. D.P. Judge: None. E. Rothenberg: None. H. Chen: None. C. Napolitano: None. S.G. Priori: Consultant/Advisory Board; Modest; medtronic, Boston scientific, Transgenomics. Consultant/Advisory Board; Significant; no, no, no. M. Delmar: None.
Key Words: Ion channels; Sodium channel; Ventricular arrhythmia; Genetics
SnoRNAs as Potential Novel Therapeutic Targets in Myocardial Ischemia/Reperfusion Injury
Dilyara Cheranova, Marianne Nsumu, Inna Sokolovsky, Dmitry Grigoryev, Daniel Heruth, Li Q Zhang, Shui Q Ye; Children's Mercy Hosps and Clinics, Kansas City, MO
Rationale: Small nucleolar RNAs (snoRNAs) are a class of small RNA molecules that primarily guide chemical modifications of other RNAs. However, emerging reports indicate that they have a wide range of functions implicated in the development and pathophysiology of many diseases. This study aims to use RNA-seq, a powerful next generation DNA sequencing strategy, to reveal new and novel transcriptional mechanisms, especially the expression of snoRNAs, underlying the role of nicotinamide phosphoribosyltransferase (NAMPT) or angiopoitin like 4 (ANGPTL4) in mouse myocardial ischemia/reperfusion injury (IRI) of either wild type or gene deficient type mice. Methods: Male C57BL/6J Nampt+/+ and Nampt+/- mice or Angptl4+/+and Angptl4+/- , 10 week-old, were subjected to either sham operation or myocardial ischemia by a 40 min occlusion of the left anterior descending coronary artery followed by 3 h reperfusion. After which mice were sacrificed and their hearts were harvested for further analyses including assessing infarct size and area at risk, assaying apoptosis and profiling their transcriptomes by RNA-seq using Illumina's HiSeq1500 next generation DNA sequencing instrument. Selected candidates, snoRNAs, from mouse IRI transcriptome were further investigated for their roles in myocardial IRI. Results: The size of myocardial infarction/area at risk or myocardial apoptosis was significantly smaller or less severe in wild type (either Nampt+/+ or Angptl4+/+) mice than in Nampt +/- or Angptl4+/- mice. RNA-seq revealed that a number of disregulated snoRNAs were shared transcriptome profiles in gene deficient mouse IRI heart, independent of mouse genotypes. Top snoRNAs (7a, 16a, 18a, 21, 31, 33, 41, 68, 69, and 81) expressed from 0.85 to 86.08 FPKM with 1.79 to 42 fold higher expression levels over their wild type mouse controls. In vitro "gain of function" and “loss of function” experiments demonstrated that overexpression or down regulation of snoRA33 significantly increased or decreased human cardiomyocyte apoptosis. Conclusion: SnoRNAs play novel roles in the cardiomyocyte apoptosis, a hallmark in the pathogenesis of myocardial IRI. SnoRNAs may represent potential novel therapeutic targets in Myocardial Ischemia/ Reperfusion Injury.
Author Disclosures: D. Cheranova: None. M. Nsumu: None. I. Sokolovsky: None. D. Grigoryev: None. D. Heruth: None. L.Q. Zhang: None. S.Q. Ye: None.
Key Words: Ischemia reperfusion; Myocardial infarction
CDC Therapeutic Efficacy is Dictated by a Stress Response that Limits Adverse Remodeling and Inflammation
David L Simpson, Ileana Valle, Agnieszka Blusztajn, Rachel R Smith; Capricor, Inc, West Hollywood, CA
Cardiosphere-derived cells (CDCs) are currently being tested in a Phase I/II clinical trial. In an effort to determine the most optimal use of procured tissue, we assessed the regenerative output of CDCs derived from the LV compared to the atria (AT). There was no difference in the proliferative capacity or surface proteome of LV-CDCs compared to AT-CDCs using three patient samples. LV-CDCs, however, were unable to promote a regenerative response when transplanted into an in vivo model of MI. Indeed, from baseline (1 day after MI) to three weeks, the ejection fraction and wall thickness of hearts treated with LV-CDCs progressively deteriorated while those treated with AT-CDCs improved, significantly (ΔEF%: -14.5 ± 4.1% vs. 7.2 ± 2.5%, p<0.001 ; ΔWall: -0.2 ± 0.1mm vs. 0.1 ± 0.1mm, p<0.01). To further understand this observation we compared gene expression between LV-CDCs and AT-CDCs cultured at high oxygen tension to mimic pathological stress. Interestingly, LV-CDCs responded more readily to stress via upregulation of the infarct remodeling components MMP7 and Collagen type I compared to AT-CDCs. Upregulation of the cardioprotective protein SDF1α, however was limited to AT-CDCs. Additionally, LV-CDCs secreted higher amounts of the pro-inflammatory cytokines IL-6 (15.9 ± 1.0 vs. 11.4 ± 1.4 RQ, p = 0.03) and GM-CSF (0.9 ± 0.1 vs. 0.4 ± 0.2 RQ, p = 0.07) compared to AT-CDCs. Thus, the lack of efficacy from engrafted LV-CDCs in vivo might be explained by an adverse stress response to pathological conditions. Interestingly, miRNAs are known to be key players in stress and disease. To determine if there was differential miRNA profiles between LV-CDCs and AT-CDCs we performed a high throughput PCR Array. miR-302a and -302b (anti-fibrotic) were upregulated in AT-CDCs while miR-146a and -126 (modulation of the SDF1α-CXCR4 axis) were upregulated in LV-CDCs suggesting that specific miRNA profiles are indicative of CDC potency. In conclusion, non-potent CDCs, those lacking cardiomyoplastic potential, promote a stress response that is detrimental to effective cardiac repair. With the observation of differential stress responses between potent and non-potent CDCs we are poised to develop strategies to optimize therapeutic efficacy and use of our clinical products.
