2017 AHA Late-Breaking Basic Science Abstracts
Late-Breaking Basic Science Oral Abstracts I
Inhibition Of Prolyl-tRNA Synthetase As A Novel Mediator Of Cardiac Fibrosis
Joon Seok Park1, Caroline H Lee1, Sungjin Yoon1, Jong Hyun Kim2, Sunghoon Kim2, Bongyong Lee1; 1Daewoong Pharmaceutical Co., Ltd., Yongin, Korea, Republic of; 2Medicinal Bioconvergence Rsch Center, Seoul National Univ, Suwon, Korea, Republic of
Introduction: Prolyl-tRNA synthetase (PRS), a member of aminoacyl tRNA synthetases (ARS), is an enzyme that conjugates amino acid proline to its cognate tRNA to generate prolyl-tRNA to be used in protein synthesis. Since ARS are essential for protein synthesis and viability, dysregulation of ARS has been proposed to many human diseases. Cardiac fibrosis is characterized by excess production and deposition of ECM proteins by activated fibroblast. Although proline is a main component of collagen and ECM proteins, the exact mechanism and involvement of PRS in cardiac fibrosis has not yet been elucidated.
Hypothesis: We hypothesized that suppression of PRS would down-regulate collagen synthesis, which could be beneficial in cardiac fibrosis.
Results: To validate our hypothesis, we investigated the expression levels of pro-fibrotic markers by overexpression or knockdown of PRS in vitro. We showed that PRS is closely related to the expression of collagen I and α-SMA. Then, we showed that treatment of DWN12088, a novel small molecular selective inhibitor of PRS, reduced expression levels of pro-fibrotic markers in TGFβ-induced fibrotic environments using various cell-lines and primary fibroblasts. Interestingly, we showed that down-regulation of pro-fibrotic markers by DWN12088 is independent of TGFβ signaling pathway, although TGFβ is required for induction of pro-fibrotic markers. In vivo study, we performed transverse aortic constriction in C57BL/6 mice to evaluate the effect of DWN12088 in cardiac fibrosis. TAC results in pressure overload-induced left ventricular hypertrophy and fibrosis, and it is one of the most widely used models to study cardiovascular diseases. 2-week oral treatment of DWN12088 markedly reduced cardiac fibrosis with ED50 of 0.4 mg/kg, based on histological examinations. In addition, we demonstrated that 2-week oral treatment of DWN12088 showed reduction in infiltration of inflammatory cells, left ventricle thickness and accumulation of collagen fiber.
Conclusion: These results suggest that inhibition of PRS attenuates pressure-overloaded cardiac fibrosis and a selective inhibitor of PRS, DWN12088, could serve as a potent anti-fibrotic agent without affecting critical cellular signaling cascade.
Author Disclosures: J. Park: None. C. Lee: None. S. Yoon: None. J. Kim: None. S. Kim: None. B. Lee: None.
Key Words: Enzyme inhibitors; Fibrosis; Heart failure
Cytosolic RBFox1 in Cardiac Fibrosis Regulation
Chen Gao, Yun-Hua Esther Hsiao, Shuxun Ren, Xinshu (Grace) Xiao, Yi Xing, Yibin Wang; UCLA-LOS ANGELES, Los Angeles, CA
RBFox1 is known to be an RNA splicing regulator with enriched expression in cardiac muscle. Loss of RBFox1 expression is a molecular hallmark associated with pathological hypertrophy and heart failure. However, much of our current knowledge about RBFox1 focuses on nuclear RBFox1 with a major impact on global alternative splicing changes in the diseased heart. Yet, RBFox1 gene also generates a cytosolic isoform through alternative splicing (RBFox1c), but the specific function of RBFox1c in heart has not been characterized.
RBFox1c expression is significantly repressed in the mouse failing heart and hypertrophic cardiomyocytes. We performed RNA-seq combined with GO and IPA analysis to determine the impact of RBFox1c expression in culture. Among the genes suppressed specifically by RBFox1c but not the nucleus RBFox1 are groups of pro-inflammatory genes. Both Motif enrichment analysis and de novo motif discovery identified significant enrichment of RBFox1 binding motif in the 3`UTRs of the RBFox1c regulated genes. Using CLIP analysis followed by RT-PCR, we observed RBFox1c, but not nuclear RBFox1 specifically interacted with targeted inflammatory gene 3`UTR. In the cardiac specific RBFox1 knockout mice, enhanced cardiac fibrosis was observed following TAC, associated with elevated expression of RBFox1c dependent inflammatory genes. In contrast, cardiac specific expression of RBFox1c significantly reduced cardiac fibrosis and inflammatory gene expression following TAC, associated with improved ejection fraction and reduced hypertrophic marker gene expression. Further, we tested the effect of RBFox1c expression on cardiac fibrosis response using NRVM conditioned media. We showed the conditioned media from the hypertrophic cardiomyocytes potently induce fibroblast proliferation. However, RBFox1c expression can suppress phenylephrine and isoproterenol induced fibroblasts proliferation.
RBFox1 regulates cardiac transcriptome reprogramming at two post-transcriptional steps. The RBFox1 nuclei isoform regulates global RNA splicing reprogramming in heart, while the RBFox1c regulates inflammatory gene expression and fibrotic remodeling potentially through potential interaction with their 3`UTR and targeted RNA degradation.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: C. Gao: None. Y. Hsiao: None. S. Ren: None. X. Xiao: None. Y. Xing: None. Y. Wang: None.
Key Words: Heart failure; Genomics; Hypertrophy
Optogenetic-induced Mitochondrial Membrane Potential Depolarization and Targeting Cell Death
Patrick Ernst1, Ningning Xu1, KahYong Goh1, Brian O’Rourke2, Jianyi J Zhang1, Margaret X Liu1, Lufang Zhou1; 1Univ of Alabama Birmingham, Birmingham, AL; 2The Johns Hopkins Univ, Baltimore, MD
Introduction: There is growing evidence that mitochondrial dysfunction is closely associated with the development of cardiovascular diseases, although the precise mechanism is not well defined. Normal functioning of mitochondria relies on maintaining the inner membrane potential (a.k.a. ΔΨm) to drive oxidative phosphorylation and redox balance. Thus, developing a tool to induce controlled mitochondrial depolarization and examine the effects on downstream intracellular processes will lead to critical information that helps to reveal the mechanisms underlying mitochondria-induced cellular dysfunction.
Methods and Results: In this study, we developed a new generation optogenetic-based technique for targeting mitochondrial depolarization with light. Particularly, a heterologous light-gated channelrhodopsin protein, named ChR2, was targeted to and expressed on the inner mitochondrial membrane (IMM). We showed that ChR2 formed functional cationic channels on IMM with properties similar to that on the plasma membrane, allowing light-induced targeting ΔΨm depolarization. We also showed that sustained moderate light illumination caused significant cell death in mitochondrial ChR2-expressing cells but not in the mock transfected or mitochondrial YFP-expressing cells. Finally, we provided evidence that the mitochondrial optogenetic-induced cell death is via apoptosis and is independent of the opening of the permeability transition pore.
Conclusion: This new generation optogenetic tool can be used to study the mechanisms how a change of mitochondrial membrane permeability influences cell and organ functions.
Author Disclosures: P. Ernst: None. N. Xu: None. K. Goh: None. B. O’Rourke: None. J.J. Zhang: None. M.X. Liu: None. L. Zhou: None.
Key Words: Cell physiology; Mitochondria; Apoptosis; Cellular Engineering; Cardioprotection
A Peptide Of The Amino-terminus Of Grk2 Induces Hypertrophy And Yet Elicits Cardioprotection After Pressure Overload.
Sarah M Schumacher, Erhe Gao, J Kurt Chuprun, Walter J Koch; Temple University School of Med, Philadelphia, PA
Heart failure (HF) is a leading cause of death worldwide and a growing burden on public health, and the underlying mechanisms of cardiac remodeling and decompensation to HF remain a focus of research efforts towards therapeutic development. Signaling via G protein-coupled receptors (GPCRs) is critical for normal heart function and is tightly controlled by GPCR kinases (GRKs) with GRK2 (originally βARK1), being intimately involved in HF progression. In addition to its well-characterized role in regulating GPCRs, ongoing research has demonstrated great diversity in the functional roles of GRK2. I have recently investigated GRK2 amino terminal binding interactions through the generation of transgenic (Tg) mice with cardiac-targeted expression of the amino-terminal peptide βARKnt (residues 50–145). In a murine model of trans-aortic constriction (TAC)-induced pressure overload, echocardiography revealed increased left ventricular (LV) posterior wall thickness (1.57 versus 1.37 ± 0.02; n = 16) and LV mass in TgβARKnt compared to non-transgenic littermate controls (NLC) 4 weeks after TAC or Sham surgery. Interestingly, despite enhanced hypertrophy at baseline and after acute pressure overload, the progression to HF was paradoxically inhibited in TgβARKnt mice during chronic pressure overload with preserved cardiac function (% Ejection Fraction 57.3 versus 37.3 ± 2.0; n = 11, 10). Further, βARKnt expression limited adverse left ventricular remodeling, with reduced interstitial fibrosis (% area fibrosis 4.1 versus 9.2 ± 0.8; n = 11, 9 hearts) and preserved β-adrenergic receptor density 4 weeks after surgery. The effect of cardiac βARKnt expression was not consistent with alterations in GRK2 activity at GPCRs as neither GRK2 peptide inhibition (TgβARKct) nor GRK2 overexpression alter hypertrophy, and overexpression hastens HF development. Further, TgβARKnt mice exhibit reduced epididymal white adipose content and altered mitochondrial respiration, suggesting altered cardiac metabolism. These data support the idea that the βARKnt peptide embodies a distinct functional interaction and that βARKnt-mediated regulation of β-adrenergic receptor density may provide a novel means of cardioprotection during pressure-overload induced HF.
Author Disclosures: S.M. Schumacher: None. E. Gao: None. J. Chuprun: None. W.J. Koch: None.
