elucidated that the 75kDa protein are located on the membrane of muscle ﬁber but the 150 kDa protein are localized at the Z-line of the sarcomere. The local- ization of these proteins are also conﬁrmed by the transfection of GFP-fusion proteins into cultured skeletal muscle cells. These insights indicate that novel proteins that transcribed from the contiguous genes of connectin gene exist in striated muscles. 2117-Pos Board B254 Skeletal Myosin Binding Protein-C Isoforms Modulate Actomyosin Contractility and are Regulated by Phosphorylation Amy Li 1,2 , Samantha Beck Previs 2 , Michael Previs 2 , Brian Lin 3 , Cristobal dos Remedios 1 , Roger Craig 4 , Sakthivel Sadayappan 3 , David Warshaw 2 . 1 Anatomy & Histology, University of Sydney, Sydney, Australia, 2 Department of Molecular Physiology & Biophysics, University of Vermont, Burlington, VT, USA, 3 Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA, 4 Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA, USA. Myosin binding protein C (MyBP-C) is a thick ﬁlament-associated protein found in striated muscle and may regulate muscle contractility. Separate genes encode the fast and slow skeletal isoforms, and there are potential PKA phos- phorylation sites in their functionally important N-terminal regions. Here, we compare mouse N-terminal fast (fC1C2) and slow (sC1C2) skeletal fragments containing the initial ~50 aa Pro/Ala-rich domain and the C1 and C2 Ig- domains that are linked by the ~100 aa M-domain. Of the known slow skeletal splice variants, we chose a highly expressed variant lacking the N-terminal 34-59 residue insert. To deﬁne the potential mechanisms by which skeletal MyBP-Cs affect contractility and whether these effects are modulated by PKA phosphorylation, we assessed the Ca 2þ -dependent motility of rabbit skeletal native thin ﬁlaments over a surface of rabbit psoas myosin in the pres- ence of C1C2 fragments. While thin ﬁlaments were fully regulated, with no motion observed at pCa > 7 in the absence of fragments, the addition of either 0.50 mM fC1C2 or sC1C2 resulted in signiﬁcant motility. This suggests that skeletal MyBP-C isoforms effectively sensitize the thin ﬁlament. Under fully activating conditions (pCa % 5), sC1C2 had little effect on motility whereas fC1C2 inhibited sliding velocities by nearly 50%. Thus, these fragments differ in their modulatory capacities with fC1C2 sensitizing the thin ﬁlament to Ca 2þ and inhibiting maximal velocities, while the sC1C2 variant exhibits only a single mode of contractile modulation; i.e. thin ﬁlament sensitization. Interestingly, PKA phosphorylation in the Pro/Ala (sC1C2) and M-domains (sC1C2 and fC1C2), as conﬁrmed by mass spectrometry, reduced both frag- ments’ Ca 2þ sensitization of the thin ﬁlament. Thus, the function of MyBP- C isoforms may be tuned to match the physiological demands of the muscle in which they are expressed. 2118-Pos Board B255 In vitro Reconstitution of Skeletal Muscle Contraction using Native Thin Filaments Augustine Cleetus, Khushboo Rastogi, Ravikrishnan Elangovan. Department of Biochemical Engg and Biotechnology, Indian Institute of Technology, Delhi, India, New Delhi, India. Intracellular Ca 2þ concentration regulate the muscle contraction by changing the conformation of Tropomyosin molecule over Actin ﬁlament. In this work we have puriﬁed native thin ﬁlaments (NTF) from Chicken Pectoralis muscle and reconstituted in in vitromotility assay (IVMA) with skeletal muscle myosin II. NTF prepared were 1.6750.14 mm in length and showed good regulation under different Ca 2þ concentrations. At pCa 4, more than 90% of NTF were continuously sliding and when solution changed to pCa 9 all the ﬁlaments were rigidly stuck to surface. Sliding velocity of NTF was V F ¼5.6351.21 mm/s at 30 o C and 17% higher compared to unregulated actin ﬁlaments. Fraction of sliding ﬁlament showed a direct correlation with pCa of buffer and pCa 50 ¼ 6.69 was obtained using Hills equation ﬁt. However the sliding ﬁlament velocity was not affected under different pCa concentration. Under varying substrate [MgATP] conditions in IVMA we found k þATP is 4.35 0.56 x 10 6 M 1 s 1 and k -ADP is 404.3352.7 s 1 at 30 o C. These kinetic parameters are comparable to values obtained using unregulated actin ﬁla- ments, suggesting the detachment kinetics of myosin II is not affected due to Ca 2þ regulation. We studied the effect of myosin density on thin ﬁlaments activation in IVMA. Even with fully activated thin ﬁlament (pCa 4.0), decrease in myosin density leads to increase in fraction of stuck ﬁlaments. Decrease in fraction of motile ﬁlaments with myosin density in IVMA suggests there is minimum number of myosin head binding required to completely activate the NTF. Supported by Department of Science and Technology, Government of India. 