Author Disclosures: D.L. Simpson: Employment; Significant; Capricor, Inc. I. Valle: Employment; Significant; Capricor, Inc. A. Blusztajn: Employment; Significant; Capricor, Inc. R.R. Smith: Employment; Significant; Capricor, Inc.
Key Words: Stem cell therapy; Myocardial infarction; Remodeling; Microrna
Functional Analysis Of Tnni3k Gene And Its Significance For Suppressing Cardiac Sudden Death
Zhong-fang Lai, Yu-Zhen Chen, Tian-Li Zhang, Nobuo Sakaguchi, Shokei Kim-Mitsuyama; Graduate Sch of Med Science, Kumamoto Univ, Kumamoto, Japan
TNNI3K is a MAP kinase specifically and continuously expressed in cardiac muscle and interacted with cardiac troponin I (cTnI). The aim of this study is to investigate role of TNNI3K gene and its mutant on modification the physiological or pathophysiological function of cardiomyocytes, and to look for some connections for developing some new therapeutic strategies.
In the in vitro experiments, 1) Availability of plasma TNNI3K level was investigated using with anti-TNNI3K monoclonal-antibodies in patients diagnosed as AMI, and in healthy volunteers. Data shown that circulating TNNI3K levels were significantly higher in AMI (p<0.001) when compared with that in the healthy volunteers group, indicating that measurement of circulating TNNI3K level may be a novel and useful tool to diagnose AMI. 2) Effects of PKC inhibitor (GF109203X) or PKA activator (Br-8-cAMP) on the beating frequency were investigated in P19CL6-derived culture beating cardiomyocytes. Results showed that TNNI3K-overexpression increased the contractility and beating frequency together with restrained phosphorylation of cTnI through activation of the PKA pathway but not by blocking of the PKC pathway.
In the in vivo experiments, a mutated TNNI3K gene obtained from substitution of serine at 835-836 sites with alanine and was directly transfected into beating hearts using an in vivo gene electroporation method. 1) Incidence of arrhythmic outbreak in control, wild TNNI3K gene, and TNNI3K-mutant gene introduction groups. Data shows that the incidences of arrhythmias including tachycardia in the TNNI3K-mutant group were significantly higher than that in other two groups. 2) Animal survival rates in the three groups were observed in a period for two months and data shown that TNNI3K-mutant increased the incidences of animal mortality.
Our above new data indicated that functional analysis of TNNI3K gene and the new remedy development using this MAP kinase would be favorable for suppressing cardiac sudden death risk.
Author Disclosures: Z. Lai: None. Y. Chen: None. T. Zhang: None. N. Sakaguchi: None. S. Kim-Mitsuyama: None.