Key Words: Heart failure; Ventricular remodeling; Cardiac hypertrophy; Cardiac metabolism
G Protein-coupled Receptor Kinase 2 Negatively Regulates Fatty Acid Utilization and Mitochondrial Bioenergetics
Jessica M Pfleger, Walter J Koch; Temple University, Philadelphia, PA
During cardiac injury or stress, G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression levels and activity are increased, leading to a desensitization of myocardial β-adrenergic receptors (βARs) and contributing to the loss of contractile reserve. Up-regulated GRK2 has been shown to be pathogenic in the post-injured heart and is involved in the promotion of heart failure (HF). There is evidence that GRK2 has other, non-GPCR dependent pathological functions within cardiomyocytes. For example, GRK2 localizes to the mitochondria following oxidative stress, where it acts as a pro-death kinase and decreases fatty acid utilization. As metabolic substrate utilization and bioenergetics are key parameters in the maintenance of cardiomyocyte contractility, our objective is to examine the role of GRK2 on metabolism and bioenergetics in the adult heart. We hypothesize that desensitization of βARs via an increase in GRK2 will result in decreased fatty acid-fueled respiration and will compromise cardiomyocyte function. Conversely, ablation of GRK2 will result in increased respiration and function, under these conditions. Our results show that basal respiration, maximal respiration, and reserve respiratory capacity (RRC) are highest in the presence of palmitate versus glucose (1.6, 3, and 7.2-fold, respectively), accompanied by increased (1.3-fold) ATP levels. Moreover, basal and maximal respiration was decreased (1.6 and 1.2-fold, respectively) cardiomyocytes isolated from cardiac-specific GRK2 transgenic mice. This correlates with a decrease in ATP levels and in vivo cardiac fatty acid uptake (1-fold and 1.6-fold, respectively). Conversely, cardiomyocytes isolated from βARKct, a peptide inhibitor of GRK2, transgenic mice or GRK2 knockout mice, show increased maximal respiration (1.5 and 1.4-fold, respectively) and RRC (2.7 and 2.2-fold, respectively) with fatty acids. This correlates with an increase in ATP levels (1.2-1.9-fold). Thus, we propose that increased GRK2, as seen during heart failure, compromises fatty acid-driven mitochondrial respiration, while GRK2 inhibition under these conditions enhances RRC, which is known to improve cellular survival during stress.
Author Disclosures: J.M. Pfleger: None. W.J. Koch: None.
Key Words: Adrenergic; Energetics; Metabolism; Mitochondria; Mitochondrial energetics; heart failure; arrhythmias
Late-Breaking Basic Science Oral Abstracts II
Exercise Instigates Apoptosis-inducing Factor Nuclear Translocation and Myocyte Death in Arrhythmogenic Cardiomyopathy
Stephen P Chelko1, Gizem Keceli1, Peter Andersen1, Nuria Amat-Codina1, Jacopo Agrimi1, Djahida Bedja1, Marcus Stahlberg2, Marc Halushka1, Cynthia A James1, Hugh Calkins1, Daniel P Judge1, Nazareno Paolocci1; 1Johns Hopkins School of Medicine, Baltimore, MD, 2Karolinska Institutet, Stockholm, Sweden
Exercise increases disease penetrance in arrhythmogenic cardiomyopathy (ACM). Yet, how exercise contributes to disease pathogenesis is unclear. Mitochondria potentiate reactive oxygen species (ROS) generation during exercise that are scavenged by antioxidants, such as thioredoxin-2 (Trx2). Here we tested if deficits in Trx2-based ROS buffering act as substrates for exercise-induced cardiac apoptosis in ACM. Homozygote Desmoglein-2 mutant mice (Dsg2mut/mut) model ACM features, thus WT and Dsg2mut/mut mice were enrolled in a 10 week swimming protocol to evaluate survival and post-effort cardiac function, ROS production by EPR, and mitochondrial ROS-gating protein levels. Survival rate after swimming was 91% (20/22) in WT, but only 60% (15/25) in Dsg2mut/mut mice (p=0.008). Of the survivors, Dsg2mut/mut mice displayed cardiac dysfunction (Ejection Fraction 57±4 vs 84±0.4% in WT; n≥14/cohort, P<0.001) and increased bouts of non-sustained VT. Swimming augmented ROS emission in Dsg2mut/mut right ventricles (RV) than in WT (25±3 vs 15±1 Gauss/total protein [G/tp]; P<0.05) and amplified ROS release from Dsg2mut/mut left ventricles (LV) (39±5 G/tp vs mutant RV; P<0.05). Isolated mutant mitochondria showed reduced NADH generation and peroxiredoxin-3, Trx2 and Trx2 reductase (Trx2R) protein levels, with impaired Trx2R activity (all P<0.05 vs WT in RV&LV). When mitochondria bound, Apoptosis-Inducing Factor (AIF) acts as a NADH oxidoreductase, yet upon oxidation, AIF translocates to the nucleus and initiates apoptosis. After exercise, Dsg2mut/mut hearts displayed marked AIF nuclear and chromatin-bound protein levels and increased AIF immunostained nuclei vs WT. Additionally, direct H2O2 exposure or Trx2 inhibition (by auranofin) in Dsg2mut/mut embryonic stem-cell derived myocytes elevated AIF-nuclear translocation and apoptosis (via AnnexinV/PI FaCS analysis). Our study reveals a novel causal link between exercise-evoked cardiac redox imbalance and aberrant AIF-Trx2 signaling in ACM, which was associated with increased apoptosis, propensity of arrhythmias and sudden cardiac death. These findings offer a new targetable mechanism for preventing one of the most cited, yet poorly understood, pathological phenotypes (apoptosis) in ACM.
Author Disclosures: S.P. Chelko: None. G. Keceli: None. P. Andersen: None. N. Amat-Codina: None. J. Agrimi: None. D. Bedja: None. M. Stahlberg: None. M. Halushka: None. C.A. James: None. H. Calkins: None. D.P. Judge: None. N. Paolocci: Ownership Interest; Significant; Cardioxyl Pharmaceuticals Inc/Bristol-Meyers Squibb.
Key Words: Apoptosis; Exercise tests and training; Cardiomyopathy; Redox; Ventricular arrhythmia
Heart Failure Induced Upregulations of MicroRNAs in the Human Sinoatrial Node Associated with Pacemaker Dysfunction
Ning Li1, Maria Petkova2, Brian J Hansen1, Prosper S Ssekayombya1, Joseph Yanni2, Brandon J Biesiadecki1, Ahmet Kilic1, Peter J Mohler1, Paul M Janssen1, Jonathan P Davis1, Federica Accornero1, Mark R Boyett2, Halina Dobrzynski2, Vadim V Fedorov1; 1The Ohio State University, Columbus, OH, 2Univ of Manchester, Manchester, United Kingdom
Background: Heart failure (HF), a leading cause of morbidity and mortality, involves significant dysfunction of the sinoatrial node (SAN). MicroRNAs (miRs) are abundant, non-coding RNAs that ultimately regulate protein expression at the post-transcriptional level and have been implicated in HF. However, nothing is known about the distribution and expression of miRs in the human SAN as well as their roles in regulating pacemaker channels and SAN dysfunction (SND) in HF.
Methods: The human SAN was isolated and cryopreserved from failing hearts with implantable pacemakers (n=5) or non-diseased donor hearts (n=3) that were cardioplegically arrested in the surgery room. Utilizing immunohistochemistry, the intact 3D intramural SAN structure was precisely identified as the fibrotic region around the SAN artery containing Connexin43-negative pacemaker cardiomyocytes (Figure). Small biopsies from the central SAN compartment were used to characterize the expression pattern of 14 miRs and their associated pacemaker channels. Ingenuity software (Qiagen) was used to predict the relationship between miRs and their targeted mRNA of SAN ion channels
Results: Out of 14 studied miRs, 3 miRs (mir-370-3p miR-133-3a and miR-1) were significantly upregulated in SAN, but not in atria, during HF. We found that mir-370-3p was predominantly expressed in the human SAN, but not atria. Whereas, both miR-133-3a and miR-1 were highly expressed in atria vs SAN. All three miRs were predicted to regulate pacemaker HCN1 and/or HCN4 channel expression. RT-PCR showed that HCN1 and HCN4 mRNA were downregulated in the HF SAN.
Conclusions: This is the first study to explore the miRs profiles in healthy and diseased human SAN with SND. We demonstrate that miR-370, miR-133a and miR-1, thought to regulate SAN pacemaker HCN channel expression, are selectively upregulated in human HF SAN. We propose that understanding the function of miRs in human SAN might lead to novel SND treatments.
Author Disclosures: N. Li: None. M. Petkova: None. B.J. Hansen: None. P.S. Ssekayombya: None. J. Yanni: None. B.J. Biesiadecki: None. A. Kilic: None. P.J. Mohler: None. P.M.L. Janssen: None. J.P. Davis: None. F. Accornero: None. M.R. Boyett: None. H. Dobrzynski: None. V.V. Fedorov: None.
Key Words: Sinoatrial node; Heart failure; MicroRNA
Machine Learning Detection of Obstructive Hypertrophic Cardiomyopathy Using a Wearable Biosensor
Eric M Green1, Reinier van Mourik2, Charles Wolfus1, Stephen B Heitner3, Onur Dur2, Marc J Semigran1; 1MyoKardia, South San Francisco, CA, 2Wavelet Health, Mountain View, CA, 3Oregon Health Sciences Univ, Portland, OR
Introduction: Hypertrophic cardiomyopathy (HCM) is a heart muscle disease characterized by left ventricular (LV) hypertrophy without a systemic etiology and is associated with heart failure, stroke and sudden death. Disease prevalence is estimated at 1:500, but ~84% remain undiagnosed. Patients with obstructive HCM (oHCM) have dynamic obstruction of the LV outflow tract and characteristic abnormalities in arterial bloodflow patterns.
Hypothesis: Arterial pulsewaves recorded with a wearable biosensor and analyzed with machine learning algorithms could identify a signature of oHCM when compared to unaffected controls.
Methods: We compared baseline arterial pulse wave morphology, obtained by photoplethysmography using an investigational wristworn biosensor (Wavelet Health, Mtn. View, CA), from oHCM patients enrolled in a digital health substudy of PIONEER HCM (NCT02842242) to unaffected controls from a Wavelet Health database. Five minute recordings were obtained at rest, and data sets were divided into training and validation cohorts. A beat-by-beat machine learning model was developed using a predefined feature set to calculate an HCM probability score, and an optimal threshold score was determined. The model was evaluated using summary statistics and an ROC area-under-curve metric.
Results: Arterial pulsewave recordings were obtained from 14 patients with oHCM at rest and 81 unaffected controls. An oHCM machine learning classifier was developed based on 42 calculated metrics. After training and cross-validation (n=9 oHCM, n=48 control), the model achieved 98% accuracy. Application of this model to a validation cohort (n=5 oHCM, n=33 control) confirmed an increased probability in oHCM patients compared to unaffected controls (0.40 ± 0.13 vs. 0.18 ± 0.10; p=0.006). Analysis of the ROC curve in the pooled cohort shows an area under the curve of 0.88.
Conclusion: This first-of-its-kind study suggests that a signature of arterial bloodflow in oHCM can be identified with the combination of a wristworn biosensor and machine learning algorithms. These data raise the possibility of a novel approach to the non-invasive detection of oHCM.
