Duchenne muscular dystrophy represents a severe inherited disease of striated muscle, caused by a mutation in the dystrophin gene and characterized by pro- gressive loss of skeletal muscle function. Most patients also develop dystrophic cardiomyopathy resulting in dilated hypertrophy and heart failure. On the cel- lular level, absence of dystrophin affects mechanical membrane stability and intracellular Ca signaling in cardiomyocytes. Cellular mechanisms leading to deterioration of cardiac function remain elusive. We tested whether defective excitation-contraction (EC) coupling contributes to impaired cardiac perfor- mance. EC-coupling gain, a measure for the effectiveness to amplify the Ca sig- nal by Ca release from the sarcoplasmic reticulum (SR), was determined from control and dystrophin-deficient mdx hearts. Ca currents were measured with the whole-cell patch-clamp technique, while Ca transients were simultaneously recorded with confocal imaging of fluo-3. Initial findings indicated subtle prob- lems of EC-coupling in mdx cells despite matched SR Ca loading. However, lowering extracellular Ca, a maneuver used to unmask latent EC-coupling problems, was surprisingly much better tolerated by mdx myocytes. Normal- ized to control conditions, the EC-coupling gain in mdx cells reached 112% compared with 31% in control cells, suggesting hypersensitive EC-coupling. Further investigation of this apparent increase in Ca sensitivity by inducing slow elevations of intracellular Ca resulted in Ca oscillations after a much shorter delay in mdx cells, consistent with enhanced Ca sensitivity of SR Ca re- lease channels (ryanodine receptors, RyRs). Elevated cellular reactive oxygen species (ROS) generation in dystrophy suggests redox-modifications on the RyR, enhancing its Ca sensitivity. Preincubation of mdx cells with a ROS scav- enger normalized the EC-coupling hypersensitivity back to control cardiomyo- cytes. Our data suggest that in dystrophin-deficient cardiomyocytes, EC-cou- pling mechanisms are altered, partly due to potentially arrhythmogenic changes in Ca sensitivity of redox-modified RyRs. Supported by SNF, MDA, SSEM. 54-Plat The Skeletal L-type Ca 2þ Current is a Major Contributor to Excitation- Coupled Ca 2þ Entry (ECCE) Roger A. Bannister, Kurt G. Beam. University of Colorado-Denver, Aurora, CO, USA. The term Excitation-Coupled Ca 2þ Entry (ECCE) designates the entry of extra- cellular Ca 2þ into skeletal muscle cells which occurs in response to prolonged depolarization or pulse trains, and which depends on the expression of both the 1,4-dihydropyridine receptor (DHPR) and the type 1 ryanodine receptor (RyR1). The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca 2þ entry, is relatively insensitive to nifedipine (1 mM), and is permeable to Mn 2þ . We have examined the effects of these agents on the L-type Ca 2þ current conducted via the DHPR. We found that the non-specific cation channel antagonists 2-APB, SKF 96356, La 3þ and Gd 3þ all inhibited the L-type current. In addition, complete (>97%) block of the L-type current re- quired concentrations of nifedipine >10 mM. Like ECCE, the L-type channel displays permeability to Mn 2þ in the absence of external Ca 2þ and produces a Ca 2þ current that persists during prolonged (~10 s) depolarization. This cur- rent appears to contribute to the Ca 2þ transient observed during prolonged KCl- evoked depolarization of intact myotubes because (i) the transients in normal myotubes decayed more rapidly in the absence of external Ca 2þ ,(ii) the tran- sients in dysgenic myotubes expressing SkEIIIK (a DHPR a 1S subunit pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca 2þ -free solution and was unaffected by Ca 2þ re- moval, and (iii) after block of SR Ca 2þ release by ryanodine, normal myotubes still displayed a large Ca 2þ transient whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Altogether, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca 2þ entry attributed to ECCE. Supported by NIH NS24444 and AR44750 to K.G.B., and MDA 4155 to R.A.B. 55-Plat Post-Tetanic Calcium Transients In Adult Skeletal Muscle Fibers Are Frequency-Dependent And Fiber Type Specific Mariana Casas, Reinaldo Figueroa, Isaac Garcia, Enrique Jaimovich. University of Chile, Santiago, Chile. Isolated adult Flexor digitorum brevis fibers from 4-6 weeks-old mice, loaded with Fluo-3 were stimulated with trains of 270, 0.3 ms pulses at different fre- quencies. We observed a fast calcium tetanus (associated with contraction) and a second, slower signal, similar to those previously described in cultured my- otubes. The slow signal (more than the fast one) was inhibited by 25 micro M nifedipine, suggesting a role for DHPR in its onset and by the IP 3 R inhibitor Xestospongin-C (5 micro M). The amplitude of post-tetanic calcium transients depended on both stimulus train frequency and duration; a bell shaped curve frequency was obtained with a maximum at 10-20 Hz. Likewise, signal ampli- tude was proportional to stimulus train duration. Fibers isolated from soleus muscle completely lack slow