Key Words: Cardiac arrest; Monoclonal antibodies; Acute heart failure; Gene mutations; Risk factors
Exome Sequencing of Atrioventricular Septal Defects Reveals Rare and de novo Variants in Genes Related to Congenital Heart Disease and Cardiac Development
James R Priest1, Santhosh Girirajan2, Frederick Dewey1, Evan E Eichler3, Bruce D Gelb4, Tom Quertermous1, Euan A Ashley1, Michael A Portman3; 1Stanford Univ Med Cntr, Stanford, CA, 2Pennsylvania State Univ, College Station, PA, 3Univ of Washington, Seattle, WA, 4Icahn Sch of Medicine at Mt. Sinai, New York, NY
Background: Despite the known association of congenital heart disease (CHD) with chromosomal abnormalities such as trisomy 21 and 22q11.2 deletions, 70% of affected infants do not have associated malformations or an identifiable genetic cause. Sporadic cases of atrioventricular septal defects (AVSDs) occur at an approximate rate of 1 in 10,000 births without chromosomal abnormalities. Familial pedigrees have identified highly penetrant variants in a handful of genes associated with related conditions, yet the genetic etiology of sporadic AVSDs remains largely unexplored. Methods and Results: We have performed exome sequencing on a discovery cohort of 41 unrelated trios of children with AVSDs and unaffected parents, and 4 affected individuals from 2 pedigrees. Two separate and robust variant calling pipelines were employed to generate a highly sensitive call set, and protein altering variants were prioritized via likely inheritance patterns and other publically available expression and phenotypic data. Variants of interest were validated by Sanger sequencing or Sequenom genotyping. Preliminary analysis of 25 probands identified likely causal variants in 60% of individuals. Protein altering variants were observed within genes recently associated with other types of CHD (MLL2, KDM5B, MYH6) or with mouse knockout data showing an AVSD phenotype (ENG), and within key signaling pathways and structural proteins expressed in the developing atrioventricular canal (IGF2R, FBN1). Additional protein altering variants were found in genes participating in cardiac development and in genes without a described cardiac function in multiple affected probands. Conclusions: We have identified novel variants in genes previously unknown to be associated with AVSDs, in addition to confirming variants in genes previously associated with other types of CHD. Functional confirmation of individual genes by TALEN engineered zebrafish knockouts is ongoing. These data support the role of rare and de novo coding variants in the pathogenesis of sporadic CHD and may implicate new genes and signaling pathways related to the pathogenesis of AVSDs. A replication cohort of targeted confirmatory sequencing of affected individuals is planned.
Author Disclosures: J.R. Priest: None. S. Girirajan: None. F. Dewey: Consultant/Advisory Board; Modest; Personalis Inc.. E.E. Eichler: None. B.D. Gelb: None. T. Quertermous: None. E.A. Ashley: Ownership Interest; Significant; Personalis Inc.. M.A. Portman: None.
Key Words: Atrioventricular canal; Genetics; Congenital heart disease; Gene mutations; Pediatric cardiology
Global Proteomics Analysis Reveals Parp9 And Parp14 As Novel Regulators Of Macrophage Activation
Hiroshi Iwata, Takuya Hara, Piero Ricchiuto, Alexander Mojcher, Sasha Singh, Masanori Aikawa; Cntr for Interdisciplinary Cardiovascular Sciences, Cardiovascular Div, Brigham and Women’s Hosp, Harvard Med Sch, Boston, MA
Background: Macrophage activation contributes to the development of atherosclerosis and metabolic disorders. A microenvironment dominant in pro-inflammatory (M1) and lacking anti-inflammatory (M2) macrophages may mediate cardiometabolic diseases. We explored key regulators of such macrophage polarization as novel therapeutic targets. Methods and Results: The tandem mass tagging strategy (TMT) was used to quantify the changes in the proteomes of mouse RAW264.7 and human THP-1 cells in response to either interferon gamma (IFNγ) or interleukin 4 (IL-4), M1 and M2 stimuli, respectively. We quantified 5816 proteins from RAW264.7 and 4723 in THP-1 (2 or more peptides, 1% peptide FDR cut-off).. Cluster analysis identified 25 molecules in RAW264.7 and 115 in THP-1, which increased in M1 macrophages but decreased in M2, as candidate regulators of the M1/M2 balance. Of these candidates, poly(ADP-ribose) polymerase 14 (PARP14) was in both RAW264.7 and THP-1 data sets. The role of PARP14 in macrophage activation was validated in both cell lines and human primary macrophages. siRNA silencing of PARP14 induced M1 genes TNF-α, IL-1β and iNOS, while decrease M2 markers Arg1 and MRC1, indicating that PARP14 suppresses M1 proinflammatory macrophage activation and promotes anti-inflammatory M2 polarization. PARP14 silencing induced phosphorylation of STAT1 and reduced STAT6 phosphorylation, suggesting their roles in the underlying signaling mechanisms. Interestingly, PARP14 silencing increased another PARP family member PARP9. In contrast to PARP14, PARP9 appears to promote M1 activation via STAT1 phosphorylation. Furthermore, in primary macrophages derived from PARP14-deficient mice, M1 genes dramatically increased (e.g., 40 times induction of TNF-α), while M2 markers decreased (Figure). Conclusions: PARP9 and PARP14 regulate macrophage polarization, offering novel therapeutic targets for inflammatory cardiovascular disorders.
