2014 AHA Late-Breaking Basic Science Abstracts
Late-Breaking Basic Science I
MAVS Mediates Protection against Myocardial Ischemic Injury with Hydrogen Sulfide
David Durrant, Adolfo G Mauro, Juan Valle Raleigh, Anindita Das, Jun He, Khoa Nguyen, Stefano Toldo, Antonio Abbate, Fadi N Salloum; Virginia Commonwealth Univ, Richmond, VA
Background: Hydrogen sulfide (H2S) has been shown to protect against myocardial ischemic and inflammatory injury in part by preserving mitochondrial integrity. Since mitochondrial antiviral signaling (MAVS) protein has been implicated in attenuating Bax-mediated cytochrome c release from mitochondria caused by oxidative stress or ischemia, we sought to determine whether MAVS mediates the cardioprotective effects of H2S. Methods and Results: After baseline echocardiography, adult male wild type (WT) or MAVS KO mice underwent myocardial infarction (MI) by coronary artery ligation for 30 min. followed by 24 h reperfusion. Mice were pretreated with Na2S (100 μg/kg; ip) or saline 1 h before MI. Infarct size, measured with TTC staining, was reduced and LV fractional shortening (FS) was preserved with Na2S at 24h post MI in WT mice as compared to saline-treated mice, but not in MAVS KO mice (Figs. A and B). The risk area was not different between the groups. Western blot analysis revealed a significant decline in myocardial MAVS expression at 24h post MI, which was preserved with Na2S (Fig. C). Another subset of mice was subjected to permanent coronary artery occlusion and treated with Na2S or saline daily for 28 days. LVFS decreased significantly at 28 days post-MI in the saline group, but was significantly preserved with Na2S (Fig. D). Moreover, LV infarct scar size, assessed by trichrome staining, was smaller in Na2S group (22.4 ± 2.7%) as compared to control (33.5 ± 2.1%, P<0.05). Survival rate was 2 fold higher with Na2S compared to saline (P<0.05). Western blot analysis confirmed a significant decrease in MAVS at 28 days after MI, which was associated with increased Bax expression (Fig. E). These changes were blunted with Na2S treatment. Conclusion: Na2S protects against acute MI and prevents MI-induced heart failure in mice possibly through a mechanism involving MAVS. We propose that preserving MAVS with H2S donors can be a promising therapeutic tool for ischemic heart failure.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: D. Durrant: None. A.G. Mauro: None. J. Valle Raleigh: None. A. Das: None. J. He: None. K. Nguyen: None. S. Toldo: None. A. Abbate: None. F.N. Salloum: None.
Key Words: Myocardial infarction; Mitochondria; Infarct size; Contractility; Apoptosis
Thioredoxin-2 Inhibits Mitochondrial Ros Generation and Ask1 Activity to Maintain Cardiac Function
Wang Min; Yale Univ, New Haven, CT
Background: Thioredoxin 2 (Trx2) is a key mitochondrial protein which regulates cellular redox and survival by suppressing mitochondrial ROS generation and by inhibiting Apoptosis Stress Kinase-1 (ASK1)-dependent apoptotic signaling. Methods and Results: Trx2 protein expression level is reduced with a concomitant increase in oxidative stress markers and increased ASK1 phosphorylation/activity in hearts from patients with dilated cardiomyopathy (DCM), when compared to healthy, non-failing hearts. Cardiac-specific Trx2 knockout mice (Trx2-cKO) develop spontaneous DCM at 1 month of age with increased heart size, reduced ventricular wall thickness, and a progressive decline in LV contractile function, resulting in mortality due to heart failure by ~4 months of age. The decline in cardiac function observed in Trx2-cKO mice was accompanied by disruption of mitochondrial ultrastructure, mitochondrial membrane depolarization, increased mitochondrial ROS generation and reduced ATP production, correlating with increased ASK1 signaling and increased cardiomyocyte apoptosis. Chronic administration of a highly selective, small molecule inhibitor of ASK1 (ASK1i) attenuated LV dilation, improved LV function and reduced mortality in Trx2-cKO mice, with significant reductions in oxidative stress, apoptosis, fibrosis and cardiac failure. Cellular data from Trx2-deficient cardiomyocytes demonstrated that ASK1 inhibition reduced apoptosis and reduced mitochondrial ROS generation. Taken together, these data indicate that ASK1 is a major target of Trx2 in the cardiomyocyte and that activation of ASK1 by Trx2 deletion is causally associated with cardiac dysfunction. Conclusion: Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial ROS production and ASK1-dependent apoptosis.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: W. Min: None.
Key Words: Cardiomyopathy; Mitochondria; Heart failure; Apoptosis; Redox
Defective Branched-chain Amino Acids Catabolism Impairs Glucose Metabolism and Exacerbates Cardiac Ischemia/reperfusion Injury
Zhen Zhang1, Tao Li1, Stephen C Kolwicz Jr.1, Haipeng Sun2, Yibin Wang2, Rong Tian1; 1Univ of Washington, Seattle, WA; 2Univ of California, Los Angeles, Los Angeles, CA
The branched-chain amino acids (BCAA), i.e. leucine, isoleucine and valine, are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. BCAA catabolism in mitochondria is regulated by the branched-chain keto acid dehydrogenase (BCKDH) complex. A mitochondria localized phosphatase 2C (PP2Cm) is a key regulator of BCKDH activity. We previously found that PP2Cm deficiency resulted in defect BCAA catabolism, increased oxidative stress, and in zebrafish caused cardiac dysfunction. The PP2Cm knock-out (KO) mice showed normal cardiac function, assessed by echocardiography at 2 months (FS: 44±3 and 43±2% for KO and WT, P=ns). Myocardial high energy phosphate content and the isovolumic contractile function assessed by 31P NMR spectroscopy in isolated perfused hearts, were also normal (PCr/ATP=1.90±0.03 (KO) vs.1.81±0.05 (WT), P =ns). However, 13C NMR isotopomer analysis revealed a significant decrease in the relative contribution of glucose to oxidative metabolism (16±3 vs. 26±2% for KO and WT, respectively, P=0.018) in the KO accompanied by an increase in fatty acid oxidation (51±4 vs. 39±3% for KO and WT, respectively, P=0.020). Glycogen content in the KO hearts was also reduced by >50% (4.4±0.5 vs. 10.9±1.8 μmol glucose/g, P=0.000). Although the KO mice at 2 months had normal blood glucose and insulin levels, 6 months old KO mice developed hyperglycemia (fasting blood glucose: 128±36 vs. 77±6 mg/dl, P=0.036) and hyperinsulinemia (serum insulin: 0.68±0.33 vs. 0.22±0.02 ng/ml, P=0.037). These findings collectively suggest an impairment of glucose metabolism in the KO. When subjected to 25 minutes low-flow ischemia (1% of baseline) and 40 minutes reperfusion, cardiac function recovered to 51±11% in WT (n=7) but only 8±3% in KO (n=11) (P=0.002). The recovery of PCr, ATP, and Pi during reperfusion in the KO also failed to reach the level of WT hearts. Increasing glucose uptake and utilization in the KO by overexpressing insulin-independent glucose transporter GLUT1 (TG) rescued the exacerbated I/R injury. Cardiac function recovered to 49±9% in KO-TG (n=6). In conclusion, our results suggest that defective BCAA catabolism impairs glucose metabolism, which contributes to insulin resistance and exacerbated I/R injury.
Author Disclosures: Z. Zhang: None. T. Li: None. S.C. Kolwicz Jr.: None. H. Sun: None. Y. Wang: None. R. Tian: None.