calcium transients. Using immunofluorescence, we have found that all three IP 3 R isoforms are present in adult muscle at dif- ferent levels and that IP 3 R-1 is differentially expressed (with a mosaic pattern) in different types of muscle fibers, being higher in a subset of fast-type fibers. ERK 1/2 phosphorylation of adult muscle fibers after tetanic stimulation ap- pears to relate slow calcium signals to transcription-related events. These results support the idea that different calcium kinetics for the slow signals me- diated by IP 3 R may exist in different types of muscle fibers and participate in the activation of specific transcriptional programs of slow and fast phenotype. FONDAP 15010006, Bicentenario-PSD24, FONDECYT 1080120 56-Plat Negative Relationship Between Fractional SR Ca 2þ Release and Stimula- tion Rate Rosana A. Bassani, Rafael A. Ricardo, Jose ´ W.M. Bassani. Universidade Estadual de Campinas, Campinas, SP, Brazil. Typically, contraction amplitude in rodent myocardium is inversely related to stimulation rate. We calculated Ca 2þ fluxes across the sarcolemma and the sarcoplasmic reticulum (SR) membrane, and SR Ca 2þ content to estimate the fraction of the SR Ca 2þ load released at a twitch (FR) during steady-state (SS) stimulation at 0.2-2 Hz in intact rat cardiomyocytes. Cytosolic [Ca 2þ ] ([Ca 2þ ] i ) was measured with indo-1. SR Ca 2þ content was determined from the transient in response to 10 mM caffeine in Na þ ,Ca 2þ -free medium. From Ca 2þ buffer parameters, and [Ca 2þ ] i values and decline kinetics during transients in which SR Ca 2þ -ATPase and/or Na þ -Ca 2þ exchanger (NCX) were inhibited, SR- and NCX-mediated Ca 2þ fluxes at each rate were estimated and integrated up to attainment of SS diastolic [Ca 2þ ] i . Assuming equal inward and outward Ca 2þ fluxes at SS, Ca 2þ influx was considered as the NCX-mediated flux, whereas SR Ca 2þ release and uptake fluxes were considered equivalent. FR was taken as the ratio of the integrated SR-mediated Ca 2þ flux and SR Ca 2þ con- tent. Increasing rate did not affect significantly SR Ca 2þ content (12854 and 13354 mM at 0.2 and 2 Hz, respectively), but decreased (p<0.01) both Ca 2þ in- flux (16.951.4 vs. 6.750.3 mM at 0.2 and 2 Hz) and SR-dependent Ca 2þ flux (9354 vs. 7753 mM at 0.2 and 2 Hz). Estimated FR showed a negative relation- ship with the stimulation rate (0.7350.03 vs. 0.5850.02, p<0.01), possibly due to decreased Ca 2þ influx, although action potential duration was increased (APD90¼ 7659 vs. 130511 ms, p<0.01). Alternatively, incomplete time-de- pendent recovery from inactivation of SR Ca 2þ channels might contribute to depress FR. These results support the proposal that changes in FR underlie the negative force-frequency relationship in rodents. (FAPESP, CNPq, FAEPEX). 57-Plat Flecainide Inhibits Cardiac Ryanodine Channels And Spontaneous Sarco- plasmic Reticulum Calcium Release In Casq2 Null Myocytes Derek Laver 1 , Nagesh Chopra 2 , Bjorn C. Knollmann 2 . 1 University of Newcastle, Callaghan, Australia, 2 Vanderbilt University, Nashville, TN, USA. Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic syndrome due to cardiac Ca 2þ release channel (RYR2) or cardiac calsequestrin (CASQ2) mutations. VT is caused by spontane- ous Ca 2þ -release from the sarcoplasmic reticulum (SR) that generates after- depolarizations and triggered beats during catecholamine surge. We recently found that flecainide, a class 1c Na þ channel blocker, suppressed ventricular arrhythmia in Casq2 null (Casq / ) mice, a model of CPVT. Here, we inves- tigated the effect of flecainide on on cardiac Ca 2þ handling in Casq2 / myo- cytes loaded with Fura-2 AM, and on sheep RyR2 RyR2 channels reconstituted in lipid bilayers. Results: In isoproterenol-stimulated Casq2 / myocytes, flecainide (6 mmol/l) reduced triggered beats by over 70% (p<0.001). Unexpected for a Na þ channel blocker, flecainide also reduced SR Ca 2þ leak (Ca 2þ fluorescence ratio: vehicle: 0.1250.01 vs. flecainide: 0.0850.01, n¼54 per group, p¼0.02) and suppressed the rate of spontaneous Ca 2þ releases (SCRs) from the SR (SCRs/min: vehicle: 4855 vs. flecainide: 2955, n¼45 per group, p¼0.006), suggesting flecainide directly inhibits SR Ca 2þ release. Lipid bilayer experiments confirmed a direct action of flecainide on RyR2 SR Ca 2þ release channels: Flecainide induced brief partial closures of channels to a substate with a conductance equal to 20% of the fully open state. On average, flecainide as low as 5 mmol/L caused a 4-fold increase in the frequency of closed events and caused a significant reduction in the open probability from control levels. The effect of flecainide was concentra- tion dependent (IC 50 ~ 50 mmol/l) and fully-reversible upon washout. Flecainide also inhibited RyR2 channels activated by high luminal Ca 2þ . Conclusion: We report a heretofore unrecognized inhibitory action of flecai- nide on RyR2 channels, which together with flecainide’s inhibition of Na þ channels may explain flecainide’s effectiveness in preventing CPVT. Supported by R01-HL88635. Sunday, March 1, 2009 11a