Author Disclosures: E.M. Green: Employment; Significant; MyoKardia. Ownership Interest; Significant; MyoKardia. R. van Mourik: Employment; Significant; Wavelet Health. Ownership Interest; Significant; Wavelet Health. C. Wolfus: Employment; Significant; MyoKardia. Ownership Interest; Significant; MyoKardia. S.B. Heitner: None. O. Dur: Employment; Significant; Wavelet Health. Ownership Interest; Significant; Wavelet Health. M.J. Semigran: Employment; Significant; MyoKardia. Ownership Interest; Significant; MyoKardia.
Key Words: Hypertrophic cardiomyopathy; mHealth; Big Data
Induced Pacemaker Spheroids As A Model To Reverse-Engineer The Native Sinoatrial Node
Sandra I Grijalva1, Jung H Sung2, Benjamin W Furman3, Pengcheng Han3, Jun Li3, Hee C Cho1; 1Emory Univ/Georgia Institute of Technology, Atlanta, GA, 2CHA Bundang Med Cntr, Seoul, Korea, Republic of3Emory Univ, Atlanta, GA
Background: The sinoatrial node (SAN) has intricate architecture, which facilitates the spontaneous action potentials generated from the SAN to pace and drive the neighboring myocardium. We sought to create an engineered SAN that recapitulates the native SAN’s ability to overcome source-sink mismatch. We hypothesized spheroids consisting of induced pacemaker cells (iPM) can pace and drive surrounding quiescent myocardium.
Methods:The iPMs were created by singular expression of a transcription factor, TBX18, to neonatal rat ventricular myocytes as we have previously demonstrated. The iPM or GFP-spheroids (control) were created by subjecting the NRVMs to the AggreWell™ plate at 1000 cells/well, and cultured for three days in suspension. Spheroids consisted of 90% NRVM and 10% cardiac fibroblast.
Results: iPM-spheroids demonstrated spontaneous pacing at 145±26 bpm compared to 66±1 bpm (p=6.5770E-9) of control, GFP-spheroids. When one iPM-spheroid was surrounded by a monolayer of quiescent NRVMs (iPM:NRVM=1:100), the iPM-spheroids were able to pace and drive the quiescent ventricular myocardium at a capture rate of 48±7%. In contrast, the random activity of control spheroids captured the myocardium at 7±4% (p=0.0078). A monolayer of TBX18 cells (1:4=TBX18:NRVM) failed to pace and drive the neighboring sheet of ventricular myocytes. iPM-spheroids had a 17-fold increase in SAN-specific gap junction, Cx45, transcripts (p=0.0001) and a 2-fold decrease in myocardial gap junction, Cx43 (p=0.0030), compared to GFP-spheroids. The iPM-spheroids have superior viability compared to control GFP-spheroids, 87±1% and 72±5% respectively (p= 0.0463). TUNEL staining confirmed apoptotic fibroblast in the periphery. When cultured for >2 weeks, iPM-spheroids demonstrated small α-sarcomeric actinin positive cells organized as a mesh in the core, similar to the pacemaker cells in the native SAN.
Conclusion: iPM-spheroids can pace and drive surrounding quiescent myocardium, overcoming the source-sink mismatch. The iPM-spheroids are viable in long-term and exhibit native SAN-like pacemaker cell organization. These data provide an in vitro platform on which the design principles of native SAN could be tested.
Author Disclosures: S.I. Grijalva: None. J.H. Sung: None. B.W. Furman: None. P. Han: None. J. Li: None. H.C. Cho: None.
Key Words: Sinoatrial node; Pacing
Regional Assessment of Pyruvate Metabolism in the Remodeled Heart Using Dynamic Nuclear Polarization Carbon13 Magnetic Resonance
James J Pilla1, Terence Gade1, Gabor Mizsei1, Norman Butler1, Yoshiaki Saito1, Akito Imai1, Keitaro Okamoto1, Christopher L Gade2, Gabrielle Pilla1, Jerry Zsido, II1, Joseph H Gorman, III1, Robert C Gorman1; 1Univ of Pennsylvania, Philadelphia, PA, 2Weill Med College of Cornell Univ, New York, NY
Introduction: After infarction compensated remodeling of the left ventricular (LV) may be followed by adverse remodeling leading to heart failure. The mechanism of adverse remodeling maybe linked to the elevated wall stress in the dysfunctional myocardium adjacent to the infarct (border zone BZ). We hypothesize that increased BZ stress results in altered metabolism which could drive the transition from compensated to adverse remodeling. To evaluate BZ and remote metabolism we compared the regional uptake and intracellular conversion of 1-13C-pyruvate using hyperpolarized (HP) 13C MR.
Methods: An established pre-clinical posterolateral infarct model of LV remodeling was used to investigate region metabolism. To accurately measure regional metabolism, we developed implantable carbon-tuned surface coils placed on the epicardium over the BZ and remote regions (Fig. Top). A coronary catheter was placed for direct injection of the HP substrate to maximize deliver and eliminate cavity blood pool signal. MR was performed at 6-weeks post infarct with a spectra acquired every 1.5s for each region simultaneously during HP infusion under physiologic and DOB stress conditions. The resulting spectra from each coil were analyzed to measure lactate, alanine, bicarb, and total flux.
Results: Under physiologic (Pre-DOB) conditions the percent difference between remote and BZ lactate, alanine, and total flux was only slightly elevated in the remote region whereas bicarb flux was greater in BZ compared to remote (Fig. Bottom). DOB stress produced an increase in remote metabolite flux compared to BZ with lactate, alanine and total flux reaching significance and bicarb flux shifting from greater in BZ Pre-DOB to greater in remote.
Conclusion: These findings demonstrate an impaired metabolic response to pharmacologic stress in BZ myocardium which may provide a mechanism for the established association of mechanical stress and adverse cardiac remodeling following infarct.
Author Disclosures: J.J. Pilla: None. T. Gade: None. G. Mizsei: None. N. Butler: None. Y. Saito: None. A. Imai: None. K. Okamoto: None. C.L.F. Gade: None. G. Pilla: None. J. Zsido: None. J.H. Gorman: None. R.C. Gorman: None.
Key Words: Ventricular remodeling; Cardiac metabolism; Cardiac MRI; Imaging agents; Ischemic heart disease
Late-Breaking Basic Science Posters
Therapeutic Effects of Human Pluripotent Stem Cell-derived Lymphatic Endothelial Cells Encapsulated With Nanomatrix Gel on Experimental Lymphedema
Shin-Jeong Lee1, Young-Doug Sohn2, Dongchan Sohn1, Young-sup Yoon3; 1Yonsei University, Seoul, Korea, Republic of; 2Emory University, Atlanta, GA, 3Emory University, Atlanta, GA
Background: Current systems generating lymphatic endothelial cell (LEC) from human induced pluripotent stem cells (hPSCs) have limited value due to low purity, the use of undefined components for differentiation, and poor cell survival in vivo. Here, we developed a fully defined system to differentiate hPSCs into LECs and evaluated their therapeutic and engraftment potential when encapsulated in a nanomatrix gel (PA-RGDS).
Methods and Results: hPSCs were cultured with GSK3β inhibitor on collagen-coated plates for 2–3 days to induce differentiation into the mesodermal lineage. The mesodermally differentiated cells were then cultured with VEGFC, VEGFA, EGF, and bFGF for another 6 days and double-sorted by PDPN and FLT4. These hPSC-PDPN+FLT4+ cells (hPSC-derived lymphatic endothelial cells, hPSC-LECs) showed highly purified and fully functional LEC characteristics in vitro. These hPSC-LECs express LEC markers such as PDPN, LYVE1, PROX1, and FLT4 at the mRNA level and the protein level, and formed tube-like structures in Matrigel. We next determined the lymphatic vascular reparative effects of engineered hPSC-LECs. After inducing lymphedema in the tail of mouse, hPSC-LECs, hPSC-LECs encapsulated with PA-RGDS, human dermal lymphatic endothelial cells (hdLEC), PA-RGDS, or PBS were injected into the tail of mouse. Tail thickness significantly decreased in the groups injected with hPSC-LECs with or without PA-RGDS compared to the other groups at day 28. At day 45, mice injected with PA-RGDS encapsulated hPSC-LECs showed significant decrease in the tail diameter compared to all other groups including those injected with hPSC-LECs. Histological examination demonstrated that the skin thickness was significantly reduced and the density of lymphatic vessels was markedly increased when the hPSC-LECs were encapsulated with PA-RGDS compared to others.
Conclusion: This study demonstrated for the first time that hPSC can be differentiated into LECs in a clinically compatible manner with a high yield. Furthermore, nanomatrix encapsulated hPSC-LECs can substantially improve lymphedema in mouse tail through enhancement of cell survival and lymphatic neovascularization. This engineered hPSC-LEC therapy represents a novel option for treating lymphedema.
Author Disclosures: S. Lee: None. Y. Sohn: None. D. Sohn: None. Y. Yoon: None.
Key Words: Stem cell therapy; Lymphatic disease; Valvular
Differential, Sex Related, Response To High-fat Diet In Mice Impairs Cardiac Autophagy And Mitophagy
Amandine Thomas, Stefanie Marek, Kyle C Tucker, Allen M Andres, Roberta A Gottlieb; Cedars Sinai Med Cntr, Los Angeles, CA
Introduction: Premenopausal women, as well as females in animal studies, have a reduced risk of cardiovascular diseases and a reduced myocardial susceptibility to ischemia/reperfusion (I/R) injury. However, with constantly increasing prevalence of obesity, the impact of diet-induced obesity on females remains unclear. Mechanisms of autophagy and mitophagy, differentially regulated between males and females, have been shown to be cardioprotective and impacted under nutritional overload.
Hypothesis: Autophagy and mitophagy pathways can be involved in both female cardio protection and deleterious effect of obesity.
Methods: Male and female C57Bl/6J mice (n =20 per group) of 8 weeks old were fed a low-fat (LFD, 10% fat) or high-fat (HFD, 60% fat) diet for 12 weeks. At 12 weeks, hearts from mice were studied under basal conditions and after global no-flow ischemia and reperfusion on a Langendorff perfusion system.
Results: In both males and females, HFD significantly increases body weight (31.4g vs 48.3g and 22.1g vs 39.9g respectively), fat mass (+200% in males and +300% in females) and blood glucose (+79mg/dl and + 54mg/dl, respectively) (all p<0.01). HFD tends to decrease flow reperfusion after global no-flow ischemia and to increase infarct size (p=0.08 in female and p=0.1 in male, n=5) in both sexes. In males and females, HFD increases pSer757-Ulk1 and decreases Parkin levels under basal conditions (all p<0.006). Moreover, HFD tends to decrease pThr172-AMPK only in females. After I/R, Parkin levels remain lower in HFD groups without sex-difference (p<0.004) but a drastic increase of LC3-II occurs only in females under HFD (p=0.047).
