Abstract P379: Inhibition Of O-glcnac Transferase Ogt Activates P38 Signaling In Neonatal Rat Cardiomyocytes

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Kyriakos Papanicolaou ◽  
Natasha Zachara ◽  
Deepthi Ashok ◽  
Agnes Sidor ◽  
D B Foster ◽  
...  
Keyword(s):  
Heart Failure ◽  
Intracellular Signaling ◽  
Ventricular Myocytes ◽  
Fold Increase ◽  
Mitogen Activated Protein Kinase ◽  
Neonatal Rat ◽  
Rat Cardiomyocytes ◽  
Creb Phosphorylation ◽  
Glcnac Transferase

The mitogen activated protein kinase (MAPK) p38 is important in cardiac hypertrophic responses and p38 inhibition has been tested as a potential therapeutic approach to heart failure. p38 is tightly regulated by upstream kinases and phosphatases. While p38 inhibitors suppress cardiac hypertrophy in vitro and in animal models, the partial efficacy of p38 inhibitors in clinical trials for heart failure illustrates the need for a deeper understanding of p38-regulatory mechanisms. O -linked N-Acetylglucosamine ( O -GlcNAc) on Ser/Thr residues is a ubiquitous intracellular modification ( O -GlcNAcylation) that participates in intracellular signaling, often occurring in counterpoint to phosphorylation. O -GlcNAcylation is catalyzed by O -GlcNAc Transferase (OGT) and removed by O -GlcNAc-Ase (OGA). Given the crucial regulation of p38 activity by phosphorylation, we hypothesized that O -GlcNAcylation regulates p38 phosphorylation during basal and hypertrophic cardiomyocyte signaling. Treating neonatal rat ventricular myocytes (NRVM) with OSMI-1 (inhibitor of OGT) significantly decreased O -GlcNAcylation (0.48 ± 0.02, P <0.001 vs. vehicle), whereas treatment with Thiamet-G (inhibitor of OGA) significantly increased O -GlcNAcylation (3.0-fold increase ± 0.35, P <0.05 vs. vehicle). OSMI1 treatment induced the phosphorylation of p38 at its activation site (3.9-fold increase ± 0.46, P <0.001 vs. vehicle) and promoted the phosphorylation of the downstream target, heat shock protein Hsp27 (8-fold increase ± 1.3, P <0.0001 vs. vehicle) and transcription factor Creb (3.3-fold increase ± 0.12, P <0.001 vs. vehicle). OSMI-1 had an additive effect in inducing p38 and Creb phosphorylation following hypertrophic stimulation by phenylephrine (3.1-fold and 1.4-fold increase vs. phenylephrine respectively, P <0.05). Treatment with the p38 inhibitor SB202190 abolished the phosphorylation of Hsp27 and Creb that was induced by OSMI-1. Canonical upstream activators of p38 include the MAP3Ks, TAK1 and ASK1. However, we found that treatment with ASK1 or TAK1 inhibitors (GS-444217 and Takinib, respectively) either alone, or in combination, did not negate the phosphorylation of p38 by OSMI-1. We conclude that regulation of p38 by OGT activity could occur at a level downstream of canonical MAP3Ks or through non-canonical pathways.

scholarly journals GH prevents apoptosis in cardiomyocytes cultured in vitro through a calcineurin-dependent mechanism

2004 ◽  
Vol 180 (2) ◽  
pp. 325-335 ◽  
Author(s):  
◽  
R Pineiro ◽  
MJ Iglesias ◽  
O Gualillo ◽  
PA Kelly ◽  
...  
Keyword(s):  
Heart Failure ◽  
Specific Binding ◽  
Primary Cultures ◽  
Mitogen Activated Protein Kinase ◽  
Neonatal Rat ◽  
Mrna Levels ◽  
Normal Medium ◽  
Rat Cardiomyocytes ◽  
Igf I

