PROJECT SUMMARY/ABSTRACT Cardiac amyloidosis can be caused by a mutation in transthyretin (TTR) (e.g. valine 122 to isoleucine, VI) [ATTR- CM] that will aggregate when taken up by the myocardium, resulting in cytotoxicity and ultimately dysfunction. The mechanisms underlying the pathogenesis of ATTR-CM remain unknown. Further, methods to enhance the degradation of dissociated and deposited transthyretin is a critical unmet need. We reported protein kinase G (PKG) can enhance protein degradation via the proteasome and lysosome to attenuate cardiac disease. We recently uncovered that PKG also phosphorylates a ubiquitin ligase/co-chaperone, Chip (carboxyl terminus of Hsc70-interacting protein), at serine 19 (human; S20, mouse). Chip is a primary mediator of cardiomyocyte proteostasis by ubiquitinating and shuttling proteins for degradation. With new and exciting pilot data we show PKG activity and Chip S19 phosphorylation (pS19) are uniquely depressed in ATTR-CM patients. We also reveal cardiomyocytes isolated from ATTR-CM patients have reduced myofibrillar function. The field has been stymied by lack of models, especially in vivo, and access to human tissue. We addressed these limitations by creating novel models and a biorepository of biopsies from ATTR-CM patients. In vitro, we developed engineered heart tissue (EHT) and cardiac organoids formed from human iPSC-derived cardiomyocytes and fibroblasts. To create ATTR-CM in vitro we incubate EHTs in TTRVI (5 µM, same as ATTR-CM plasma) or culture cardiac organoids with TTRVI hepatic organoids (excretes TTRVI at a similar concentration) in an interconnected microphysiological device for 14 days, resulting in cellular uptake, protein aggregation, lower PKG activity, cell death, and (in EHTs) reduced function. Our new TTRVI knock in mouse develops diastolic dysfunction, increased expression of fibrotic genes, and decreased PKG signaling. Males and ovariectomized female mice, but not intact females, develop ATTR-CM, similar to human ATTR-CM which affects men and post-menopausal women. Our pilot data shows activating PKG or expressing a Chip pS19-mimic (ChipSE) facilitates the clearance of TTRVI to enhance cardiac function (mice and EHTs) and reduce cytotoxicity (organoids). This project will provide new mechanistic insight into ATTR-CM by testing the impact of PKG activity and Chip pS19 in vitro and in vivo, tests a new therapeutic strategy, and determines the translational relevance in human patients. Aim 1 tests if PKG stimulation or ChipSE attenuates markers of ATTR-CM in vitro and if chaperone-mediated autophagy is the degradative process utilized. We also developed and will further test a novel tool (PROTAC) specifically targeting Chip for TTR to enhance TTRVI removal. In Aim 2, we test the ability of various PKG activators protect against ATTR-CM and if this occurs in a Chip pS20 dependent manner. In Aim 3, we will test the relevance of PKG signaling and Chip pS19 in human ATTR-CM. We further interrogate myofilaments isolated from ATTR-CM human myocardium and test if PKG activation can improve contraction and relaxation. The work is highly translational, as it provides key mechanistic insight into and potentially identifies a new therapeutic target for ATTR-CM patients.

Cardiac amyloidosis results in a restrictive cardiomyopathy that is often caused by a mutation in the protein transthyretin, resulting in dissociation from its complex and uptake into the myocardium where it forms large protein aggregates and amyloid fibrils. The mechanisms that regulate the degradation of transthyretin in the heart and methods to enhance this were unknown until now. This proposal investigates unexplored underpinnings of cardiac amyloidosis and tests a novel mechanism in which increasing Chip phosphorylation via gene mutation and pharmacological protein kinase G activation enhances the removal of mutant transthyretin and improve cardiac function, potentially providing a new and viable therapeutic target for cardiac amyloidosis.