Key Words: Metabolism; Cardiovascular disease; Glucose; Ischemia reperfusion; Insulin resistance
SCN8A Mutation From a Patient With Sudden Unexplained Death in Epilepsy Displays Alterations in Cardiac Excitability in a Mouse Model
Chad R Frasier, Yangyang Bao, Jacy Wagnon, Miriam H Meisler, Lori L Isom; Univ of Michigan, Ann Arbor, MI
Mutations in voltage-gated Na+ channels (VGSCs) are associated with epileptic encephalopathies, and affected individuals are at increased risk for Sudden Unexpected Death in Epilepsy, or SUDEP. While the underlying mechanisms of SUDEP remain elusive, a growing body of evidence suggests that cardiac arrhythmias resulting from mutant VGSCs expressed in autonomic nerve and/or cardiac myocytes play an important role. Members of our group previously reported a de novo mutation, SCN8A-N1768D, in a patient with epileptic encephalopathy and SUDEP. Comparison of expressed Na+ currents (INa) from wildtype (WT) and mutant Nav1.6 in a heterologous system showed that this mutation causes gain of function in both transient and persistent INa densities. In addition to neurons, Nav1.6 is expressed in heart, where it is preferentially expressed at the T-tubules and contributes to tetrodotoxin-sensitive (TTX-S) INa. To ask whether the SCN8A-N1768D mutation alters cardiac excitability, we generated a SCN8A-N1768D/+ (D/+) mouse. Current clamp recordings in acutely isolated ventricular myocytes revealed an increased incidence of delayed afterdepolarizations (4/5 in D/+ vs. 0/5 in WT cells). Overall action potential (AP) duration was unaltered in mutant myocytes, however, the early repolarization phases of the AP (APD30% and APD50%) were prolonged. The AP upstroke and peak amplitude were unaltered, consistent with the minor contribution of TTX-S VGSCs to the total INa in ventricular myocytes. Surface electrocardiograms exhibited a trend toward decreased heart rate for D/+ mice vs. littermate controls (P=0.06; N≥3/group). Following i.p. administration of 2 mg/kg norepinephrine, this apparent difference in heart rate dissipated. However, following i.p. administration of 120 mg/kg caffeine, 3/3 D/+ mice, but 0 WT animals, showed ECG morphology similar to accelerated idioventricular rhythm. In ex-vivo heart preparations, the decrease in heart rate was no longer seen, suggesting that the observed bradycardia was due to increased parasympathetic drive in mutant mice. We conclude that SCN8A plays a critical role in cardiac excitability and that mutations in this gene result not only in epilepsy but also in cardiac arrhythmias that contribute to the mechanism of SUDEP.
Author Disclosures: C.R. Frasier: None. Y. Bao: None. J. Wagnon: None. M.H. Meisler: None. L.L. Isom: None.
Key Words: Ion channels; Ion currents; Electrophysiology
Late-Breaking Basic Science II
TRPV4 Channel Deletion Improves Cardiac Function and Remodeling Following Myocardial Infarction and Transverse Aortic Constriction via Modulation of Rho/MRTF-A Pathway
Ravi K Adapala1, Roslin Thoppil1, Holly Cappelli1, Vahagn A Ohanyan1, Jordan Luli1, Daniel J Luther1, Sailaja Paruchuri2, William M Chilian1, J. Gary Meszaros1, Charles K Thodeti1; 1Northeast Ohio Med Univ, Rootstown, OH; 2Univ of Akron, Akron, OH
Cardiac remodeling following myocardial injury involves the differentiation of cardiac fibroblasts (CF) into highly contractile and hypersecretory myofibroblasts which secrete extracellular matrix (ECM) components to form a scar. However, excessive contraction and ECM turnover often contributes to cardiac dysfunction and heart failure. We have recently shown TRPV4, a mechanosensitive ion channel, plays a pivotal role in mediating CF differentiation in-vitro through integration of mechanical and soluble signals. In the present study, we investigated the physiological role of TRPV4 during cardiac remodeling following myocardial infarction (MI) or pressure overload (transverse aortic constriction,TAC), in wild type (WT) and TRPV4 knockout (KO) mice. Survival rates following TAC showed TRPV4 KO mice to have improved survival rates compared to WT, but was insignificant after MI. Cardiac function analysis showed preserved ejection fraction in TRPV4 null mice, post MI and TAC surgeries. Further, cardiac fibrosis was analyzed with picrosirius red staining which revealed TRPV4 null mice to have significantly less fibrosis compared to its WT counterpart. To explore the molecular mechanism downstream of TRPV4, we focused on small GTPase Rho. We found that TGF-β1-treatment significantly increased Rho activity, which was inhibited by pre-treatment of the cells with TRPV4 antagonist, AB159908, suggesting that Rho is downstream of TRPV4 in CF differentiation to myofibroblasts. Next, we focused on myocardin-related transcription factor-A (MRTF-A) which was shown to be activated in response to Rho mediated stress fiber formation. Our recent results found that both TGF-β1 and pharmacological TRPV4 activator GSK1016790A induced MRTF-A activation (nuclear translocation), which was abolished when TRPV4 was inhibited. Finally, we found that both TGF-β1 and GSK1016790A induced α-SMA and col1a promoter activities indicating CF differentiation. Taken together, these findings suggest that the TRPV4 channels mediate cardiac fibrosis through a Rho-dependent MRTF-A pathway and the absence of TRPV4 improves cardiac function and remodeling following myocardial injury possibly via down-regulation of this mechanotranscription pathway.
Author Disclosures: R.K. Adapala: None. R. Thoppil: None. H. Cappelli: None. V.A. Ohanyan: None. J. Luli: None. D.J. Luther: None. S. Paruchuri: None. W.M. Chilian: None. J. Meszaros: None. C.K. Thodeti: None.
Key Words: Myocardial infarction; Cardiac hypertrophy; Extracellular matrix; Fibrosis; Ion channels
Identification of a Novel Pathway of Parkin-Mediated Mitochondrial Clearance in Cardiac Myocytes
Babette C Hammerling, Melissa Q Cortez, Rita A Hanna, Rebecca E Jimenez, Asa B Gustafsson; UCSD, La Jolla, CA
Although the primary function of cardiac mitochondria is to provide energy, they can quickly change into death-promoting organelles during stress. Not surprisingly, myocytes have developed a defense mechanism against such aberrant mitochondria. We have previously identified the E3 ubiquitin ligase Parkin as an important regulator of mitochondrial clearance via autophagy in the myocardium. Here, we report that Parkin also induces clearance of mitochondria via an autophagy-independent pathway in cells. We discovered that Parkin promotes clearance of damaged mitochondria via the endosomal-lysosomal pathway in both wild type (WT) and autophagy-deficient Atg5 knockout mouse embryonic fibroblasts (MEFs) treated with the mitochondria uncoupler FCCP. Immunofluorescence analysis revealed that FCCP causes a significant increase in Rab5-positive endosomes with subsequent sequestration of mitochondria inside the endosomes in both WT and Atg5-/- MEFs. The presence of mitochondria inside endosomes was confirmed by transmission electron microscopy (TEM). Thus, these data confirm that the endosomal-mediated mitochondrial clearance is also activated in autophagy competent cells. Interestingly, the activation of the endosome pathway occurs more rapidly than activation of autophagy, indicating that the endosomal pathway acts as the first line of defense against damaged mitochondria. This pathway is ULK1 independent and the endosomes are Rab9 negative, confirming that this pathway is distinct from alternative autophagy. We further found that the endosomal pathway is rapidly activated and clears mitochondria in response to FCCP and simulated ischemia/reperfusion in neonatal myocytes. As in the MEFs, this activation occurs before the activation of autophagy. Pharmacological and genetic inhibition of the endosomal pathway results in increased cell death in both MEFs and myocytes confirming that this pathway is activated as a protective response to clear damaged mitochondria during stress. Thus, we hypothesize that endosomal pathway is the first line of defense against dysfunctional mitochondria, but when the number of damaged mitochondria exceeds the degradation capacity of this pathway, the cells activate autophagy.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: B.C. Hammerling: None. M.Q. Cortez: None. R.A. Hanna: None. R.E. Jimenez: None. A.B. Gustafsson: None.
