XB-ART-56688
Front Physiol
2019 Jan 01;10:1612. doi: 10.3389/fphys.2019.01612.
Show Gene links
Show Anatomy links
Familial Dilated Cardiomyopathy Associated With a Novel Combination of Compound Heterozygous TNNC1 Variants.
Landim-Vieira M
,
Johnston JR
,
Ji W
,
Mis EK
,
Tijerino J
,
Spencer-Manzon M
,
Jeffries L
,
Hall EK
,
Panisello-Manterola D
,
Khokha MK
,
Deniz E
,
Chase PB
,
Lakhani SA
,
Pinto JR
.
???displayArticle.abstract???
Familial dilated cardiomyopathy (DCM), clinically characterized by enlargement and dysfunction of one or both ventricles of the heart, can be caused by variants in sarcomeric genes including TNNC1 (encoding cardiac troponin C, cTnC). Here, we report the case of two siblings with severe, early onset DCM who were found to have compound heterozygous variants in TNNC1: p.Asp145Glu (D145E) and p.Asp132Asn (D132N), which were inherited from the parents. We began our investigation with CRISPR/Cas9 knockout of TNNC1 in Xenopus tropicalis, which resulted in a cardiac phenotype in tadpoles consistent with DCM. Despite multiple maneuvers, we were unable to rescue the tadpole hearts with either human cTnC wild-type or patient variants to investigate the cardiomyopathy phenotype in vivo. We therefore utilized porcine permeabilized cardiac muscle preparations (CMPs) reconstituted with either wild-type or patient variant forms of cTnC to examine effects of the patient variants on contractile function. Incorporation of 50% WT/50% D145E into CMPs increased Ca2+ sensitivity of isometric force, consistent with prior studies. In contrast, incorporation of 50% WT/50% D132N, which had not been previously reported, decreased Ca2+ sensitivity of isometric force. CMPs reconstituted 50-50% with both variants mirrored WT in regard to myofilament Ca2+ responsiveness. Sinusoidal stiffness (SS) (0.2% peak-to-peak) and the kinetics of tension redevelopment (kTR) at saturating Ca2+ were similar to WT for all preparations. Modeling of Ca2+-dependence of kTR support the observation from Ca2+ responsiveness of steady-state isometric force, that the effects on each mutant (50% WT/50% mutant) were greater than the combination of the two mutants (50% D132N/50% D145E). Further studies are needed to ascertain the mechanism(s) of these variants.
???displayArticle.pubmedLink??? 32038292
???displayArticle.pmcLink??? PMC6990120
???displayArticle.link??? Front Physiol
Species referenced: Xenopus tropicalis
Genes referenced: tnnc1
GO keywords: calcium ion sensor activity
???displayArticle.disOnts??? cardiomyopathy
???attribute.lit??? ???displayArticles.show???
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Figure 1. (A,B) Echocardiography images and (C) electrocardiogram. Still images obtained from echocardiogram of the proband at ages 2 weeks (A) and 12 months (B). Note particularly the progressive dilation and prominent rounding of the left ventricle (LV) in the later image. (C) Electrocardiogram at 19 months of age demonstrating sinus rhythm and left ventricular enlargement. |
|
Figure 2. (A) Pedigree and (B) Sanger sequencing. (A) Family pedigree showing the proband and similarly affected brother (black shading). There was a prior loss of a severely premature live birth and three prior failed IVF pregnancies. (B) Sanger sequencing data from the four available family members. Red arrows indicate residue of interest. The left side shows residues around the c.394G>A change that results in D132N in one of the two alleles for TNNC1 for three of four individuals (mother is reference, other three are heterozygous for the variant). The right side shows residues around the c.435C>A change that results in D145E in one of the two alleles for TNNC1 for three of four individuals (father is reference, other three are heterozygous for the variant). |
|
