fatal arrhythmia and ventricular ischemia due to the restricted ventricles and enlarged atria. The transgenic mouse model provides us with a good tool to study the mechanisms and the cause of the death of RCM, which will be useful for the prevention and treatment of the disease. 2456-Pos Board B442 Both PKA Treatment and Cardiac Troponin-I N-Terminal Phosphoryla- tion Alone Decrease Ca-Sensitivity and Eliminate Length-Dependent Activation in Skinned Cardiac Muscle Donald A. Martyn, Steven Korte, Maria Razumova, Erik Feest, Thomas Irving, Michael Regnier. Protein kinase A (PKA) phosphorylation of myofibrillar proteins constitutes an important pathway for b-adrenergic modulation of cardiac contractility. PKA tar- gets the cardiac troponin I (cTnI) N-terminus, cardiac myosin-binding protein C (cMyBP-C) and titin. To isolate cTnI and cMyBP-C /titin phosphorylation effects on force-[Ca 2þ ] relations, endogenous cardiac troponin (Tn) was exchanged in rat, skinned trabeculae with either WT Tn or Tn containing a non- phosphorylatable mutant cTnI(S23/24A) or phosphomimetic cTnI(S23/24D). PKA cannot phosphorylate either cTnI mutant, leaving cMyBP-C and titin as sole PKA targets. Force-[Ca 2þ ] relations and Ca 2þ -sensitivity (pCa50) were mea- sured at 2.3 and 2.0 mm SL. Decreasing steady SL reduced maximal force (Fmax) and pCa50 similarly with WT Tn and Tn containing cTnI(S23/24A). PKA treat- ment of native, WT and cTnI(S23/24A) exchanged trabeculae reduced pCa50 at 2.3, but not 2.0 um SL, eliminating SL-dependence of pCa50. Reconstitution with Tn containing cTnI(S23/24D) reduced pCa50 at both SL (compared to WT and cTnI(S23,24A) and eliminated pCa50 SL-dependence; PKA did not significantly alter pCa50 at either SL. At each SL Fmax was similar with WT and mutant tro- ponins, and was unaffected by PKA. Low angle x-ray diffraction experiments were performed to determine whether shifts in pCa50 were associated with changes in myofilament spacing (D1,0) or interaction. D1,0 at 2.3 um SL was sim- ilar in native trabeculae, with WT Tn and Tn containing either cTnI(S23,24A) or cTnI(S23,24D); PKA increased D1,0 in all cases. The results suggest that PKA phosphorylation of either cTnI or cMyBP-C /titin reduced the Ca 2þ -sensitivity of force and length-dependent activation. Supported by NIH HL067071-06. 2457-Pos Board B443 Reducing Thin Filament Ca 2þ Affinity with a cTnC Variant (L57Q) Re- duces Force but Enhances Cross-Bridge Dependence of Cooperative Activation in Demembranated Rat Trabeculae Cameron W. Turtle, Frederick S. Korte, Maria V. Razumova, Michael Regnier. Activation of cardiac contraction is initiated by binding of Ca2þ to troponin C (cTnC) and regulated by cooperative strong cross-bridge binding. We previ- ously showed that passive exchange with cTn containing a cTnC variant, L48Q, increased Ca2þ sensitivity of force development and eliminated sarco- mere length (SL) dependence of Ca2þ sensitivity of force in rat cardiac trabec- ulae. This was shown to be due to decreased reliance on strong cross-bridge binding for full thin-filament activation, possibly due to stronger cTnC-cTnI in- teraction. We also showed that PKA phosphorylation decreased Ca2þ sensitiv- ity of force and eliminated SL dependence of force-pCa relations, by unknown specific mechanisms. Here we test the hypothesis that incorporation of a cTnC variant with decreased Ca2þ binding affinity, L57Q cTnC, will result in in- creased reliance on cross-bridge binding for full activation, thus increasing SL-dependence of Ca2þ sensitivity. As expected, results indicate trabeculae passively exchanged with L57Q cTnC-cTn displayed decreased Ca2þ sensitiv- ity and rate of force production compared to WT cTnC at a given [Ca2þ]. In- terestingly, preliminary results indicate L57Q cTnC-cTn mildly increased the SL-dependence of Ca2þ sensitivity of force and also significantly decreased maximal force. Both of these effects were not observed in PKA-treated trabec- ulae, which had a comparable reduction in Ca2þ sensitivity of force. These findings suggest that reducing cTnC Ca2þ affinity per se can reduce Ca2þ sen- sitivity of contractile activation to the point of limited overall force production, which may enhance cross-bridge dependence of cooperative thin filament acti- vation. Current experiments aim to increase force in L57Q cTnC (to near WT cTnC) by increasing cross-bridge attachment using 2’-deoxy-ATP, which has previously been shown to increase force and Ca2þ sensitivity while maintain- ing SL dependence. NIH-HL65497(MR), AHA-2310117(FSK). 2458-Pos Board B444 Fluorescence Measurements Using Rhodamine-Labeled cTnC Mutants In- dicate Little Cooperative Interaction Between Cardiac Thin Filament Regulatory Units Don Martyn, Vijay Rao, Michael Regnier. Exchange of mixtures of WT cTnC and mutant cTnC(D65A), which cannot bind Ca 2þ at N-terminal site II (‘‘dead’’ cTnC), reduced maximal Ca 2þ acti- vated force (Fmax) with little effect on force-Ca 2þ relations and force kinetics in skinned cardiac trabeculae (Gillis et al., J Physiol. 