CASQ2-knockout animals (Knollmann et al., 2006. J Clin Invest 116:2510). Lack of both CASQ isoforms was confirmed by western blot. The double- null mice are viable and breed normally, however the rate of spontaneous mor- tality of male animals is higher than CASQ1-null animals. Whereas the overall phenotype of mice is similar to that of CASQ1-null mice, significant differ- ences are found in Soleus. From the structural point of view, in Soleus muscle we find many fibers (about 30%) with severe structural damage that were not found in CASQ1-null animals. Functional studies indicate significant prolonga- tion in twitch time parameters, increased twitch tension and impaired tension generation during prolonged tetani both in EDL and Soleus, likely related to abnormal calcium release kinetics. These findings suggest that: a) expression of CASQ2 is essential for the maintenance of a subpopulation of Soleus fibers; and b) lack of both CASQ1 and 2 exacerbates the overall phenotype of CASQ1- null mice. 2821-Pos Refractoriness of Sarcoplasmic Reticulum Calcium Release in Cardiac Muscle Due to Calsequestrin Oleksiy Gryshchenko, Dmytro Kryshtal, Bjorn C. Knollmann. Vanderbilt University, Nashville, TN, USA. In cardiac excitation-contraction coupling, L-type Ca 2þ current (I Ca ) triggers Ca 2þ release from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca 2þ release channels. It is unclear why SR Ca 2þ release cannot be eli- cited by premature stimuli, even though I Ca is fully recovered. Here, we use cal- sequestrin null mice (Casq2 KO) and wild-type littermates (WT) to test the hy- pothesis that calsequestrin (Casq2) determines refractoriness of SR Ca 2þ release. Ca 2þ release refractoriness was measured in voltage-clamped myo- cytes dialyzed with Fluo-4 by applying premature extrastimuli (S2) at succes- sively shorter S1-S2 coupling intervals following a 1 Hz train (S1 stimuli). To maintain constant trigger, Ca 2þ release was activated with I Ca tail currents that elicited maximal Ca 2þ release during the S1 train. WT S2 Ca 2þ release was sig- nificantly depressed with short coupling interval whereas Casq2 KO cardio- myocytes exhibit no refractoriness of Ca 2þ release (Figure, n=11 WT, 12 KO, p ¼ 0.01). At the same time, I Ca current density, SR Ca 2þ content, and steady-state Ca 2þ transients (S1) were not significantly different from WT-myocytes. We conclude that cal- sequestrin is a critical determinant of SR Ca 2þ release refractoriness in car- diac muscle (Supported by NIH- R01HL71670, R01HL88635). 2822-Pos Effect of Triadin on Retrograde and Orthograde Signaling between RyR1 and DHPR in Cultured Myotubes Jose M. Eltit, Hongli Li, Paul D. Allen, Claudio F. Perez. Brigham and Women’s Hospital, Boston, MA, USA. Using pan triadin-null mice we previously showed that triadins ablation did not disrupt EC-coupling in muscle cells. However, calcium imaging studies in cul- tured myotubes did reveal that triadin-null myotubes had slightly smaller depo- larization-induced Ca 2þ transients than Wt cells. Here, using whole-cell volt- age clamp, we analyze the effect of triadin ablation in skeletal EC-coupling by characterizing the retrograde and orthograde signaling between RyR1 and DHPR of triadin-null myotubes. Calcium currents elicited by 200ms depolar- ization steps in Wt and triadin-null cells showed slow kinetics of activation and peak current at approximately þ30 mV. Although, the overall voltage de- pendence was preserved between Wt and triadin-null cells a leftward shift in the I/V curve was observed in triadin-null cells (V 1/2 , 22.350.8 mV in Wt vs 16.651.1 mV in triadin-null cells, p<0.05). In addition, kinetic analysis of the DHPR Ca 2þ current shows that the activation time constant of the slow component (t slow ) was slightly decreased from 3752.4 ms in Wt to 2652.6 ms (p<0.05) in triadin-null cells. The voltage-evoked Ca 2þ transient, on the other hand, showed a small but signif- icant reduction of the peak fluorescence amplitude of triadin-null cells (DF /F max , 0.7250.2 in Wt vs 0.6150.1 in triadin-null) with no differences in voltage de- pendence (V m , 7.251.1 mV in Wt vs 10.151.9 mV in null cells). Our results suggest that the absence of triadin expression preserves the orthograde and ret- rograde signaling between DHPR and RyR1 nearly intact and that the effect of triadin ablation on DF/F max would be secondary to the dysregulation of calcium homeostasis observed in triadin-null cells. These data give further support to the idea that skeletal triadins do not play a direct role in skeletal EC-coupling. Supported by NIH Grants 5K01AR054818-02 (to CFP) and 1P01AR044750 (to PDA). 