Figure 3. (A–D) Sequence and structure analyses of cTnC surrounding divalent cation-binding site IV. (A) Primary sequence of human cTnC (NP_003271.1). Divalent cation-coordinating regions (sites II, III, and IV) are highlighted in bold, and amino acids that comprise α-helical regions (N-helix and α-helices A–H) are underlined [annotated according to Li and Hwang (2015)]. Site II [EF-hand comprised of a-helices C–D and joining residues] is in the N-domain of cTnC and is the regulatory “trigger” site that activates contraction when Ca2+ binds during the systolic Ca2+ transient. Sites III and IV (EF-hands comprised of α-helices E–F or G–H, respectively, along with the respective residues that join each pair of α-helices) are in the C-domain and can bind Mg2+ or Ca2+. Note that all 161 amino acids of the primary sequence are intentionally shown as a single row to illustrate the relative sizes and locations of annotated regions. (B,C) Ribbon structures of the Ca2+-saturated C-domain of human cTnC in complex with a cardiac troponin I peptide (cTnI128–147), illustrating the locations of D132 and D145 (red arrows). NMR structure adapted from PDB 1OZS model 1 (Lindhout and Sykes, 2003) using MATLAB (ver. R2018b, The MathWorks, Inc.) molviewer. cTnC90–161 (dark red) and cTnI128–147 (blue) are shown as backbone ribbons, with space-filled atoms to highlight cTnC residues D132 and D145 (CPK colors). Ca2+ ions at sites III and IV are large gray spheres (gray arrows); note that, physiologically, it is likely that Mg2+ rather than Ca2+ would be bound at most sites III and IV in the sarcomeres of living cardiomyocytes. Labels in either panel (B) or (C) apply to the locations of structural elements in both panels. Note that only one affected amino acid is highlighted in each of panels (B,C), corresponding to the occurrence of only one of the two variants in each TNNC1 allele of the proband’s genome (Figure 2B). (D) Multiple alignments of residues around site IV demonstrate a remarkable degree of conservation of amino acids D132 and D145 across a wide range of species. Conserved amino acids are shown in red. Affected residues D132 and D145 are identified by underlines (human sequence) and arrows (above). D132 is within the G-helix (refer to panel A) that is part of site IV EF-hand. D145 is at the +Z location within the divalent cation-coordinating region of site IV; all 12 residues of this region are highlighted in bold in the human sequence (top sequence), as in panel (A), and the coordinating residues (+X, +Y, +Z, –Y, –X, and –Z) within site IV are annotated (arrows) below the aligned sequences. |
|
Figure 4. (A,B) Brightfield microscopy and (C,D) Optical Coherence Tomography (OCT) imaging of Control and TNNC1 F0 knockout tadpoles. As compared to control tadpoles (A), TNNC1 knockout tadpoles (B) show normal gross morphology, but significant total body edema (white arrows) secondary to severe dilated cardiomyopathy leading to congestive heart failure. Red arrows indicate plane of OCT images in (C,D). OCT images demonstrate normal size and wall thickness of cardiac chambers in control tadpole (C), with severe dilation and enlargement of the ventricle in TNNC1 knockout tadpole (D), indicated with white arrowheads. V = ventricle, LA = left atrium, RA = right atrium. |
|
Figure 5. Ca2+ dependence of steady-state isometric tension in porcine CMPs reconstituted with exogenous cTnC WT (100% WT) or variants (50% WT/50% D132N, 50% WT/50% D145E, or 50% D132N/50% D145E). (A) Relative steady-state isometric force as a function of pCa. The force values were normalized to the maximal steady-state isometric force in the same preparation. (B) Normalized steady-state isometric force as a function of pCa. The force values were normalized to the maximal steady-state isometric force generated by WT. Data are shown as mean ± S.E. and best fit parameter estimates from non-linear least squares regression on the Hill equation are summarized in Table 2 (n = 4–8). |
|
