365-369 POSTERS Sunda as a cold-sensitive allele in vivo. Our earlier analyses have the relaxed fiber at an ionic strength of 30 mM. Despite differences revealed that: (1) G680V exhibits reduced basal and actin-activated in magnitude, in all cases the frequency dependent stiffness ATPase activities; (2) it cannot move actin filaments and inhibits showed similar characteristics. The stiffness of the relaxed fiber at movement by wild type myosin in mixed assays; (3) it cosediments low ionic strength and the rigor stiffness after unloading at 30 mM with actin even in the presence of ATP, but not in the presence of closely coincided. No indication of attachment and/or detachment ATPyS; (4) its defects in vivo are suppressed by combining with a of cross bridges was observed in the spectrum of stiffness of the second mutation that accelerates Pi release. Here we report that relaxed fiber (in agreement with Bagni et al, G680V SI was unable to quench fluorescence of pyrene-actin in J.Electromyogr.Kinesiol.,1999). The present results indicate that the presence of ATP. In contrast, it quenched the pyrene the remainder of stiffness of the unloaded rigor is carried by fluorescence in the presence of ADP, indicating that an extended unloaded cross bridges. ADP-bound state cannot account for its excessive actin binding in the presence of ATP. Taken together, it was suggested that the Muscle Regulatory Proteins ATPase cycle of acto-G680V is blocked in a strongly bound 1 A.S .ADP.Pi state, or the "A state" of the 3G model (Geeves et al., 368-Pos Board # B224 1984). Negatively stained acto-G680V SI crossbridges in the THE presence of ATP appeared similar to DIFFERENCES IN MECHANISMS BY WHICH typical rigor, except that they TROPONIN C EXTRACTION AND THE TNI-IP INHIBIT were more disordered. Further structural analyses on acto-G680V THIN FILAMENT S1 should shed light on the nature of the yet elusive prestroke ACTIVATION conformation of myosin. Fred Schachate, Philip W Brandt2; 'Duke University Medical Center, 353 Sands Building, Durham, NC 27710, Columbia University Medical School, 630 W. 168th Street, New York, NY 366-Pos Board # B222 10032 WHERE DOES THE ENERGY GO IN MUSCLE DURING A RAMP STRETCH? Troponin C (TnC) plays a central role in both Ca2+ and rigor crossbridge (RXB) activation. Partial TnC extraction reduces the Marco Linaril, Roger C. Woledge2, Nancy A. Curtin3; 'University maximum tension of Florence, Viale G.B. Morgagni, 63, Florence, 50134 and cooperativity of activation by disrupting the Italy, cooperative unit on the thin filament. To determine whether any 2University College London, Brockley Hill, Stanmore, HA7 4LP inhibition of United Kingdom, 3Imperial College School of Medicine, London, TnC function has similar consequences, we SW7 2AZ United Kingdom investigated the effect of the troponin 1-inhibitory peptide (TnI-ip). TnI-ip inhibits RXB activation by shifting the pS/tension During stretch muscles absorb more energy than they release (Hill relationship to higher activator concentrations, without reducing its and Howarth, Proc. R. Soc. London Ser B 151:169, 1959). We cooperativity or maximum tension. This is simply explained by reinvestigated this effect on single frog muscle fibers (1 C, 2.1 Sun stabilization of the relaxed state. Tnl-ip's effect on Ca2+-activation sarcomere length). Fibers were mounted between a motor and a is also consistent with stabilization of the relaxed state, as TnI-ip force transducer in a drop of Ringer solution on a metal-film increases the (Ca2+] required for maximal tension, but does not thermopile, which has an unprecedented spatial resolution. reduce the cooperativity of Ca2+ activation. Interestingly, Tnl-ip Constant velocity stretches were applied at the plateau of the also reduces the maximal tension of Ca2+ activation. These isometric tetanus. During the stretch there was a net energy observations, as well as studies on the effect of TnI-ip following absorption by the fibers. Two types of fibers could be TnC extraction, demonstrate that TnI-ip does not disrupt the distinguished on the basis of how the heat rate and the energy (E=- cooperative unit. Moreover, because TnI-ip inhibits maximal Ca2- work+heat) depended on stretch. in the fibers of 18' type, for tension without reducing cooperativity, it suggests that tension moderate lengthening velocities, an initial decrease in E was generation is not essential for the transmission of cooperativity followed by no further change. In the fibers of 2nd type, E along the thin filament. continued to decrease during the entire period of lengthening. These results can be explained assuming that during lengthening, following the initial energy storage due to cross-bridges dragged 369-Pos Board # B225 into a high energy state (Piazzesi et al., J. Physiol. 