580:561-76, 2007), sug- gesting interaction between structural regulatory units (RUs; 7 actins, 1 tropo- nin, 1 tropomyosin) along cardiac thin filaments is less than in skeletal muscle (Regnier et al., J Physiol. 540:485-97, 2002). To more directly test that this finding, we exchanged skinned cardiac trabeculae with mixtures of mutant cTnC(C35S) and cTnC(C35S,D65A), with one or the other labeled at Cys 84 with 5’tetramethyl rhodamine (IATR) for dichroism measurements. In trabec- ulae exchanged with 100% cTnC(C35S)-IATR, dichroism increased in re- sponse to both Ca 2þ and rigor crossbridges, while trabeculae with 100% Tn containing (cTnC(C35S,D65A)-IATR) had no response to Ca 2þ , but retained a strong response to rigor crossbridge binding. This response to strong cross- bridges allows use of cTnC(C35S,D65A)-IATR to determine if isolated regu- latory units containing cTnC(C35S,D65A)-IATR are perturbed by Ca 2þ - induced active contraction in adjacent ‘‘live’’ RUs. To test this, trabeculae were exchanged with a mixture of 20% functional cTnC(C35S)-IATR and 80% unlabeled cTnC(C35S,D65A), to isolate functional RUs. Fmax decreased but there was little change in the Ca 2þ -dependence of dichroism compared to trabeculae exchanged with 100% functional cTnC(C35S)-IATR. These data in- dicate minimal or no apparent spread of activation between adjacent RUs in cardiac muscle, indicating that the apparent cooperativity of force production in cardiac muscle results from interactions between myosin and thin filaments within a thin filament structural regulatory unit. Supported by NIH RO1- HL65497 (Regnier). 2459-Pos Board B445 Combination of Phosphomimetic Substitutions within Cardiac Troponin I Cause Functional Cross-Talk Sarah E. Kampert, Tamara K. Stevenson, Mark A. Jensen, Erin M. Keyes, Gail L. Romanchuk, Margaret V. Westfall. Protein kinase C (PKC) phosphorylates 3 clusters of residues within cardiac tro- ponin I (cTnI) and yet, it is unclear whether phosphorylation at multiple sites pro- duces additive and/or divergent functional modifications. Our goal was to evaluate the influence of cTnI with phosphomimetic substitutions on contractile performance under basal conditions and in response to PKC activation by endo- thelin. Endogenous cTnI was replaced with phosphomimetic substitutions using adenoviral-mediated gene transfer into adult rat cardiac myocytes. Phosphomi- metics of Ser43/45, Ser43/45 plus Thr144, and Ser23/24 plus Ser43/45 were substituted with Asp to form AdcTnISer43/45Asp, cTnIAsp Triple (e.g. cTnISer43/45AspThrT144Asp), and cTnIAsp Quad (cTnISer23/24/43/45Asp). Isolated myocytes were electronically paced and studied 4 days after gene trans- fer. Gene transfer of epitope-tagged versions of each construct resulted in 30- 40% replacement after 2 days and >65% replacement of endogenous cTnI 4 days after gene transfer without significant alterations in the expression of other myofilament proteins. In functional studies, peak shortening amplitude was sig- nificantly decreased in myocytes expressing cTnISer43/45Asp or cTnIAsp Quad, while peak shortening in myocytes expressing cTnIAsp Triple was not signifi- cantly different from controls. Relaxation was accelerated in myocytes express- ing cTnIAsp Quad , but was not different from controls in myocytes expressing cTnIAsp Triple or cTnISer43/45Asp. Together, these results suggest the Ser23/ 24 and Ser43/45 sites have an additive influence on shortening, while substitu- tion at Thr144 attenuates the influence of Ser43/45 on peak shortening. To fur- ther determine whether multiple phosphomimetic substitutions within cTnI influence myocyte shortening, we studied the change in peak shortening and re- laxation produced by the PKC agonist, endothelin (10 nM). In preliminary stud- ies, the increased peak amplitude and accelerated relaxation observed in control myocytes is not significantly different in myocytes expressing cTnISer43/ 45Asp. However, there is a trend for myocytes expressing cTnIAsp Quad to show an attenuated amplitude and relaxation response to ET. 2460-Pos Board B446 The Motif of Myosin Binding Protein-C is Mechanically Weak and Extensible Arpad Karsai, Samantha P. Harris, Miklo ´s Kellermayer. Cardiac myosin binding protein-C (cMyBP-C) is a member of the immuno- globulin (Ig) superfamily of proteins and consists of 8 Ig- and 3 fibronectin (Fn)-like domains along with a unique regulatory sequence referred to as the MyBP-C ‘‘motif’’ or M-domain. The structure of the M-domain is not known, but small angle X-ray scattering experiments suggest that it adopts a compact shape in solution and that its overall dimensions are similar to other Ig-like do- mains. To investigate whether the M-domain behaves similarly to an Ig domain under mechanical stress or load, we used atomic force microscopy (AFM) to investigate single molecule elasticity and mechanical properties of recombinant full-length mouse cardiac cMyBP-C and smaller proteins containing just the M- domain and flanking Ig- sequences. Force-extension curves of full-length cMyBP-C showed unfolding of individual Ig or Fn-like domains at forces Tuesday, March 8, 2011 453a