longitudinal section is reduced (5457 vs. 4356), suggesting a remodelling/fu- sion of these organelles. Finally, we have assessed the positioning of mitochon- dria in respect to myofibrils and triads: a) the number of mitochondria at the A band (misplaced) slightly increases with age (9% vs 3%), whereas the number of triads-mitochondria couples is significantly reduced: 3955 vs. 2654. Our ob- servations indicates: a) a age-related partial disarrangement and spatial re-orga- nization of EC coupling/mitochondrial apparatuses; and b) a decreased percent- age of mitochondria functionally tethered to calcium release sites. This could in part explain the decline of muscle performance associated to increasing age. 2826-Pos Knockdown of TRIC-B from tric-a -/- mice Alters Intracellular Ca 2þ Signaling in Skeletal and Cardiac Muscles Ki Ho Park 1 , Xiaoli Zhao 1 , Daiju Yamazaki 2 , Samantha Pitt 3 , Andoria Tjondrokoesoemo 1 , Shinji Komazaki 4 , Jae-Kyun Ko 1 , Pei-Hui Lin 1 , Marco Brotto 5 , Rebecca Sitsapesan 3 , Noah Weisleder 1 , Hiroshi Takeshima 2 , Jianjie Ma 1 . 1 University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA, 2 Kyoto University, Kyoto, Japan, 3 University of Bristol, Bristol, United Kingdom, 4 Saitama Medical University, Kawagoe, Japan, 5 University of Missouri-Kansas City, Kansas City, MO, USA. Trimeric intracellular cation (TRIC) channel subtypes are present in the endo/ sarcoplasmic reticulum (SR) and nuclear membranes of muscle cells and other tissues. Knockout mice lacking both TRIC-A and TRIC-B channels suffer le- thal embryonic cardiac failure due to dysfunctional intracellular Ca 2þ signaling in the mutant cardiomyocytes (Yazawa et al., Nature 448, 78-82). The lethality associated with double knockout of tric-a and tric-b prevents physiological as- sessment of TRIC channels in adult tissues. Here we took advantage of the vi- able tric-a -/- mice and employed RNAi-mediated knockdown of tric-b, in order to examine the physiological function of TRIC channels in adult muscle cells. We used electroporation-mediated delivery of shRNA against tric-b into the flexor digitorum brevis (FDB) muscles of living tric-a -/- mice. Individual FDB fibers with knockdown of TRIC-B were used to examine the Ca 2þ sparks properties in response to osmotic stress, and voltage-induced Ca 2þ release un- der voltage clamp. Compared with the tric-a -/- muscle treated with control shRNA, acute knockdown of TRIC-B leads to significant reduction of the am- plitude of Ca 2þ sparks accompanied with prolongation of the duration of Ca 2þ sparks. In neonatal cardiomyocytes isolated from the tric-a -/- mice, knockdown of TRIC-B led to significant perturbation of Ca 2þ signaling from the SR, evi- denced by irregular intracellular Ca 2þ signaling and reduced frequency of spontaneous Ca 2þ oscillations. These results indicate that disruption of TRIC function can alter intracellular Ca 2þ signaling in skeletal and cardiac muscles and this may underlie an increased susceptibility of these tissues to various physiological stresses. 2827-Pos Local Ca 2þ Releases Enable Rapid Heart Rates in Developing Cardiomyo- cytes Topi Korhonen 1 , Risto Rapila 1 , Veli-Pekka Ronkainen 2 , Pasi Tavi 1 . 1 A. I. Virtanen Institute for Molecular Sciences, Department of Biotechnology and Molecular Medicine, Kuopio, Finland, 2 Institute of Biomedicine, Department of Physiology, University of Oulu, Kuopio, Finland. Homogeneous intracellular Ca 2þ release repeated with high frequency is the basis of the rhythmic contractions of cardiac myocytes. In adult ventricular my- ocytes, the t-tubular system enables transient homogeneous Ca 2þ signals. Inter- estingly, the developing cardiomyocytes do not have t-tubuli and Ca 2þ signal propagation in the cytosol is based on the relatively slow diffusion of Ca 2þ ions. This is likely to result in spatiotemporal heterogeneity of Ca 2þ , which limits the maximal frequency of the Ca 2þ signals. We observed that intracellu- lar Ca 2þ signals of 12.5 days old mouse embryonic ventricular myocytes are more homogeneous than expected if the Ca 2þ signals would propagate by pure diffusion. To study the propagation more accurately, we injected a small amount of Ca 2þ to a single point in the cytosol via patch-clamp pipette while performing the line-scan imaging of the intracellular Ca 2þ . With this method we found that inhibition of the sarcoplasmic reticulum (SR) Ca 2þ release chan- nels results in 3-fold slowing of Ca 2þ signal propagation (control: 10.1 5 2.7 ms/mm vs. ryanodine (50 mM): 33.6 5 9.2 ms/mm, P < 0.05). This suggested that the propagation of Ca 2þ signals is amplified with local SR Ca 2þ releases. Immunolabeling of SR Ca 2þ release and uptake proteins revealed a regular structure throughout the cytosol at ~2 mm intervals. These extensions of SR were equally functional in all parts of the cytosol. To further study the role of these local Ca 2þ release sites in developing cardiomyocytes, we imple- mented a model of them into the previously published mathematical model of an embryonic cardiomyocyte. The computer simulations showed that the lo- cal Ca 2þ releases are prerequisite for synchronizing the global intracellular Ca 2þ releases upon electrical excitation and maintaining the capability of de- veloping cardiomyocytes to generate spontaneous pacemaking at a sufficiently high frequency. 