The use of GH to treat heart failure has received considerable attention in recent years. Although the mechanisms of its beneficial effects are unknown, it has been implicated in the regulation of apoptosis in several cell types, and cardiomyocyte apoptosis is known to occur in heart failure. We therefore decided to investigate whether GH protects cardiomyocytes from apoptosis. Preliminary experiments confirmed the expression of the GH receptor (GHR) gene in primary cultures of neonatal rat cardiomyocytes (PC), the specific binding of GH by HL-1 cardiomyocytes, and the GH-induced activation of GHR and its classical downstream effectors in the latter. That GH prevented the apoptosis of PC cells deprived of serum for 48 h was shown by DNA electrophoresis and by Hoechst staining assays in which GH reduced the percentage of cells undergoing apoptosis. Similarly, the TUNEL-evaluated pro-apoptotic effect of cytosine arabinoside (AraC) on HL-1 cells was almost totally prevented by pre-treatment with GH. Fluorescence-activated cell sorter (FACS) analysis showed apoptosis in 9.7% of HL-1 cells growing in normal medium, 21.1% of those treated with AraC and 13.9% of those treated with AraC+GH, and that GH increased the percentage of AraC-treated cells in the S/G(2)/M phase from 36.9% to 52.8%. GH did not modify IGF-I mRNA levels or IGF-I secretion in HL-1 cells treated with AraC, and the protection afforded by GH against AraC-induced apoptosis in HL-1 cells was not affected by the presence of anti-IGF-I antibodies, but was largely abolished by the calcineurin-inhibiting combination cyclosporin+FK506. GH also reduced AraC-induced phosphorylation of mitogen-activated protein kinase p38 (MAPK p38) in HL-1 cells. In summary, GH protects PC and HL-1 cells from apoptosis. This effect is not mediated by IGF-I and may involve MAPK p38 as well as calcineurin.

Abstract 208: Mitochondrial-targeted Grk2 Increases Superoxide Formation And Impairs Cardiomyocyte Respiratory Reserve Capacity

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Priscila Y Sato ◽  
J K Chuprun ◽  
Jessica Ibetti ◽  
John W Elrod ◽  
Walter J Koch
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Permeability Transition ◽  
Fold Increase ◽  
Confocal Imaging ◽  
Neonatal Rat ◽  
Redox Stress ◽  
Mitochondrial Oxidative Stress ◽  
Neonatal Rat Ventricular Myocytes ◽  
Cellular Dysfunction

β-adrenergic receptors (βARs) are powerful regulators of cardiovascular function and are impaired in heart failure (HF). Signal transduction of βARs is canonically shut down by phosphorylation via G protein-coupled receptor kinase 2 (GRK2) and the subsequent binding of β-arrestins. This process of receptor desensitization is enhanced in HF via the up-regulation of GRK2 and contributes to disease progression. We have recently reported non-canonical actions of GRK2, which contribute to the development of HF independent of βAR desensitization. We have previously shown that GRK2 can act as a pro-death kinase in cardiomyocytes bytranslocating to mitochondria and activating mitochondria permeability transition. This study was designed to gain more understanding of the mitochondrial function of GRK2. We isolated adult cardiomyocytes from cardiac-specific transgenic mice overexpressing GRK2 at levels found in human HF (TgGRK2), and examined superoxide production using the redox sensitive reporter MitoSox Red. Confocal imaging revealed a 4.6 fold increase in superoxide levels in cardiomyocytes overexpressing Grk2 as compared to non-transgenic (NLC) cardiomyocytes (corrected total cell fluorescence 11.59±1.06, TgGRK2 (n= 3 hearts, 88 cells) vs 2.54±0.02 NLC (n=3 hearts, 52 cells), (p<0.001). This indicates that the chronic elevation of GRK2 induces mitochondrial oxidative stress priming the myocyte for enhanced injury. To further explore the mitochondrial actions of GRK2 and consequences of redox stress we examined oxidative phosphorylation by performing oxygen consumption measurements in neonatal rat ventricular myocytes overexpressing GRK2 or GFP-expressing control myocytes. Seahorse analysis showed that cells overexpressing GRK2 have a significant decrease in spare respiratory capacity indicating that cells with elevated GRK2 levels have an impaired capacity to generated ATP during times of stress. Further studies with mutants that limit GRK2 kinase activity or mitochondrial localization demonstrate that mitochondrial GRK2 may be a significant contributor to cellular dysfunction as seen in heart failure.

