Functional characterization of the two isoforms of troponin C from the arthropodBalanus nubilus

Abstract

Two isoforms of troponin C (BTnC1 and BTnC2) from the striated muscle of the arthropod Balanus nubilus Darwin (giant barnacle) have been purified (Potter et al., 1987; Collins et al., 1991). Both isoforms were present in all of the white striated muscle fibres studied but not in the red fibres. The ratio of BTnC2 to BTnC1 in different fibre types varied between 3:1 and 1:1. Both forms of TnC could be readily extracted from myofibrillar bundles of barnacle muscle in low ionic strength EDTA solutions, reducing force activation to less than 10%. Both forms either separately or together reassociated with the TnC-depleted fibres in a relaxing (LR) solution (pCa greater than 8.0, [Mg2+] free = 1 mM, I = 0.15 M), and the reconstituted fibres could be subsequently activated in contraction (LA) solution (pCa = less than 3.8, [Mg2+] free = 1 mM, I = 0.15 M). The dissociation of BTnC 1 + 2 is blocked in low ionic strength solutions containing Mg2+ (greater than or equal to 10 mM). The two isoforms of crayfish TnC (CrTnC1 and CrTnC2) were also found to be equivalent to the barnacle TnCs in their ability to reactivate TnC-depleted barnacle myofibrillar bundles. Similar experiments using rabbit skeletal muscle TnC (STnC) (I = 0.15 M) in BTnC-depleted myofibrillar bundles of barnacle showed considerable variability. STnC could associate, although weakly, with the depleted bundles in either LR or LA, and force could be partially restored. In neither situation was it as effective as either BTnC or CrTnC. Interestingly, bovine cardiac TnC (CTnC), although it did not associate at pCa greater than 7.0, did associate and effectively activate force at pCa less than 3.8, but dissociated on return to pCa greater than 7.0 (LR). Neither barnacle TnC isoform associated with TnC-depleted skinned fibres from rabbit skeletal muscle at pCa greater than 7.0, but did associate and activate these fibres at pCa less than 3.8. Once these fibres were returned to LR and then placed in LA at pCa 3.8 all BTnC-restored force was lost, indicating a dissociation of BTnC once the Ca2+ is lowered, as observed with CTnC in barnacle myofibrillar bundles. Finally, the inhibitory effect of BTnI on force and the absence of an effect of calmodulin, trifluoperazine or ATP-gamma-S on force were all taken as evidence for a thin filament regulated Ca2+ control system.

The origin of passive force enhancement in skeletal muscle

“…To determine the effects of calcium concentration on passive (titin) stiffness and passive force enhancement without inducing cross-bridge formation and active force production, TnC was extracted from actin by using standard procedures (2,3,9,24,27).…”

“…To achieve this aim, tests were performed in myofibrils isolated from rabbit psoas muscle before and after elimination of cross-bridge formation and active force production through the deletion of troponin C (TnC) (2,3,9,24,27). In this way, the effects of increasing calcium concentrations on passive forces during and after stretching could be determined without the confounding effects of actin-myosin cross-bridge formation.…”

Invertebrate muscles: Thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

“…Crustacean actomyosin has a thin filament regulatory system (Maruyama et al, 1968a;Benzonana et al, 1974;Regenstein and Szent-Györgyi, 1975;Lehman and SzentGyörgyi, 1975;Chantler and Szent-Györgyi, 1978;Wnuk et al, 1984;Shima et al, 1984;Shinoda et al, 1988;Kobayashi et al, 1989;Wnuk, 1989;Nishita and Ojima, 1990;Kambara et al, 1990;Garone et al, 1991;Collins et al, 1991;Ashley et al, 1991;Miegel et al, 1992;Royuela et al, 1999;Allhouse et al, 1999c;Royuela et al, 2000b;Koenders et al, 2004). Early data suggested that some, but not all, crustacean muscles also have parallel myosin based regulation (Lehman and Szent-Györgyi, 1975;Lehman, 1977;Chantler and Szent-Györgyi, 1978;Sellers et al, 1980;Simmons and Szent-Györgyi, 1980;Watanabe et al, 1982;Ojima and Nishita, 1989).…”

Ca2+ and Sr2+ activation properties of skinned muscle fibres with different regulatory systems from crustacea and rat.

“…The muscle fibres from decapod crustacea are interesting preparations in which to study contractile activation because the regulatory system in these muscle fibres is known to consist of different isoforms of troponin C (Wnuk et al 1986; Kobayashi, Takagi, Konishi & Wnuk, 1989;Wnuk, 1989;Garone, Theibert, Miegel, Maeda, Murphy & Collins, 1991;Ashley, Lea, Hoar, Kerrick, Strang & Potter, 1991;Collins, Theibert, Francois, Ashley & Potter, 1991). The two major isoforms (az-TnC and y-TnC) of troponin C from the short-sarcomere fibres in the crayfish tail muscle have different calcium binding properties (Wnuk, 1989).…”