Conclusion: All together, these results suggest an impairment of autophagy and mitophagy pathways under HFD. A more drastic change occurs in female mice and may imply a more important response of female mice to HFD, although further studies are needed. However, this result suggests that cardiovascular disease may be more deleterious in women despite their lower cardiovascular risk; additional studies examining sex differences in the response to metabolic syndrome and ischemic heart disease are warranted.
Author Disclosures: A. Thomas: None. S. Marek: None. K.C. Tucker: None. A.M. Andres: None. R.A. Gottlieb: None.
Key Words: Ischemia reperfusion; Obesity; Sex differences; Autophagy; Mitochondria
ZMYND8 Regulates the Hypoxic Response in Cardiomyocytes
Kathryn J Schunke, Chad B Walton, Ralph V Shohet; John A Burns Sch of Medicine, Honolulu, HI
Introduction: The Zmynd8 gene contains a variety of recognized motifs including a bromodomain and PHD finger, which are implicated in cooperative binding to acetylated nucleosomes. It was recently identified as a key regulator of cancer progression and transcriptional repression by directing modification of histone methylation in the enhancer regions of genes. Mutations and dysregulation of Zmynd8 have been found in multiple cancer types, however its role in the cardiac myocardium is unexplored. Hypoxia-inducible factor 1α (HIF-1α) upregulation and stabilization is a common feature of both cancer and myocardial ischemia, promoting cellular functions such as proliferation, glucose metabolism and angiogenesis. Therefore, we investigated the role of Zmynd8 in the pathophysiology of cardiac ischemia in a model of HIF-1 activation.
Hypothesis: Zmynd8 modulates the HIF-1 response in ischemic heart disease by inhibiting enhancer activity of HIF-1 target genes.
Methods & Results: We expressed an inducible cardiac-specific, oxygen-stable form of HIF-1α in a mixed strain of mice that genetically express increased Zmynd8 (Z+). Compared to HIF-induced wild-type (WT) mice, these mice did not exhibit the expected HIF-1 phenotype of increased heart weight to body weight (6.4±0.3 vs. 4.8±0.2 vs. 5.1±0.3 mg/g, WT vs. Z+ vs. uninduced; p<0.05) with reduced ventricular function and associated chamber dilation. Semi-quantitative qPCR analysis of HL-1 and H9C2 cardiac cell lines transfected with CMV-expression plasmids for Zmynd8 and oxygen-stable HIF-1α resulted in striking reduction of multiple HIF-1 target genes such as PDK1 (45% reduction) compared to the HIF-1α plasmid alone. RNAi mediated knockdown of Zmynd8 alleviated this negative regulation (65% increase). Bioinformatic analysis of human Zmynd8 and HIF-1α ChIP-seq data indicates that Zmynd8 binds to the enhancer of 78% of HIF-1 regulated genes. This further supports our observation that Zmynd8 modulates HIF-1 activity in the heart.
Conclusion: We have discovered a new regulator of HIF-1 action that modifies the hypoxic response, likely through chromatin remodeling. We suggest that this new form of regulation can modify the pathophysiology of ischemia and potentially provide new targets for therapy.
Author Disclosures: K.J. Schunke: None. C.B. Walton: None. R.V. Shohet: None.
Key Words: Hypoxia; Epigenetics
Alpha Calcitonin Gene-Related Peptide (CGRP) Protects Against Pressure-induced Heart Failure
Ambrish Kumar1, Scott Supowit1, Jay D Potts1, Donald J DiPette2; 1Dept of Cell Biology and Anatomy, Sch of Medicine, Univ of South Carolina, Columbia, SC, 2Dept of Internal Medicine, Sch of Medicine, Univ of South Carolina, Columbia, SC
Introduction: Calcitonin gene-related peptide (CGRP), a 37 amino acid neuropeptide, is a potent vasodilator, and plays a critical role in the efferent functions of the sensory nervous system. A protective role for CGRP in cardiovascular diseases (hypertension, cardiac ischemia, and failure) has been well established by our laboratory and others. In the present study we determined whether long-term exogenous administration of α-CGRP protects against pressure-induced heart failure.
Method: Three groups of nine-week-old C57/BL6 mice were studied: one group received a sham procedure (n= 4) and two groups underwent transverse aortic constriction (TAC). Two days after TAC, one group had CGRP-filled osmotic pumps (4 mg/kg bwt/day) implanted subcutaneously (n= 7) while the second group was TAC-only (n=6). At day 28, all groups had echocardiography performed and were sacrificed and heart tissue collected.
Results: Echocardiographic and histological data showed that TAC markedly decreased fractional shortening (FS) and ejection fraction and increased heart and lung weight, cardiac hypertrophy, and fibrosis compared to sham. However, the TAC-CGRP mice had preserved cardiac function and less cardiac fibrosis (FS ±SEM: sham 46.2±1.8% vs TAC 25.4±1.1%, p< 0.001; and TAC 25.4±1.1% vs TAC-CGRP 36.6±1.2%, p< 0.001). CGRP significantly reduced apoptotic cell death and lipid peroxidation (an oxidative stress marker measured by malondialdehyde and 4-HNE staining) in the TAC hearts [malondialdehyde (nmol/mg protein) ±SEM: sham 3.5±0.19 vs TAC 14.3±0.57, p< 0.05; and TAC 14.3±0.57 vs TAC-CGRP 5.5±0.12, p< 0.05]. TAC alone decreased the level of p-ERK1/2 and increased p-JNK compared to sham. CGRP-TAC hearts had higher p-ERK1/2 levels but equal p-JNK levels compared to the TAC hearts. HIF1α and nrf2 protein levels were not different between experimental groups. Compared to TAC hearts, TAC-CGRP hearts had lower p-AMPK and nuclear Sirt1 level, regulatory proteins of energy metabolism.
Conclusion: Our results suggest that CGRP, mediated through energy metabolic, and oxidative stress pathways, decreases myocyte apoptosis and is protective in pressure-induced heart failure. Thus, CGRP is a potential therapeutic agent in preventing the progression of heart failure.
Author Disclosures: A. Kumar: None. S. Supowit: None. J.D. Potts: None. D.J. DiPette: None.
Key Words: Heart failure; Cardioprotection; Cardiac hypertrophy; Cardioprotective drugs
Targeting Trpv4 Channels Protects Heart From Pathological Remodeling Following Myocardial Infarction
Ravi K Adapala1, Ashot Minasyan2, Anantha K Kanugula1, Holly C Cappelli1, Sailaja Paruchuri3, Gary J Meszaros1, Charles K Thodeti1; 1North East Ohio Medical University, Rootstown, OH, 2North East Ohio Med Univ(NEOMED), Rootstown, OH, 3Univ of Akron, Akron, OH
Heart failure is one of the leading causes of death which is often characterized by pathological fibrosis. Cardiac remodeling following myocadial infarction is a multiphase reparative process which involves replacement of damaged tissue with physiological (reparative) fibrosis to form scar that limit the expansion of the infarct. Myofibroblasts are critical mediator of this reparative fibrosis, however, hyperactivation of these cells can cause pathological fibrosis leading to heart failure. We have previously demonstrated that the mechanosensitive ion channel TRPV4 (transient receptor potential vanilloid channel 4) regulates cardiac fibroblast differentiation into myofibroblasts. However, the physiological or translational significance of TRPV4 in cardiac remodeling following MI is unknown. To explore this, we have induced MI (permanent LAD ligation) in WT and TRPV4KO mice and measured cardiac function for 8 weeks. Separately, WT mice were given an orally active TRPV4 antagonist GSK2193874, immediately after MI surgery and followed for 5 weeks. 2D-echocardiography revealed that the cardiac function (ejection fraction and fractional shortening) is preserved post-MI in both TRPV4KO and GSK2193874-treated WT mice compared to either WT or vehicle treated mice. Further, we found reduced cardiac fibrosis at infarcted and remote zones in TRPV4KO and GSK2193874-treated WT mice compared to their MI counter parts. Furthermore, TRPV4KO hearts exhibited decreased cardiomyocyte apoptosis (TUNEL assay) and increased capillary density (CD31 staining) post-MI compared to WT hearts. Our results thus suggest that targeting TRPV4 protects heart from myocardial infarction-induced damage by preserving cardiac structure and function via reduced myocyte apoptosis, diminished fibrosis and increased revascularization, and identifies TRPV4 as a novel therapeutic target for heart failure.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: R.K. Adapala: None. A. Minasyan: None. A.K. Kanugula: None. H.C. Cappelli: None. S. Paruchuri: None. G.J. Meszaros: None. C.K. Thodeti: None.
Key Words: Ion channels; Fibrosis; Myocardial infarction; Cardioprotection
Titin Truncating Variants Predict Life-threatening Arrhythmias in Patients With Dilated Cardiomyopathy
Ben Corden, Julian Jarman, Nicola Whiffin, Upasana Tayal, Rachel Buchan, Joban Sehmi, Andrew Harper, William Midwinter, Karen Lascelles, Vias Markides, Mark Mason, Dudley J Pennell, Paul J Barton, Sanjay K Prasad, Tom Wong, Stuart A Cook, James S Ware; Imperial College London, London, United Kingdom
Introduction: There is an urgent need for better arrhythmic risk stratification in non-ischaemic dilated cardiomyopathy (DCM), where the benefit of ICD implantation is unclear. Titin truncating variants (TTNtv) are the commonest genetic cause of DCM and are associated with early onset non-sustained ventricular tachycardia (NSVT) and atrial fibrillation (AF) in these patients.
Hypothesis: We hypothesize that TTNtv status can predict potentially life threatening ventricular tachycardia (VT) or fibrillation (VF) and development of new persistent AF in DCM patients with CRT-D or ICD devices.
Methods: We studied 117 DCM patients with an ICD or CRT-D and documented device-recorded arrhythmia over a median period of 4.2 years. Patients were stratified by TTN genotype (28 positive for a TTNtv, 89 negative). The primary outcome was time to first device-treated VT >200bpm or VF. Secondary outcome measures included time to first development of persistent AF.
Results: TTNtv predicted the risk of receiving an appropriate ICD therapy for VT/VF (hazard ratio [HR] = 4.9, 95% confidence interval [CI]=2.3–10.7, P<0.0001). This association was independent of all covariates, including replacement fibrosis measured by late-gadolinium enhancement (LGE), (adjusted HR = 8.2, 95% CI 1.9–36.5, P=0.005). Individuals with both a TTNtv and fibrosis had a markedly greater risk for appropriate device therapy than those with neither (HR = 16.6, CI 3.5–79.3, P<0.0001). TTNtv were also a risk factor for developing new persistent AF (HR = 4.4, 95% CI = 1.45–13.1, P=0.006).
