The calcium dependent interactions between troponin C (TnC) and other thin and thick filament proteins play a key role in regulation of cardiac muscle contraction. Five hydrophobic residues Phe 20 , Val 44 , Met 45 , Leu 48 and Met 81 in the regulatory domain of TnC were individually substituted with polar Gln, to examine the effect of these mutations that sensitized isolated TnC to calcium on: 1) calcium binding and exchange with TnC in increasingly complex biochemical systems and 2) calcium sensitivity of actomyosin ATPase. The hydrophobic residue mutations drastically affected calcium binding and exchange with TnC in increasingly complex biochemical systems, indicating that side chain intra-and inter-molecular interactions of these residues play a crucial role in determining how TnC responds to calcium. However, the mutations that sensitized isolated TnC to calcium did not necessarily increase the calcium sensitivity of the troponin (Tn) complex or reconstituted thin filaments with or without myosin S1. Furthermore, the calcium sensitivity of reconstituted thin filaments (in the absence of myosin S1) was a better predictor of the calcium dependence of actomyosin ATPase activity than that of TnC or the Tn complex. Thus, both the intrinsic properties of TnC and its interactions with the other contractile proteins play a crucial role in modulating calcium binding to TnC in increasingly complex biochemical systemsThe processes of cardiac muscle contraction and relaxation can be regulated by multiple physiological and patho-physiological stimuli. It is clear that protein alterations associated with heart disease, isoform switching and post translational modifications can affect both the Ca 2+ sensitivity of muscle force generation and relaxation kinetics (for review, see (1-4)). Since cardiac troponin C (TnC) 1 is the Ca 2+ sensor responsible for initiating the contraction / relaxation cycle (for review, see (5,6)), a potentially important mechanism to alter cardiac † This research was funded by NIH grants 5R00HL087462 (to S.B.T) and 5R01HL073828 (to D.R.S.); by Predoctoral Fellowship Award from the American Heart Association (to B. L.) and by National Scientist Development Award from the American Heart Association (to J.P.D).*Address correspondence to: Jonathan P. Davis, Department of Physiology and Cell Biology, The Ohio State University, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, Tel. 614-247-2559; Fax. 614-292-4888; davis.812@osu.edu. ‡ These two authors contributed equally to the manuscript NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 March 9. Published in final edited form as:Biochemistry. 2010 March 9; 49(9): 1975-1984. doi:10.1021/bi901867s. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript muscle performance is through directly modifying the properties of TnC. As it has been difficult to find specific pharmacological modulators of TnC, we have taken a genetic approach to modify Ca 2+ binding and exchange with TnC.I...

The role of the C-domain sites of cardiac troponin C in the modulation of the calcium signal remains unclear. In this study we investigated the effects of hypertrophic cardiomyopathy linked mutations A8V, E134D and D145E in cardiac troponin C on the properties of the C-domain sites. The A8V mutation had essentially no effect on the calcium or magnesium binding properties of the C-domain sites, while E134D mutation moderately decreased calcium and magnesium binding affinities. On the other hand, D145E mutation affected cooperative interactions between sites III and IV, significantly reducing calcium binding affinity of both sites. Binding of the anchoring region of cardiac troponin I (corresponding to residues 34-71) to cardiac troponin C with D145E mutation was not able to recover normal calcium binding to the C-domain. Experiments utilizing the fluorescent hydrophobic probe bis-ANS suggest that D145E mutation dramatically reduced the extent of calcium-induced hydrophobic exposure by the C-domain. At high non-physiological calcium concentration, A8V, E134D and D145E mutations minimally affected the affinity of cardiac troponin C for the regulatory region of cardiac troponin I (corresponding to residues 128-180). In contrast, at lower physiological calcium concentration, D145E mutation led to ~8-fold decrease in the affinity of cardiac troponin C for the regulatory region of cardiac troponin I. Our results suggest that calcium binding properties of the C-domain sites might be important for the proper regulatory function of cardiac troponin C.

The objective of this work was to investigate the effect of hypertrophic cardiomyopathy-linked A8V and E134D mutations in cardiac troponin C (cTnC) on the response of reconstituted thin filaments to calcium upon phosphorylation of cardiac troponin I (cTnI) by protein kinase A. The phosphorylation of cTnI at protein kinase A sites was mimicked by S22D/S23D mutation in cTnI. Our results demonstrate that the A8V and E134D mutations had no effect on the extent of calcium desensitization of reconstituted thin filaments induced by cTnI pseudo-phosphorylation. However, the A8V mutation enhanced the effect of cTnI pseudo-phosphorylation on the rate of calcium dissociation from reconstituted thin filaments and on calcium dependence of actomyosin ATPase. Consequently, while the A8V mutation still led to a slower rate of calcium dissociation from reconstituted thin filaments upon pseudo-phosphorylation of cTnI, the ability of the A8V mutation to decrease the rate of calcium dissociation was diminished. In addition, the ability of the A8V mutation to sensitize actomyosin ATPase to calcium was diminished after cTnI was replaced by the phosphorylation mimetic of cTnI. Consistent with the hypothesis that the E134D mutation is benign, it exerted minor to no effect on the rate of calcium dissociation from reconstituted thin filaments, and on calcium sensitivity of actomyosin ATPase, regardless of cTnI phosphorylation status. In conclusion, our study enhances understanding of how cardiomyopathy-linked cTnC mutations affect the response of reconstituted thin filaments to calcium upon cTnI phosphorylation.

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The objective of this work was to investigate the role of acidic residues within the exposed middle segment of the central helix of cTnC in (1) cTnC-cTnI interactions, (2) Ca2+ binding and exchange with the regulatory N-domain of cTnC in increasingly complex biochemical systems, and (3) ability of the cTn complex to regulate actomyosin ATPase. In order to achieve this objective, we introduced the D87A/D88A and E94A/E95A/E96A mutations into the central helix of cTnC. The D87A/D88A and E94A/E95A/E96A mutations decreased affinity of cTnC for the regulatory region of cTnI. The Ca2+ sensitivity of the regulatory N-domain of isolated cTnC was decreased by the D87A/D88A, but not E94A/E95A/E96A mutation. However, both the D87A/D88A and E94A/E95A/E96A mutations desensitized the cTn complex and reconstituted thin filaments to Ca2+. Decreases in the Ca2+ sensitivity of the cTn complex and reconstituted thin filaments were, at least in part, due to faster rates of Ca2+ dissociation. In addition, the D87A/D88A and E94A/E95A/E96A mutations desensitized actomyosin ATPase to Ca2+, and decreased maximal actomyosin ATPase activity. Thus, our results indicate that conserved acidic residues within the exposed middle segment of the central helix of cTnC are important for the proper regulatory function of the cTn complex.