Background Dilated and hypertrophic cardiomyopathy mutations in troponin can blunt effects of protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI) decreasing myofilament Ca2+-sensitivity; however this effect has never been tested for restrictive cardiomyopathy (RCM) mutants. cTnI (cTnI-SS/DD.cTnC). Fibers displaced with cTnT-WT reconstituted with cTnI-SS/DD.cTnC decreased Ca2+-sensitivity of pressure (pCa50 = 5.61) compared to control cTnI-WT. cTnC (pCa50 = 5.75) similarly affecting cTnT-��E96 (pCa50 MK-3102 = 6.03) compared to control cTnI-WT.cTnC (pCa50 = 6.14). Fluorescence studies measuring cTnCIAANS Ca2+-affinity changes due to cTnT-��E96 indicated higher complexity (thin filament) better recapitulates skinned fiber Ca2+ sensitive changes. Circular Dichroism revealed reduced ��-helicity and earlier thermal unfolding for cTnT-��E96 compared to WT. Conclusions 1 although ineffective in MK-3102 decreasing myofilament Ca2+-sensitivity to normal levels cTnT-��E96 does not interfere with PKA cTnI phosphorylation mediated effects; 2) cTnT-��E96 requires actin to increase cTnC Ca2+-affinity; and 3) deletion of E96 reduces cTnT stability likely disrupting crucial thin filament interactions. General Significance The pathological effect of cTnT-��E96 is largely manifested by dramatic myofilament Ca2+-sensitization which still persists even after PKA phosphorylation mediated Ca2+-desensitization. and genes. MK-3102 The first RCM mutation reported in the cTnT MK-3102 (gene) was a deletion of glutamic acid found at position 96 (cTnT-��E96) in a pediatric patient [1]. Cardiac Tn has an important role in regulating cardiac contractility therefore amino acid deletions or substitutions that disrupt its function can lead to dysregulation of interactions between the thin and thick filaments [8 9 The cTn complex is usually constituted by three subunits: troponin C (cTnC) confers the Ca2+ sensitive properties to striated muscle; cTnI prevents interactions of myosin with actin at subthreshold Ca2+ levels; cTnT has a key role in activation of muscle contraction and actually links the Tn complex with tropomyosin (Tm) in the thin filament [10-12]. A more refined view of cTnT function has been derived from studying cardiomyopathic mutations in cTnT which appears to MK-3102 have additional nuanced functions in muscle contraction including modulation of actomyosin ATPase activity and the kinetics of contraction Ca2+ sensitivity of contraction TRAC1 as well as maximal pressure [6 13 14 Previously our group has performed studies that elucidated the functional defects caused by the associated RCM mutation the cTnT-��E96. Functional parameters of the mutant cTnT-��E96 were greatly altered along with early presentation in the proband both indicating the severity of the disease phenotype [1 15 Skinned fibers reconstituted with the cTnT-��E96 mutant protein showed a large increase in Ca2+ sensitivity of pressure and an inability to fully relax; reconstituted assays also revealed that the mutant troponin complex was unable to fully inhibit myosin-actin-tropomyosin ATPase activity [15]. Our findings were further corroborated by another study that recapitulated the increased Ca2+ sensitivity of contraction in skinned fibers containing cTnT-��E96 using a different protocol for incorporation of exogenous proteins [16]. Although little is known about the pathological mechanisms underlying RCM mutations it has been previously suggested that this pathogenesis associated with RCM mutations involves drastic sensitization of the myofilament MK-3102 to Ca2+ [17 18 Developmentally important is the switching of TnI isoforms from the fetal (slow skeletal TnI) to the the adult isoform (cTnI) during embryogenesis and postnatal development [19-21]. Since the patient had a severe onset of disease shortly after birth we evaluated whether the deletion of amino acid E96 in cTnT futher altered regulatory mechanisms that modulate the contractile response in cTnI that contains the PKA target sites in the N-terminal extension. During ��-adrenergic stimulation of the heart cTnI is usually phosphorylated at serines 23 and 24 by PKA which decreases the Ca2+ sensitivity of contraction and enhances the relaxation rate of the heart [22-24]. Therefore sarcomeric protein phosphorylation is a prominent mechanism for maintenance of cardiac function and homeostasis [25]. The rationale to study the effects of PKA phosphorylation in the presence of a RCM mutant is that.