Figure 6. Sinusoidal stiffness analysis in porcine CMPs reconstituted with exogenous cTnC WT (100% WT) or variants (50% WT/50% D132N, 50% WT/50% D145E, or 50% D132N/50% D145E). (A) Ca2+ dependence of steady-state sinusoidal stiffness. (B) Normalized force vs. steady-state sinusoidal stiffness. The force values were normalized to the maximal steady-state isometric force generated by WT. (B) Dashed lines were drawn to connect the points. Data are shown as mean ± S.E. and maximum SS values are summarized in Table 3. |
|
Figure 7. Rate of tension redevelopment (kTR) analysis in porcine CMPs reconstituted with exogenous cTnC WT (100% WT) or variants (50% WT/50% D132N, 50% WT/50% D145E, or 50% D132N/50% D145E). (A) kTR as a function of increasing Ca2+ concentration. Solid lines were drawn to connect the points. (B) Relative force vs. kTR. The force values were normalized to the maximal steady-state isometric force for each preparation. Data are shown as mean ± S.E. and maximum kTR values are summarized in Table 3. Dashed lines were obtained from fits to the 3-state model, which take into account the changes in Ca2+ sensitivity (Table 2 and Figures 5A,B) as described in section “Experimental Procedures”; best fit parameter estimates are summarized in Table 4. |
|
FIGURE 1. (A,B) Echocardiography images and (C) electrocardiogram. Still images obtained from echocardiogram of the proband at ages 2 weeks (A) and 12 months (B). Note particularly the progressive dilation and prominent rounding of the left ventricle (LV) in the later image. (C) Electrocardiogram at 19 months of age demonstrating sinus rhythm and left ventricular enlargement. |
|
FIGURE 2. (A) Pedigree and (B) Sanger sequencing. (A) Family pedigree showing the proband and similarly affected brother (black shading). There was a prior loss of a severely premature live birth and three prior failed IVF pregnancies. (B) Sanger sequencing data from the four available family members. Red arrows indicate residue of interest. The left side shows residues around the c.394G>A change that results in D132N in one of the two alleles for TNNC1 for three of four individuals (mother is reference, other three are heterozygous for the variant). The right side shows residues around the c.435C>A change that results in D145E in one of the two alleles for TNNC1 for three of four individuals (father is reference, other three are heterozygous for the variant). |
|
FIGURE 3. (A–D) Sequence and structure analyses of cTnC surrounding divalent cation-binding site IV. (A) Primary sequence of human cTnC (NP_003271.1). Divalent cation-coordinating regions (sites II, III, and IV) are highlighted in bold, and amino acids that comprise α-helical regions (N-helix and α-helices A–H) are underlined [annotated according to Li and Hwang (2015)]. Site II [EF-hand comprised of a-helices C–D and joining residues] is in the N-domain of cTnC and is the regulatory “trigger” site that activates contraction when Ca2+ binds during the systolic Ca2+ transient. Sites III and IV (EF-hands comprised of α-helices E–F or G–H, respectively, along with the respective residues that join each pair of α-helices) are in the C-domain and can bind Mg2+ or Ca2+. Note that all 161 amino acids of the primary sequence are intentionally shown as a single row to illustrate the relative sizes and locations of annotated regions. (B,C) Ribbon structures of the Ca2+-saturated C-domain of human cTnC in complex with a cardiac troponin I peptide (cTnI128–147), illustrating the locations of D132 and D145 (red arrows). NMR structure adapted from PDB 1OZS model 1 (Lindhout and Sykes, 2003) using MATLAB (ver. R2018b, The MathWorks, Inc.) molviewer. cTnC90–161 (dark red) and cTnI128–147 (blue) are shown as backbone ribbons, with space-filled atoms to highlight cTnC residues D132 and D145 (CPK colors). Ca2+ ions at sites III and IV are large gray spheres (gray arrows); note