Conclusion: TTNtv status is an important risk factor for clinically significant arrhythmia in patients with DCM and CRT-D or ICD devices. TTNtv status alone, or more powerfully in combination with fibrosis imaging by MRI, may provide an effective approach for risk stratifying the need for ICD therapy in DCM patients.
Author Disclosures: B. Corden: None. J. Jarman: None. N. Whiffin: None. U. Tayal: None. R. Buchan: None. J. Sehmi: None. A. Harper: None. W. Midwinter: None. K. Lascelles: None. V. Markides: Consultant/Advisory Board; Modest; Biosense Webster. M. Mason: None. D.J. Pennell: Other Research Support; Modest; Siemens. Ownership Interest; Modest; Cardiovascular Imaging Solutions. P.J. Barton: None. S.K. Prasad: None. T. Wong: None. S.A. Cook: None. J.S. Ware: None.
Key Words: Genetics; Ventricular arrhythmia; Cardiomyopathy; Heart failure; adult; Ventricular tachycardia
Microrna-125b-5p Protects The Heart From Acute Myocardial Infarction By Repressing Pro-apoptotic Bak1 And Klf13 In Cardiomyocytes
Tatsuya Aonuma, Ahmed S Bayoumi, Il-man Kim; Augusta Univ, Augusta, GA
Cardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs), small non-coding RNAs that post-transcriptionally regulate target genes. MiR-125b-5p is downregulated in patients with end-stage dilated and ischemic cardiomyopathy and has been proposed as a biomarker of heart failure. We previously showed using the β-arrestin-biased β-blocker, carvedilol that β1-adrenergic receptor-mediated cardioprotective signaling through β-arrestin1 stimulates processing of miR-125b-5p in the mouse heart (Figure A-C). We hypothesize that β1-adrenergic receptor/β-arrestin1-responsive miR-125b-5p confers cardioprotection against acute myocardial infarction. Using cultured cardiomyocyte (CM) and in vivo approaches, we show that miR-125b-5p is an ischemic stress-responsive protector against CM apoptosis. CMs lacking miR-125b-5p exhibit an increased sensitivity to stress-induced apoptosis, while CMs overexpressing miR-125b-5p have increased phospho-AKT pro-survival signaling. Moreover, we demonstrate that loss-of-function of miR-125b-5p in the mouse heart causes abnormalities in cardiac structure and function after acute myocardial infarction. Mechanistically, cardioprotection elicited by miR-125b-5p is in part attributed to repression of the pro-apoptotic genes Bak1 and Klf13 in CMs (Figure D). In conclusion, these findings reveal a pivotal role for miR-125b-5p in regulating CM survival during acute myocardial infarction.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: T. Aonuma: None. A.S. Bayoumi: None. I. Kim: None.
Key Words: MicroRNA; Cardioprotection; Receptor-mediated signaling; Apoptosis
Naturally Occurring Hypertension is Related to Cardiac Diastolic Dysfunction in Rhesus Monkeys
Yinan Liang1, Zunyuan Yang2, Zunwei Yao2, Li Gong2, Zhigang Liang2, Yubo Shen2, Yuanhai Chen2, Wen Zeng2, Barbara C Hansen3; 1Sichuan Primed Shines Bio-tech Co., Ltd, Chengdu, FL, 2Sichuan Primed Shines Bio-tech Co., Ltd, Chengdu, China, 3University of South Florida, Tampa, FL
Introduction: The lack of effective treatment for Diastolic Dysfunction (DD) is partially due to the differences between widely used preclinical rodent models and humans in the physiology and function of the heart. In previous studies, we have demonstrated that rhesus monkeys with naturally occurring adult onset Type 2 Diabetes (T2DM) frequently have DD that is similar in characteristics to DD in diabetic patients. To further characterize DD in rhesus monkeys, we studied the relationship between hypertension and DD, and evaluated the response of rhesus monkeys with DD to Entresto (sacubitril/valsartan).
Methods: Blood pressure, fasting plasma glucose and cardiac function were measured in 322 adult rhesus monkeys (Macaca mulatta, 7–22 yrs) under light anesthesia with ketamine. Monkeys with LV hypertrophy, e’<8 cm/s and E/e’>10 were defined as DD. Ten monkeys with DD were enrolled in the validation study and divided into the Entresto group (n=5) and the vehicle group (n=5). Cardiac function and blood pressure were measured before and at the end of 13 weeks of treatment.
Results: Among the 322 adult rhesus monkeys studied, 53 monkeys (16.46%) had SBP>140 mm Hg or DBP>90 mm Hg. Among the 174 monkeys with fasting glucose >80 mg/dL, 67 monkeys had isolated DD, and 8 had DD+ SD (systolic dysfunction). The incidence of isolated DD was 31% in monkeys with SBP<140 mm Hg and 74% in monkeys with SBP>140 mm Hg. Following Entresto administration (1.66 to 13.33 mg/kg) for 13 weeks, DD and BP evaluation showed an increase of e’ (5.27±0.51 to 6.43±1.27 cm/s), a decrease of E/e’ (12.74±2.23 to 10.53±2.09) and a decrease of SBP (128±5 to 113±16 mm Hg). These parameters remained stable and unchanged in the vehicle group.
Conclusions: The incidence of naturally occurring hypertension in adult rhesus monkeys was similar to that in adult humans. Entresto reduced blood pressure, but led to no significant improvement of DD in monkeys. The extent of change in rhesus monkeys was similar to that observed in clinical trials. In rhesus monkeys, as in patients, hypertension is significantly related to cardiac diastolic dysfunction. These monkeys, therefore, provide important new opportunities to understand the pathogenesis of DD, as well as to predict the human response to new therapeutic agents.
Author Disclosures: Y. Liang: None. Z. Yang: Employment; Significant; Sichuan Primed Shines Biotech Co., Ltd. Z. Yao: Employment; Significant; Sichuan Primed Shines Biotech Co., Ltd. L. Gong: Employment; Significant; Sichuan Primed Shines Biotech Co., Ltd. Z. Liang: Employment; Significant; Sichuan Primed Shines Biotech Co., Ltd. Y. Shen: Employment; Significant; Sichuan Primed Shines Biotech Co., Ltd. Y. Chen: Employment; Significant; Sichuan Primed Shines Biotech Co., Ltd. W. Zeng: Employment; Significant; Sichuan Primed Shines Biotech Co., Ltd. B.C. Hansen: None.
Key Words: Diastolic function; Hypertension; Drugs; Diabetes (Type II)
Longitudinal Evaluation of the Associations between Severe Hypertriglyceridemia and Cardiovascular Features
Samuel C Gidanian, Uddhav Chaudhari, Jennifer D Newcomb, Rahul D Mhaskar, Barbara C Hansen; 1University of South Florida, Tampa, FL
Introduction: For at least 35 years the associations between insulin resistance, hyperinsulinemia, hypertriglyceridemia and hypertension have been described. Nevertheless, the existence of an independent association between hypertriglyceridemia (HTG) and cardiovascular diseases (CVD) remains controversial. This may be due to the insufficiency of longitudinal study data that include severe elevations of TG. The purpose of this study was to characterize the associations between systolic blood pressure and various cardiovascular features under different degrees of naturally-occurring HTG severity and to determine the independent effect of severe HTG.
Methods: We characterized TG in a large cohort of longitudinally studied nonhuman primates (n=227; 164 males) maintained for their lifetimes under constant environmental and dietary conditions. The TG ranged from normal TG levels <100 mg/dl, to mild HTG 100–200 mg/dl, high HTG 200–500 mg/dl, very high 500–1000 mg/dl and severe >1000 mg/dl. The Kruskal Wallis H test was applied, as the data samples were not normally distributed.
Results: Severe hypertriglyceridemia was statistically significantly related to systolic blood pressure and was significantly higher (p<0.0001) at the severe HTG levels of 500–1000 mg/dl and ≥1000 mg compared to all other TG levels. Diastolic blood pressure and mean arterial blood pressure were not significantly different among the normal to severe HTG groups. HDL cholesterol was significantly lower (p<0.0001) at all levels of HTG (TG 100–200, 200–500, 500–1000 and ≥1000mg/dl) compared to normal TG <100 mg/dl, and was not related to the severity of the HTG, a finding similar to the relationship with body weight. By contrast, LDL cholesterol was significantly higher (p<0.05) in monkeys with severe HTG >500–1000 mg/dl, as was systolic blood pressure.
Conclusions: In conclusion, much of the association between triglyceride levels and cardiovascular features, including blood pressure, may be principally determined by the severely elevated triglyceride levels, possibly highlighting the importance of longitudinal within subject evaluation of such associations for break points in the interactions of these features.
Author Disclosures: S.C. Gidanian: None. U. Chaudhari: None. J.D. Newcomb: None. R.D. Mhaskar: None. B.C. Hansen: None.
Key Words: Triglycerides; Hyperlipidemia; Hypertension
Selective Inhibition Of Hdac3 Prevents Diabetic Cardiomyopathy In Ove26 Mice Via Mir-200a-mediated Nrf2 Activation
Zheng Xu1, Yang Zheng1, Zhiguo Zhang1, Jian Sun1, Lu Cai2; 1First Hosp of Jilin Univ, Changchun, China; 2Pediatric Rsch Institute, Dept of Pediatrics, the Univ of Louisville, Louisville, KY
Although histone deacetylases (HDACs) was are the promising target for the initiation and development of diabetic cardiomyopathy (DCM), which isoform of them plays the key role remains unclear. The present study was designed to determine whether DCM could be prevented by selective inhibition of HDAC3 and the underlying mechanism. Male type 1 diabetic OVE26 and age-matched wild-type mice were given the selective HDAC3 inhibitor (HDAC3i) RGFP966 or vehicle for 3 months before the cardiac function was examined with Echo. Results indicated that HDAC3i treatment improved cardiac function in the diabetes group. HDAC3 activity was significantly increased in the heart of diabetic mice, which was blocked by the treatment of HDAC3i. Oxidative stress as a major cause of DCM is also inhibited by HDAC3i. Mechanistically cardiac miR-200a, which targets and destabilizes kelch-like ECH-associated protein 1 (KEAP1) mRNA, was significantly up-regulated and KEAP1 expression was markedly inhibited by HDAC3i in the diabetic mouse. Immunoprecipitation shows that the binding of KEAP1 in the diabetic heart was decreased by the treatment of HDAC3i when we pull down nuclear factor-E2-related factor 2 (NRF2). Meanwhile, the nuclear localization of NRF2 and its downstream anti-oxidative stress genes, NADPH quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), were markedly up-regulated in the HDAC3i treated diabetic mouse group. These results suggest that HDAC3i prevents DCM likely via miR-200a-mediated degradation of KEAP1 and consequently activation of the Nrf2-regulated antioxidant pathway.
Author Disclosures: Z. xu: None. Y. zheng: None. Z. zhang: None. J. Sun: None. L. Cai: None.
