downregulation of g-protein coupled receptor signaling in the pathogenesis of viral myocarditis a.b....
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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 CenterWashington, D.C.
AFMR Eastern Regional Annual Meeting
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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
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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
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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
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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
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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
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Microarray of Cardiac Myocytes Infected with Reovirus at Early Timepoint Post-infection
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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.
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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
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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
GPCRSIGNALINGPATHWAY
ANTI-APOPTOTICPATHWAY
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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
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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
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NPY1R
Non-myocarditic Day 6 Myocarditic Day 7
Myocarditic Day 3Non-myocarditic Day 3__________________
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OLF-49
Myocarditic Day 7Non-myocarditic Day 5
Non-myocarditic Day 3
Myocarditic Day 3
____________________
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GPR-88
Non-myocarditic Day 3 Myocarditic Day 3
Myocarditic Day 7Non-myocarditic Day 6
___________________
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P2YR4 – Myocarditic Virus Infection: Time Course
Day 2
Day 6
Day 7
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RGS 16: Negative Regulator
Early non-myocarditic Early myocarditic
Late non-myocarditic Late myocarditic
______________________
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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
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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
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Special Thanks
Dr. Roberta DeBiasi – Mentor
Sally Maikarfi – Research Assistant
Children’s Research Institute
Children’s National Medical Center