Increasing mammalian cardiomyocyte contractility with residues identified in trout troponin C

2005 ◽  
Vol 22 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Todd E. Gillis ◽  
Bo Liang ◽  
Franca Chung ◽  
Glen F. Tibbits
Keyword(s):  
Amino Acid ◽  
Cardiac Myocytes ◽  
Cardiac Troponin ◽  
Troponin C ◽  
Force Generation ◽  
Wild Type ◽  
Cardiac Troponin C ◽  
Cardiomyocyte Contractility

The Ca2+ sensitivity of force generation in trout cardiac myocytes is significantly greater than that from mammalian hearts. One mechanism that we have suggested to be responsible, at least in part, for this high Ca2+ sensitivity is the isoform of cardiac troponin C (cTnC) found in trout hearts (ScTnC), which has greater than twice the Ca2+ affinity of mammalian cTnC (McTnC). Here, through a series of mutations, the residues in ScTnC responsible for its high Ca2+ affinity have been identified as being Asn2, Ile28, Gln29, and Asp30. When these residues in McTnC were mutated to the trout-equivalent amino acid, the Ca2+ affinity of the molecule, determined by monitoring the fluorescence of a Trp inserted for a Phe at residue 27, is comparable to that of ScTnC. To determine how a McTnC mutant containing Asn2, Ile28, Gln29, and Asp30 (NIQD McTnC) affects the Ca2+ sensitivity of force generation, endogenous cTnC in single, chemically skinned rabbit cardiomyocytes was replaced with either wild-type McTnC or NIQD McTnC. Our results demonstrate that the cardiomyocytes containing NIQD McTnC were approximately twice as sensitive to Ca2+, illustrating that a McTnC mutant with similar Ca2+ affinity as ScTnC can be used to sensitize mammalian cardiac myocytes to Ca2+.

Familial hypertrophic cardiomyopathy-related cardiac troponin C mutation L29Q affects Ca2+ binding and myofilament contractility

2008 ◽  
Vol 33 (2) ◽  
pp. 257-266 ◽  
Author(s):  
Bo Liang ◽  
Franca Chung ◽  
Yang Qu ◽  
Dmitri Pavlov ◽  
Todd E. Gillis ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Cardiac Myocytes ◽  
Cardiac Troponin ◽  
Sarcomere Length ◽  
Troponin C ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Force Generation ◽  
Binding Characteristics ◽  
Cardiac Troponin C ◽  
Myocyte Contractility

The cardiac troponin C (cTnC) mutation, L29Q, has been found in a patient with familial hypertrophic cardiomyopathy. We previously showed that L29, together with neighboring residues, Asp2, Val28, and Gly30, plays an important role in determining the Ca2+ affinity of site II, the regulatory site of mammalian cardiac troponin C (McTnC). Here we report on the Ca2+ binding characteristics of L29Q McTnC and D2N/V28I/L29Q/G30D McTnC (NIQD) utilizing the Phe27 → Trp (F27W) substitution, allowing one to monitor Ca2+ binding and release. We also studied the effect of these mutants on Ca2+ activation of force generation in single mouse cardiac myocytes using cTnC replacement, together with sarcomere length (SL) dependence. The Ca2+-binding affinity of site II of L29Q McTnCF27W and NIQD McTnCF27W was ∼1.3- and ∼1.9-fold higher, respectively, than that of McTnCF27W. The Ca2+ disassociation rate from site II of L29Q McTnCF27W and NIQD McTnCF27W was not significantly different than that of control (McTnCF27W). However, the rate of Ca2+ binding to site II was higher in L29Q McTnCF27W and NIQD McTnCF27W relative to control (∼1.5-fold and ∼2.0-fold respectively). The Ca2+ sensitivity of force generation was significantly higher in myocytes reconstituted with L29Q McTnC (∼1.4-fold) and NIQD McTnC (∼2-fold) compared with those reconstituted with McTnC. Interestingly, the change in Ca2+ sensitivity of force generation in response to an SL change (1.9, 2.1, and 2.3 μm) was significantly reduced in myocytes containing L29Q McTnC or NIQD McTnC. These results demonstrate that the L29Q mutation enhances the Ca2+-binding characteristics of cTnC and that when incorporated into cardiac myocytes, this mutant alters myocyte contractility.

Sequence mutations in teleost cardiac troponin C that are permissive of high Ca2+ affinity of site II

2003 ◽  
Vol 284 (5) ◽  
pp. C1176-C1184 ◽  
Author(s):  
Todd E. Gillis ◽  
Chris D. Moyes ◽  
Glen F. Tibbits
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cardiac Troponin ◽  
Troponin C ◽  
Fluorescent Reporter ◽  
Cardiac Troponin C ◽  
The Difference ◽  
Higher Sensitivity

Cardiac myofibrils isolated from trout heart have been demonstrated to have a higher sensitivity for Ca2+ than mammalian cardiac myofibrils. Using cardiac troponin C (cTnC) cloned from trout and mammalian hearts, we have previously demonstrated that this comparatively high Ca2+ sensitivity is due, in part, to trout cTnC (ScTnC) having twice the Ca2+ affinity of mammalian cTnC (McTnC) over a broad range of temperatures. The amino acid sequence of ScTnC is 92% identical to McTnC. To determine the residues responsible for the high Ca2+ affinity, the function of a number of ScTnC and McTnC mutants was characterized by monitoring an intrinsic fluorescent reporter that monitors Ca2+ binding to site II (F27W). The removal of the COOH terminus (amino acids 90–161) from ScTnC and McTnC maintained the difference in Ca2+ affinity between the truncated cTnC isoforms (ScNTnC and McNTnC). The replacement of Gln29 and Asp30 in ScNTnC with the corresponding residues from McNTnC, Leu and Gly, respectively, reduced Ca2+ affinity to that of McNTnC. These results demonstrate that Gln29 and Asp30 in ScTnC are required for the high Ca2+ affinity of site II.