Author Disclosures: H. Iwata: Employment; Significant; Takuya Hara. Research Grant; Significant; Masanori Aikawa. T. Hara: Employment; Significant; Kowa Company LTD. P. Ricchiuto: None. A. Mojcher: None. S. Singh: None. M. Aikawa: Research Grant; Significant; Kowa Company LTD.
Key Words: Vascular; Inflammation; Arteriosclerosis
Cardiomyogenesis in the Mouse Heart
Barbara Ogorek, Ewa Wybieralska, Yingnan Bai, Giulia Borghetti, Kazuya Isobe, Antonio Canata, Polina Goichberg, Marcello Rota, Annarosa Leri, Piero Anversa, Jan Kajstura; Brigham and Women's Hosp, Harvard Med Sch, Boston, MA
Cardiomyogenesis in the mouse heart has been documented by the presence of cell cycle proteins, BrdU incorporation, and aurora B kinase localization. However, these determinants of myocyte growth are influenced by the length of the phases of the cell cycle. For example, an extended S phase would increase the fraction of BrdU-positive cells, implying a higher turnover rate. To establish the number of left ventricular (LV) myocytes being formed in the young-adult, 2 months, and senescent, 30 months, mouse heart, a double “pulse” labeling protocol was used. Mice were injected with BrdU, and 4 hours later with EdU to measure the proportion of cells tagged by both thymidine analogs, and cells tagged by either BrdU or EdU. BrdU- and EdU-positive cells corresponded to cells in S phase, and cells positive for BrdU only reflected cells that entered G2-M. The BrdU-positive cells in G2-M defined the myocytes generated during the 4 hour interval between the delivery of BrdU and EdU, regardless of the length of the cell cycle and its two major phases, G1 and S.
The nucleotide analogs employed could not discriminate whether DNA replication was associated with karyokinesis in the absence of cytokinesis, or ploidy formation; both would mimic myocyte growth resulting in an overestimation of cell turnover. These variables were measured in isolated cells: 89% of myocytes were binucleated, and mononucleated and multinucleated cells constituted 11% of myocytes in the young and old LV. Moreover, at the two ages, essentially all myocyte nuclei showed a 2n diploid DNA content, 98%; only a small fraction of nuclei was tetraploid, 2%, and octaploid nuclei were not detected.
Based on this premise, we established that in the 2 month-old heart 19.000±7,000 (0.6%), and 580,000±210,000 (18%), new myocytes were formed per day and per month, respectively. Corresponding values at 30 months were 50,000±18,000 (1.8%) and 1.5±0.5 million (54%). Therefore, LV myocytes were replaced entirely in 6 and 2 months in the young and old heart, respectively. Importantly, these rates of myocyte turnover were not influenced by changes in the number of nuclei per cell and polyploidy. In conclusion, the adult mouse heart is a highly dynamic organ that renews its myocyte compartment several times during the course of life.
Author Disclosures: B. Ogorek: None. E. Wybieralska: None. Y. Bai: None. G. Borghetti: None. K. Isobe: None. A. Canata: None. P. Goichberg: None. M. Rota: None. A. Leri: None. P. Anversa: None. J. Kajstura: None.