Key Words: Autophagy; Mitochondria; Ischemia reperfusion
Myocardial Edema After Ischemia/reperfusion is Not Stable and Follows a Bimodal Pattern: Advanced Imaging and Histological Tissue Characterization Study to Challenge a Classical Dogma
Rodrigo Fernandez-Jimenez, Javier Sanchez-Gonzalez, Jaume Aguero, Leticia Fernandez-Friera, Jaime Garcia-Prieto, Carlos Galan-Arriola, Gonzalo Lopez-Martin, Xavier Rossello, María Del Trigo, Gonzalo Pizarro, Jose Manuel Garcia-Ruiz, Ana Garcia-Alvarez, David Sanz-Rosa, Valentin Fuster, Borja Ibanez; Spanish National Cntr for Cardiovascular Rsch (CNIC), Madrid, Spain
Background and Objectives: Accepted paradigm states that after myocardial ischemia/reperfusion (I/R) there is a stable edematous reaction lasting ≈ 1 week. However, most of studies have focused in a single time-point and there are no longitudinal studies covering from very early (minutes) to late (one week) reperfusion stages. Here we comprehensively explore this phenomenon and advance into the pathophysiology behind this edematous reaction. Methods and Results: Closed-chest 40min I/R was performed in 15 pigs, which were sacrificed at 120 minutes (n=5), 24 hours (n=5) and 7 days (n=5) after reperfusion for the quantification of myocardial water content. Cardiac magnetic resonance (CMR) with T2W-STIR and T2-map sequences were performed at every follow-up until sacrifice (i.e. animals sacrificed at day7 underwent baseline, 120min, 24h and day7 CMR). In all animals, edema initially increased abruptly at early reperfusion, exhausted at24 hours, and increased again at day 7 (Fig 1A and 1B). To better understand the mechanism underlying the consistent two separated edematous waves, 10 additional pigs underwent infarction. Five animals underwent ischemia without reperfusion and were sacrificed after the 120min-CMR study (like former group but with no reperfusion). Edema was significantly less than in reperfused animals, suggesting that the initial wave of edema is mostly due to reperfusion itself (Fig 1C). Additional 5 pigs underwent I/R (40min/7 days) with immunosuppression therapy started after 120min-CMR to abrogate the inflammatory reaction. In all pigs, the second wave of edema was significantly attenuated (Fig 1D). Conclusions: Contrary to accepted view, myocardial edema after I/R during the first week follows a bimodal pattern. The first wave of edema peaks in early reperfusion, disappearing at 24 hours and is due to reperfusion itself. Second wave of edema appears days after ischemia/reperfusion and is secondary to inflammatory mechanisms and tissue healing.
Author Disclosures: R. Fernandez-Jimenez: None. J. Sanchez-Gonzalez: None. J. Aguero: None. L. Fernandez-Friera: None. J. Garcia-Prieto: None. C. Galan-Arriola: None. G. Lopez-Martin: None. X. Rossello: None. M. Del Trigo: None. G. Pizarro: None. J. Garcia-Ruiz: None. A. Garcia-Alvarez: None. D. Sanz-Rosa: None. V. Fuster: None. B. Ibanez: None.
Key Words: Ischemia reperfusion; Cardiac MRI; Infarction; Reperfusion
Treatment and Diagnosis of Vascular Thromboembolism Using Thrombus-specific Drug Delivery System Using Nano-polymers
Joo Heung Yoon1, Chang-Sun Kang1, On Hwang1, Soochan Bae1, Byung-Ryul Cho1, Dongwon Lee2, Peter M Kang1; 1Beth Israel Deaconess Hosp, Boston, MA; 2Chonbuk National Univ, Jeonju, Republic of Korea
Treatments and diagnosis of vascular thrombosis are often limited by bleeding complications and lack of specificity. In this study, we develop a novel theranostic application to effectively deliver drugs to the targeted area and specifically image actively forming thrombus using a nano-delivery system. We first generated polymer based nano-micelles that consisted of hydrophilic-hydrophobic interactions of mPEG-PCL (methoxy poly(ethylene glycol)-poly(caprolactone)) and pluronic F-127. A pentapeptide CREKA (H-Cys-Arg-Glu-Lys-Ala-OH), which showed a high affinity to fibrinogen-fibrin complex, was covalently bounded to the micelle. For thrombus specific therapy, we used tirofiban, a glycoprotein IIb-IIIa inhibitor. The half-life of micelle complex was about 15 hours. For therapeutic efficacy of drug-loaded micelle complex, inferior vena cava (IVC) was banded to make an 80-90% stenosis, which resulted in subacute thrombus formation within 24-48 hours. In animals with IVC stenosis, treatment with micelle containing CREKA and loaded with 10% of weight-adjusted therapeutic dose of tirofiban (M/T10/CREKA) showed 65% reduction in thrombus weight compared to 10% of therapeutic tirofiban dose without micelle complex (p<0.05). M/T10/CREKA in fact demonstrated a similar effectiveness as 100% of therapeutic tirofiban dose without micelle suggesting that therapeutic dose is achieved with 10% the amount of tirofiban. For actively forming thrombus imaging, we loaded the nano-polymer with indocyanine green (ICG). There was highly sensitive and specific imaging, as evidenced by near infrared fluorescence, of active thrombogenic area at both 6 (no visible thrombus) and 24 hours (maximal amount of thrombus) after IVC stenosis ICG alone or micelle without CREKA did not show any significant thrombus imaging both at 6 and 24 hours. Treatment of 10% tirofiban loaded micelle prior to imaging also eliminated thrombus imaging. We conclude that our novel nano-polymer drug-delivery system using micelle complex and CREKA targeting may be used for theronostic application for thromboembolism diseases with greater potency and with less bleeding risk.
Author Disclosures: J. Yoon: None. C. Kang: None. O. Hwang: None. S. Bae: None. B. Cho: None. D. Lee: None. P.M. Kang: None.
