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(Circulation Research. 2007;101:1081.)
© 2007 American Heart Association, Inc.

Report

Cardiac Troponin I Threonine 144

Role in Myofilament Length–Dependent Activation

Kittipong Tachampa, Helen Wang, Gerrie P. Farman, Pieter P. de Tombe

From the Department of Physiology and Biophysics and Center for Cardiovascular Research, University of Illinois, Chicago.

Correspondence to Pieter P. de Tombe, Department of Physiology and Biophysics, University of Illinois, 835 S Wolcott, Chicago IL 60612. E-mail pdetombe{at}uic.edu

	Abstract

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Myofilament length–dependent activation is the main cellular mechanism responsible for the Frank–Starling law of the heart. All striated muscle display length-dependent activation properties, but it is most pronounced in cardiac muscle and least in slow skeletal muscle. Cardiac muscle expressing slow skeletal troponin (ssTn)I instead of cardiac troponin (cTn)I displays reduced myofilament length–dependent activation. The inhibitory region of troponin (Tn)I differs by a single residue, proline at position 112 in ssTnI versus threonine at position 144 in cTnI. Here we tested whether this substitution was important for myofilament length–dependent activation; using recombinant techniques, we prepared wild-type cTnI, ssTnI, and 2 mutants: cTnI_Thr>Pro and ssTnI_Pro>Thr. Purified proteins were complexed with recombinant cardiac TnT/TnC and exchanged into skinned rat cardiac trabeculae. Force–Ca²⁺ relationships were determined to derive myofilament Ca²⁺ sensitivity (EC₅₀) at 2 sarcomere lengths: 2.0 and 2.2 µm (n=7). Myofilament length-dependent activation was indexed as EC₅₀, the difference in EC₅₀ between sarcomere lengths of 2.0 and 2.2 µm. Incorporation of ssTnI compared with cTnI into the cardiac sarcomere reduced EC₅₀ from 1.26±0.30 to 0.19±0.04 µmol/L. A similar reduction also could be observed when Tn contained cTnI_Thr>Pro (EC₅₀=0.24±0.04 µmol/L), whereas the presence of ssTnI_Pro>Thr increased EC₅₀ to 0.94±0.12 µmol/L. These results suggest that Thr144 in cardiac TnI modulates cardiac myofilament length–dependent activation.

Key Words: skinned muscle • rat • Frank–Starling relationship

	Introduction

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The Frank–Starling "Law of the Heart" describes the relationship between ventricular end-systolic pressure and end-systolic volume. It is well established that the cellular basis for this phenomenon is the modulation of myofilament calcium sensitivity with sarcomere length.¹ Although all mammalian striated muscle display myofilament length–dependent activation properties, it is most pronounced in cardiac muscle and least in slow skeletal muscle.¹ The molecular mechanisms that underlie myofilament length–dependent activation are incompletely understood. The mammalian heart expresses slow skeletal troponin (ssTn)I during development and, in many species, also during the early neonatal state.² Replacement of endogenous cardiac troponin (cTn)I by ssTnI by transgenesis has been shown to be sufficient to reduce myofilament-length dependence.¹ Those results indicate that TnI plays a pivotal role in modulating the response of the cardiac sarcomere to changes in sarcomere length and, moreover, that the extent of this modulation depends on the structure of TnI. We found in preliminary experiments that a specific region of cTnI, located between the inhibitory region and the C terminus of the molecule, may be of specific importance for myofilament length–dependent activation. Inspection of the sequence differences between cTnI and ssTnI in the inhibitory region of TnI reveals a substitution of a single residue, threonine, in cTnI (144) by a proline in ssTnI (112). Accordingly, in the present study, we investigated the role of Thr144 in length dependence. We found that the presence of threonine at position 144 (in cTnI) or 112 (in ssTnI) is sufficient to impart length dependence onto the cardiac sarcomere.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

