Monday, May 12, 2025
5/12/2025
Mutation-Specific Calmodulin Kinase II Activation in Hypertrophic Cardiomyopathy - Project Summary: Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiomyopathy, affecting ~1/300-1/500 individuals worldwide. In the clinic, HCM displays a high degree of phenotypic variability which presents major challenges regarding targeted therapeutics and determining the mechanisms which drive disease progression. HCM has been definitively linked to mutations in genes encoding the protein components of the cardiac sarcomere, including the cardiac thin filament (CTF). The CTF is an essential regulator of sarcomeric contraction and couples the availability of cytoplasmic calcium to force development. Previous work has shown that in human late-stage HCM samples, there are increased levels of Ca2+/Calmodulin-dependent kinase IIδ (CaMKIIδ) phosphorylation and oxidation. Both post-translational modifications, along with several others, render the kinase autonomous of its physiological regulation via calcium, increasing kinase activity. As CaMKIIδ targets several proteins which maintain electromechanical and calcium homeostasis, increased activity has high pathophysiological potential and has been noted in several cardiac diseases in addition to HCM. Our lab has demonstrated that this increase in autonomous CaMKIIδ activity is dependent on the specific point mutation in the CTF. Specifically, increased CaMKIIδ is observed in mice expressing the R92W mutation in cardiac troponin T (cTnT), but not in mice expressing the R92L mutation. This mutation-specificity suggests that there are specific primary alterations in biophysical properties of the CTF, which can be altered in differing fashions by mutations, that act as triggers for increased CaMKIIδ activity. To the best of our knowledge, these specific alterations, and the mechanisms by which they increase CaMKIIδ activity, have yet to be investigated. We hypothesize that increased CaMKIIδ activity is a result of early, myofilament-based calcium dysregulation, specifically accelerated calcium dissociation kinetics from the CTF, and that the increased activity drives pathological remodeling and thus can be a therapeutic target in a subset of HCM. In Aim 1 we will perform experiments utilizing mass spectrometry, a novel CaMKIIδ biosensor, and sarcoplasmic reticulum calcium uptake techniques to define CaMKIIδ activity and the state of calcium dysregulation in three separate mouse models of HCM which express mutations in cTnT associated with HCM: R94H, I79N, and R92W. We will use these data to determine whether calcium dysregulation stemming from acceleration of calcium dissociation kinetics is strictly associated with increased CaMKIIδ activity. In Aim 2 we will assess the efficacy of the small molecule CaMKIIδ inhibitor ruxolitinib, currently FDA-approved for various bone marrow and skin diseases as a Janus kinase 1/2 inhibitor to alter pathogenic remodeling in HCM in vivo. We will treat cTnT-R92W mice at early- and late-stage disease to determine if CaMKIIδ inhibition can be a viable treatment option at any point in disease progression or only at specific times. The data from these experiments will identify new links between molecular phenotypes, provide new insight into mechanisms of HCM progression, and describe a novel therapeutic target for cTnT-linked HCM.
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