Cardiac Troponin I Threonine 144

Materials and Methods

An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

Results

We used the “whole Tn exchange technique” to introduce either wild-type or truncated cTnI into the cardiac sarcomere. As we reported previously,^3,4 this procedure caused no major alterations in the structure and properties of the fiber bundles. That is, exchanged skinned muscles retained a clearly detectable laser diffraction pattern. In addition, maximum calcium-saturated force-development reduction resulting from Tn exchange was <10% in all 4 groups (Figure 1A). The amount of recombinant Tn replacement of endogenous Tn in the 4 groups was 75% to 98% (Figure 1B). The principal aim of the present study was to examine the role of a subdomain of cTnI on myofilament length–dependent activation. Differential exchange for recombinant Tn in only part of the thin filament could, potentially, artificially affect the contractile response to changes in sarcomere length. Figure 1C demonstrates that the Tn exchange occurred uniformly along the thin filament in the 4 groups studied. That is, the confocal signals obtained for actin (red) and myc-TnT (green) were striated in appearance and, moreover, the signals colocalized as shown by the merged images displayed in the bottom images (yellow).

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Figure 1. Recombinant Tn exchange. A, Maximum Ca²⁺-saturated force development in exchanged skinned trabeculae relative to preexchange force. B, Amount of recombinant Tn exchange in the 4 groups, as determined by SDS-PAGE analysis. C, The distribution of recombinant Tn exchange in skinned trabeculae in the 4 groups was analyzed by confocal imaging for actin (green) and myc-TnT (red). The merged images are shown in yellow.

To characterize myofilament length–dependent activation properties following Tn exchange, force–Ca²⁺ relationships were determined in the skinned cardiac trabeculae at 2 sarcomere lengths (Figure 2). Examples of original force recordings as well as summarized Hill fit parameters are presented in the online data supplement. Exchange for Tn containing cTnI resulted in force–Ca²⁺ relationships similar to those obtained in nonexchanged muscles,¹ albeit with a reduced level of cooperativity; importantly, myofilament length–dependent activation was not affected by recombinant Tn exchange (see on-line data supplement). Exchange for cTnI_Thr>Pro Tn resulted in a reduction in overall Ca²⁺ sensitivity concomitant with a significant reduction of the influence of sarcomere length on Ca²⁺ sensitivity, similar to the result obtained on ssTnI Tn exchange. Exchange for ssTnI_Pro>Thr Tn induced an increase in overall myofilament Ca²⁺ sensitivity, an enhanced response to changes in sarcomere length and a decrease in cooperative activation, albeit only at the long sarcomere length. To more directly quantify myofilament length–dependent activity, we computed ΔEC₅₀, the difference between myofilament Ca²⁺ sensitivity recorded at the 2 sarcomere lengths as indexed by EC₅₀. As shown in Figure 3, average ΔEC₅₀ was similar in cTnI and ssTnI_Pro>Thr Tn-exchanged muscles. Likewise, average ΔEC₅₀ in both ssTnI and cTnI_Thr>Pro Tn-exchanged muscles was significantly reduced, indicating very little if any myofilament length–dependent activation properties. The level of cooperativity at a sarcomere length of 2.2 μm was significantly higher in cTnI Tn exchange (Thr or Pro at position 144) compared with ssTnI Tn exchange (Thr or Pro at position 112).

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Figure 2. Impact of Tn exchange on force–Ca²⁺ relationships. Following recombinant Tn exchange, force–Ca²⁺ relationships were determined in skinned trabeculae at sarcomere lengths of 2.0 μm (open circles) and 2.2 μm (closed circles) in the 4 groups (Figure 1) as indicated.

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Figure 3. Impact of Thr144 on myofilament-length dependence. Myofilament length–dependent activation was indexed as ΔEC₅₀, the difference in EC₅₀ between sarcomere lengths 2.0 and 2.2 μm. Control represents unexchanged skinned trabeculae. The presence of Thr144 in TnI was associated with a significantly enhanced cardiac myofilament-length dependence.

Discussion

In the present study, we found that presence of a threonine residue at position 144 in cTnI significantly modulated myofilament length–dependent properties of the cardiac sarcomere. The molecular mechanisms responsible for the change in Ca²⁺ affinity on a change in sarcomere length are not well understood.⁵ A role for interfilament spacing in the phenomenon⁵ is not supported by direct x-ray diffraction measurements.¹ Consistent with this notion, the change in lattice spacing between sarcomere lengths 2.0 and 2.2 μm was not different in the 4 groups studied (online data supplement). What could be the molecular mechanism by which the Thr144 residue confers length-dependent properties onto the sarcomere? Thr144 is located in the middle of the inhibitory region of TnI.² Phosphorylation of this residue reduces the affinity of cTnI for cTnC and depresses sliding velocity in the motility assay, albeit this effect is most pronounced when serines 43/45 are also phosphorylated.² Protein kinase A–mediated phosphorylation of cTnI is associated with an increase in myofilament length–dependent activation.¹ Thr144 phosphorylation may also affect this parameter, but this awaits further study. A partial structure of cTn has been determined by crystallography.⁶ Unfortunately, the structure of the inhibitory region of cTnI in the complex is still unresolved; hence few molecular cues exist as to the potential structural role of Thr144 in cTnI. The general molecular mechanism^2,7 of cardiac muscle activation involves binding of a Ca²⁺ ion to the regulatory lobe of cTnC, a movement of cTnI away from cTnT toward cTnC, movement of tropomyosin into the actin groove, followed by binding of myosin to the exposed actin sites to form active cycling cross-bridges. End-to-end interactions between tropomyosin molecules along the thin filament are believed to aid in the cooperative spread of activation. Support exists for additional binding between Tn subunits and actin, including domains within cTnI²; binding of myosin heads has been shown to promote further activation of the thin filament, as well as increase the binding affinity of cTnC to Ca²⁺. The cardiac thin filament may be less activated than the skeletal thin filament, even under conditions of Ca²⁺ saturation.⁷ How sarcomere length and cTnI-Thr144 affects any of these processes cannot be determined from the present study. It is possible that Thr144 residue itself is a length sensor or that absence of Thr144 simply masks the length sensing mechanisms. Modulation of Ca²⁺ sensitivity by sarcomere length may involve regulation of Ca²⁺ transduction by Tn possibly via the cooperative spread of thin filament activation communicated by tropomyosin, and this phenomenon may require the presence of Thr144 in TnI. Introduction of Pro144 in cTnI not only interrupts this process but also reduces the gain of Ca²⁺ transduction as evidenced by the reduction in myofilament Ca²⁺ sensitivity and the nonparallel changes in cooperativity on Tn exchange. The recombinant Tns used here are unphosphorylated, and this may explain the observed changes in cooperative activation.

Myofilament-length dependence is affected by mechanical strain on titin, a large sarcomeric protein known to make multiple interactions with both thin and thick filament proteins.⁸ It is possible that titin strain affects the interaction between cTnI and actin only in the presence of Thr144 within cTnI. Arguing against this hypothesis is the lack of any significant changes in passive force development among the 4 groups of Tn-exchanged skinned trabeculae (online data supplement). Finally, the finding that presence of cTnI-Pro144 in Tn greatly diminishes length-dependent properties of the sarcomere provides for a novel investigative avenue to determine the molecular mechanisms that underlie myofilament length–dependent activation.