Key Words: Thrombosis; Venous thrombosis; Anticoagulants; Cardiovascular imaging; Glycoprotein iib/iiia platelet inhibitors
Late-Breaking Basic Science Posters and Reception
p53 Regulates the Myocardial Recovery with Diabetes
Ramaswamy Kannappan1, Eric Y Zhang1, Sergio Signore1, Giorgia Palano1, Joao D Pereira1, Silvana Bardelli2, Marco Moccetti2, Alex Matsuda1, Polina Goichberg1, Marcello Rota1, Piero Anversa1, Tiziano Moccetti2, Annarosa Leri1; 1Brigham and Women's Hosp, Harvard Med Sch, Boston, MA; 2Univ of Zurich, Lugano, Switzerland
The current study had two objectives: a) to determine whether the DNA repair machinery and the growth of cardiac stem cells (CSCs) can be enhanced following DNA damage by modulating the intracellular level of p53; and b) to determine whether the expression of p53 in CSCs conditions their engraftment and survival improving the diabetic myopathy. CSCs were isolated from the heart of WT and superp53 (Sp53) mice, which carry an additional normal-p53 allele. The frequency of gH2A.X foci, which reflects the sites of DNA damage, was 2.6-fold lower in Sp53 CSCs and was coupled with increased cell proliferation, and lower expression of the cell cycle inhibitor p21Cip1 and the senescence-associated protein p16INK4a. Following doxorubicin (doxo) induced oxidative stress, WT and Sp53 CSCs exhibited different adaptations in p53 and p53-target genes. Phosphorylation of p53 at serine-18 was seen in both cell groups, but p53 serine-34 phosphorylation and P53 serine-389 phosphorylation was detected in Sp53 and WT CSCs, respectively. The pro-apoptotic genes Noxa and PUMA were upregulated in WT CSCs only, while a 5-fold increase in p21Cip1 was observed in both Sp53 and WT CSCs. Sp53 CSCs showed an accumulation of the DNA repair protein PCNA and a marked increase in the frequency of gH2A.X foci, indicative of activation of DNA repair mechanisms. Seventy-two hours after doxo removal, proliferation was restored in Sp53 CSCs, but cellular senescence occurred in WT CSCs. To assess the relevance of these results in an in vivo system of enhanced oxidative stress and DNA damage, diabetes was induced by streptozotocin administration in mice and one month later Sp53 and WT CSCs were injected intramyocardially. Sp53 CSCs nested in large number within the diabetic myocardium and differentiated into myocytes and coronary vessels replacing extensive areas of tissue damage. Conversely, cardiac repair was dramatically attenuated after the delivery of WT CSCs. In conclusion, our results strongly suggest that the genetic regulation of p53 function has beneficial effects on the DNA repair response of CSCs reestablishing cell division, which has important consequences on the survival, growth and lineage specification of CSCs potentiating their reparative role in the diabetic heart.
Author Disclosures: R. Kannappan: None. E.Y. Zhang: None. S. Signore: None. G. Palano: None. J.D. Pereira: None. S. Bardelli: None. M. Moccetti: None. A. Matsuda: None. P. Goichberg: None. M. Rota: None. P. Anversa: None. T. Moccetti: None. A. Leri: None.
Key Words: Cardiac regeneration; Diabetes (Type I); Cell signaling; Stem/progenitor cells; Reactive oxygen intermediates
Pharmacologic Blockade and Genetic Deletion of Androgen Receptors Attenuates Abdominal Aortic Aneurysm Formation
John P Davis, Morgan Salmon, Nicolas H Pope, Guanyi Lu, Gang Su, Gorav Ailawadi, Gilbert R Upchurch, Jr; Univ of Virginia Health System, Charlottesville, VA
Background: Testosterone is theorized to play a major role in the pathophysiology of abdominal aortic aneurysms (AAAs) as this disease occurs primarily in males. However, the role of the androgen receptor (AR) in the formation of AAAs has not been well elucidated. It is hypothesized that androgen blockade will attenuate experimental aneurysm formation. Methods: Aortas of 8- to 12-week-old male C57Bl/6 wild type (WT) mice or male androgen receptor knockout (ARKO) mice were perfused with purified porcine pancreatic elastase (0.35 units/ml) to induce AAA formation. Two groups of WT male mice were treated with flutamide or ketoconazole (both AR blockers, 50mg/kg and 150mg/kg) twice daily via intraperitoneal injection. Aortas were harvested on day 14 after video micrometry was used to measure AAA diameter. Cytokine arrays and histologic analysis were performed on aortic tissue. Groups were compared using an ANOVA and a Tukey’s post hoc test. Results: Mean aortic dilation in the elastase WT group was 121±13.8% (mean±SD) compared to ARKO mice which showed marked attenuation of AAA growth diameter (*64.4±22.7% P< 0.0001, Figure). Flutamide and ketoconazole treatment also attenuated AAA formation in WT mice (**84.24±22.8%, P=0.009, and ***91.51±18.24%, P=0.037). Cytokine arrays of aortic tissue revealed decreased levels of proinflammatory cytokines interleukin (IL)-1α, IL-6, and IL-17 in both flutamide-treated and ARKO groups. Conclusions: These data exhibit attenuation of AAA formation after pharmacologic and genetic AR blockade. Therapies for AR blockade commonly utilized in prostate cancer may provide insight into medical treatment to halt the progression of AAAs in humans.
Author Disclosures: J.P. Davis: None. M. Salmon: None. N.H. Pope: None. G. Lu: None. G. Su: None. G. Ailawadi: None. G.R. Upchurch: None.
Key Words: Abdominal aortic aneurysm; Vascular surgery; Vascular disease
Transplantation of Adipose Tissue Mesenchymal Cells Overexpressing Telomerase and Myocardin Promotes Revascularization and Tissue Repair in a Murine Model of Myocardial Infarction
Rosalinda Madonna1, Maria Anna Teberino2, Lyubomir Petrov3, Peter Ferdinandy4, Raffaele De Caterina2; 1Texas Heart Institute - Houston and Univ of Chieti - Italy, Houston, TX; 2Univ of Chieti - Italy, Chieti Scalo, Italy; 3Biocenter Kuopio, A. I. Virtanen Institute for Molecular Sciences, Kuopio, Finland; 4Dept of Pharmacology and Pharmacotherapy, Semmelweis Univ, Budapest, Hungary
Rationale: The success of stem cell therapy is hampered by poor engraftment and survival of transplanted stem cells in the harmful microenvironment of the host tissue. This may be improved by genetically reprogramming the stem cells to delay apoptosis and enhance their regenerative properties. Myocardin (MYOCD), a promyogenic transcription factor with anti-apoptotic activity, and telomerase (TERT), an anti-senescence protein, may synergize to promote survival and cardiomyogenesis of adult mesenchymal cells. Objectives: We examined the therapeutic efficacy of transplanted adipose tissue-derived mesenchymal stromal cells overexpressing MYOCD and TERT (AT-MSCs/TERT-MYOCD) in a murine model of myocardial infarction (MI). Methods: AT-MSCs from adipose tissues of aged (12-month-old) male C57BL/6 mice were efficiently transduced with lentiviral vectors encoding TERT and/or MYOCD. Twelve month-old C57 mice underwent coronary artery ligation (Lig), followed by randomization into 4 groups (n=5/group): Sham operation, MI control (saline 20 μL), MI followed by intramyocardial injection with mock-transduced aged AT-MSCs (2.5x10^5 cells/20 μL), or aged AT-MSCs/TERT-MYOCD (2.5x10^5 cells/20 μL). Results: AT-MSCs/TERT-MYOCD decreased the area of fibrosis (Figure A-D) and increased arteriogenesis (Figure A’-D’) and myocardial fractional shortening when transplanted into the infarcted hearts of C57 mice (n=5, P<0.05, by ANOVA). These effects were accompanied by increased number of Ki-67+ and ckit+ cells (n=5, P<0.05, by ANOVA), and enhanced expression of cardiac actin and promyogenic nuclear transcription factors such as GATA4, Nkx2.5, MEF2c and MYOCD (Figure E). Conclusions: The delivery of the TERT and MYOCD genes into AT-MSCs promoted activation of the cardiomyogenic program, along with vasculogenesis and stem cell survival, and may have applications for use in treating patients with MI.
Author Disclosures: R. Madonna: None. M. Teberino: None. L. Petrov: None. P. Ferdinandy: None. R. De Caterina: None.