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

Exchange of Recombinant cTn Into Skinned Rat Trabeculae
Right ventricular rat trabeculae were dissected, chemically permeabilized with Triton X-100, and attached to T clips as described.³ Exogenous troponin (Tn) was exchanged for endogenous Tn by slight modification of previously described methods.³

Confocal Analysis of Tn Exchange
Recombinant TnT in the present study included an NH₂ terminus myc tag to allow for confocal analysis of recombinant Tn exchange and colocalization with actin (model 2100 LSM, Bio-Rad). Previously, we have demonstrated that the presence of the myc tag does not affect myofilament function.⁴

Measurement of Isometric Tension
The measurement of steady-state isometric tension at varied free Ca²⁺ was conducted as described.³ Briefly, sarcomere length was set at 2.2 µm by laser diffraction. Trabeculae were activated over a range of free [Ca²⁺] to measure steady-state isometric tension. Only muscles that maintained >80% maximal tension were included for analysis.

Data Processing and Statistical Analysis
Force–Ca²⁺ relationships were fit to a modified Hill equation.³ Statistical analyses were performed by ANOVA. P<0.05 was considered statistically significant; data are presented means±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
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).

View larger version (67K): [in this window] [in a new window]   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 EC₅₀, the difference between myofilament Ca²⁺ sensitivity recorded at the 2 sarcomere lengths as indexed by EC₅₀. As shown in Figure 3, average EC₅₀ was similar in cTnI and ssTnI_Pro>Thr Tn-exchanged muscles. Likewise, average EC₅₀ 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).

View larger version (16K): [in this window] [in a new window]   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.

View larger version (13K): [in this window] [in a new window]   Figure 3. Impact of Thr144 on myofilament-length dependence. Myofilament length–dependent activation was indexed as EC50, 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

	Acknowledgments

 
We thank Dr Katherine Sheehan for help with the confocal images and Drs Tom Irving and David Gore for assistance with the x-ray diffraction experiments. Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. W-31-109-ENG-38. BioCAT is a NIH-supported Research Center (RR-08630). The content of this report is the sole responsibility of the authors and does not necessarily reflect the official views of the National Center for Research Resources or the NIH.

Sources of Funding

This study was supported by AHA pre-doctoral Fellowship 0615597Z and NIH grants PO1-HL62426, RO1-HL75494.

Disclosures

None.

	Footnotes

 
Original received March 23, 2007; resubmission received October 4, 2007; revised resubmission received October 17, 2007; accepted October 18, 2007.

	References

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2. Kobayashi T, Solaro RJ. Calcium, thin filaments, and the integrative biology of cardiac contractility. Annu Rev Physiol. 2005; 67: 39–67.[CrossRef][Medline] [Order article via Infotrieve]

3. Sumandea MP, Pyle WG, Kobayashi T, de Tombe PP, Solaro RJ. Identification of a functionally critical protein kinase C phosphorylation residue of cardiac troponin T. J Biol Chem. 2003; 278: 35135–35144.[Abstract/Free Full Text]

4. Chandra M, Rundell VL, Tardiff JC, Leinwand LA, De Tombe PP, Solaro RJ. Ca(2+) activation of myofilaments from transgenic mouse hearts expressing R92Q mutant cardiac troponin T. Am J Physiol. 2001; 280: H705–713.

5. Fuchs F, Smith SH. Calcium, cross-bridges, and the Frank-Starling relationship. News Physiol Sci. 2001; 16: 5–10.[Abstract/Free Full Text]

6. Vassylyev DG, Takeda S, Wakatsuki S, Maeda K, Maeda Y. Crystal structure of troponin C in complex with troponin I fragment at 2.3-A resolution. Proc Natl Acad Sci U S A. 1998; 95: 4847–4852.[Abstract/Free Full Text]

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This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 101/11/1081    most recent CIRCRESAHA.107.165258v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tachampa, K. Articles by de Tombe, P. P. Search for Related Content PubMed PubMed Citation Articles by Tachampa, K. Articles by de Tombe, P. P. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL Related Collections Structure Contractile function