2823-Pos Altering Skeletal Muscle EC Coupling by Ablating the Sarcoplasmic Retic- ulum Protein JP45 Affects Both Metabolism and Muscle Performance in Old Mice Osvaldo Delbono 1 , Zhong-Min Wang 1 , Jackson Taylor 1 , Maria Laura Messi 1 , Susan Treves 2 , M. Nishi 3 , H. Takeshima 3 , Francesco Zorzato 2 . 1 Wake Forest University School of Medicine, Winston-Salem, NC, USA, 2 Basel University Hospital, Basel, Switzerland, 3 Kyoto University, Kyoto, Japan. JP45, a sarcoplasmic reticulum protein, appears to be mainly expressed in skel- etal muscle. In mice, its expression is down-regulated during aging, and it in- teracts with the a1.1 subunit of the dihydropyridine receptor (Cav1.1) and cal- sequestrin, two key components of the excitation-contraction (EC) coupling machinery. We examined 12- and 18-month-old JP45 knock-out mice and compared them with age-matched, wild-type littermates. The JP45 KO mice exhibit a pheno- type consistent with impaired skeletal muscle EC coupling, confirming our pre- vious results in young JP45 KO mice. Spontaneous motor activity assessed with a running wheel revealed that the older JP45 KO group runs less and much more slowly than age-matched WT and young JP45 KO mice. In vitro muscle contractile property analysis showed lower twitch and tetanic absolute and spe- cific force, evident mostly in the EDL of aged JP45 KO mice compared to age- matched WT, which correlates with type-II fiber atrophy. Cav1.1 expression and SR Ca 2þ release in voltage-clamped flexor digitorum brevis muscle fibers of aged JP45 KO mice were reduced compared to age-matched WT. Addition- ally, aged JP45 KO mice exhibited decreased food intake and body weight. Our results show that JP45 plays a role in EC coupling and regulation of body metabolism. Supported by NIH/NIA, Japanese Science Foundation, M.U.R.S.T., A.F.M., and Swiss Muscle Foundation. 2824-Pos Characterization of Calumenin-RyR2 Interaction in Murine Heart Sanjaya K. Sahoo, Taeyong Kim, Do Han Kim. Gwangju Institute of Science and Technology, Gwangju, Korea, Republic of. Calumenin is a multiple EF-hand Ca 2þ -binding protein localized in the sarco- plasmic reticulum (SR). In our recent study, we showed that calumenin-knock- down (KD) of HL-1 cells led to enhanced Ca 2þ release and Ca 2þ uptake in the SR (Sahoo et. al. J. Biol. Chem., 2009). To elucidate the underlying mecha- nisms responsible for the enhanced Ca 2þ release from the SR in calumenin- KD samples, the possible interaction between calumenin and RyR2 was exam- ined by various methods. GST pull-down assay showed a direct interaction be- tween calumenin and RyR2. We have further found that the middle region of calumenin (aa 132-222) interacts with RyR2. GST pull-down assay also shows that RyR2 intra luminal loop-I region (aa 4519-4576) is the binding site for cal- umenin. Immunofluorescence study shows that RyR2 and calumenin are co-lo- calized in the junctional region of SR in rat ventricular cardiomyocytes. The detailed amino acid residues involved in the interaction between calumenin and RyR2 are currently under investigation. (This work was supported by the Korean Ministry of Science and Technology grant, Systems Biology Research Grant, M1050301001-6N0301-0110, and the 2009 GIST Systems Biology In- frastructure Establishment Grant). 2825-Pos Progressive Triad-Mitochondria Un-Coupling in Aging Alessandra D’Incecco, Marco Dainese, Feliciano Protasi, Simona Boncompagni. University of G. D’Annunzio, Chieti, Italy. An impairment of the mechanisms controlling the release of calcium from in- ternal stores (excitation-contraction (EC) coupling) has been proposed to con- tribute to the age-related decline of muscle performance that accompanies ag- ing (EC un-coupling theory). EC coupling in muscle fibers occurs at specialized intracellular junctions called calcium release units, or triads, which are specif- ically placed at sarcomere’s I-A band transition. In recent publications we have shown that: a) in human muscle, the frequency of triads decreases significantly with age (Boncompagni et al., 2006; J Gerontol 61:995); and b) in mice, triads are tethered to mitochondria placed at the I band (Boncompagni et al., 2009; MBC 20:1059). Here we have studied the frequency, sarcomeric-localization, ultrastructure, and coupling of triads/mitochondria in EDL from male WT mice using transmission electron microscopy (TEM). Preliminary results indicates that the number of triads/100mm2 of longitudinal section in aging mice (n=4, 25-35 months of age) decreases compared to the adult mice (n=5, 3-12 months of age): 9259 vs. 7958. In addition, the percent- age of abnormally positioned triads (longitudinal and/or oblique) increases. On the other hand, the total volume of mitochondria does not change significantly with age. However, the number of mitochondria-profiles/100mm2 of Tuesday, February 23, 2010 547a