Key Words: Adipose; Myocardin; Aging; Myocardial infarction; Cardiac regeneration
The Novel PPARα Selective Agonist K-877 Suppresses Pro-inflammatory Pathways and Experimental Arterial Lesion Formation
Hiroshi Iwata, Kentaro Murakami, Piero Ricchiuto, Sasha Singh, Peter Libby, Elena Aikawa, Masanori Aikawa; Brigham and Women's Hosp, Boston, MA
Inflammatory cell activation contributes to the pathogenesis of atherosclerosis. This study tested the hypothesis that activation of the nuclear receptor PPARα exerts anti-inflammatory effects and attenuates arterial disease using the novel selective agonist K-877. Methods and Results: Computational network-based analysis, using disease genes from public databases revealed significant relationship between the PPARα gene network and the coronary artery disease and diabetes module (p<0.001). In human macrophage cell line THP1, silencing PPARα by siRNA induced expression of TNFα and IL-1β, markers of a pro-inflammatory macrophage slant (“M1”), indicating the anti-inflammatory role of PPARα. In mouse and human macrophages, K-877 suppressed IFNγ or LPS-induced proteins associated with M1 polarization (e.g., TNFα, IL-1β, iNOS, IL-6) at lower concentrations than the conventional PPARα agonist fenofibric acid. The effects of K-877 on the recruitment of cofactors (e.g., PGC-1α/β, SRC) to PPARα at lower concentrations (< 1/1000-7000) than fenofibric acid may contribute to its potent action. K-877 rescued IFNγ-induced suppression of NcoR1 and NcoR2 (SMRT), co-repressors of pro-inflammatory cytokines. In addition, K-877 suppressed other pro-inflammatory pathways, including IFNγ-induced endothelial cell activation, as gauged by decreased expression of proinflammatory VCAM-1 and MCP-1. K-877 administration in Ldlr-/- mice also suppressed hepatic expression of the pro-inflammatory apolipoprotein apoC-III (p<0.01, n=7-8/group, Figure A), which has been linked with coronary risk. To examine its effects on arterial disease, we administered K-877 in LDL receptor deficient (Ldlr-/-) mice. K-877 reduced macrophage accumulation in femoral arteries 28 days after mechanical injury (p<0.05, n=5-7/group, Figure B). Conclusion: The potent PPARα activator K-877 can attenuate experimental arterial diseases in association with anti-inflammatory actions.
Author Disclosures: H. Iwata: None. K. Murakami: Employment; Modest; Kowa Company. P. Ricchiuto: None. S. Singh: None. P. Libby: None. E. Aikawa: None. M. Aikawa: Research Grant; Modest; Kowa Company.
Key Words: Atherosclerosis; Vascular; Inflammation; Ppar
Heart-derived Cell Therapy for Duchenne Cardiomyopathy: Cardiosphere-derived Cells and their Exosomes Improve Function, Restore Mitochondrial Integrity and Reverse Degenerative Changes in the Hearts of Mdx Mice
Mark A Aminzadeh, Rachel Tobin, Rachel Smith, Linda Marbán, Eduardo Marbán; Cedars-Sinai Heart Institute, Los Angeles, CA
Introduction: Cardiosphere-derived cells (CDCs) promote cardiomyogenesis and angiogenesis, while inhibiting oxidative stress, inflammation and fibrosis, in both ischemic and nonischemic cardiomyopathy. The mdx mouse model of Duchenne muscular dystrophy develops cardiomyopathy due to dystrophin deficiency and the resultant intense oxidative stress, inflammation and apoptosis. Here we tested the hypothesis that transplantation of CDCs or of exosomes derived from CDCs (CDC-XO) may be beneficial in mdx mice. Methods and Results: A total of 78 mice were studied at a point when global cardiac dysfunction was already evident by echocardiography. Wild-type syngeneic mouse CDCs (105 cells total), CDC-XO (70μg), or vehicle only were injected intramyocardially in 5 left ventricular (LV) sites in 10-month old mdx mice. LV ejection fraction markedly improved over 3 months after treatment either with CDC or CDC-XO compared to vehicle-treated mice (60.4±1.6 vs 48.1±2.2; p<0.005). The functional improvement was associated with enhanced Nrf2 activation, upregulation of Nrf2 downstream gene products, increased expression of mitochondrial transcription factor A and cellular mitochondrial content, restored expression of mitochondrial respiratory chain subunits, reduced collagen I and III deposition, and attenuated infiltration of inflammatory cells [CD68+ macrophages and CD3+ T cells] in the CDC or CDC-XO-treated mouse hearts. Mitochondrial swelling and disorganization of cristae, prominent in vehicle-treated hearts by electron microscopy, were reversed by CDC treatment. Concomitantly, CDC-treated mdx mice exhibited higher maximal exercise capacity compared to vehicle-treated mice over 3 months of follow up (p<0.05). Conclusions: Cardiac function and exercise capacity improved in mdx mice treated with either CDCs or CDC-XO, accompanied by enhanced activation of the antioxidative Nrf2 pathway, attenuation of inflammation, reduction in collagen content and fibrosis, and augmented cardiomyogenesis in CDC-treated mdx hearts. The findings raise the possibility that CDCs and/or CDC-derived exosomes may be useful therapeutically in patients with Duchenne cardiomyopathy.
Author Disclosures: M.A. Aminzadeh: None. R. Tobin: None. R. Smith: Employment; Significant; Capricor. L. Marbán: Consultant/Advisory Board; Significant; Capricor. E. Marbán: Ownership Interest; Significant; Capricor.
Key Words: Cardiac regeneration; Cardiomyopathy; Cardioprotection
Rnd3/RhoE Plays an Essential Role in the Transition of Heart to Failure
Xiaojing Yue, Xiangsheng Yang, Xi Lin, Tingli Yang, Xin Yi, Yuan Dai, Jiang Chang; Texas A&M University Health Science Cntr, Houston, TX
Introduction: Rho family guanosine triphosphatase (GTPase) 3 (Rnd3, also called RhoE), a member of the small Rho GTPase family, has been suggested to regulate cell actin cytoskeleton dynamics, cell migration, and apoptosis through the Rho kinase-dependent signaling pathway. The biological function of Rnd3 in the heart is unknown. The downregulation of small GTPase Rnd3 transcripts was found in patients with end-stage heart failure.Hypothesis: The loss of Rnd3 has pathological significance in the transition to heart failure. Methods and Results: To investigate the functional consequence of Rnd3 downregulation and the associated molecular mechanism, we generated Rnd3+/- haploinsufficient mice to mimic the downregulation of Rnd3 observed in the failing human heart. Rnd3+/- mice were viable; however, the mice developed heart failure after pressure overload by transverse aortic constriction (TAC). Remarkable apoptosis, increased caspase-3 activity, and elevated Rho kinase activity were detected in the Rnd3+/- haploinsufficient animal hearts. Pharmacological inhibition of Rho kinase by Fasudil treatment partially improved Rnd3+/- mouse cardiac functions and attenuated myocardial apoptosis. To determine if Rho associated coiled-coil kinase 1 (ROCK1) was responsible for Rnd3 deficiency-mediated apoptotic cardiomyopathy, we established a double knockout mouse line, the Rnd3 haploinsufficient mice with ROCK1-null background (Rnd3+/-/ROCK1-/-). Again, genetic deletion of ROCK1 partially but not completely rescued Rnd3 deficiency-mediated heart failure phenotype. Conclusion: In summary, Rnd3 is an essential factor for normal cardiac functions in mice. Animals with Rnd3 downregulation are predisposed to hemodynamic stress and develop heart failure. Activation of ROCK1 is partially responsible for Rnd3 deficiency-mediated cardiomyopathy. Downregulation of Rnd3 correlates with cardiac loss of function as in heart failure patients.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: X. Yue: None. X. Yang: None. X. Lin: None. T. Yang: None. X. Yi: None. Y. Dai: None. J. Chang: None.