Key Words: Cardioprotection; Diabetes (Type I); Antioxidant
Muscle-specific A-Kinase Anchoring Protein Polymorphisms Pre-dispose Humans to Cardiovascular Diseases by Affecting Cyclic AMP/PKA Signaling
Santosh Suryavanshi1, Shweta Jadhav1, Kody Anderson2, Panagiotis Katsonis3, Olivier Lichtarge3, Bradley K McConnell1; 1Univ of Houston College of Pharmacy, Houston, TX, 2Univ of Houston Cullen College of Engineering, Houston, TX, 3Baylor College of Medicine, Houston, TX
In-depth understanding of key cardiac signaling pathways is crucial in finding new targets for cardiovascular diseases (CVDs), the no. 1 cause of deaths globally. One of such pathways is cAMP-dependent PKA signaling which is modulated by scaffold proteins, A-kinase anchoring proteins (AKAPs). Muscle-specific AKAP (mAKAP) regulates expression of hypertrophic factors partly by controlling cardiac cAMP levels. Furthermore, published literature revealed a strong correlation between single nucleotide polymorphisms (SNPs) in proteins and risk of developing CVDs by varying cAMP/PKA signaling. This aspect of AKAPs has been largely unexplored. Hence, we hypothesized that mAKAP SNPs alter cAMP/PKA signaling making individuals susceptible to CVDs. We analyzed selected mAKAP SNPs found often in human patients with CVDs through multiple online tools that predict functional effects of SNPs to finalize two SNPs, Ser(S)1653Arg(R) in PDE4D3 binding domain and Glu(E)2124Gly(G) in 3`-PKA binding domain of mAKAP. After making both mAKAP mutant plasmids from WT using site-directed mutagenesis, we studied them in HEK 293T cells. Four separate samples were used for each experiment. In immunoprecipitation studies, S1653R mutant showed increased binding to PDE4D3 at baseline but significantly reduced binding after stimulation with 1 µM isoproterenol as compared to WT. Similarly, E2124G mutant exhibited significantly lower PKA binding at baseline and higher binding after stimulation. cAMP levels and PKA activity were significantly lower at baseline but higher after stimulation in S1653R mutant cells. Also, E2124G expressed cells showed no significant change in cAMP levels when compared to WT but PKA activity was significantly lower at baseline followed by abrupt increase after stimulation. PDE activity assay was in congruent with cAMP changes in S1653R mutant cells. Fluorometric assay showed higher intracellular calcium in E2124G mutant cells after stimulation. Lastly, immunoblotting data showed altered phosphorylation of hypertrophic markers in both mutants. To conclude, human mAKAP SNPs may pre-dispose humans to the risk of developing CVDs by affecting cAMP/PKA signaling and thus confirming the clinical significance of PKA-mAKAP-PDE4D3 interaction.
Author Disclosures: S. Suryavanshi: None. S. Jadhav: None. K. Anderson: None. P. Katsonis: None. O. Lichtarge: None. B.K. McConnell: None.
Key Words: Cardiovascular disease; Cell signaling; Cardiovascular health; Molecular biology; Gene mutations
Developing a Cardiomyocyte Pipeline for Gene Edited hiPSCs
Kaytlyn Gerbin, Angelique Nelson, Brock Roberts, Joy Arakaki, Tanya Grancharova, Colette DeLizo, Ru Gunawardane; Allen Institute for Cell Science, Seattle, WA
The Allen Institute for Cell Science (AICS) is creating an open source collection of fluorescently tagged human induced pluripotent stem cell (hiPSC) lines to model cell organization and dynamics of stem cells and differentiated hiPSC-cardiomyocytes. Understanding the sub-cellular organization and the structure/function relationships of organelles within cardiomyocytes will contribute to the development of better disease models, therapies, and regenerative medicine approaches for cardiac disease. Using the WTC human iPSC line and the CRISPR/Cas9 system, we have fluorescently tagged ~20 target genes representing key cellular organelles including a few cardiac-specific genes. Edited iPSC lines are differentiated into cardiomyocytes using established protocols with either small molecules or a combination of cytokines and small molecules, which produce high-purity monolayers of beating cardiomyocytes within 1–2 weeks. Differentiation into cardiomyocytes serves as an important quality control criterion for our gene editing efforts, but also comprises an important aspect of our predictive cell modeling efforts. We plan to study the changes in localization and organization of these tagged organelles as the stem cells differentiate into cardiomyocytes using live fluorescent cell imaging. Here, we present our cardiac differentiation methods for multiple edited hiPSC lines and the quantitative and qualitative assays used to determine the efficacy of differentiation, including myofibril contraction, cardiac protein expression, and transcriptome profiling by bulk and single cell RNAseq. Additionally, we confirm the localization of cardiac proteins such as troponin T and alpha-actinin to the myofibrils in differentiated cells using image-based assays. In experiments initiated to date we have successfully differentiated multiple gene edited iPSC lines representing major cellular structures, 2 of which are specific to cardiomyocytes (ssTNNi1 and ACTN2). Some of the gene edited hiPSC lines are fluorescently-tagged for structures including focal adhesions, actin and microtubule cytoskeleton, mitochondria, nuclear envelope, desmosomes, and endoplasmic reticulum, which are all publically available to the community.
Author Disclosures: K. Gerbin: None. A. Nelson: None. B. Roberts: None. J. Arakaki: None. T. Grancharova: None. C. DeLizo: None. R. Gunawardane: None.
Key Words: Stem cells; Cellular Engineering; Cardiac development; Stem cell biology
Heart Fields Are Induced by Coordinated Activity of Wnt and Bmp Signaling and Identified by CD184 and EphA2 in PSC-Derived Organoids
Peter Andersen, Dennisse Jimenez-Cyrus, Stephen P Chelko, Suraj Kannan, Emmanouil Tampakakis, Amir Saberi, Sean Murphy, Matthew Miyamoto, Chulan Kwon; Johns Hopkins University, Baltimore, MD
Over the past few decades, major advances have been made in identifying the origins of cardiac cells from developing embryos. In particular, the discovery of the first heart field (FHF) and the second heart field (SHF), led us to understand how diverse lineages and different anatomical structures of the heart arise during cardiogenesis. However, it remains unknown how the two heart fields are specified and segregated, a fundamental step toward understanding heart formation and developing pluripotent stem cell (PSC)-based therapeutic strategies. Here, we generated 3D organoids with mouse PSCs that harbor green and red fluorescent protein (GFP and RFP) reporters under the control of the FHF marker Hcn4 and the SHF marker Tbx1, respectively. We demonstrate how GFP+ cells and RFP+ cells appear from two distinct areas of mesodermal cells and develop in a complementary fashion, similar to the in vivo process. Consistently, these populations exhibit a high degree of similarities with FHF/SHF cells isolated from early embryos, determined by RNA-sequencing analysis. Through a series of bioinformatics approaches, we found that Bmp and Wnt are among the most differentially regulated pathways in the two populations. Importantly, an increased activity of Bmp or Wnt signaling resulted in selective induction of GFP+ or RFP+ cells from mesodermal cells, enabling us to generate heart field-specific cells from PSCs. We further found that FHF/SHF cells can be distinguished and isolated by the surface proteins CD184 and EphA2. This study provides fundamental insights into understanding the specification of two cardiac origins that enable generation of chamber-specific populations for studying heart field/chamber-specific heart disease in cell culture.
Author Disclosures: P. Andersen: None. D. Jimenez-Cyrus: None. S.P. Chelko: None. S. Kannan: None. E. Tampakakis: None. A. Saberi: None. S. Murphy: None. M. Miyamoto: None. C. Kwon: None.
Key Words: Stem cell biology; Cardiac development; Stem cells; Progenitor cell
A Combined Basic Science and PopulationScience Approach Demonstrating the Potential for Simvastatin to Mitigate Cardiovascular Disease after Lower Hemi Body Radiotherapy
Jessica Olson1, Reiauna Taylor2, Rodney Sparapani1, Marek Lenarczyk3, John Baker3; 1Institute for Health and Equity, Med College of Wisconsin, Milwaukee, WI, 2Univ of Wisconsin-Madison, Madison, WI, 3Dept of Surgery, Med College of Wisconsin, Milwaukee, WI
Radiation is a cornerstone of successful cancer treatment, with one-half to two-thirds of all patients receiving radiotherapy. Survivors of cancer treated with radiation are at increased risk for cardiovascular disease (CVD). Understanding how radiation causes CVD will allow development of novel therapies. Irradiation of the lower hemi body, but not the upper hemi body, with 10 Gy in rats increases risk factors for CVD and results in cardiac fibrosis quantitatively similar to total body irradiation, suggesting radiation injury to the heart can be indirect. Simvastatin, an inhibitor of liver cholesterol synthesis administered continuously to rats (10 mg/kg/day) after 10 Gy lower hemi body irradiation mitigated against increased blood cholesterol and cardiac fibrosis. These findings indicate simvastatin limits transmission of a signal from the lower hemi body that decreases risk for and occurrence of CVD independent of any direct exposure of the heart to radiation. Bivariate examination of 3,607 patients following therapeutic lower hemi body irradiation using Chi-square, Wilcoxon rank-sum and t-tests was used to examine risk factors for CVD in patients diagnosed with congestive heart failure, myocardial infarction, atrial fibrillation, and cardiomyopathy before 80 years of age. We found that 47.4% of patients age 70–80 developed CVD compared to 29.7% who received simvastatin (p= < 0.001, n = 293 and 361, respectively). Patients who were male, overweight, smokers, and had a diagnosis of chronic kidney disease and diabetes also had significantly higher risk of CVD. Race and hypertension were not indicative of increased risk for CVD. These clinical findings, taken together with the results from our animal studies, support a new research paradigm where radiation-induced heart disease can be indirect, with abdominal organs exporting factors that cause CVD. Simvastatin can be developed to mitigate and treat CVD after therapeutic radiation.
Author Disclosures: J. Olson: None. R. Taylor: None. R. Sparapani: None. M. Lenarczyk: None. J. Baker: None.
Key Words: Statins; Radiation; Heart failure; Cardiooncology; Cardiovascular disease
Human iPSC-Derived Endothelial Cells Predict Predilection to Atherogenesis by Endothelial Proinflammatory Activation
David T Paik, Youngkyun Kim, June-Wha Rhee, Hyoju Yi, Ridhima Mishra, Joseph C Wu; Stanford Univ, Stanford, CA
Introduction: Coronary artery disease (CAD), the direct outcome of atherosclerosis, is the leading cause of death in the United States. Previous studies demonstrated that impaired function of aldehyde dehydrogenase 2 (ALDH2), a key enzyme for alcohol metabolism, is linked to increased susceptibility to CAD. A single-nucleotide polymorphism that generates E487K mutation (ALDH2*2) reduces enzymatic activity of ALDH2 to less than 40% of the wild type (WT) and is present in ~560 million people. However, it remains unclear how ALDH2 regulates atherosclerotic progression.