Abstract 356: KChIP2 Mediates Ion Channel Dysregulation by Novel Transcriptional Regulation of miR-34s

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Drew Nassal ◽  
Haiyan Liu ◽  
Xiaoping Wan ◽  
Angelina Ramirez-Navarro ◽  
Eckhard Ficker ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ion Channel ◽  
Transcriptional Control ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Physical Interaction ◽  
Protein Patterns ◽  
Protein Levels ◽  
Disease States

Introduction: Cardiac ion channel dysregulation is a hallmark of heart failure. Consistently, the disease yields dramatic decline in Ito through loss in Kv4 and its auxiliary partner KChIP2. Notably, transcriptional changes in heart failure can be elicited through KChIP2 silencing without disease signaling, suggesting potential transcriptional capacity for KChIP2. Further, disparity between resulting transcript and protein patterns suggests a mechanism compatible with modified miRNA activity. Considering other members of the KChIP family behave as transcriptional repressors, we hypothesize that KChIP2 regulates discrete miRNAs which in turn regulate cardiac excitability. Methods and Results: A miRNA microarray was conducted on neonatal rat ventricular myocytes (NRVM) following in vitro silencing of KChIP2 by siRNA, identifying the miR-34 family as potential transcriptional targets of KChIP2. Regulation, confirmed by quantitative PCR, showed reduction in miR-34a/b/c when over-expressing KChIP2 and increase following silencing. Luciferase assays were performed on the cloned promoter for miR-34b/c which reinforced direct KChIP2 repression on the miR-34b/c promoter. Furthermore, chromatin immunoprecipitation followed by PCR identified physical interaction of KChIP2 to the promoter site. Previous studies show modified expression of KChIP2 can lead to changes in several ion channel subunits. Therefore, we investigated if this was the consequence of KChIP2 regulation via miR-34. miR-34a/b/c precursors were expressed in NRVM which reduced transcript levels of Nav1.5 and Navβ1, and reduced protein levels for Kv4.3. Reflecting these changes, peak INa was reduced following miR precursor treatment. NRVMs were exposed to 100 μM phenylephrine for 48 hrs, significantly reducing KChIP2, Nav1.5, Navβ1, and Kv4.3, while elevating miR-34b/c. Returning KChIP2 expression by adenovirus normalized these changes back towards baseline, implicating the physiologic relevance of this pathway. Conclusion: These observations describe a novel mechanism where KChIP2 regulates a host of cardiac genes through transcriptional control of miRNAs, potentially explaining electrical remodeling observed in disease states where KChIP2 is reduced.

Abstract 17473: GRK2 in the Mitochondria: Uncovering Novel Mechanisms in Heart Failure

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Kimberly M Ferrero ◽  
Gizem Kayki Mutlu ◽  
Jessica M Pfleger ◽  
Douglas G Tilley ◽  
Walter J Koch
Keyword(s):  
Heart Failure ◽  
Mitochondrial Dysfunction ◽  
Ventricular Myocytes ◽  
Atp Synthesis ◽  
Neonatal Rat ◽  
Negative Effects ◽  
Protein Kinase C Inhibitor ◽  
Neonatal Rat Ventricular Myocytes

Introduction: During heart failure, levels and activity of G protein-coupled receptor kinase 2 (GRK2) increase. GRK2 is canonically studied in the phosphorylation of GPCRs and β-adrenergic desensitization. Noncanonical activities of GRK2 are being uncovered, however. Our lab has recently discovered that in cardiac myocytes, GRK2 translocates to the mitochondria ( mtGRK2 ) following injury and is associated with negative effects on metabolism and cell survival. Hypothesis: GRK2 plays a role in regulating mitochondrial function following cardiac stress and contributes to HF pathogenesis in a novel manner, by interacting with a novel group of mitochondrial proteins involved in pro-death signaling, bioenergetics and substrate utilization. Methods: Mitochondrial translocation of GRK2 was validated with either protein kinase C inhibitor (chelerythine) administration or hypoxia/reoxygenation stress in primary neonatal rat ventricular myocytes or a cardiac-like cell line. Immunoprecipitation of the GRK2 interactome basally and under stress conditions was conducted endogenously in vitro, in vivo , and with purified recombinant GRK2 peptides. Proteins were separated via SDS-PAGE and potential binding partners were identified by mass spectroscopy (LCMS) and proteomics analysis conducted with Ingenuity Pathway (IPA; Qiagen) software to determine which partners in the GRK2 interactome were potentially involved in mitochondrial dysfunction. Results: Subunits of Complexes I, II, IV and V of the electron transport chain were identified as potential mtGRK2 interacting partners. Several mtGRK2-ETC interactions were increased following oxidative stress-induced translocation of GRK2. Finally, mtGRK2 appears to phosphorylate some of the interactome partners identified in mitochondrial dysfunction. Conclusions: The phosphorylation of subunits of the ATP synthesis machinery by mtGRK2, or other mechanisms of interaction between these proteins, may be regulating some of the phenotypic effects of HF previously observed by our lab, such as increased ROS production and reduced fatty acid metabolism. Further research is essential to elucidate the novel role of GRK2 in regulating mitochondrial bioenergetics and cell death in failing hearts.