that, physiologically, it is likely that Mg2+ rather than Ca2+ would be bound at most sites III and IV in the sarcomeres of living cardiomyocytes. Labels in either panel (B) or (C) apply to the locations of structural elements in both panels. Note that only one affected amino acid is highlighted in each of panels (B,C), corresponding to the occurrence of only one of the two variants in each TNNC1 allele of the proband’s genome (Figure 2B). (D) Multiple alignments of residues around site IV demonstrate a remarkable degree of conservation of amino acids D132 and D145 across a wide range of species. Conserved amino acids are shown in red. Affected residues D132 and D145 are identified by underlines (human sequence) and arrows (above). D132 is within the G-helix (refer to panel A) that is part of site IV EF-hand. D145 is at the +Z location within the divalent cation-coordinating region of site IV; all 12 residues of this region are highlighted in bold in the human sequence (top sequence), as in panel (A), and the coordinating residues (+X, +Y, +Z, –Y, –X, and –Z) within site IV are annotated (arrows) below the aligned sequences. |
|
FIGURE 4. (A,B) Brightfield microscopy and (C,D) Optical Coherence Tomography (OCT) imaging of Control and TNNC1 F0 knockout tadpoles. As compared to control tadpoles (A), TNNC1 knockout tadpoles (B) show normal gross morphology, but significant total body edema (white arrows) secondary to severe dilated cardiomyopathy leading to congestive heart failure. Red arrows indicate plane of OCT images in (C,D). OCT images demonstrate normal size and wall thickness of cardiac chambers in control tadpole (C), with severe dilation and enlargement of the ventricle in TNNC1 knockout tadpole (D), indicated with white arrowheads. V = ventricle, LA = left atrium, RA = right atrium. |
|
FIGURE 5. Ca2+ dependence of steady-state isometric tension in porcine CMPs reconstituted with exogenous cTnC WT (100% WT) or variants (50% WT/50% D132N, 50% WT/50% D145E, or 50% D132N/50% D145E). (A) Relative steady-state isometric force as a function of pCa. The force values were normalized to the maximal steady-state isometric force in the same preparation. (B) Normalized steady-state isometric force as a function of pCa. The force values were normalized to the maximal steady-state isometric force generated by WT. Data are shown as mean ± S.E. and best fit parameter estimates from non-linear least squares regression on the Hill equation are summarized in Table 2 (n = 4–8). |
|
FIGURE 6. Sinusoidal stiffness analysis in porcine CMPs reconstituted with exogenous cTnC WT (100% WT) or variants (50% WT/50% D132N, 50% WT/50% D145E, or 50% D132N/50% D145E). (A) Ca2+ dependence of steady-state sinusoidal stiffness. (B) Normalized force vs. steady-state sinusoidal stiffness. The force values were normalized to the maximal steady-state isometric force generated by WT. (B) Dashed lines were drawn to connect the points. Data are shown as mean ± S.E. and maximum SS values are summarized in Table 3. |
|
FIGURE 7. Rate of tension redevelopment (kTR) analysis in porcine CMPs reconstituted with exogenous cTnC WT (100% WT) or variants (50% WT/50% D132N, 50% WT/50% D145E, or 50% D132N/50% D145E). (A) kTR as a function of increasing Ca2+ concentration. Solid lines were drawn to connect the points. (B) Relative force vs. kTR. The force values were normalized to the maximal steady-state isometric force for each preparation. Data are shown as mean ± S.E. and maximum kTR values are summarized in Table 3. Dashed lines were obtained from fits to the 3-state model, which take into account the changes in Ca2+ sensitivity (Table 2 and Figures 5A,B) as described in section “Experimental Procedures”; best fit parameter estimates are summarized in Table 4. |
References [+] :
Alves,
Early sensitization of myofilaments to Ca2+ prevents genetically linked dilated cardiomyopathy in mice.
