The role of thin filament cooperativity in cardiac length-dependent calcium activation

Abstract

Length-dependent activation (LDA) is a prominent feature of cardiac muscle characterized by decreases in the Ca(2+) levels required to generate force (i.e., increases in Ca(2+) sensitivity) when muscle is stretched. Previous studies have concluded that LDA originates from the increased ability of (strong) cross-bridges to attach when muscle is lengthened, which in turn enhances Ca(2+) binding to the troponin C (TnC) subunit of the troponin complex. However, our results demonstrate that inhibition of strong cross-bridge attachment with blebbistatin had no effect on the length-dependent modulation of Ca(2+) sensitivity (i.e., EC(50)) or Ca(2+) cooperativity, suggesting that LDA originates upstream of cross-bridge attachment. To test whether LDA arises from length dependence of thin-filament activation, we replaced native cTnC with a mutant cTnC (DM-TnC) that is incapable of binding Ca(2+). Although progressive replacement of native cTnC with DM-TnC caused an expected monotonic decrease in the maximal force (F(max)), DM-TnC incorporation induced much larger increases in EC(50) and decreases in Ca(2+) cooperativity at short lengths than at long lengths. These findings support the conclusion that LDA arises primarily from the influence of length on the modulation of the Ca(2+) cooperativity arising from interaction between adjacent troponin-tropomyosin complexes on the thin filament.

Figure 1

Impact of blebbistatin on strong binding cross-bridges and the Force-Ca relationship. Panels A and B illustrate the impact of increasing blebbistatin concentration (0 mM circle/solid line, 1 μM squares/dashed line) on the force calcium relationship as a function of the maximally calcium activated force. Blebbistatin induced a similar rightward shift in the Force-Ca relationship at both SL's indicating no SL dependent shift, in contrast to the results observed in DM-cTnC exchanged fibers (c.f. Fig. 3). Blebbistatin induced an equal decline in the maximally activated force production (F_max) at both short (circles) and long (squares) lengths (Panel C) that smoothly declined up to the maximum amount used (1 μM). As with force, the impact of blebbistatin on EC₅₀ (Panel D) was to decrease calcium sensitivity with increasing concentration yet demonstrating no SL dependent shifts in the EC₅₀ as opposed to that observed in DM-cTnC exchanged fibers. Error bars represent the mean ±SE.

Figure 2

Incorporation of DM-cTnC into the myofilament lattice. Panel A illustrates a typical example of SDS-PAGE gel analysis using Blue Silver staining to visualize TnC protein. Due to the fluorescent label, DM-TnC migrated slower allowing separation from WT-TnC. Note that exchange with 100% DM-TnC revealed a small (∼5%) amount endogenous TnC remaining in the fiber (consistent with the virtual complete elimination of active force development in 100% DM-TnC exchanged fibers, c.f. Fig. 3). Fig. 2B illustrates the percent of DM-cTnC incorporated into the myofilament (y axis) as a function of the percent DM-cTnC used in the exchange solution (x axis). Error bars represent the mean ±SE. As shown, the percent of DM-cTnC incorporated into the myofilament was linearly related to the amount of DM-cTnC in the exchange solution. Fig. 2C illustrates confocal images of two fibers labeled with fluorescent WT-cTnC or fluorescent DM-cTnC (i and iv) or rhodamine phalloidin (ii and v), as well as the merge images (iii and vi). Note the sarcomeric pattern of the TnC labeling in comparison to the rhodamine phalloidin staining. Merging the images demonstrates that both bacterially expressed proteins localize to the thin filament when they are allowed to incubate with skinned fibers.

Figure 3

Impact of DM-cTnC incorporation on the force-Ca relationship. (A and B) Impact of increasing DM-cTnC (circles, 0%; squares, 10%; diamonds, 50%) on the force-Ca relationship as a function of the maximally calcium-activated force. The impact of DM-cTnC is greater at the short SL (2.0 μm) (A) than at the long SL (2.2 μm) (B), with a larger shift in the relationship to the right (lower Ca²⁺ sensitivity) at the short SL with a concomitant shift in the slope (Hill coefficient). (C) The impact of DM-cTnC incorporation into the myofilament caused an equal shift in the maximally activated force production (F_max) at both short (circles) and long (squares) SLs that reached an approximate maximum at 25% DM-cTnC incorporation. (D) The impact of DM-cTnC incorporation on the EC₅₀ illustrates that the reduction of force had a greater impact at short SLs (circles) than at long SLs, indicating a large calcium dependence for activation at short SL. Error bars represent the mean ±SE.

Figure 4

(A and B) Impact of reducing F_max on the calcium sensitivity (EC₅₀) (A) and cooperative activation (n) (B) of isolated myocardial tissue. As demonstrated, the use of blebbistatin at short (green) and long (red) SLs reduced F_max while having no significant impact on either the EC₅₀ (A) or the Hill coefficient (B), as illustrated by the shallow slopes. The use of DM-cTnC at long sarcomere lengths (gray) mimics the impact of blebbistatin, indicating that the loss of thin-filament activity at long SLs did not have a great impact on either the calcium sensitivity or the cooperativity; meanwhile, at short SLs (blue), the impact of DM-cTnC exchange on calcium sensitivity (A) and cooperative activation (Hill coefficient) (B) was severe, causing a significantly larger slope (p < 0.05) in both cases than that observed in any of the other three groups. Error bars represent the mean ±SE.

Figure 5

Schematic drawing of the proposed mechanism of LDA, showing tropomyosin (gray lines), actin (ovals), and cardiac troponin units (tricolored rectangles). In our version of the three-state model, the impact of calcium on the activation of the neighboring units (dashed black line) at short SL works to activate the neighboring cTn unit, and thus, calcium needs to bind to more Tn units (arrows), whereas at longer SL, the activation of one cTn unit, due to the proposed stiffening of the tropomyosin, is able to activate more cTn units, thus requiring less calcium to activate the same number of cTn units (arrows). In other words, stretching the SL theoretically shifts the requirement for calcium binding from every 1–2 troponins to approximately every 3–4 troponins, allowing for more binding sites to be activated with the same levels of calcium activation.