scholarly journals Cell stress-induced phosphorylation of ATF2 and c-Jun transcription factors in rat ventricular myocytes

1997 ◽  
Vol 325 (3) ◽  
pp. 801-810 ◽  
Author(s):  
Angela CLERK ◽  
Peter H. SUGDEN
Keyword(s):  
Transcription Factors ◽  
Okadaic Acid ◽  
Ventricular Myocytes ◽  
Fold Increase ◽  
Neonatal Rat ◽  
Western Blots ◽  
Hyperosmotic Shock ◽  
Nuclear Extracts

Ventricular myocytes are exposed to various pathologically important cell stresses in vivo. In vitro,extreme stresses (sorbitol-induced hyperosmotic shock in the presence or absence of okadaic acid, and anisomycin) were applied to ventricular myocytes cultured from neonatal rat hearts to induce a robust activation of the 46 and 54 kDa stress-activated protein kinases (SAPKs). These activities were increased in nuclear extracts of cells in the absence of any net import of SAPK protein. Phosphorylation of ATF2 and c-Jun was increased as shown by the appearance of reduced-mobility species on SDS/PAGE, which were sensitive to treatment with protein phosphatase 2A. Hyperosmotic shock and anisomycin had no effect on the abundance of ATF2. In contrast, cell stresses induced a greater than 10-fold increase in total c-Jun immunoreactivity detected on Western blots with antibody to c-Jun (KM-1). Cycloheximide did not inhibit this increase, which we conclude represents phosphorylation of c-Jun. This conclusion was supported by use of a c-Jun(phospho-Ser-73) antibody. Immunostaining of cells also showed increases in nuclear phospho-c-Jun in response to hyperosmotic stress. Severe stress (hyperosmotic shock+okadaic acid for 2 h) induced proteins (migrating at approx. 51 and 57 kDa) that cross-reacted strongly with KM-1 antibodies in both the nucleus and the cytosol. These may represent forms of c-Jun that had undergone further modification. These studies show that stresses induce phosphorylation of transcription factors in ventricular myocytes and we suggest that this response may be pathologically relevant.

scholarly journals LCZ696 Ameliorates Oxidative Stress and Pressure Overload-Induced Pathological Cardiac Remodeling by Regulating the Sirt3/MnSOD Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Shi Peng ◽  
Xiao-feng Lu ◽  
Yi-ding Qi ◽  
Jing Li ◽  
Juan Xu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heart Failure ◽  
Cardiac Hypertrophy ◽  
Pressure Overload ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Rat Cardiomyocytes ◽  
Pathological Cardiac Hypertrophy

Aims. We aimed to investigate whether LCZ696 protects against pathological cardiac hypertrophy by regulating the Sirt3/MnSOD pathway. Methods. In vivo, we established a transverse aortic constriction animal model to establish pressure overload-induced heart failure. Subsequently, the mice were given LCZ696 by oral gavage for 4 weeks. After that, the mice underwent transthoracic echocardiography before they were sacrificed. In vitro, we introduced phenylephrine to prime neonatal rat cardiomyocytes and small-interfering RNA to knock down Sirt3 expression. Results. Pathological hypertrophic stimuli caused cardiac hypertrophy and fibrosis and reduced the expression levels of Sirt3 and MnSOD. LCZ696 alleviated the accumulation of oxidative reactive oxygen species (ROS) and cardiomyocyte apoptosis. Furthermore, Sirt3 deficiency abolished the protective effect of LCZ696 on cardiomyocyte hypertrophy, indicating that LCZ696 induced the upregulation of MnSOD and phosphorylation of AMPK through a Sirt3-dependent pathway. Conclusions. LCZ696 may mitigate myocardium oxidative stress and apoptosis in pressure overload-induced heart failure by regulating the Sirt3/MnSOD pathway.

