Myocarditis Research Presented by Candace Moose & Dr. Leslie Cooper

Our Grant History The foundation will have awarded eleven research fellowship grants by year end Each grant is awarded in the amount of $30,000 to $35,000 dollars. They have been awarded to doctors in University Hospitals across the US and in Canada

Application Process 1.Applicants send their grant applications to the foundation by December 1 st. 2.The foundation’s Medical Advisory Board scores the proposals and make a recommendation to the Board of Directors as to which grants should be funded. 3.Applicants are notified and we begin negotiations on their contracts. 4.When Contracts are signed, research begins on July 1 st through June 30 th of the following year. 5.We receive updates on their research and maintain contact even after their research has been completed

Application Overview The MYOCARDITIS FOUNDATION (MF) awards funds to support research related to all forms of myocarditis research. The goal of MF’s research program is to advance medical knowledge on the disease and develop more accurate diagnostic methods and life-saving therapies with the goal of saving more lives. TYPES OF PROPOSALS SOUGHT -The Myocarditis Foundation (MF) accepts fellowship grant applications on an annual basis for innovative basic, clinical or translational research relevant to the cause or treatment of myocarditis. MF’s fellowship grant program is designed to provide seed funding to investigators for the testing of initial hypotheses and collecting preliminary data to help secure long-term funding by the National Institute of Health (NIH) and other major granting institutions. Funding is available at $35,000 USD for salary only. -Grant award decisions are made through a peer review process by our Medical Advisory Board. Scientific excellence and relevance to myocarditis are the basic criteria for selecting the supported research project. The award is designed to support training and career development of physician-scientists in myocarditis research. AVAILABLE FUNDING The MF research grant provides salary support for 1 year of full-time research. The stipend is $35,000. No additional funds for benefits, travel, or indirect costs, etc. are available. ELIGIBILITY REQUIREMENTS Candidates may apply up to 10 years following receipt of an MD, PhD, or an equivalent degree and plan to perform the funded work in the United States or Canada, in order to apply for the Fellowship. All applicants must select a preceptor with a proven track record of research in myocarditis. In addition to providing a letter of recommendation, the preceptor is expected to assist in preparing the application. For applicants wishing advice in the selection of a preceptor, a list of potential preceptors is available from the MF. APPLICATION PROCEDURE The MF will issue one request for proposals in September The deadline for grant submission is December 1, 2012 with final award decisions made by December 31, The research plan should be limited to 5 pages and must include the following: hypothesis, specific aims, background/significance, preliminary data, methods and expected results. The applicant should include a cover letter, supporting letter from the preceptor, and applicant and preceptor biographical sketches. Upon receipt of a signed letter of agreement from the selected candidate the MF will disburse the funds in installments during the research year. FINAL REPORT A final report will be required upon completion of the research year. The Myocarditis Foundation reserves the right to cite the research in all/any of our printed materials and on our website. The Myocarditis Foundation must be acknowledged in all publications resulting from the research.

Grant Recipients Dr. Daniela Cihakova at Johns Hopkins University Dr. Bettina Heidecker at the University of Miami Dr. Susan Wollersheim at the University of California in Los Angeles Dr. Silvio Antoniak at University of North Carolina in Chapel Hill Dr. David Marchant at the University of British Columbia in Vancouver, Canada Dr. Byung Kwan Lim at the University of California in San Diego Dr. Kathleen Simpson at Washington University Dr. Alan Valaperti at the University of Toronto in Canada Dr. Laure Case at the University of Vermont in Burlington Dr. Kevin Quinn at the University of California in Los Angeles

Grant Recipients! We are happy to announce this year’s grant recipients: Dr. Laure Case of the University of Vermont in Burlington Department of Medicine “Chromosome Y Regulates Susceptibility to Coxsackievirus B3-induced Myocarditis” Dr. Kevin Quinn of the University of California in Los Angeles Department of Pediatrics “Identifying Antiviral Agents to Treat Coxsackievirus Myocarditis”

