1. GRK2 Inhibition Reduces Post-Myocardial Infarction Cardiac Fibroblast-Mediated Adverse Remodeling Jennifer L. Philip 1, Xianyao Xu 1,Mei Han 1, Jinju Li 2, Abdur Razzaque 1, Shahab A. Akhter 1 AATS Lillehei Resident Forum April 27,2015 Divisions of Cardiothoracic Surgery 1 University of Wisconsin & 2 University of Chicago

2. Disclosures None

3. Post-Infarction Remodeling Increased Interstitial collagen Adverse Remodeling Heart Failure Adapted From: Zouein FA et al. Post-infarct biomaterials, left ventricular remodeling, and heart failure: is good good enough? Congest Heart Fail. 2012 Sep-Oct;18(5):284-90. Konstam MA et al. Left ventricular remodeling in heart failure: current concepts in clinical significance and assessment. JACC Cardiovasc Imaging. 2011 Jan;4(1):98-108.

4. Adapted From: Porter KE, Turner NA. Cardiac fibroblasts: at the heart of myocardial remodeling. Pharmacol Ther. 2009 Aug, 123(2):255-78. β-agonist Adapted from: Paul A Insel, Fiona Murray, Utako Yokoyama, Silvia Romano, Hongruo Yun, Loren Brown, Aaron Snead, David Lu, Nakon Aroonsakool. cAMP and Epac in the regulation of tissue fibrosis. Br J Pharmacol. 2012 May; 166(2): 447–456. Cardiac Fibroblasts Cardiac Fibroblasts (CF) make up 60-70% of cells in the heart CF play a critical role in adverse remodeling HF phenotype characterized by: differentiation into myofibroblasts and increased collagen production

5. Adapted From: Howard A. Rockman, Walter J. Koch & Robert J. Lefkowitz. Seven-transmembrane-spanning receptors and heart function. Nature. 2002 Jan 10;415(6868):206-12. GPCR uncoupling and endocytosis GPCR Phosphorylation and Desensitization Prolonged Agonist Stimulation cAMP Karen M. D'Souza et al. J. Biol. Chem. 2011 GRK2 Control Vimentin HF Rhodopsin (38kD) * ControlHF The Role of β-ARs and GRK2 in CF β-adrenergic receptor (β-AR) signaling/cAMP inhibits CF-mediated fibrosis G protein-coupled receptor kinase-2 (GRK2) uncouples β-AR signaling GRK2 upregulated in human HF

6. Hypotheses GRK2 is upregulated in cardiac fibroblasts post-MI and is a primary etiology of maladaptive ventricular remodeling via uncoupling of β-ARs and decreased intracellular cAMP production Inhibition of GRK2 in vivo can decrease post-MI ventricular remodeling and cardiac dysfunction by inhibiting cardiac fibroblast activation and collagen synthesis

7. Whole Heart Infarct Area Remote Territory Picrosirius red stain: Collagen stained Red Control * * * * * # # * 2 4 8 12 Weeks Post-MI *p<0.01 vs. Control; n=3-8 *p<0.04 vs. Control, #p<0.01 vs. Control &vs. 2wk Post-MI; n=3-8 Post-MI Ventricular Remodeling

8. Development of Failing CF Phenotype Post-MI * Collagen I Green α-SMA Red ControlPost-MI Vimentin Red Nuclei stained Blue with DAPI Collagen I (134 kDa) Post-MI Control * * *p<0.0001 vs. Control; n=4 *p<0.0002 vs. Control; n=3-4 α-SMA (42 kDa) GAPDH (37 kDa)

9. * Increased CF Collagen Synthesis and Uncoupled β–AR signaling Post-MI * * * ** # *p<0.02 vs. Control + No Drug, **p<0.005 vs. Control + Iso, #p<0.01 vs. Control + TGF-β; n=7-9 *p<0.02 vs. Control; n=3

10. * GRK2 is Upregulated Post-MI GRK2 (80 kDa) GAPDH (37 kDa) ControlPost-MI Vimentin Red Nuclei stained Blue with DAPI GRK2 Green Post-MI Control * *p<0.02 vs. Control; n=4

11. GRK2 (80 kDa) GAPDH (37kDa) α-SMA (42 kDa) GRK2ct (27 kDa) ControlMI+Ad-NullMI+Ad-GRK2ct GAPDH (37kDa) * Collagen I (134kDa) GAPDH (37kDa) * ControlMI+Ad-NullMI+Ad-GRK2ct Control MI+ Ad-Null MI+ Ad-GRK2ct Inhibition of GRK2 in vivo Decreases Post-MI Activation of Cardiac Fibroblasts *p<0.02 vs. Control & vs. MI+Ad-GRK2ct, n=5 *p<0.04 vs. Control & vs. MI+Ad-GRK2ct, n=5

12. * ** Control MI+Ad-NullMI+Ad-GRK2ct * ** Whole Heart Infarct Area Remote Territory Inhibition of GRK2 in vivo Decreases Post-Infarction Ventricular Fibrosis Picrosirius red stain: Collagen stained Red *p<0.001 vs. Control, **p<0.002 vs. Ad-Null & vs. Control; n=3-5 *p<0.001 vs. Control, **p<0.002 vs. Ad-Null & vs. Control; n=3-5

13. * ** ControlMI+Ad-NullMI+Ad-GRK2ct Inhibition of GRK2 Preserves Ventricular Function *p<0.01 vs. Control, **p<0.03 vs. Ad-Null; n=4-5

14. Conclusions Post-MI LV remodeling is characterized by significant remote territory fibrosis and CF differentiation and activation Uncoupling of β-AR signaling via increased GRK2 appears to be an important mechanism of CF-mediated myocardial fibrosis Inhibition of GRK2 decreases cardiac fibroblast transformation and collagen synthesis resulting in decreased maladaptive ventricular remodeling

15. Future Directions Investigation of the potential role of GRK2 in MMP and TIMP biology Studying additional regulators of GPCR signaling including β-arrestin in CF biology Creation of a fibroblast-specific transgenic mouse model for inhibition of GRK2 to further explore CF-specific inhibition of GRK2 as a potential therapeutic strategy to prevent maladaptive ventricular remodeling

16. Acknowledgements PI – Shahab Akhter, MD Lab members – Xianyao Xu – Mei Han – Abdur M. Razzaque, PhD – Tiju Theccanat – Jinju Li, PhD University of Wisconsin Department of Surgery University of Chicago Department of Surgery Funding: – Howard Hughes Medical Institute – National Institutes of Health