DOWNREGULATION OF G-PROTEIN COUPLED RECEPTOR SIGNALING IN THE PATHOGENESIS OF VIRAL MYOCARDITIS A.B. Patel, S. Maikarfi, R.L. DeBiasi Ankita Patel, M.D. Pediatrics Resident Children’s National Medical Center Washington, D.C. AFMR Eastern Regional Annual Meeting

Viral Myocarditis  High morbidity and mortality  Of those infected, death occurs in 75% newborns and % older children  Cardiac transplantation in 10-20% of affected children  Dilated cardiomyopathy and chronic myocarditis  Multiple viruses:  Enterovirus (Coxsackie B>A, Echo)  Adenovirus (Type C)  Influenza, Parainfluenza  Herpesviruses (CMV, EBV, HHV-6, Varicella)  Hepatitis B and C, HIV  Parvovirus B19  Measles, Mumps, Rubella

Pathogenesis of Viral Myocarditis  Mechanisms of virus-induced damage to target cells/tissues:  Early: Direct virus-mediated  Late: Indirect Immune-mediated  Precise pathophysiologic mechanism in humans uncharacterized  Therapies sub-optimal and poorly studied

REOVIRUS  Respiratory Enteric Orphan Virus  Well-characterized in vitro and in vivo murine models of viral myocarditis  Strains vary in myocarditic potential:  Myocarditic strains - 8B  Non-myocarditic strain - T3D

Reovirus-induced Apoptosis H&ECaspase 3Viral Antigen Murine cardiac cross-sections, 7 days post-infection with myocarditic Reovirus strain, co-localization of tissue injury, apoptosis, and Reovirus

Differential Gene Expression Reovirus-infected Primary Cardiac Myocytes  Primary Murine Cardiac Myocytes  Single early time point at 18 hours post-infection  Panel of viruses of varying myocarditic potential:  Mock – infected  Non – myocarditic (T3D and T1L)  Myocarditic (8B and T3A)  G-protein Coupled Receptors shown to be significantly altered in expression

Microarray of Cardiac Myocytes Infected with Reovirus at Early Timepoint Post-infection

Hypothesis GPCR signaling components are differentially expressed at the protein level in cardiac tissue in the setting of myocarditic viral infection, when compared to non-myocarditic viral infection.

G-Protein Coupled Receptors  Large family of proteins whose primary function transduction of extracellular stimuli into intracellular signals  GPCR are seven-transmembrane proteins; ubiquitously expressed  Involved in a variety of physiologic and pathologic processes

GPCR and Apoptosis  Activated G-proteins regulate downstream cell-signaling effectors, including cascades modulating cell proliferation and death  GPCR capable of simultaneously coupling to pro/anti-apoptotic pathways  May serve as flexible regulators of the fate of the cell depending on environment in which activated GPCR SIGNALING PATHWAY ANTI- APOPTOTIC PATHWAY

Methods  Immunohistochemical staining on paraffin- embedded neonatal murine cardiac tissue  Myocarditic (8B) and non-myocarditic (T3D) viruses  Various timepoints post infection  Early – Day 2-3  Late – Day 6-7

Results  In vivo down-regulation of receptors in myocarditic virus infection at late time point  NPY1R  P2YR4  OLF-49  GPR-88  Up-regulation of inhibitory regulator - RGS-16  Most prominent in regions of histologic tissue injury

NPY1R Non-myocarditic Day 6Myocarditic Day 7 Myocarditic Day 3Non-myocarditic Day 3 __________________

OLF-49 Myocarditic Day 7Non-myocarditic Day 5 Non-myocarditic Day 3Myocarditic Day 3 ____________________

GPR-88 Non-myocarditic Day 3Myocarditic Day 3 Myocarditic Day 7Non-myocarditic Day 6 ___________________

P2YR4 – Myocarditic Virus Infection: Time Course Day 2 Day 6 Day 7

RGS 16: Negative Regulator Early non-myocarditicEarly myocarditic Late non-myocarditicLate myocarditic ______________________

Conclusions  Downregulation of 4 GPCR’s and upregulation of 1 inhibitory regulator in setting of myocarditic virus infection in vivo  Alterations in GPCR signaling likely plays a significant role in pathogenesis of reovirus- induced myocarditis  Tipping the balance of cell survival/death signals toward death by inhibiting a protective GPCR pathway

Future Direction  Injection of mice with pharmacologic regulator of G-protein coupled receptor signaling  Analysis of differential expression of these receptors in myocarditic vs. non-myocarditic viral infection  Determine novel therapeutic options for viral myocarditis by targeting these pathways

Special Thanks  Dr. Roberta DeBiasi – Mentor  Sally Maikarfi – Research Assistant  Children’s Research Institute  Children’s National Medical Center