Key Words: Cardiac regeneration; Cells; Myocardium
Absence of TRPV4 Channels Improves Cardiac Function and Remodeling Following Myocardial Infarction and Transverse Aortic Constriction
Ravi K Adapala1, Daniel J Luther1, Vahagn A Ohanyan1, Jordan Luli1, Roslin Thoppil1, Holly Cappelli1, Sailaja Paruchuri2, William Chilian1, J. Gary Meszaros1, Charles Thodeti1; 1Northeast Ohio Med Univ, Rootstown, OH, 2Univ of Akron, Akron, OH
A major phenomenon that occurs during cardiac remodeling involves the differentiation of cardiac fibroblasts (CF) into highly contractile and hypersecretory myofibroblasts. However excessive contraction and extracellular matrix (ECM) turnover often contributes to cardiac dysfunction and heart failure. In light of the robust mechanical environment present in the heart, our lab recently showed TRPV4, a mechanosensitive ion channel, to play a pivotal role in mediating CF differentiation into myofibroblasts in-vitro, by integrating mechanical and soluble signals. Our current study focuses on the physiological role of TRPV4 during cardiac remodeling, in wild type (WT) and TRPV4 knock out (KO) mice, when subjected to either myocardial infarction (MI) or pressure overload. MI surgeries in the two groups of mice were performed by ligating the left anterior descending (LAD) artery and pressure overload was induced by transverse aortic constriction (TAC). Post-surgery survival studies showed TRPV4 KO mice had improved survival rates compared to the WT mice, while both groups subjected to the MI surgery demonstrated insignificant changes in rates of survival. Cardiac function analysis using 2D and PW Doppler echocardiography, post-MI and TAC surgeries, showed ejection fraction was preserved in TRPV4 KO mice compared to WT mice. Further, cardiac fibrosis assessment using picrosirius red staining depicted TRPV4 KO mice had significantly less fibrosis compared to its WT mice. A comparative gene expression analysis of whole heart tissue showed downregulation of pro-fibrotic genes such as col1A1, col1A2 and TGF-β1 in TRPV4 KO mice compared to the wild type. Interestingly, we found gene expression levels of Rho and a mechanosensitive transcription factor MRTF-α were significantly decreased in TRPV4 KO hearts. Finally, expression of these pro-fibrotic and mechanosensitive genes remained low in TRPV4 KO hearts even after exposure to MI and TAC surgeries. Taken together, these findings suggest the absence of TRPV4 channels improves cardiac function and remodeling following myocardial injury possibly through the inhibition of fibrotic and mechanotransduction pathways.
Author Disclosures: R.K. Adapala: None. D.J. Luther: None. V.A. Ohanyan: None. J. Luli: None. R. Thoppil: None. H. Cappelli: None. S. Paruchuri: None. W. Chilian: None. J. Meszaros: None. C. Thodeti: None.
Key Words: Ion channels; Myocardial infarction; Remodeling; Calcium; Fibrosis
Adventitial Progenitor Cells Are Therapeutically Equipotent To Exogenous Cardiac Progenitor Cells And Interact With Resident Progenitor Cells For Cardiac Repair
Paolo Madeddu, Elisa Avolio, Giuseppe Mangialardi, Helen Spencer; Univ of Bristol, Bristol, United Kingdom
Background: Cardiac progenitor cells (CPCs) are foundation to spontaneous and therapeutic cardiac repair. We investigate if progenitor cells from accessible human tissues exert benefits similar to human CPCs and interact with endogenous CPCs to improve recovery in a murine myocardial infarction (MI) model. Methods&Results: CPCs were isolated from discarded atrial specimens of transplanted hearts and adventitial progenitor cells (APCs) were immunosorted from vein leftovers of patients undergoing coronary artery bypass surgery. Both CPCs and APCs express mesenchymal (CD44/CD90/CD105/vimentin) and pericyte markers (NG2/PDGFRb), but are negative for endothelial and hematopoietic antigens. They secrete similar paracrine factors, including HGF, SCF, VEGF, FGF and angiopoietin1. Moreover, APCs acquire cardiomyocyte antigens (alpha-sarcomeric actinin/NKX2.5/TNNI3/CACNAIC) using 2 different differentiation protocols. Transplantation of APCs or CPCs (300,000/heart) in immunodeficient mice with operative MI improves volume, contractility and pressure indexes at 2 weeks post-MI compared to vehicle, with no additive effect by combined therapy. This was associated to similar improvements of arteriologenesis, cardiomyocyte proliferation and apoptosis and fibrosis. At 6 weeks, however, APCs surpassed CPCs with regard to cardiac output and fractional shortening outcomes. Finally, single or combined therapy equally increased the abundance of endogenous ckit+/αSA-/Tryptase- CPCs in the infarct border zone. The APC conditioned medium (CM) attracts human and murine CPCs. This in vitro promigratory effect was abrogated by proteinase K, but resisted to heating, ribonuclease, inhibitors of tyrosine kinase (genistein), Ser/Thr kinase (staurosporine) and PI3K (LY294002) or blocking antibodies against HGF, SCF, FGF, VEGF and Tie2. The promigratory factor has a MW>10kD and is not associated to secreted exosomes. Proteomics analysis of selected fractions from anion exchange chromatography of APC-CM identified 3 promigratory candidates. Conclusions: APCs provide an alternative means to encourage post-MI cardiac repair and secrete soluble attractive molecules different from those previously implicated in CPC migration.
Author Disclosures: P. Madeddu: None. E. Avolio: None. G. Mangialardi: None. H. Spencer: None.
Key Words: Stem cell therapy; Myocardial infarction; Ischemic heart disease
- © 2013 American Heart Association, Inc.