Identification and characterization of a cardiac-specific transcriptional regulatory element in the slow/cardiac troponin C gene

1992 ◽  
Vol 12 (5) ◽  
pp. 1967-1976
Author(s):  
M S Parmacek ◽  
A J Vora ◽  
T Shen ◽  
E Barr ◽  
F Jung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Protein Binding ◽  
Cardiac Myocytes ◽  
Cardiac Troponin ◽  
Nuclear Protein ◽  
Troponin C ◽  
Cardiac Troponin C ◽  
Transcriptional Regulatory ◽  
Skeletal Myotubes

The slow/cardiac troponin C (cTnC) gene has been used as a model system for defining the molecular mechanisms that regulate cardiac and skeletal muscle-specific gene expression during mammalian development. cTnC is expressed continuously in both embryonic and adult cardiac myocytes but is expressed only transiently in embryonic fast skeletal myotubes. We have reported previously that cTnC gene expression in skeletal myotubes is controlled by a developmentally regulated, skeletal muscle-specific transcriptional enhancer located within the first intron of the gene (bp 997 to 1141). In this report, we show that cTnC gene expression in cardiac myocytes both in vitro and in vivo is regulated by a distinct and independent transcriptional promoter and enhancer located within the immediate 5' flanking region of the gene (bp -124 to +32). DNase I footprint and electrophoretic mobility shift assay analyses demonstrated that this cardiac-specific promoter/enhancer contains five nuclear protein binding sites (designated CEF1, CEF-2, and CPF1-3), four of which bind novel cardiac-specific nuclear protein complexes. Functional analysis of the cardiac-specific cTnC enhancer revealed that mutation of either the CEF-1 or CEF-2 nuclear protein binding site abolished the activity of the cTnC enhancer in cardiac myocytes. Taken together, these results define a novel mechanism for developmentally regulating a single gene in multiple muscle cell lineages. In addition, they identify previously undefined cardiac-specific transcriptional regulatory motifs and trans-acting factors. Finally, they demonstrate distinct transcriptional regulatory pathways in cardiac and skeletal muscle.

The amino acid sequence of human cardiac troponin-C

1986 ◽  
Vol 9 (1) ◽  
pp. 73-77 ◽  
Author(s):  
Alex Roher ◽  
Norman Lieska ◽  
Werner Spitz
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cardiac Troponin ◽  
Troponin C ◽  
Cardiac Troponin C ◽  
Human Cardiac Troponin C

scholarly journals A Novel Mutant Cardiac Troponin C Disrupts Molecular Motions Critical for Calcium Binding Affinity and Cardiomyocyte Contractility

2008 ◽  
Vol 94 (9) ◽  
pp. 3577-3589 ◽  
Author(s):  
Chee Chew Lim ◽  
Haijun Yang ◽  
Mingfeng Yang ◽  
Chien-Kao Wang ◽  
Jianru Shi ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Calcium Binding ◽  
Cardiac Troponin ◽  
Troponin C ◽  
Molecular Motions ◽  
Cardiac Troponin C ◽  
Cardiomyocyte Contractility

scholarly journals Cardiomyopathy-Related Mutations in Cardiac Troponin C, L29Q and G159D, Have Divergent Effects on Rat Cardiac Myofiber Contractile Dynamics

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Sampath K. Gollapudi ◽  
Murali Chandra
Keyword(s):  
Papillary Muscle ◽  
Cardiac Troponin ◽  
Thin Filament ◽  
Muscle Fibers ◽  
Troponin C ◽  
Wild Type ◽  
Cycling Rate ◽  
Cardiac Troponin C ◽  
Maximal Tension ◽  
Kinetics Of

Previous studies of cardiomyopathy-related mutations in cardiac troponin C (cTnC)—L29Q and G159D—have shown diverse findings. The link between such mutant effects and their divergent impact on cardiac phenotypes has remained elusive due to lack of studies on contractile dynamics. We hypothesized that a cTnC mutant-induced change in the thin filament will affect global myofilament mechanodynamics because of the interactions of thin filament kinetics with bothCa2+binding and crossbridge (XB) cycling kinetics. We measured pCa-tension relationship and contractile dynamics in detergent-skinned rat cardiac papillary muscle fibers reconstituted with the recombinant wild-type rat cTnC (cTnCWT),cTnCL29Q, andcTnCG159Dmutants.cTnCL29Qfibers demonstrated a significant decrease inCa2+sensitivity, butcTnCG159Dfibers did not. Both mutants had no effect onCa2+-activated maximal tension. The rate of XB recruitment dynamics increased incTnCL29Q(26%) andcTnCG159D(25%) fibers. The rate of XB distortion dynamics increased incTnCG159Dfibers (15%). Thus, thecTnCL29Qmutant modulates the equilibrium between the non-cycling and cycling pool of XB by affecting theon/offkinetics of the regulatory units (Tropomyosin-Troponin); whereas, thecTnCG159Dmutant increases XB cycling rate. Different effects on contractile dynamics may offer clue regarding howcTnCL29QandcTnCG159Dcause divergent effects on cardiac phenotypes.

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