Key Words: Apoptosis; Heart failure; Cardiomyopathy; Ventricular remodeling
Pim1 Kinase Functional Effect Depends Upon Intracellular Localization
Kaitlen Samse, Nirmala Hariharan, Pearl Quijada, Mirko Völkers, Lucia Ormachea, Shabana Din, Roberto Alvarez, Jacqueline Emathinger, Sadia Mohsin, Mark A Sussman; San Diego State Univ, San Diego, CA
Background: Human c-kit+ cardiac progenitor cells (hCPC) improve heart function after autologous transfer in heart failure patients. Regenerative potential of hCPCs is severely limited with age, requiring genetic modification to enhance therapeutic potential. A legacy of work from our lab with Pim1 kinase reveals effects on proliferation, survival, metabolism, rejuvenation and regeneration of the heart. We hypothesize that subcellular targeting of Pim1 preferentially bolsters the cardioprotective effects in hCPCs to increase proliferation and survival, supporting the reversion of a senescent phenotype. Methods and Results: Adult hCPCs isolated from patients undergoing left ventricular assist device implantation were engineered to overexpress Pim1 in the whole cell, mitochondria or nuclei. Nuclear-targeted Pim1 preferentially supports phenotypic and biological changes in senescent hCPCs to enhance stem cell youthfulness associated with decreased senescence associated β-galactosidase activity (-30.25%, p<0.001), increased telomere length (1.43 fold, p<0.05) and reduced protein expression of senescence markers, p16 (-49%, p<0.001) and p53 (-31.1%, p<0.001) relative to whole-cell Pim1 overexpression (PimWT). Alternately, mitochondrial-targeted Pim1 works to enhance survival by increasing expression of Bcl-2 and Bcl-XL (2.05 fold, p<0.001 and 1.73 fold, p<0.01 respectively) and decreasing cell death after H2O2 treatment (-9.92%, p<0.01), thereby preserving mitochondrial integrity over PimWT alone. Mitochondrial localization also results in increased proliferation rate (2.78 fold, p<0.001) and subsequent upregulation of cell cycle modulator protein Cyclin D (2.44 fold, p<0.001) and phosphorylated Rb (1.94 fold), correlating with stabilization of hCPC energy metabolism. Changes in proliferation, survival and youthful properties of aged hCPCs are more substantial when expression of Pim1 is localized to the mitochondrial or nuclear compartment, as compared to whole-cell overexpression. Conclusion: Targeted Pim1 overexpression enhances regenerative potential of hCPCs, with the opportunity to select for desired phenotypic properties to overcome patient variability and improve specific stem cell characteristics.
Author Disclosures: K. Samse: None. N. Hariharan: None. P. Quijada: None. M. Völkers: None. L. Ormachea: None. S. Din: None. R. Alvarez: None. J. Emathinger: None. S. Mohsin: None. M.A. Sussman: None.
Key Words: Stem/progenitor cells; Aging; Apoptosis; Heart failure; Cell signaling
Oxidative Stress Modulation of IKACh via PKC Epsilon: Implications for Atrial Fibrillation in the Aging Heart
Guillaume Bassil1, Marian Haburcak1, Diana Slough2, Bo Wang1, Jonas Galper1, Richard Karas1, Yu-Shan Lin2, Sami Noujaim1; 1Tufts Med Cntr, Boston, MA; 2Tufts Univ, Medford, MA
BACKGROUND: Atrial fibrillation (AF) incidence increases with age. Yet, the molecular role of aging in AF remains unclear. It was shown that the constitutively active acetylcholine sensitive inward rectifier potassium current (ca-IKACh), and the activation of PKC epsilon (PKCε) are hallmarks of remodeling in the fibrillating atria. Additionally, cardiac oxidative stress increases in aging. However, we lack direct mechanistic links between aging, ca-IKACh, and AF. We tested the hypothesis that oxidative stress phosphorylates Kir3.1 at residue S185 via PKCε, which leads to ca- IKACh, shortening of action potential duration, and AF perpetuation. METHODS: Oxidative stress (OS) was induced by 100 μM H2O2 treatment for 1 hour. Phospho-S185 Kir3.1 antibody, HEK293 cells transfected with Kir3.1/3.4 and aged (24 months) and young (4 months old) mice were used. We tested our hypothesis with a combination of numerical, molecular and electrophysiological approaches. RESULTS: In HEK cells, OS activated PKCε by promoting its translocation to the membrane, phosphorylated Kir3.1, and increased IKACh from -16.1pA/pF±5.1, n=3 to -34.2pA/pF±3.8, n=6 p<0.01. PKCε silencing prevented OS phosphorylation of Kir3.1, and IKACh increase. In young atrial myocytes, OS shortened APD90 from 98ms±13, n=6 to 37.9±5.3, n=13, p<0.01. TertiapinQ, a selective IKACh blocker, significantly prolonged the APD90 of the stressed but not the unstressed myocytes. Voltage imaging in isolated hearts demonstrated that in the young mouse, OS promoted the formation of rotors and AF. In aged versus young atria, phosphorylation of Kir3.1 was increased by 4 folds, and APD90 was shorter in the aged versus young myocytes (57ms±7, n=6 vs. 98ms±13, n=6 p<0.05). TertiapinQ prolonged the aged, but not the young APD90. Molecular simulations using the chimeric Kir3.1 crystal structure suggested that phosphorylation of S185 leads to structural rearrangements in the intracellular domain of the channel, causing its opening. CONCLUSIONS: Oxidative stress can promote APD shortening and AF, in part through the development of ca-IKACh via activation of PKCε and Kir3.1 phosphorylation. These results could shed light on the complex role of aging in AF and could help in the development of new anti AF agents.
Author Disclosures: G. Bassil: None. M. Haburcak: None. D. Slough: None. B. Wang: None. J. Galper: None. R. Karas: None. Y. Lin: None. S. Noujaim: None.
Key Words: Aging; Atrial fibrillation; Potassium channel; Protein kinase C; Oxidative stress
Mechanisms of Direct PCSK9 Effect on Atherosclerosis
Ilaria Giunzioni1, Hagai Tavori1, Irene M Predazzi1, Lei Ding2, Youmin Zhang3, MacRae F Linton3, Sergio Fazio1; 1Oregon Health and Science Univ, Portland, OR; 2Vanderbilt University, Nashville, TN; 3Vanderbilt Univ, Nashville, TN
Proprotein Convertase Subtilisin Kexin type 9 (PCSK9) interacts with LDLR and causes its intracellular degradation. The regulation of plasma lipid levels by PCSK9 is of obvious relevance to cardiovascular risk, but it is yet unknown if and how LDLR removal from atheroma cells contributes to atherogenesis. We aimed to study the effects of PCSK9 on atherosclerosis in transgenic mice expressing human PCSK9 (hPCSK9tg) on WT, LDLR-/- and apoE-/- background. After high fat diet administration for 8 weeks, we measured the extent of atherosclerosis. As expected, PCSK9 overexpression in WT was associated with a 2-fold increase in cholesterol levels (325±64 vs. 158±44 mg/dl, p<0.05) and increased atherosclerosis development. In the absence of LDLR, hPCSK9 also increased serum cholesterol (1294±91 vs. 1052±233 mg/dl, p<0.05) and triglyceride levels (275±89 vs. 210±29 mg/dl, p<0.05), as well as lesion size (241,013 vs. 159,570 um2/section, p<0.05). We have shown that even in the absence of LDLR, PCSK9 increases VLDL production. Here we found that HepG2 cells overexpressing WT-PCSK9 or the gain of function mutant D374Y have a 2-fold increase in fatty acid synthase mRNA and protein expression, and a 1.5-2.0-fold increase in MTP protein expression and activity (without changes in mRNA levels), suggesting transcriptional and posttranscriptional effects of PCSK9 unrelated to LDLR. Interestingly, hPCSK9 expression increased plaque size also in apoE-/- mice (268,700±26180 vs 189,400±17560 um2 in apoE-/-, p<0.05) in the absence of changes in cholesterol (1066±188 vs. 1039±286 mg/dl) and triglyceride levels (107±9 vs. 91±10 mg/dl). Lesion composition analysis showed increased inflammatory Ly6Chi positive cells in hPCSK9tg/apoE-/- mice relative to apoE-/- controls (6.7±0.2% vs. 5.7±0.4%, p<0.05). Also, increased numbers of splenic CD11b/Ly6Chi positive cells were found in hPCSK9tg/apoE-/- mice and these effects were abolished in the absence of LDLR. Our results show that hPCSK9 expression aggravates atherosclerosis both via increased plasma lipid levels (not exclusively dependent on LDLR) and activation of inflammatory responses (exclusively dependent on LDLR). These observation gives further support to the value of anti-PCSK9 therapies to control CVD risk.
