downregulation of g-protein coupled receptor signaling in the pathogenesis of viral myocarditis

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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. - PowerPoint PPT Presentation

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  • DOWNREGULATION OF G-PROTEIN COUPLED RECEPTOR SIGNALING IN THE PATHOGENESIS OF VIRAL MYOCARDITISA.B. Patel, S. Maikarfi, R.L. DeBiasi

    Ankita Patel, M.D.Pediatrics ResidentChildrens National Medical CenterWashington, D.C.AFMR Eastern Regional Annual Meeting

  • Viral MyocarditisHigh morbidity and mortalityOf those infected, death occurs in 75% newborns and 10-25% older childrenCardiac transplantation in 10-20% of affected childrenDilated cardiomyopathy and chronic myocarditisMultiple viruses: Enterovirus (Coxsackie B>A, Echo)Adenovirus (Type C)Influenza, ParainfluenzaHerpesviruses (CMV, EBV, HHV-6, Varicella)Hepatitis B and C, HIVParvovirus B19Measles, Mumps, Rubella

  • Pathogenesis of Viral Myocarditis

    Mechanisms of virus-induced damage to target cells/tissues:Early: Direct virus-mediated Late: Indirect Immune-mediatedPrecise pathophysiologic mechanism in humans uncharacterizedTherapies sub-optimal and poorly studied

  • REOVIRUSRespiratory Enteric Orphan Virus

    Well-characterized in vitro and in vivo murine models of viral myocarditis

    Strains vary in myocarditic potential:Myocarditic strains - 8BNon-myocarditic strain - T3D

  • Reovirus-induced ApoptosisH&ECaspase 3Viral AntigenMurine 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 MyocytesPrimary Murine Cardiac MyocytesSingle early time point at 18 hours post-infectionPanel of viruses of varying myocarditic potential:Mock infectedNon 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

  • HypothesisGPCR 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 ReceptorsLarge 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 ApoptosisActivated 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 activatedGPCRSIGNALINGPATHWAYANTI-APOPTOTICPATHWAY

  • MethodsImmunohistochemical staining on paraffin-embedded neonatal murine cardiac tissue

    Myocarditic (8B) and non-myocarditic (T3D) viruses

    Various timepoints post infectionEarly Day 2-3Late Day 6-7

  • ResultsIn vivo down-regulation of receptors in myocarditic virus infection at late time pointNPY1RP2YR4OLF-49GPR-88Up-regulation of inhibitory regulator - RGS-16Most prominent in regions of histologic tissue injury

  • NPY1RNon-myocarditic Day 6Myocarditic Day 7Myocarditic Day 3Non-myocarditic Day 3__________________

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

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

  • P2YR4 Myocarditic Virus Infection: Time CourseDay 2Day 6Day 7

  • RGS 16: Negative RegulatorEarly non-myocarditicEarly myocarditicLate non-myocarditicLate myocarditic______________________

  • ConclusionsDownregulation of 4 GPCRs 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 DirectionInjection 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

    Childrens Research Institute

    Childrens National Medical Center

    ******************

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