445:659, 1992), MYOCARDIAL ISCHEMIA UNCOUPLES THE (1) in the fibers of I" type work is fully converted into heat as if FORCEIATPASE RELATION THROUGH MYOFILAMENT cross-bridges acted as a brake; (2) in the fibers of 2nd type energy PROTEIN ALTERATIONS OTHER THAN TNI continued to be stored presumably as the elasticity in parallel with DEGRADATION. weak sarcomeres is loaded. Supported by the Wellcome Trust. Jason Leo McDonough, Jennifer E Van Eyk; Queen's University, Rm 414 Botterell Hall, Kingston, Ontario K7L 3N6 Canada 367-Pos Board # B223 Brief periods of ischemia(l)/reperfusion(R) produce myocardial FREQUENCY DEPENDENT STIFFNESS OF THE stunning, in which the maximum force of triton-skinned muscle UNSTRAINED RIGOR AND OF THE WEAKLY BOUND fiber bundles is decreased compared to control perfused or CROSS BRIDGE CLOSELY COINCIDES. ischeniic only hearts. While TnI degradation during stunning may Evert de Beer', Ben Treijtel2, Tugenhold Blang62; 'University account for this contractile dysfunction, its effect on the economy Utrecht, PO Box 85060, Utrecht, 3508 AB Netherlands, of force production is not known. Hence, myofibrillar ATPase 2Academic Medical Cemter, University of Amsterdam, PO Box activity and TnI degradation were assessed in isolated rat hearts 22700, Amsterdam, 1100 DA Netherlands following control perfusion (45"), ischemia alone (15"I), and stunning Recently we proved that (15"V45"R). Ischemia alone produced depressed unloading of skeletal muscle in rigor maximal and minimal myofibillar ATPase activities compared to results in a fall of frequency dependent stiffness. After unloading control the fiber by a (83±2 vs 125±2, and 62±1 vs 91±1 nmolPiWmin/mg, shortening length adjustment of 0.6 %, a remaining respectively), without substantial TnI degradation (7+2%), despite stiffness was observed, while the cross bridges stayed attached. To maintained explore the nature of force production. Stunned myocardium demonstrated a this stiffness we measured the in-phase and similar depression in maximal and minimal myofibrillar ATPase out-of-phase stiffness under three experimental conditions by activities (87±2 and 68+1 releasing and stretching step changes in length of 0.025 %. From nmolPi/min/mg, respectively), as well as the induced the expected degradation of Tnl (21±t6%). The presence of other tension transients the normalized (for length and modified myofilament proteins that may diameter) stiffness was estimated from 10 Hz to 50 kHz. We account for this behavior compared is being assessed using a proteomic approach. These results rigor stiffness before and after unloading, at an ionic suggest that myocardial ischemia strength of 160 mM and at an ionic strength of 30 mM, and that of increases the economy of the 82a SundavLO LI&I 1%&464 y POSThERSX %., U.0 A. '169_174I IT force/ATPase relation, while subsequent reperfusion re-couples the TROPONIN-I REDUCES THE TROPONIN-T MEDIATED relationship at a lower economy. ACTIVATION OF MYOSIN S1-THIN FILAMENT ATPASE ACTIVITY. 370-Pos Board # B226 Yin Luo, Guang Yang, Knut Langsetmo, John Gergely, Terence BIOCHEMICAL STUDIES OF HUMAN CARDIAC TNI 1- Tao; Boston Biomedical Research Institute, 64 Grove St., 192: IMPLICATIONS FOR THE MECHANISM OF Watertown, MA 02472 MYOCARDIAL STUNNING The actin-activated skeletal myosin S1 ATPase activity is Ca(2+)- D. Brian Foster', Ann M. Murphy2, Jennifer E. Van Eyk'; regulated via the thin filament components tropomyosin (Tm) and 'Queen's University, Kingston, Ontario K7L 3N6 Canada, 2Johns troponin (Tn), which is composed of troponin-C (TnC), troponin-I Hopkins University, Baltimore, Maryland (TnI) and troponin-T (TnT). TnI alone can fully inhibit SloTmoF- Contractility of the heart is depressed following bouts of ischemia actin ATPase activity (inhibition); Ca2+-bound TnC reverses this (X) and subsequent reperfusion (RP) and the primary lesion occurs inhibition (deinhibition); the presence of TnT further elevates the within the myofilament proteins. Specifically, in the isolated rat activity (activation). We found that stoichiometric labeling of one heart, the first protein changes upon I/RP are phosphorylation and of the three endogenous Cys of Tnl, Cys64, using a mono-Cys TnI proteolysis of Tnl. Triton-skinned muscle fibers from the I/RP mutant (ThI64), with iodoacetamide or 4-benzophenone- hearts display decreased maximum force production and altered iodoacetamide reduced the TnT-mediated activation by 73 or 87%, Ca2+-sensitivity. The primary proteolytic product, TnI residues 1- respectively, without affecting the inhibition and the deinhibition. 193, lacks 17 amino acids at its C-terminus. To determine how There was no measurable effect on the ability of the labeled TnI64 truncated TnI contributes to contractile dysfunction, we have to form the ternary Tn complex. Labeling at other Cys of TnI did undertaken in vitro analysis of recombinant human cardiacTnll- not have any effect. Since Cys64 is in the TnT-binding region of 192. TnII-192 and wtcTnl bind to actin or actin-tropomyosin with Tnl, our results suggest that the attached probes disrupt the equivalent affinity. Both proteins inhibit actoTMSl ATPase conformation of Tn at the TnlI-TnT interface that is critical for activity to a similar extent and potency.