Dr. Daniela Cihakova of Johns Hopkins University Awarded grant in 2007 “The Role of Monocytes in Autoimmune Myocarditis “We have assembled evidence that EAM is driven in part by the Th17 pathway in the mouse; however, in the absence of IL-17A an equally severe disease can be driven through the Th1 pathway. It is not yet known whether IL-17 plays a similarly dominant role in the pathogenesis of human myocarditis. Given a lack of immunomodulatory treatments for myocarditis, blocking of the IL- 23/IL-17 pathway could be very attractive option. Thus, in myocarditis different pathways can result in comparable disease (Cihakova et al., 2008)’’

Dr. Bettina Heidecker of the University of Miami Awarded grant in 2007 “Gene Expression Profiling for Detection of Myocarditis” “The overall goal of this study is to improve diagnostic sensitivity for detection of myocarditis by endomyocardial biopsy derived transcriptomic biomarkers and to evaluate peripheral blood mononuclear cells as possible surrogates of heart tissue.”

Dr. Susan Wollersheim of the University of California in Los Angeles Awarded grant in 2008 “Cellular and Viral Determinants of Neonatal Group B Coxsackievirus Myocarditis” “Sequence comparisons of more recently circulating CVB1 and CVB3 virus 5’UTR and stemloop 2 folding patterns reveal that more recently circulating CVB1 and CVB3 viruses are more similar to each other than to their prototype viruses from the 1940’s. The area of highest divergence from protoype of each CVB was within the stemloop 2 structure, which has previously been identified as a genomic determinant in CVB cardiovirulence [11]; this may indicate why the newer strain of CVB1 was more cardiovirulent than ever described previously. This new CVB1-Chi07 also has larger plaque size than CVB1-Conn5 which correlates with a higher viral replication and titer [12]. The CVB1-Chi07 shows a slower rate of growth and lower peak titer than all of the CVB3 viruses assessed. In vitro mouse models to compare these CVB1 viruses have been initiated with one experiment which indicates that CVB1-Chi07 is much more virulent than CVB1-Conn5, however further studies and data will be collected to verify this. IRES transactivating protein identification is preliminary at this time, but the three proteins that have been identified are involved in cellular translation. Further studies of confirmation are underway and future experiments will be performed with mouse myocyte proteins.”

Dr. Silvio Antoniak of the University of North Carolina in Chapel Hill Awarded grant in 2009 “Thrombin-PAR-1 Signaling in Viral Myocarditis” “I conclude that PAR-1 activation is essential in the early phase of virus infection to manage an effective immune response against CVB3. This response is maintained by a successful recognition of virus’ antigens by the TLR-3 pathway and a PAR-1 dependent NK cell activation. The altered immune reaction in PAR-1 -/- mice leads to uncontrolled virus replication and later to an over-bursting immune response, aberrant cardiac remodeling leading to cardiac dysfunction after CVB3 infection.”

Dr. David Marchant of the University of British Columbia in Vancouver, Canada Awarded grant in 2009 “Discovering and Understanding Virus-Host Factor Interactions for Treatment of Viral Myocarditis” “We showed that virus signaling during infection originated from, and converge upon, dominant signaling ‘nodes.” This work, which employed several signaling inhibitors to investigate signaling ‘networks,” identified those target whose inhibition most effectively inhibited viral replication; the ‘nodes.’ We concluded that by targeting the dominant signaling nodes that are required for viral replication we could, hypothetically, discover the most effective target for treatment of CVB3 infection. This is the first work of its kind and should provide new avenues for investigation of the most effective antivirals for treatment of virally induced myocarditis.”

Dr. Byung Kwan Lim of the University of California in San Diego Awarded grant in 2009 “The Role of Dystrphin in Enterovirus Induced Viral Myocarditis” Specific Aim1. Determine whether cardiac myocyte-specific, inducible expression of enterovirus protease 2A in the dystrophin knock-in adult mouse heart can prevent myocyte sarcolemmal membrane disruption and the subsequent development of cardiomyopathy. Hypothesis: Dystrophin knock-in can prevent mycoyte sarcolemmal membrane disruption through inhibition of dystrophin cleavage by enterovirus protease 2A. Expected Results: We will generate Dys KI /2A/MCM triple transgenic mice to determine the effect of cleavage- resistant dystrophin in cardiac-specific inducible expression of enterovirus protease 2A. Specific Aim2. Determine whether knock-in of dystrophin that prevent protease 2A cleavage of dystrophin can protect against CVB3 infection mediated cardiac myocyte damage and myocarditis. Hypothesis: Dystrophin knock-in reduces dystrophin cleavage and sarcolemmal membrane disruption after CVB3 infection. Expected Results: We generated inbred Balb/C Dys KI mice to determine whether Dys KI mice have less sarcolemmal membrane disruption and myocyte damage in CVB3 infection. Preliminary data showed no significant difference between Dys KI and littermate controls. There are similar virus titer and Evans blue dye uptake percent. However, Balb/C mice have high susceptibility to CVB3 and immune response. To prove a mechanical effect of virus infection we will generate less susceptible inbred C3H background Dys KI mice.