2017, Pubmed
Alves, Early sensitization of myofilaments to Ca2+ prevents genetically linked dilated cardiomyopathy in mice. 2017, Pubmed
Alves, Desensitization of myofilaments to Ca2+ as a therapeutic target for hypertrophic cardiomyopathy with mutations in thin filament proteins. 2014, Pubmed
Asumda, Nuclear cardiac troponin and tropomyosin are expressed early in cardiac differentiation of rat mesenchymal stem cells. 2012, Pubmed
Badr, Fluorescent Protein-Based Ca2+ Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C. 2016, Pubmed
Bhattacharya, CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus. 2015, Pubmed , Xenbase
Biesiadecki, The troponin C G159D mutation blunts myofilament desensitization induced by troponin I Ser23/24 phosphorylation. 2007, Pubmed
Brenner, Effect of Ca2+ on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction. 1988, Pubmed
Burke, Clinical and Mechanistic Insights Into the Genetics of Cardiomyopathy. 2016, Pubmed
Chase, Isometric force redevelopment of skinned muscle fibers from rabbit activated with and without Ca2+. 1994, Pubmed
Deniz, CRISPR/Cas9 F0 Screening of Congenital Heart Disease Genes in Xenopus tropicalis. 2018, Pubmed , Xenbase
Dieseldorff Jones, Pathogenic troponin T mutants with opposing effects on myofilament Ca2+ sensitivity attenuate cardiomyopathy phenotypes in mice. 2019, Pubmed
Dipchand, The Registry of the International Society for Heart and Lung Transplantation: Sixteenth Official Pediatric Heart Transplantation Report--2013; focus theme: age. 2013, Pubmed
Dweck, Expanding the range of free calcium regulation in biological solutions. 2005, Pubmed
Dweck, A dilated cardiomyopathy troponin C mutation lowers contractile force by reducing strong myosin-actin binding. 2010, Pubmed
Dweck, Challenging current paradigms related to cardiomyopathies. Are changes in the Ca2+ sensitivity of myofilaments containing cardiac troponin C mutations (G159D and L29Q) good predictors of the phenotypic outcomes? 2008, Pubmed
Dweck, Long term ablation of protein kinase A (PKA)-mediated cardiac troponin I phosphorylation leads to excitation-contraction uncoupling and diastolic dysfunction in a knock-in mouse model of hypertrophic cardiomyopathy. 2014, Pubmed
Dyer, Functional analysis of a unique troponin c mutation, GLY159ASP, that causes familial dilated cardiomyopathy, studied in explanted heart muscle. 2009, Pubmed
Farah, The troponin complex and regulation of muscle contraction. 1995, Pubmed
Freeman, Progression from hypertrophic to dilated cardiomyopathy in mice that express a mutant myosin transgene. 2001, Pubmed
Fujino, A novel mutation Lys273Glu in the cardiac troponin T gene shows high degree of penetrance and transition from hypertrophic to dilated cardiomyopathy. 2002, Pubmed
Fujino, Cardiac troponin T Arg92Trp mutation and progression from hypertrophic to dilated cardiomyopathy. 2001, Pubmed
Gonzalez-Martinez, Structural and functional impact of troponin C-mediated Ca2+ sensitization on myofilament lattice spacing and cross-bridge mechanics in mouse cardiac muscle. 2018, Pubmed
Hancock, Models of calcium activation account for differences between skeletal and cardiac force redevelopment kinetics. 1997, Pubmed
Hershberger, Dilated cardiomyopathy: the complexity of a diverse genetic architecture. 2013, Pubmed
Hershberger, Coding sequence rare variants identified in MYBPC3, MYH6, TPM1, TNNC1, and TNNI3 from 312 patients with familial or idiopathic dilated cardiomyopathy. 2010, Pubmed
Ho, Conditional antisense-knockdown of zebrafish cardiac troponin C as a new animal model for dilated cardiomyopathy. 2009, Pubmed
Holroyde, The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase. 1980, Pubmed
Johnston, Troponin through the looking-glass: emerging roles beyond regulation of striated muscle contraction. 2018, Pubmed
Landesberg, Coupling calcium binding to troponin C and cross-bridge cycling in skinned cardiac cells. 1994, Pubmed
