downregulation of g-protein coupled receptor signaling in the pathogenesis of viral myocarditis a.b....
TRANSCRIPT
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
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
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&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
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
GPCRSIGNALINGPATHWAY
ANTI-APOPTOTICPATHWAY
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 6 Myocarditic Day 7
Myocarditic Day 3Non-myocarditic Day 3__________________
OLF-49
Myocarditic Day 7Non-myocarditic Day 5
Non-myocarditic Day 3
Myocarditic Day 3
____________________
GPR-88
Non-myocarditic Day 3 Myocarditic 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-myocarditic Early myocarditic
Late non-myocarditic Late 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