Author Disclosures: I. Giunzioni: None. H. Tavori: None. I.M. Predazzi: None. L. Ding: None. Y. Zhang: None. M.F. Linton: Research Grant; Modest; FH Foundation. Consultant/Advisory Board; Modest; KOWA, Amgen, Merck, LipoScience. Research Grant; Significant; NIH, NHLBI, Merck, Sanofi, Regeneron, ISIS, Genzyme. S. Fazio: None.
Key Words: Atherosclerosis; PCSK9
Role of LDL in PCSK9-mediated Degradation of LDL Receptors
Hagai Tavori1, Ilaria Giunzioni1, Irene M Predazzi1, Sotirios Tsimikas2, P. B Duell1, MacRae F Linton3, Sergio Fazio1; 1Oregon Health & Science Univ, Portland, OR; 2Univ of California San Diego, San Diego, CA; 3Vanderbilt Univ, Nashville, TN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulatory ligand that terminates the lifecycle of low-density lipoprotein (LDL) receptor (LDLR) thus affecting serum LDL-cholesterol (LDL-C) levels. Although recent evidence shows that PCSK9 can be found in direct association with LDL particles in serum, the physiological significance of this finding is not clear. We aimed to study the activity of LDL-bound versus apoB-free PCSK9 and developed a high-throughput method to quantify LDL-bound PCSK9 in serum. We isolated LDL and apoB-free fractions from human serum and serum from PCSK9 transgenic and knockout mice, and determined the degree of: 1. binding to the epidermal growth factor-like repeat A (EGF-A) of the LDLR (in vitro ELISA); and 2. degradation of LDLR in HEK293T cells, which do not produce PCSK9. Our results show that LDL-bound PCSK9 is the only form of serum PCSK9 that directly (Figure 1A, transgenic mice) and competitively (Figure 1B, human subjects) binds EGF-A, and induces LDLR-mediated degradation (Figure 1, insert). Measurement of serum PCSK9 levels is a research method that strives to reach practical applicability, but currently available ELISA methods do not distinguish LDL-bound from apoB-free PCSK9. Thus, we developed an ELISA method to directly measure LDL-bound PCSK9 in serum. We studied serum from 12 subjects (3 with genetic low cholesterol, 6 with normal cholesterol, and 3 with genetic high cholesterol) exhibiting a wide range of PCSK9 levels. Our results show that LDL-bound PCSK9 levels correlate with LDL levels more strongly (r2=0.53, p=0.0075) than do total PCSK9 levels (r2=0.21, p=0.027). Our results suggest that LDL-bound PCSK9 is the active form of PCSK9 in serum and support the notion that measuring levels of LDL-bound PCSK9 is a more precise indicator of PCSK9 effect.
Author Disclosures: H. Tavori: None. I. Giunzioni: None. I.M. Predazzi: None. S. Tsimikas: None. P.B. Duell: None. M.F. Linton: Research Grant; Modest; FH Foundation. Consultant/Advisory Board; Modest; KOWA, Amgen, Merck, LipoScience. Research Grant; Significant; NIH, NHLBI, Merck, Sanofi, Regeneron, ISIS, Genzyme. S. Fazio: None.
Key Words: PCSK9; LDL; Atherosclerosis
A Novel Long Non-coding RNA Contributes to Cardiac Hypertrophy via Epigenetic Remodeling
Zhihua Wang1, Xinghua Wang2, Iris Chen1, Chen Gao1, Ashley Cass3, Xinshu G Xiao3, Shuxun V Ren1, Guangping Li2, Yibin Wang1; 1Div of Molecular Medicine, Depts of Anesthesiology, Physiology and Medicine, David Geffen Sch of Medicine, Univ of California at Los Angeles, Los Angeles, CA; 2Dept of Cardiology, Tianjin Institute of Cardiology, Second Hosp of Tianjin Med Univ, Tianjin, China; 3Molecular Biology Institute, Dept of Integrative Biology and Physiology, College of Life Sciences, David Geffen Sch of Medicine, Univ of California at Los Angeles, Los Angeles, CA
Rationale: Emerging studies have identified thousands of long non-coding RNAs (lncRNAs) in human, while only a few are functionally annotated. Objective: We identified a novel lncRNA, named by us as Cardiac Epigenetics Remodeling Non-coding RNA (Cern), with dynamic expression during pressure-overload-induced heart failure. This study aims to investigate its role in cardiac hypertrophy and the underlying mechanism. Methods and Results: We examined the sub-cellular localization of Cern using single molecule fluorescent in situ hybridization in neonatal rat ventricular myocytes (NRVMs) and RT-PCR following fractionation in mouse hearts. Both showed that Cern was predominantly located in nucleus. Cern knockdown with siRNA significantly suppressed phenylephrin- (PE, 50 μM) induced hypertrophy in NRVM based on hypertrophy marker genes expression and cell size. On the other hand, expression of Cern in NRVM led to hypertrophic growth. The transcriptome changes after Cern knockdown was examined by RNA sequencing which revealed a number of targets commonly regulated by epigenetic modifications. Immunoblotting analysis for histone H3 methylations revealed that methylation at H3 Lys27 (K27) was specifically enhanced by Cern knockdown and reduced by Cern overexpression. RNA co-immunoprecipitation and tagged-RNA pull-down analyses showed that Cern directly bound to enhancer of zeste homolog 2 (Drosophila), a key component of the polycomb repressive complex 2 (PRC2) responsible for H3K27 methylation. This interaction was dramatically enhanced by hypertrophic stimulation. Interestingly, Cern-PRC2 interaction affected other PRC2-binding lncRNAs in a competition manner. Consistently, Cern expression altered H3K27 tri-methylation level at the promoter regions of hypertrophy related genes. Conclusions: Cern is a novel heart-specific and nucleus-localized lncRNA that contributes to cardiac transcriptome reprogramming during cardiac hypertrophy. Cern specifically modifies H3K27 methylation at promoters of targeted genes via competing with other PRC2-binding lncRNAs and negatively regulates PRC2 function. Our findings provide novel insights into lncRNA biology, and identify a novel target for cardiomyopathy therapies.
Author Disclosures: Z. Wang: None. X. Wang: None. I. Chen: None. C. Gao: None. A. Cass: None. X.G. Xiao: None. S.V. Ren: None. G. Li: None. Y. Wang: None.