Landstrom, Molecular and functional characterization of novel hypertrophic cardiomyopathy susceptibility mutations in TNNC1-encoded troponin C. 2008, Pubmed
Lee, Pediatric Cardiomyopathies. 2017, Pubmed
Li, Structure and function of cardiac troponin C (TNNC1): Implications for heart failure, cardiomyopathies, and troponin modulating drugs. 2015, Pubmed
Li, Correcting diastolic dysfunction by Ca2+ desensitizing troponin in a transgenic mouse model of restrictive cardiomyopathy. 2010, Pubmed
Lindhout, Structure and dynamics of the C-domain of human cardiac troponin C in complex with the inhibitory region of human cardiac troponin I. 2003, Pubmed
Lipshultz, The incidence of pediatric cardiomyopathy in two regions of the United States. 2003, Pubmed
Loong, Slowed Dynamics of Thin Filament Regulatory Units Reduces Ca2+-Sensitivity of Cardiac Biomechanical Function. 2013, Pubmed
Marques, Allosteric Transmission along a Loosely Structured Backbone Allows a Cardiac Troponin C Mutant to Function with Only One Ca2+ Ion. 2017, Pubmed
Memo, Familial dilated cardiomyopathy mutations uncouple troponin I phosphorylation from changes in myofibrillar Ca²⁺ sensitivity. 2013, Pubmed
Messer, Investigating the role of uncoupling of troponin I phosphorylation from changes in myofibrillar Ca(2+)-sensitivity in the pathogenesis of cardiomyopathy. 2014, Pubmed
Mogensen, Severe disease expression of cardiac troponin C and T mutations in patients with idiopathic dilated cardiomyopathy. 2004, Pubmed
Nanni, Hypertrophic cardiomyopathy: two homozygous cases with "typical" hypertrophic cardiomyopathy and three new mutations in cases with progression to dilated cardiomyopathy. 2003, Pubmed
Parvatiyar, A mutation in TNNC1-encoded cardiac troponin C, TNNC1-A31S, predisposes to hypertrophic cardiomyopathy and ventricular fibrillation. 2012, Pubmed
Pinto, Strong cross-bridges potentiate the Ca(2+) affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments: a fast kinetic approach. 2011, Pubmed
Pinto, Functional characterization of TNNC1 rare variants identified in dilated cardiomyopathy. 2011, Pubmed
Pinto, Will you still need me (Ca2+ , TnT, and DHPR), will you still cleave me (calpain), when I'm 64? 2017, Pubmed
Pinto, A functional and structural study of troponin C mutations related to hypertrophic cardiomyopathy. 2009, Pubmed
Ploski, Evidence for troponin C (TNNC1) as a gene for autosomal recessive restrictive cardiomyopathy with fatal outcome in infancy. 2016, Pubmed
Ran, Genome engineering using the CRISPR-Cas9 system. 2013, Pubmed
Regnier, Regulation of skeletal muscle tension redevelopment by troponin C constructs with different Ca2+ affinities. 1999, Pubmed
Robinson, Dilated and hypertrophic cardiomyopathy mutations in troponin and alpha-tropomyosin have opposing effects on the calcium affinity of cardiac thin filaments. 2007, Pubmed
Takeda, Structure of the core domain of human cardiac troponin in the Ca(2+)-saturated form. 2003, Pubmed
Veltri, Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca2+-binding affinity in a C-domain mutant of cardiac troponin C. 2017, Pubmed
Veltri, Hypertrophic Cardiomyopathy Cardiac Troponin C Mutations Differentially Affect Slow Skeletal and Cardiac Muscle Regulation. 2017, Pubmed
Wang, Generation and functional characterization of knock-in mice harboring the cardiac troponin I-R21C mutation associated with hypertrophic cardiomyopathy. 2012, Pubmed
Willott, Mutations in Troponin that cause HCM, DCM AND RCM: what can we learn about thin filament function? 2010, Pubmed
Wu, Epigenetic Regulation of Phosphodiesterases 2A and 3A Underlies Compromised β-Adrenergic Signaling in an iPSC Model of Dilated Cardiomyopathy. 2015, Pubmed
Zhang, Calpain inhibition rescues troponin T3 fragmentation, increases Cav1.1, and enhances skeletal muscle force in aging sedentary mice. 2016, Pubmed
van der Velden, Cardiac Disorders and Pathophysiology of Sarcomeric Proteins. 2019, Pubmed