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DOWNREGULATION OF G-PROTEIN COUPLED RECEPTOR SIGNALING IN THE PATHOGENESIS OF VIRAL MYOCARDITIS - PowerPoint PPT Presentation


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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.

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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
Viral Myocarditis

  • High morbidity and mortality

    • Of those infected, death occurs in 75% newborns and 10-25% 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
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
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
Reovirus-induced Apoptosis

H&E

Caspase 3

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


Hypothesis
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
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
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
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
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
NPY1R

__________________

Non-myocarditic Day 3

Myocarditic Day 3

Non-myocarditic Day 6

Myocarditic Day 7


Olf 49
OLF-49

____________________

Non-myocarditic Day 3

Myocarditic Day 3

Non-myocarditic Day 5

Myocarditic Day 7


Gpr 88
GPR-88

___________________

Non-myocarditic Day 3

Myocarditic Day 3

Non-myocarditic Day 6

Myocarditic Day 7



Rgs 16 negative regulator
RGS 16: Negative Regulator

______________________

Early non-myocarditic

Early myocarditic

Late non-myocarditic

Late myocarditic


Conclusions
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
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
Special Thanks

  • Dr. Roberta DeBiasi – Mentor

  • Sally Maikarfi – Research Assistant

  • Children’s Research Institute

  • Children’s National Medical Center


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