Key Words: Cardiac hypertrophy; Heart failure; Heart disease; Cardiomyopathy; Molecular biology
Cortical Bone Stem Cells Preserve Cardiac Structure and Function in the Swine Heart after Myocardial Infarction
Thomas E Sharp, III, Remus M Berretta, Timothy Starosta, Sadia Moshin, Hajime Kubo, Steven R Houser; Temple Univ Sch of Medicine, Philadelphia, PA
Introduction: Novel therapies are needed to improve cardiac function after MI; one strategy is to replace lost myocardium. Despite the success of bone marrow- and cardiac- stem cell clinical trials, we’re still searching for the optimal stem cell type most suitable for cardiac regeneration. Previously, we described a novel cell population derived from the cortical bone (CBSCs) which repaired the heart post MI via transdifferentiation and paracrine signaling mechanisms in a mouse model. In the present study, we evaluate the translational potential of CBSCs in swine post MI. Hypothesis: Intramyocardial injection of CBSCs into the MI border zone preserves cardiac structure and pump function via cell retention and transdifferentiation, while enhancing endogenous repair through secretion of paracrine factors. Methods and Results: MI was induced in female Göttingen minipigs by occlusion of the left anterior descending coronary artery (LAD) using an inflated percutaneous transluminal coronary angioplasty (PTCA) balloon for 90 minutes, followed by reperfusion. Animals received either 20 million CBSCs (via 10 intramyocardial injections) or saline injections. Cardiac structure and function was evaluated using echocardiography at baseline and 4 weeks post MI. During the 4 weeks after MI there was depression if pump function in saline treated group (EF: 69.27% ± 1.92 [baseline] to 46.305% ± 3.53 [4 weeks post MI] p<0.0001) and dilation of the LV (EDV: 39.01 ml ± 3.47 [baseline] to 51.5 ml ± 5.67 [4 weeks post MI] and ESV was 13.71 ml ± 2.47 to 27.72 ml ± 3.86 p= 0.0136). The MI + CBSC group had no significant change in pump functional and remodeling, while demonstrating preservation of cardiac structure which recapitulated the inhibition of ventricular remodeling/scar formation. Electromechanical assessment demonstrated regional changes within the LV pre- vs. post-MI; with restoration back towards baseline in the MI + CBSCs group. Conclusion: These data show that administration of CBSCs preserved cardiac structure, therefore improved cardiac function, while altering the regional electromechanical properties 4 weeks post MI. These results suggest that CBSCs are a potential candidate as a novel cell-based therapy for the heart in a preclinical large animal model.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: T.E. Sharp: None. R.M. Berretta: None. T. Starosta: None. S. Moshin: None. H. Kubo: None. S.R. Houser: None.
Key Words: Heart failure; Myocardial infarction; Cardiac regeneration; Stem cell therapy
Harnessing Autophagy to Treat Diabetes Related Cardiac Fibrosis
Yangxin Li1, Zhenya Shen1, Peng-li Xiao1, Bei-bei Lan1, Zhi-wei Zhang1, Xue-yan Jiang2, Jia-ming Xu2, Mang Zhu2, Qiu-xiong Lin3, Yong-jian Geng2, Xi-yong Yu3; 1Institute for Cardiovascular Science & Dept of Cardiovascular Surgery of the First Affiliated Hosp of Soochow Univ, Suzhou, China; 2Sch of Medicine, Univ of Texas Health Science Cntr, Houston, TX; 3Med Rsch Cntr, Guangdong General Hosp, Guangzhou, China
Introduction: Cardiac fibrosis is the leading cause of cardiovascular diseases in patients with diabetes. Excessive accumulation of collagen I secreted by fibroblast is the hallmark of cardiac fibrosis. However, the mechanisms underlying hyperglycemia-induced secretion of collagen I is unknown. Autophagy is the major intracellular degradation system. Here, we test whether autophagy is involved in diabetes related cardiac fibrosis. Methods and Results: To induce diabetes, we injected streptozotocin in C57BL/6 mice for 5 days. One week after injection, blood glucose levels significantly increased in diabetic C57BL/6 mice compared to control. The increased blood glucose was accompanied by increased cardiac fibrosis (Massion Tricrime), and decreased autophagy (increased p62 and decreased LC3-II expression). To explore the mechanisms, we isolated fibroblasts from the hearts of diabetic or control mice. We found no significant difference on fibroblast proliferation (BrdU assay) and fibroblast migration (wound scratch migratory assay) between diabetes and control group. However, primary fibroblasts from diabetic hearts showed decreased autophagy compared to control. The secreted collagen I protein levels were also increased in diabetic primary fibroblast as determined by ELISA. To confirm the role of autophagy, the primary fibroblasts from non-diabetic hearts were treated with either normal levels of glucose (5 mM) or high glucose (HG, 25 mM) for 24 hours. HG treatment led to similar results as primary diabetic fibroblast (decreased autophagy and increased collagen secretion). To decipher the mechanisms mediating HG induced collagen secretion, the primary diabetic fibroblasts were treated with an autophagy inducer rapamycin which inhibits mTORC1 signaling pathway. We found that rapamycin significantly attenuated the secretion of collagen I. However, transfection of the diabetic fibroblasts with siRNA to Rictor, a component of the rapamycin-insensitive mTORC2 signaling has no effects on collagen I secretion. Conclusion: This study demonstrates that fibroblasts contribute to cardiac fibrosis via down regulation of autophagy, and suggests that diabetes related cardiac fibrosis may be prevented by harnessing autophagy with rapamycin.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: Y. Li: None. Z. Shen: None. P. Xiao: None. B. Lan: None. Z. Zhang: None. X. Jiang: None. J. Xu: None. M. Zhu: None. Q. Lin: None. Y. Geng: None. X. Yu: None.
Key Words: Autophagy; Fibrosis; Cardiovascular disease
Fate Mapping of Bone Marrow Cell Transdifferentiation In Vivo
Anna Czarna1, Fumihiro Sanada1, Toru Hosoda2, Sergio Signore1, Piero Anversa1, Marcello Rota1, Annarosa Leri1; 1Brigham and Women's Hosp, Boston, MA; 2Tokai Univ, Isehara, Japan
Lineage tracing protocols can be used to identify the fate of bone marrow cells (BMCs) in the infarcted heart. This technique, however, requires immunolabeling and microscopic imaging, yielding results of difficult interpretation because of the tissue autofluorescence and potential staining artifacts. Additionally, conventional lineage tracing provides information at the cell population level, i.e., the pool of BMCs sharing the fluorescent protein, but leaves unanswered important questions concerning: a) the properties of single BMCs; and b) the functional heterogeneity of the BMC pool. These issues were addressed here by employing viral gene tagging, a methodology widely utilized for the recognition of the origin of circulating blood cells. c-kit-positive BMCs (c-kit-BMCs) were infected with a lentiviral vector carrying EGFP. Lentiviruses integrate permanently in the genome of the transduced cells. The specific site of viral insertion in the DNA of the infected c-kit-BMC is inherited by its progeny and can be utilized as fate mapping marker. EGFP-positive c-kit-BMCs were injected in the border zone of acutely infarcted mice. Two weeks later, infarcted hearts were enzymatically dissociated and EGFP-labeled myocytes, endothelial cells, and c-kit-cells were sorted. In our study, the native fluorescence of EGFP was used exclusively for cell sorting by FACS. DNA was extracted separately from each cell population. By employing a PCR-based method, viral tags were identified in distinct sites of the DNA of the isolated cells. Common insertion sites were found in the DNA of c-kit-BMCs and specialized cells, documenting that single c-kit-BMCs transdifferentiated into multiple cell lineages. However, only specific subsets of c-kit-BMCs generated cardiomyocytes and/or vascular cells, indicating that the ability to transdifferentiate is not common to the entire pool of c-kit-BMCs. In fact, subsets of c-kit-BMCs failed to adopt the myogenic and vasculogenic lineages and maintained their primitive phenotype. In conclusion, our results document that c-kit-BMCs constitute a functionally heterogeneous population. These observations may lead to the identification of the c-kit-BMC with higher regenerative potential for the diseased heart.
Author Disclosures: A. Czarna: None. F. Sanada: None. T. Hosoda: None. S. Signore: None. P. Anversa: None. M. Rota: None. A. Leri: None.
Key Words: Regenerative medicine stem cells; Stem cell therapy; Cardiac regeneration; Heart disease
- © 2014 American Heart Association, Inc.