Hypothesis: Recent studies suggest a critical role of ALDH2 in plaque development and endothelial activation. Therefore, we hypothesize that endothelial cells of ALDH2*2 carriers possess greater susceptibility to proinflammatory activation, whereby endothelial cells recruit immune cells, leading to increased risk of atherogenesis.
Methods: To study the patient-specific effects of ALDH2*2 mutation on endothelial proinflammatory activation, we generated and characterized iPSC-derived endothelial cells (iPSC-ECs) from 5 WT subjects and 5 ALDH2*2 carriers. We exposed the iPSC-ECs to pro-inflammatory conditions and assessed the level of endothelial proinflammatory activation by gene expression analysis and monocyte adhesion assay.
Results: Our preliminary data show ALDH2*2-iPSC-ECs exhibit impaired ALDH2 function resulting in metabolic dysregulation compared to WT. Presence of ALDH2*2 mutation resulted in enhanced inflammatory response in the iPSC-ECs when treated with proinflammatory cytokines such as TNFα and IL-1β, as evidenced by up-regulation of cell adhesion molecules and augmented adherence to monocytes. The ALDH2*2-iPSC-ECs also exhibited an increased basal expression of vascular endothelial growth factor receptor 1 (FLT1) gene, which was further augmented upon inflammatory stimulation. FLT1 is a receptor for vascular endothelial growth factor ligands, playing a critical role in endothelial homeostasis and biology.
Conclusion: Taken together, we elucidate the effects of impaired ALDH2 function on increased susceptibility to atherogenesis by endothelial proinflammatory activation using patient-derived iPSC-ECs.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: D.T. Paik: None. Y. Kim: None. J. Rhee: None. H. Yi: None. R. Mishra: None. J.C. Wu: None.
Key Words: Endothelial function; Inflammation and inflammatory markers; Stem cell biology; Stem cells; Stem cell and atherosclerosis
Sacubitril/Valsartan Attenuates Fibrosis and Improves Left Ventricular Function in a Rabbit Model of HFrEF
Juan Torrado, Chad Cain, Adolfo G Mauro, Ramzi A Ockaili, Francisco Romeo, John Nestler, Teja Devarakonda, Anindita Das, Fadi N Salloum; Virginia Commonwealth Univ, Richmond, VA
Background: Sacubitril/Valsartan (SAC/VAL), a drug combining a neprilysin inhibitor and an angiotensin receptor blocker, was shown to reduce myocardial infarct size and left ventricular (LV) dysfunction in preclinical models of myocardial infarction (MI). In the PARADIGM-HF trial, SAC/VAL prevented the clinical progression of patients with heart failure (HF) more effectively than enalapril. Whether SAC/VAL attenuates cardiac fibrosis and improves LV function in a rabbit model of MI-induced HF with reduced ejection fraction (rEF) is unknown.
Methods: Anesthetized adult male NZW rabbits (~2.5kg) underwent left thoracotomy and the left anterior descending (LAD) coronary artery was identified and occluded for 45 min followed by reperfusion. Weekly echocardiography was performed to confirm reduced EF (~40%), which was uniformly achieved at 5 weeks post MI. Subsequently, rabbits were randomized to orally receive placebo (volume-matched water, BID), SAC/VAL (10 mg/kg, BID) or VAL (9.1mg/kg/day) starting on week 6. At 10 weeks post MI, rabbits were sacrificed and hearts were harvested, fixed with 10% formalin and embedded in paraffin to assess myocardial fibrosis (Picrosirius red staining). Operators performing echocardiography, Picrosirius red staining and analysis were blinded to treatment allocation.
Results: Two weeks after treatment initiation, a significant improvement in LVEF was observed in the SAC/VAL group compared to both placebo and VAL, a benefit that lasted throughout the entire study (Fig. A). The functional improvement observed was associated with a significant reduction in LV scar size compared to placebo at week 10 (Fig. B). However, when compared to VAL, the decrease in scar size did not reach statistical significance despite a clear trend.
Conclusion: Our results suggest that SAC/VAL may offer superior benefits compared to equivalent dose of stand-alone VAL in attenuating LV scar size and improving LVEF in a rabbit model of ischemic HFrEF.
Author Disclosures: J. Torrado: None. C. Cain: None. A.G. Mauro: None. R.A. Ockaili: None. F. Romeo: None. J. Nestler: None. T. Devarakonda: None. A. Das: None. F.N. Salloum: Research Grant; Significant; Novartis Pharmaceuticals.
Key Words: Heart failure; Ischemia reperfusion; Natriuretic peptide; Ejection fraction; Fibrosis
Need of Treating Residual Inflammatory Activity in Coronary Heart Disease: The Value of High Sensitive CRP and LDL in a Real World Cohort
Alexander Peikert1, Klaus Kaier2, Julian Merz1, Ibrahim Ahmed Fadel Ali Hassanin1, Dennis Wolf1, Ingo Hilgendorf1, Florian Willecke1, Peter Stachon1, Andreas Zirlik1; 1Univ Heart Cntr Freiburg, Freiburg im Breisgau, Germany; 2Univ Freiburg, Freiburg im Breisgau, Germany
Introduction: Inflammation drives atherosclerosis and its complications. Thus, CANTOS as the first anti-inflammatory outcome trial in this population produced positive results. However, it is unclear how many patients qualify for an anti-inflammatory therapy in everyday practice.
Hypothesis: This study analyzes how many patients with coronary heart disease (CHD) on guideline conform therapy show an increased residual inflammatory as opposed to an increased residual lipid risk in order to define the need for an anti-inflammatory treatment in a real world setting.
Methods: High sensitive C-reactive protein (hsCRP) and low density lipoprotein (LDL) levels were determined in 700 all comer patients between June 2016 and June 2017 in our center. Patients lacking CHD, such with chronic-inflammatory diseases, acute inflammation, and on immunosuppressive medication were excluded. Patients were divided in the following groups: elevated hsCRP (≥2mg/dl), normal hsCRP (<2mg/dl), off target LDL-cholesterol (≥70mg/dl), on target LDL-cholesterol (<70mg/dl). Univariate logistic regression and backward selection was performed in order to define factors influencing hsCRP.
Results: From 700 patients 221 fulfilled the inclusion and exclusion criteria. HsCRP was increased in 45% of these patients. Patients with on target LDL Levels showed lower hsCRP concentrations than those with off target values of LDL confirming a positive association between both (1,92mg/dl vs. 3,15mg/dl, p=0.005). However, despite guideline-conform LDL-control 34% of patients with a LDL-cholesterol <70mg/dl had elevated levels of hsCRP (≥2mg/dl) suggestive of residual inflammation. After logistic univariate regression LDL-cholesterol ≥70mg/dl (OR 2.15, p=0.014), heart failure (OR 3.07, p<0.001) and diabetes mellitus (OR 2.22, p=0.021) independently predicted elevated levels of hsCRP. Heart failure (OR 4.56, p<0.001) and diabetes together (OR 3.04, p=0.012) identified as co-predictors increased hsCRP following backward selection.
Conclusions: A substantial part of patients with CHD shares a residual inflammatory risk defining a need for an anti-inflammatory therapy. Residual inflammation is particularly prevalent in patients with heart failure and diabetes.
Author Disclosures: A. Peikert: None. K. Kaier: None. J. Merz: None. I. Hassanin: None. D. Wolf: None. I. Hilgendorf: None. F. Willecke: None. P. Stachon: None. A. Zirlik: None.
Key Words: Arteriosclerosis; Inflammation and inflammatory markers; LDL; Cardiovascular disease; Cardiovascular therapeutics
Cardiac-Specific Overexpression Of Caveolin-3 Expedites Cardiac Relaxation After Adrenergic Stimulation
Alice E Zemljic-Harpf1, Anna R Busija2, Mugdha A Joshi3, Basheer F Alas4, Jan M Schilling1, David M Roth1, Paul A Insel5, Hemal H Patel1; 1VA San Diego Healthcare System, and Univ. of California, San Diego, Dep. of Anesthesiology, La Jolla, CA, 2Univ. of California, San Diego, Dep. of Anesthesiology, La Jolla, CA, 3Univ. of California, San Diego, La Jolla, CA, 4VA San Diego Healthcare System, La Jolla, CA, 5Univ. of California, San Diego, Dep. of Medicine, and Dep. of Pharmacology, La Jolla, CA
Introduction: Caveolae are membrane localized signaling platforms that compartmentalize signal transduction proteins such as GPCRs. Previous studies from our laboratory have demonstrated that overexpression of caveolin-3 in cardiac myocytes (Cav3 OE) protects against pressure-overload induced heart failure. Additionally, Cav3 OE mice present increased heart rate variability with lower nocturnal heart rates, but the mechanisms behind these phenotypes remain unclear.
Hypothesis: Since Cav3 OE mice are protected from stressful cardiac stimuli, we tested the rationale that Cav3 OE hearts may show altered parasympathetic control of cardiac responses to adrenergic stimulation.
Methods: Cav3 OE mice and transgene negative littermate controls (Ctrl) (12-16-week-old, n=10–11 each) were anesthetized with isoflurane and cardiac contractility was assessed by echocardiography at baseline. Isoprenaline (Isuprel USP [Iso], 300 nM i.p.) was administered ~10 min after baseline recordings and cardiac function recorded at 2 min, 5 min, and 10–15 min after Iso. The same animal cohort received a single atropine injection (2 mg/kg i.p) 20 min before isoflurane anesthesia and Iso challenge echocardiography was performed as described above.
Results: At baseline, no differences in cardiac contractility (% ejection fraction, %EF±SD) were detected between the two groups ~68±8 %EF. After Iso injection cardiac contractility was increased to ~91±3 %EF in both genotypes at 2 min; however, in Cav3 OE mice cardiac contractility recovered to 83±3 %EF by 5 min post Iso challenge whereas Ctrl animals maintained increased contractility of 92±3 %EF (p < 0.001). Importantly, Cav3 OE mice pre-treated with atropine no longer showed increased recovery at 5 min or 10–15 post Iso (p = 0.007). A three-way ANOVA of the time-course after Iso injection found a significant effect of atropine on the responses of Cav3 OE versus Ctrl hearts to Iso (p = 0.007).
Conclusion: We show for the first time that Cav3 OE mice show a faster recovery from hypercontractility after isoproterenol stimulation. Since atropine abrogated this recovery, these data suggest that Cav3 OE mice exhibit increased parasympathetic tone that may be responsible for improved stress adaptation and heart rate variability.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: A.E. Zemljic-Harpf: None. A.R. Busija: None. M.A. Joshi: None. B.F. Alas: None. J.M. Schilling: None. D.M. Roth: None. P.A. Insel: None. H.H. Patel: None.