scholarly journals CTRP5-Overexpression Attenuated Ischemia-Reperfusion Associated Heart Injuries and Improved Infarction Induced Heart Failure

2020 ◽  
Vol 11 ◽  
Author(s):  
Meng Peng ◽  
Yuan Liu ◽  
Xiang-qin Zhang ◽  
Ya-wei Xu ◽  
Yin-tao Zhao ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Mouse Heart ◽  
Related Protein ◽  
Rat Cardiomyocytes ◽  
Knock Out ◽  
Cardiac Functions

Aims: C1q/tumor necrosis factor (TNF)-related protein 5 (CTRP5) belongs to the C1q/TNF-α related protein family and regulates glucose, lipid metabolism, and inflammation production. However, the roles of CTRP5 in ischemia/reperfusion (I/R) associated with cardiac injuries and heart failure (HF) needs to be elaborated. This study aimed to investigate the roles of CTRP5 in I/R associated cardiac injuries and heart failure.Materials and Methods: Adeno-associated virus serum type 9 （AAV9）vectors were established for CTRP5 overexpression in a mouse heart (AAV9-CTRP5 mouse). AAV9-CTRP5, AMPKα2 global knock out （AMPKα2−/−）and AAV9-CTRP5+ AMPKα2−/− mice were used to establish cardiac I/R or infarction associated HF models to investigate the roles and mechanisms of CTRP5 in vivo. Isolated neonatal rat cardiomyocytes (NRCMS) transfected with or without CTRP5 adenovirus were used to establish a hypoxia/reoxygenation (H/O) model to study the roles and mechanisms of CTRP5 in vitro.Key Findings: CTRP5 was up-regulated after MI but was quickly down-regulated. CTRP5 overexpression significantly decreased I/R induced IA/AAR and cardiomyocyte apoptosis, and attenuated infarction area, and improved cardiac functions. Mechanistically, CTRP5 overexpression markedly increased AMPKα2 and ACC phosphorylation and PGC1-α expression but inhibited mTORC1 phosphorylation. In in vitro experiments, CTRP5 overexpression could also enhance AMPKα2 and ACC phosphorylation and protect against H/O induced cardiomyocytes apoptosis. Finally, we showed that CTPR5 overexpression could not protect against I/R associated cardiac injuries and HF in AMPKα2−/− mice.Significance: CTRP5 overexpression protected against I/R induced mouse cardiac injuries and attenuated myocardial infarction induced cardiac dysfunction by activating the AMPKαsignaling pathway.

scholarly journals Human Neonatal Thymus Mesenchymal Stem Cells Promote Neovascularization and Cardiac Regeneration

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Shuyun Wang ◽  
Shan Huang ◽  
Lianghui Gong ◽  
Zhize Yuan ◽  
Joshua Wong ◽  
...  
Keyword(s):  
Heart Failure ◽  
Congenital Heart Disease ◽  
Heart Disease ◽  
Congenital Heart ◽  
Neonatal Rat ◽  
Vascular Density ◽  
Rat Cardiomyocytes ◽  
Coronary Ligation ◽  
Thymus Tissue

Newborns with critical congenital heart disease are at significant risk of developing heart failure later in life. Because treatment options for end-stage heart disease in children are limited, regenerative therapies for these patients would be of significant benefit. During neonatal cardiac surgery, a portion of the thymus is removed and discarded. This discarded thymus tissue is a good source of MSCs that we have previously shown to be proangiogenic and to promote cardiac function in an in vitro model of heart tissue. The purpose of this study was to further evaluate the cardiac regenerative and protective properties of neonatal thymus (nt) MSCs. We found that ntMSCs expressed and secreted the proangiogenic and cardiac regenerative morphogen sonic hedgehog (Shh) in vitro more than patient-matched bone-derived MSCs. We also found that organoid culture of ntMSCs stimulated Shh expression. We then determined that ntMSCs were cytoprotective of neonatal rat cardiomyocytes exposed to H2O2. Finally, in a rat left coronary ligation model, we found that scaffoldless cell sheet made of ntMSCs applied to the LV epicardium immediately after left coronary ligation improved LV function, increased vascular density, decreased scar size, and decreased cardiomyocyte death four weeks after infarction. We conclude that ntMSCs have cardiac regenerative properties and warrant further consideration as a cell therapy for congenital heart disease patients with heart failure.