2828-Pos Ca 2þ Transients and Myosin Heavy Chain (MHC) Composition in Murine Enzymatically Dissociated Fibers Juan C. Caldero ´n 1,2 , Pura Bolan ˜os 1 , Carlo Caputo 1 . 1 Instituto Venezolano de Investigaciones Cientı ´ficas, Caracas, Venezuela, Bolivarian Republic of, 2 Universidad de Antioquia, Medellı ´n, Colombia. Single and tetanic Ca 2þ transients reported with MagFluo4-AM were obtained together with MHC electrophoretic patterns in enzymatically dissociated fibres from adult mice soleus and extensor digitorum longus (EDL) muscles. Kinetics of transient rise (Ca 2þ release) and decay (Ca 2þ clearance) of both twitch and tetanic responses showed a continuum from the slowest records obtained in fi- bers type I, to the fastest obtained in fibers IIX/D and IIB. Fibers IIA were fast regarding Ca 2þ release but slow regarding Ca 2þ clearance. Single transients decay was described by a double exponential function with time constants (t 1 and t 2 , ms) of 3.2 and 49.5 in soleus (types I and IIA, n=23) and 1.6 and 10.5 in EDL fibres (types IIX/D and IIB, n=16). These time constants were associated with components A1 and A2 (%) of 28.1 and 71.9 for soleus, and 35.8 and 64.2 for EDL. For all fiber types, after few repetitive stimuli at 100 Hz there was a big change of decay kinetics compared to single transients and then mild changes were seen in records lasting from 50 to 350 ms. In EDL tetanic transients, the fast component A1 almost disappeared, leaving the A2 and a much slower third one (A3) with t 2 and t 3 of 14.6 and 1259.7 (n=6). In soleus the A1 disappeared, while A2 increased with a t 2 of 74.6 (n=5). Preliminary experiments using CPA (1-2 mM) and FCCP (2-4 mM) have shed some light into the mechanisms in- volved in relaxation of tetanic transients in different fiber types. In conclusion, we show for the first time the diversity of Ca 2þ transients in the whole spectrum of fibre types and correlate it with the structural and biochemical diversity of mammalian skeletal muscle fibres. (FONACIT G-2001000637). 2829-Pos Effects of g-Ketoaldehydes on Ca 2þ Cuttrent Induced SR Ca 2þ Release in Ventricular Myocytes Hyun Seok Hwang, Oleksiy Gryshchenko, Sean S. Davies, Bjorn C. Knollmann. Vanderbilt University Medical Center, Nashville, TN, USA.  Oxidation increases RyR2 channel activity, enhances cardiac SR Ca 2þ release and causes spontaneous SR Ca 2þ waves. Isoprostanes have become a recog- nized marker of oxidative stress in rodents and humans. g-ketoaldehydes(g- KAs) are the most reactive product of the isoprostane pathway. Recently, we found that lipophilic pyridoxamine analogues, salicylamine(SA) scavenge g- KAs and thereby prevent formation of g-KA protein adducts in response to ox- idative stress. We hypothesized that g-KAs are potential mediators of oxidant- induced RyR2 channel dysfunction and spontaneous SR Ca 2þ waves, and that SA would prevent oxidant-induced spontaneous SR Ca 2þ waves(SCW) in the ventricular myocytes.  We compared the effect of g-KAs(1uM) or H 2 O 2 (10uM) and the effect of SA on Ca-current induced Ca release(CICR) in murine ventricular myocytes loaded with Fura-2AM or Fluo-4. All data are expressed relative to vehicle (Mean5SEM, n=15-50 per group).  Acute exposure(3 min) to g-KAs(1 uM) or H 2 O 2 (10 uM) increased the am- plitude of Ca 2þ transients, and the fraction of Ca 2þ released from the SR(g- KAs130510%*, H 2 O 2 120510%, *p<0.05) during each beat. Furthermore, the rate of SCW was significantly increased(g-KAs 42%*, H 2 O 2 33%*, *p<0.05) and SR Ca 2þ content was reduced. In voltage-clamped myocytes, di- alysis with g-KAs enhanced Ca 2þ release without changing L-type Ca 2þ cur- rent, demonstrating that the effect of g-KAs is the result of RyR2 modification. However, after chronic exposure(30 min) to g-KAs(1 uM) or H 2 O 2 (10 uM), Ca 2þ transients(g-KAs 0.5350.1*, H 2 O 2 0.750.1*, *p<0.05) and SR Ca 2þ contents decreased, and SCW remained elevated. Pre-treatment(3 days) of sal- icylamine reduced H 2 O 2 -induced spontaneous Ca 2þ waves(SCWs/sec, H 2 O 2 1.250.3*, SA-H 2 O 2 0.450.2*, *p<0.05) preserved with SR Ca 2þ con- tent in ventricular myocytes.  We found that H 2 O 2 and g-KAs have analogous biphasic effects on SR Ca 2þ release in ventricular myocytes. The protective effect of g-KA scavengers sug- gests that g-KAs are possible mediators of oxidant-induced RyR2 channel dys- function. 2830-Pos CamkII Phosphorylation of RyRs: a Mechanistic Mathematical Model Yasmin L. Hashambhoy, Raimond L. Winslow, Joseph L. Greenstein. Johns Hopkins University, Baltimore, MD, USA. 548a Tuesday, February 23, 2010