Project Summary:Hypertrophic cardiomyopathy (HCM) is a complex genetic cardiac disorder that affects ~1/300 1/500individuals worldwide. A common clinical manifestation of patients with HCM is an impairment in left ventricularrelaxation (diastolic dysfunction). Beta-adrenergic stimulation is a key regulator of diastolic performance.During beta- adrenergic stimulation protein kinase A (PKA) mediates phosphorylation of a variety of sarcomerictargets including cardiac troponin I (cTnI) at serine 23/24 (Ser23/24). This phosphorylation event results in asignificant increase in relaxation (or de-activation) rates at the myofilament level. Previous work has shown thatthis observation is due to increases in calcium dissociation rate from the cardiac thin filament. Additionallysome thin filament HCM mutations have been shown to exhibit an impaired response to phosphorylation ofSer23/24 in ATPase assays and force-pCa measurements. While extensive work by several groups hasinvestigated the structural basis for this increase in calcium dissociation rate all previous studies to the best ofour knowledge lack the key thin filament binding partners actin and tropomyosin crucial components forallosteric regulation of relaxation. In this proposal we will perform TR-FRET experiments to assess bothintramolecular and intermolecular interactions between the N-terminus of cTnI and C-terminus of cTnI and theN-terminus of cTnI and Site II of cTnC in the presence and absence of phosphorylation at Ser23/24. Theexperimental design will provide distances that we will employ in our atomistic thin filament model. We will thenuse stopped flow fluorescence anisotropy in order to probe transitions in dynamic behavior in the C-terminus ofcTnI when Ser23/24 is phosphorylated as calcium dissociates from the cardiac thin filament. We hypothesizethat phosphorylation of Ser23/24 will alter the rate at which these transitions occur and that these mechanismsmay be altered by HCM causative mutations. To explore the possibility that the degree of observed diastolicimpairment (and potentially the severity of the end HCM phenotype) may be mutation-specific we propose toinvestigate the molecular effects of 3 independent known cTnI mutations at residue R145 in cTnI. Thismutational hotspot includes HCM- linked mutations R145G R145Q and the restrictive cardiomyopathy (RCM)mutation R145W. We will couple structural data from TR-FRET experiments to changes in calcium dissociationrate to investigate how structural changes impact function and if the diastolic dysfunction is additive in thepresence of Ser23/24 phosphorylation. We will employ metadynamics simulations to obtain free energychanges and identify specific changes in interactions that occur from these mutations and phosphorylation aswell as in the two calcium states (on and off). The in-vitroin-silico coupled approaches proposed in thisapplication will provide atomic level resolution of the structural changes that occur upon Ser23/24phosphorylation in the WT state and in the context of known cTnI-linked HCM/RCM mutations with a long-termgoal of identifying targetable disease mechanisms.