Identification of hypertrophy- and heart failure-associated genes by combining in vitro and in vivo models

2012 ◽  
Vol 44 (8) ◽  
pp. 443-454 ◽  
Author(s):  
Bo Lu ◽  
Hongjuan Yu ◽  
Maarten Zwartbol ◽  
Willem P. Ruifrok ◽  
Wiek H. van Gilst ◽  
...  
Keyword(s):  
Heart Failure ◽  
Stress Responses ◽  
Differentially Expressed ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Novel Genes ◽  
In Vivo Models ◽  
Rat Cardiomyocytes

Heart failure (HF) is a complex disease involving multiple changes including cardiomyocyte hypertrophy (growth). Here we performed a set of screens in different HF and hypertrophy models to identify differentially expressed genes associated with HF and/or hypertrophy. Hypertensive Ren2 rats and animals with postmyocardial infarction (post-MI) HF were used as in vivo HF models, and neonatal rat cardiomyocytes treated with hypertrophy inducing hormones phenylephrine, endothelin-1, and isoproterenol were used as in vitro models. This combined approach revealed a robust set of genes that were differentially expressed both in vitro and in vivo. This included known genes like NPPA (ANP) and FHL1, but also novel genes not previously associated with hypertrophy/HF. Among these are PTGIS, AKIP1, and Dhrs7c, which could constitute interesting targets for further investigations. We also identified a number of in vivo specific genes and these appeared to be enriched for fibrosis, wounding, and stress responses. Therefore a number of novel genes within this in vivo specific list could be related to fibroblasts or other noncardiomyocytes present in the heart. We also observed strong differences between the two HF rat models. For example KCNE1 was strongly upregulated in Ren2, but not in post-MI HF rats, suggesting possible etiology-specific differences. Moreover, Gene Ontology analysis revealed that genes involved in fatty acid oxidation were specifically down regulated in the post-MI group only. Together these results show that combining multiple models, both in vivo and in vitro, can provide a robust set of hypertrophy/HF-associated genes. Moreover it provides insight in the differences between the different etiology models and neurohormonal effects.

Abstract 128: Network-based Approaches to Identify Novel Regulators of Heart Failure

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Christoph D Rau ◽  
Milagros C Romay ◽  
Jessica Wang ◽  
Shuxun Ren ◽  
Yibin Wang ◽  
...  
Keyword(s):  
Heart Failure ◽  
Gene Networks ◽  
Structural Equation ◽  
Cardiac Fibrosis ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Heart Weight ◽  
Equation Modeling ◽  
Phenotypic Traits

Heart failure is a highly heterogeneous disorder characterized by the interactions of multiple environmental and genetic factors. While reductionistic approaches have made significant inroads into characterizing the pathophysiology of the syndrome, they are unable to properly dissect the complex interactions between sets of genes and pathways which result in the emergent phenotypes . Systems genetics offers a means by which these interactions may be identified and explored. We have developed a resource, the Hybrid Mouse Diversity Panel (HMDP) to perform systems-level analyses in mice. Nine week old female mice from 93 unique inbred lines of the HMDP were give 30 ug/g/day of isoproterenol through an abdominally implanted Alzet micropump. After three weeks, mice were sacrificed along with age-matched controls. A portion of the left ventricle was arrayed on an Illumina Mouse Ref 8.0 platform. Maximal Information Component Analysis was used to construct gene networks, and a module of 41 genes was identified which shows strong correlation to a number of important phenotypic traits, including heart weight and cardiac fibrosis. This module contains a number of genes of interest, including Lgals3, a diagnostic marker for heart failure. Through the use of structural equation modeling, we identified several key genes within the module for further analysis, the most important of which is the metalloprotease Adamts2. We have performed a series of in vitro analyses demonstrating the important role of Adamts2 in this module using neonatal rat ventricular myocytes. Knockout of Adamts2 results in an amelioration of the hypertrophic response to catecholamine stimulation as well as a reduction of hypertrophic markers such as Nppa and Nppb. Furthermore, we observe that other genes in the module no longer respond to catecholamine stimulation after knockdown of Adamts2.

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