The green tea polyphenol (-)-epigallocatechin-3-gallate inhibits magnesium binding to the C-domain of cardiac troponin C.

Cardiac muscle contraction is activated via the single Ca(2+)-binding site (site II) in the N-domain of troponin C (cTnC). The two Ca(2+)/Mg(2+) binding sites in the C-domain of cTnC (sites III and IV) have been considered to play a purely structural role in anchoring cTnC to the thin filament. However, several recent discoveries suggest a possible role of this domain in contractile regulation. The green tea polyphenol (-)-epigallocatechin 3-gallate (EGCg), which binds specifically to the C-domain of cTnC, reduces cardiac myofilament Ca(2+) sensitivity along with maximum force and acto-myosin ATPase activity. We have determined the effect of EGCg on Ca(2+) and Mg(2+) binding to the C-domain of cTnC. In the absence of Mg(2+) there was no significant effect of EGCg on the Ca(2+)-cTnC affinity. Surprisingly, in the presence of Mg(2+) EGCg caused an increase in Ca(2+) affinity for sites III and IV of cTnC. However, in the absence of Ca(2+) the addition of EGCg caused a significant reduction in Mg(2+)-cTnC affinity. This reduction is presumably responsible for the increase in Ca(2+)-cTnC affinity produced by EGCg in the presence of Mg(2+). We propose that the inhibitory effect of EGCg on myofilament Ca(2+) activation may be related to an enhanced Ca(2+)-Mg(2+)exchange at sites III and IV of cTnC, which might reduce the myosin crossbridge dependent component of thin filament activation.

Tropomyosin is in a reduced state in rabbit psoas muscle.

Tropomyosin (Tm) purified from skeletal and cardiac muscle often contains disulfide bonds due to oxidation of cysteine groups that are in close proximity in the coiled-coil structure. Are these disulfide crosslinks present in the muscle or produced by oxidation during preparation? To answer this question we reacted one part of freshly dissected rabbit psoas muscle fibers, which was permeabilized with Triton X-100, with N-ethyl maleimide (NEM) to block cysteine groups and another part with 5,5'-dithiobis(2-nitro benzoate) (DTNB) to facilitate disulfide bond formation by interchain sulfhydryl-disulfide exchange. We found, by high resolution gradient SDS polyacrylamide gels, that the NEM-treated muscle was only composed of uncrosslinked Tm and the DTNB treated muscle was composed of disulfide-crosslinked Tm. This work indicates that Tm exists in a reduced state in rabbit psoas muscle.

The Ca2+/Mg2+ sites of troponin C modulate crossbridge-mediated thin filament activation in cardiac myofibrils.

The Ca(2+)/Mg(2+) sites (III and IV) located in the C-terminal domain of cardiac troponin C (cTnC) have been generally considered to play a purely structural role in keeping the cTnC bound to the thin filament. However, several lines of evidence, including the discovery of cardiomyopathy-associated mutations in the C-domain, have raised the possibility that these sites may have a more complex role in contractile regulation. To explore this possibility, the ATPase activity of rat cardiac myofibrils was assayed under conditions in which no Ca(2+) was bound to the N-terminal regulatory Ca(2+)-binding site (site II). Myosin-S1 was treated with N-ethylmaleimide to create strong-binding myosin heads (NEM-S1), which could activate the cardiac thin filament in the absence of Ca(2+). NEM-S1 activation was assayed at pCa 8.0 to 6.5 and in the presence of either 1mM or 30 µM free Mg(2+). ATPase activity was maximal when sites III and IV were occupied by Mg(2+) and it steadily declined as Ca(2+) displaced Mg(2+). The data suggest that in the absence of Ca(2+) at site II strong-binding myosin crossbridges cause the opening of more active sites on the thin filament if the C-domain is occupied by Mg(2+) rather than Ca(2+). This finding could be relevant to the contraction-relaxation kinetics of cardiac muscle. As Ca(2+) dissociates from site II of cTnC during the early relaxing phase of the cardiac cycle, residual Ca(2+) bound at sites III and IV might facilitate the switching off of the thin filament and the detachment of crossbridges from actin.

Length-dependent Ca(2+) activation in cardiac muscle: some remaining questions.

The steep relationship between systolic force and end diastolic volume in cardiac muscle (Frank-Starling relation) is, to a large extent, based on length-dependent changes in myofilament Ca(2+) sensitivity. How sarcomere length modulates Ca(2+) sensitivity is still a topic of active investigation. Two general themes have emerged in recent years. On the one hand, there is a large body of evidence indicating that length-dependent changes in lattice spacing determine changes in Ca(2+) sensitivity for a given set of conditions. A model has been put forward in which the number of strong-binding cross-bridges that are formed is directly related to the proximity of the myosin heads to binding sites on actin. On the other hand, there is also a body of evidence suggesting that lattice spacing and Ca(2+) sensitivity are not tightly linked and that there is a length-sensing element in the sarcomere, which can modulate actin-myosin interactions independent of changes in lattice spacing. In this review, we examine the evidence that has been cited in support of these viewpoints. Much recent progress has been based on the combination of mechanical measurements with X-ray diffraction analysis of lattice spacing and cross-bridge interaction with actin. Compelling evidence indicates that the relationship between sarcomere length and lattice spacing is influenced by the elastic properties of titin and that changes in lattice spacing directly modulate cross-bridge interactions with thin filaments. However, there is also evidence that the precise relationship between Ca(2+) sensitivity and lattice spacing can be altered by changes in protein isoform expression, protein phosphorylation, modifiers of cross-bridge kinetics, and changes in titin compliance. Hence although there is no unique relationship between Ca(2+) sensitivity and lattice spacing the evidence strongly suggests that under any given set of physiological circumstances variation in lattice spacing is the major determinant of length-dependent changes in Ca(2+) sensitivity.

Length dependence of cardiac myofilament Ca(2+) sensitivity in the presence of substitute nucleoside triphosphates.

Although ATP is the immediate source of energy for muscle contraction other nucleoside triphosphates (NTP) can substitute for ATP as substrates for myosin and as sources of energy for contraction of skinned muscle fibers. However, experiments with skinned skeletal muscle fibers in the presence of substitute NTP indicate significant differences with respect to cross-bridge kinetics, force generation, and Ca(2+) regulation. In this study the length dependence of Ca(2+) sensitivity of skinned bovine cardiac muscle was analyzed in the presence of MgATP, MgCTP, MgUTP, and MgITP. Ca(2+) regulation in the presence of MgCTP and MgUTP was essentially the same as in the presence of MgATP, although the maximum force generated (at sarcomere length 2.4 microm) was about 25% less. However, the length dependence of Ca(2+) sensitivity was eliminated in the presence of MgUTP. With MgITP the maximum force generated (at sarcomere length 2.4 microm) was about the same as in the presence of MgATP, but there was an impairment of relaxation such that at pCa 8 the force developed was about 50-60% of that developed at pCa 5. Moreover, the Ca(2+)-dependent component showed no length-dependent sensitivity. Thus length modulation of Ca(2+) sensitivity is a function of the myosin substrate. Taken in conjunction with other data, the results are consistent with the hypothesis that length-dependence of Ca(2+) sensitivity is modulated at a step upstream from the force-generating reaction.