Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle

John D. Strauss, Jennifer E. Van Eyk, Zacharias Barth, Lan Kluwe, Rudolf J. Wiesner, K. Maéda, J. Caspar Rüegg

Research output: Contribution to journalArticle

Abstract

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca2+-dependent regulation. Force then depended on the negative logarithm of Ca2+ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa50, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca+, but restoration of Ca2+ dependence required incubation with both TnI and TnC. Relaxation at low Ca2+ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20-150 μM), while Ca2+ sensitivity, i.e. the pCa50, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pCa50 approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G104-K-F-K-R-P-P-L-R-R-V-R115-). The four mutants produced by substitution of T for P110, G for P110, G for L111 and G for K105 were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-Si, ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca2+ dependence could be restored whengly110sTnI,thr110sTnI orgly111sTnI was used for reconstitution. The mutantgly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.

Original languageEnglish (US)
Pages (from-to)853-862
Number of pages10
JournalPflugers Archiv European Journal of Physiology
Volume431
Issue number6 Supplement
DOIs
StatePublished - Nov 1996
Externally publishedYes

Fingerprint

Troponin I
Muscle
Myocardium
Substitution reactions
pioglitazone
Calcium
Fibers
Protein Isoforms
Troponin C
Rabbits
Peptides
Vanadates
Amino Acid Substitution
Site-Directed Mutagenesis
Point Mutation
Heart Ventricles
Adenosine Triphosphatases
Actins
Skeletal Muscle
Plasmids

Keywords

  • Calcium sensitivity
  • Cardiac muscle contraction
  • Site-directed mutagenesis
  • Skinned fibers
  • Troponin I

ASJC Scopus subject areas

  • Physiology

Cite this

Strauss, J. D., Van Eyk, J. E., Barth, Z., Kluwe, L., Wiesner, R. J., Maéda, K., & Rüegg, J. C. (1996). Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle. Pflugers Archiv European Journal of Physiology, 431(6 Supplement), 853-862. https://doi.org/10.1007/BF02332169

Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle. / Strauss, John D.; Van Eyk, Jennifer E.; Barth, Zacharias; Kluwe, Lan; Wiesner, Rudolf J.; Maéda, K.; Rüegg, J. Caspar.

In: Pflugers Archiv European Journal of Physiology, Vol. 431, No. 6 Supplement, 11.1996, p. 853-862.

Research output: Contribution to journalArticle

Strauss, JD, Van Eyk, JE, Barth, Z, Kluwe, L, Wiesner, RJ, Maéda, K & Rüegg, JC 1996, 'Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle', Pflugers Archiv European Journal of Physiology, vol. 431, no. 6 Supplement, pp. 853-862. https://doi.org/10.1007/BF02332169
Strauss, John D. ; Van Eyk, Jennifer E. ; Barth, Zacharias ; Kluwe, Lan ; Wiesner, Rudolf J. ; Maéda, K. ; Rüegg, J. Caspar. / Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle. In: Pflugers Archiv European Journal of Physiology. 1996 ; Vol. 431, No. 6 Supplement. pp. 853-862.
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AU - Strauss, John D.

AU - Van Eyk, Jennifer E.

AU - Barth, Zacharias

AU - Kluwe, Lan

AU - Wiesner, Rudolf J.

AU - Maéda, K.

AU - Rüegg, J. Caspar

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N2 - Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca2+-dependent regulation. Force then depended on the negative logarithm of Ca2+ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa50, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca+, but restoration of Ca2+ dependence required incubation with both TnI and TnC. Relaxation at low Ca2+ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20-150 μM), while Ca2+ sensitivity, i.e. the pCa50, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pCa50 approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G104-K-F-K-R-P-P-L-R-R-V-R115-). The four mutants produced by substitution of T for P110, G for P110, G for L111 and G for K105 were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-Si, ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca2+ dependence could be restored whengly110sTnI,thr110sTnI orgly111sTnI was used for reconstitution. The mutantgly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.

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KW - Calcium sensitivity

KW - Cardiac muscle contraction

KW - Site-directed mutagenesis

KW - Skinned fibers

KW - Troponin I

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