Dr. Kathleen Simpson of Washington University in St. Louis, MO Awarded grant in 2010 “Autoimmunity in Pediatric Myocarditis: A Pilot Study” “The relationship of autoimmunity in the pathophysiology and outcomes of pediatric myocarditis is unknown. Through the collaboration of regional pediatric hospitals encompassing the states of Missouri, Oklahoma, and Nebraska, we plan to determine the relationship of autoimmunity to outcomes in previously and newly diagnosed pediatric myocarditis. In addition, we will use MRI of the heart to determine specific findings in children and the role of MRI in disease progression and prognosis for children with myocarditis. Improving the understanding of patient immune reaction in myocarditis may lead to improvement in prognosis and treatment in children.”

Dr. Alan Valaperti of the University of Health Network at the University of Toronto in Canada Awarded grant in 2011 “IL-1 Receptor-associated Kinase 4 (IRAK4) Epigentically Modulates Nod2- and MDA5- dependent Protection in Viral Myocarditis” “First we observed that the major cell population infiltrating into the inflamed heart of Coxsackie virus B3 (CVB3)- inoculated IRAK4 deficient mice were macrophages. We were able to show that upon in vitro infection with Coxackievirus B3 (CVB3), IRAK4 down-regulated IFN-α and IFN-γ, but not IFN-β, exclusively in macrophages, but not in myeloid dendritic cells or plasmacytoid dendritic cells. Simultaneous down-regulation of IFN-α and IFN-γ, which means up-regulation of IFN-α and IFN-γ in IRAK4 deficient cells, was enough to dramatically reduce viral replication in infected macrophages.” “In addition, IRAK4 showed unique functions that were only partially shared with its upstream molecule MyD88. In particular, IRAK4 deficiency showed better stability of MDA5, which is an important cytoplasmic receptor to recognize Coxackievirus genome to mount an interferon-dependent anti-viral response. On the contrary, CVB3-infected wild- type macrophages showed a relevant degradation of MDA5 few hours after infection, whereas MyD88 deficient cells showed degradation of MDA5 in a later stage. This resulted in higher viral protection in IRAK4 deficient cells compared to wild-type or MyD88 deficient counterparts.”

Dr. Laure Case of the University of Vermont Awarded grant in 2012 “Chromosome Y Regulates usceptibility to Coxsackievirus B3-induced Myocardtis” “The experiments proposed in this fellowship will identify the critical cell types, for example cardiomyocytes and/or immune cells, which influence myocarditis susceptibility in the consomic mice. Thisdata will provide the necessary cellular link for future work that tests the hypothesis that ChrY dependent regulation of genes on other chromosomes is evolutionarily conserved, and that this mechanism influences myocarditis susceptibility among male mice. This is a very novel and exciting area of genetic research that will reveal ChrY’s contribution to phenotypic differences that directly impact susceptibility to myocarditis and allow for the identification of genes and pathways that can be targeted for mechanistic studies and therapeutic intervention.”

Dr. Kevin Quinn of the University of California in Los Angeles Awarded grant in 2012 “Identifying Antiviral Agents to Treat Coxsackievirus Myocarditis” “In support of a long-term goal of identifying potent and tolerable antiviral drugs to prevent enteroviral myocarditis, our project has the following 3 goals: 1) To identify the compounds which most actively inhibit coxsackievirus replication in tissue culture cells 2) To identify the mechanism of action that allows these viral inhibitors to prevent coxsackievirus from completing its lytic cycle 3) To test the most active inhibitors in a mouse model of coxsackievirus myocarditis. Those effective in inhibiting coxsackievirus infection in animal models could in turn be evaluated towards application in human patients.”

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