2119-Pos Board B256 Direct Troponin-Myosin Interaction Enhances ATPase Activity of Cardiac HMM Nazanin Bohlooli Ghashghaee 1 , King-Lun Li 1 , Wen-Ji Dong 1,2 . 1 The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State univeristy, Pullman, WA, USA, 2 The Department of Integrative Physiology and Neuroscience, Washington State univeristy, Pullman, WA, USA. Phenomenological evidence has long suggested that the strongly bound cross- bridges exert a positive feedback on myoﬁlament regulation, which increases the sensitivity of thin ﬁlament to Ca 2þ and achieves full activation. This posi- tive feedback mechanism is uniquely important to the beat-to-beat regulation of cardiac output. Despite its importance, the molecular mechanism remains elusive. The current wisdom implies that the positive feedback mechanism is mediated by speciﬁc movement of tropomyosin on actin surface caused by actin-myosin interaction. Since the Ca 2þ -regulated interactions between car- diac thin ﬁlament and myosin can be considered as an allosteric system which has long-range coupling between distinct components of the system, multiple protein-protein interactions at the interface between thin and thick ﬁlament may provide alternative mechanism for the feedback effects on myoﬁlament activation. In this study, we investigated the potential interaction between troponin and myosin, known as troponin-bridge, and how the interaction affects function of myosin and thin ﬁlament regulation. The direct interaction between myosin and cardiac troponin was monitored using a sedimentation assay. Different reconstituted troponin complexes were incubated with myosin fol- lowed by ultra-centrifugation to pull down the myosin and the bound proteins. Western blot was used to identify the troponin subunits that were bound to myosin. Our results suggest that both N-cTnI(1-129) and C-cTnT (T2) bind to myosin, suggesting a possible direct interaction between myosin and IT arm of troponin. ATPase assay showed that the HMM ATPase activity was signiﬁcantly enhanced by the presence of troponin regardless of the presence of actin and/or tropomyosin. In addition, truncation of the C-domain (residues 129-212) of cTnI also increased the HMM ATPase activity. The results of our study indicate that troponin-bridge may be a crucial component of thin ﬁlament regulation and play an important role in actomyosin interaction and muscle function. 2120-Pos Board B257 The Regulation of Actomyosin ATPase in Cardiac Muscle by the N-Termi- nal Extension of Cardiac Troponin T Laura Gunther 1 , Hanzhong Feng 2 , Hongguang Wei 2 , Justin Raupp 1 , Jian-Ping Jin 2 , Takeshi Sakamoto 1 . 1 Physics and Astronomy, Wayne State University, Detroit, MI, USA, 2 Physiology, Wayne State University School of Medicine, Detroit, MI, USA. Cardiac troponin T (cTnT) and cardiac troponin I (cTnI) both have an amino- terminal variable region that plays a role in modifying the overall protein conformation and functions in the calcium-dependent regulation of cardiac muscle function. Previous studies have showed that in comparison with wild type controls, transgenic mouse hearts over-expressing cTnT lacking the N-ter- minal variable region (cTnT-ND) had an increased rate of relaxation. In contrast, transgenic mouse heart over-expressing cTnI lacking the N-terminal extension (cTnI-ND) had lower sensitivity to calcium activation of ATPase, re- sulting enhanced ventricular relaxation and Frank-Starling response. Recently, we demonstrated that the second order mant-dATP binding rate of cardiac myo- ﬁbrils containing cTnI-ND was three-fold as fast as that of wild type myoﬁbrils in low [Ca 2þ ]. The ADP dissociation rate of cTnI-ND myoﬁbrils was positively dependent on calcium concentrations, while the wild type controls were not signiﬁcantly affected. These results from native cardiac myoﬁbrils under phys- iological conditions indicate that the N-terminal extension of cTnI plays a role in the calcium regulation of the kinetics of actomyosin ATPase. In the present study, we employed the same techniques in order to investigate which step(s) of the ATPase cycle is regulated by the N-terminal variable region of cTnT. mant- ATP binding, mant-ADP/ADP dissociation, and phosphate releasing rate of cTnT-ND myoﬁbrils are examined and the results will provide novel informa- tion dissecting the function of the two troponin subunits and their posttransla- tional modiﬁcations. 2121-Pos Board B258 Pseudo-Acetylation of Actin Residues K326 and K328 Disrupts Drosophila Flight Performance and Muscle Structure William M. Schmidt, Meera Cozhimuttam Viswanathan, Anna C. Blice-Baum, D. Brian Foster, Anthony Cammarato. Cardiology, Johns Hopkins University, Baltimore, MD, USA. Striated muscle contraction is driven by cyclical interaction between myosin- containing thick and actin-containing thin ﬁlaments and is regulated by the Tuesday, February 10, 2015 421a