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