Key Words: Heart function tests; Adrenergic; Pharmacology; Heart rate/Heart rate variability; Cardioprotection
MFG-E8 Fragment Medin and Arterial Aging
Yushi Wang; NIH, Baltimore, MD
Background: Aging increases milk fat globule EGF-VIII (MFG-E8) expression in the rat, nonhuman primate and human aortic walls, facilitating the invasion, proliferation, and proinflammation of vascular smooth muscle cells (VSMCs). The MFG-E8 C-terminal fragment medin has been reported to be involved in the necrosis of VSMCs at the inflammatory sites of aortic dissection, however, its cleavage processes, aortic wall levels with aging and bio-role in VSMCs remains to be determined.
Material and Methods: In this study, immunofluorescence, immunohistochemistry and western blot analysis demonstrate that MFG-E8 and its fragment medin (Figure), activated matrix metalloproteinase type II (MMP-2) and platelet derived growth factor receptor type-beta (PDGFR-β) protein levels were markedly increased in human grossly normal aortic walls from old (n=10, >50 yrs) vs. younger donors (n=10, <25 yrs). Importantly, exposure of medin peptide (20 to100nM) to primary cultured VSMCs isolated from young (8 mo) and old (30 mo) FXBN rat aortae significantly increased MMP-2 activation, PDGFR-β expression, and migratory capacity measured by a modified Boyden chamber in a dose-dependent manner in both also. Separate exposure of activated MMP-2 to recombinant human MFG-E8 protein and also to MFG-E8 enriched old human aortic protein markedly increased the cleavage product medin in both, which was substantially inhibited by an MMP inhibitor, GM6001. Exposure of medin to VSMCs did not significantly affect the expression of cell cycle related proteins. In addition, PDGF-BB treatment markedly activated MMP-2 in both young and old VSMCs, which was substantially reduced by a PDGFR-β inhibitor, RTK.
Conclusion: Taken together, targeting MFG-E8 or its cleavage product medin is a novel approach to the prevention or treatment of large arterial aging or age-associated disease.
Author Disclosures: Y. Wang: None.
Key Words: Aging; Arteries; Cardiovascular disease
Utilizing Telemedicine to Construct Population-Based STEMI Systems of Care in Developing Countries
Sameer Mehta1, Roberto Botelho2, Francisco Fernandez1, Sunil Agarwal1, Marco Perin3, Rodolfo Cardoso4, Cesar Dusilek5, Marco A Gamba6, Juan C Perez-Alva7, Mario Torres1, Daniel Rodriguez1; 1Lumen Foundation, Miami, FL, 2Triangulo Heart Institute and Eurolation Med Rsch, Uberlandia, Brazil, 3Hosp Santa Marcelina, Hosp 9 de Julio and Hosp Sirio Libanes., Sao Paulo, Brazil, 4Hosp do Coração de Duque de Caxias., Rio de Janeiro, Brazil, 5Hosp Do Rocio, Campo Largo, Brazil, 6Heart Institute of Queretaro, Queretaro, Mexico, 7Cardiovascular Institute of Puebla, Puebla, Mexico
Background: Major disparities exist between developed and developing countries in the outcomes of patients presenting with Acute Myocardial Infarction (AMI). Telemedicine has emerged as a powerful, cost-efficient, and scalable tool. Whether telemedicine can improve AMI results remains unclear.
Methods: We incorporated a hub and spoke strategy for Latin America Telemedicine Infarct Network (LATIN) to expand access to care in regions in Brazil, Colombia, and Mexico that lacked AMI care. Small clinics and primary care health centers in remote areas (spokes) were strategically connected to hubs that could perform 24/7 primary PCI. Experts at 3 central sites in Uberlandia, Sao Paulo, and Bogota, provided urgent EKG diagnosis and tele-consultation for the entire LATIN network by triggering ambulance dispatch and implementing standardized AMI protocols.
Results: A total of 257 LATIN centers (Brazil 95, Colombia 113, Mexico 49) were networked using similar telemedicine protocols. In Colombia, LATIN coverage was established to cover 31% of the nation’s 48 million population. With this expanded geographic reach, 4.694 (1.2%) of the 401,095 screened patients were diagnosed as having STEMI. A total of 2,041 (43.5%) STEMI were urgently reperfused. Primary PCI was performed in the majority of patients - 1,578 (77.3%) that were referred for urgent perfusion. The major reasons for non-treatment included insurance denials, lack of ICU beds and chest pain >12 hours. Time to Telemedicine Diagnosis (TTD) was 5.1 minutes, and tele-ECG accuracy was 98%. D2B time for the cohort was 53 minutes, but chest pain to treatment time was >6 hours. Overall, in-hospital mortality was 5.8%.
Conclusion: LATIN demonstrates the feasibility of creating a population based and telemedicine-guided AMI management strategy that can hugely expand access to reperfusion strategies. Telemedicine has important public health implications as a global approach to urgent AMI care in developing countries.
Author Disclosures: S. Mehta: None. R. Botelho: None. F. Fernandez: None. S. Agarwal: None. M. Perin: None. R. Cardoso: None. C. Dusilek: None. M.A. Gamba: None. J.C. Perez-Alva: None. M. Torres: None. D. Rodriguez: None.
Key Words: Myocardial infarction; Telemedicine; STEMI; Systems of care
Genome-Wide Association Study of Vasodilator Response in Pulmonary Arterial Hypertension
Jason H Karnes1, Ken Batai1, Amit Arora1, Justin Kaye1, Heidi Steiner1, Vineet Nair1, Joe GN Garcia1, Jason Yuan1, Mike Pauciulo2, William Nichols2, Ankit Desai1; 1University of Arizona, Tucson, AZ, 2Cincinnati Children’s Hosp, Cincinnati, OH
Introduction: Pulmonary arterial hypertension (PAH) is a rare and fatal disease associated with variable therapeutic response, suggesting a genetic contribution. Vasodilator-responsive PAH accounts for a minority of PAH cases and is associated with dramatically improved survival over vasodilator-nonresponsive PAH. The objective of our study was to identify genetic influences on vasodilator drug response in PAH.
Methods: Two cohorts of patients with Group I PAH confirmed by right heart catheterization were derived from the PAH Biobank (NHLBI R24HL105333), representing over 40 US institutions. Differences between hemodynamics at rest (baseline mean pulmonary arterial pressure, mPAP) and after vasodilator (nitric oxide, prostacyclin) administration were determined to define acute vasodilator drug response (with unchanged cardiac index) as a continuous measure. All cases were genotyped using HumanOmni5 with single nucleotide polymorphism (SNP) call rate >99%, minor allele frequency (MAF)>3%, and Hardy-Weinberg p-value>0.01. Analysis was restricted to cases with European ancestry. We performed linear regressions in an additive model for acute vasodilator drug response with adjustment for baseline mPAP, age, gender, and first 3 principal components. A Bonferroni-corrected alpha=5x10-8 was used in the discovery cohort and alpha=0.05 was used in the replication cohort.
Results: The discovery cohort included 434 PAH cases and the replication cohort included 49 less severe PAH cases. QQ-plots showed no evidence of genomic inflation (lambda=1.00). Association of the intronic SNP rs8057488 (MAF 0.05) in the sorting nexin 29 gene (SNX29) with acute vasodilator drug response reached genome-wide significance in the discovery cohort (beta= -7.03 mmHg, p=3.39x10-8). A significant association between rs8057488 and acute vasodilator drug response was also observed in the replication cohort (beta = -6.48 mmHg, p=0.03).
Conclusion: These findings implicate a novel association between SNX29 variation and differential responses to vasodilator treatment in Group I PAH. While requiring further replication in a larger independent cohort, these observations advance our understanding of the molecular underpinnings in PAH.
Author Disclosures: J.H. Karnes: None. K. Batai: None. A. Arora: None. J. Kaye: None. H. Steiner: None. V. Nair: None. J. Garcia: None. J. Yuan: None. M. Pauciulo: None. W. Nichols: None. A. Desai: None.
Key Words: Pulmonary circulation; Pulmonary hypertension; Genome-wide association studies (GWAS)
Inducible Depletion of Calpain-2 Attenuates Obesity-accelerated Abdominal Aortic Aneurysms in Mice
Aida Javidan, Weihua Jiang, Jessica J. Moorleghen, Venkateswaran Subramanian; University of Kentucky, Lexington, KY
Background and Objective: Recent clinical studies demonstrated that abdominal adiposity is associated with increased risk of abdominal aortic aneurysm (AAA) development. Calpains are non-lysosomal calcium dependent cysteine proteases that are highly expressed in human and experimental AAAs. Using a pharmacological inhibitor and genetically deficient mice, we identified that calpain-2 (a major ubiquitous isoform) plays a critical role in Angiotensin II (AngII)-induced AAA formation in mice. In addition, calpain inhibition strongly suppressed adipose tissue inflammation in obese mice. The purpose of this study was to determine the functional contribution of calpain-2 in obesity-accelerated AAA.
Methods and Results: Calpain-2 floxed mice that were hemizygous for β-actin Cre-ERT2 were produced by breeding male Cre-ERT2 to female calpain-2 floxed mice. At 8 weeks of age, male non-Cre littermates (Cre-) and Calp-2 x Cre-ERT2 (Cre+) mice were injected with tamoxifen (25 mg/kg, i.p.) daily for 5 consecutive days. After 2 weeks, Western blot analyses showed a complete depletion of calpain-2 protein in the aorta and periaortic adipose tissue from Cre+ mice compared to non-Cre littermates. Mice were fed a high fat diet (60% Kcal) for 20 weeks. After 16 weeks of diet feeding, mice were infused with AngII (1,000 ng/kg/min) by osmotic minipumps for 4 weeks. Depletion of calpain-2 had no effect on high fat diet-induced body weight gain, fat mass, glucose and insulin tolerance. Interestingly, calpain-2 depletion significantly attenuated AngII-induced expansion of ex-vivo maximal diameter of abdominal aortas in obese mice (Cre-: 1.4 ± 0.14; Cre+: 0.9 ± 0.04 mm; P<0.001). In addition, calpain-2 depletion significantly reduced the incidence of AngII-induced AAAs in mice (Cre-: 75%, Cre+: 7%; P< 0.001).
Conclusion: These findings suggest that calpain-2 plays a critical role in AngII-induced AAA development in diet-induced obese mice.
This research has received full or partial funding support from the American Heart Association.
Author Disclosures: A. Javidan: None. W. Jiang: None. J. Moorleghen: None. V. Subramanian: None.
Key Words: Abdominal aortic aneurysm; Angiotensin II
- © 2017 American Heart Association, Inc.