Exploring the role of Sirtuin 3 gene polymorphisms and oxidative stress markers in the susceptibility to coronary artery disease.

OBJECTIVE: Coronary artery disease (CAD) is a complex disorder influenced by genetic and environmental factors. This case-control study investigated the association between Sirtuin SIRT3 gene polymorphisms, serum malondialdehyde (MDA) levels, and CAD susceptibility. METHODS: Blood samples were collected from 70 CAD cases and 30 controls at the Cardiac Center, Azadi Teaching Hospital, Duhok, Iraq. Genomic DNA was extracted, and PCR-based allele genotyping determined SIRT3 rs11246029 T/C polymorphisms. Serum MDA levels were measured using ELISA. Statistical analysis included t-tests, Mann-Whitney tests, and Spearman correlations. Odds ratios (OR) with 95% confidence intervals (CI) assessed genotypes/alleles and CAD associations. The accuracy of serum MDA in predicting the severity of CAD was evaluated using receiver operating characteristic (ROC) curve analysis. RESULTS: There were no significant variations in serum MDA levels between controls and CAD patients in the study. The diagnostic accuracy of serum MDA for CAD severity prediction was modest (Area Under Curve (AUC) = 0.56). Correlations revealed associations between MDA and total bilirubin (negative) and Troponin (positive). CRP correlated positively with LDH, glucose, cholesterol, LDL, CKmB, and Troponin. CKmB and Troponin are positively associated with clinical characteristics. Genotype analysis identified a significantly higher CAD risk with the CC genotype compared to controls. CONCLUSION: These findings shed light on the potential role of SIRT3 gene polymorphisms and serum MDA levels in CAD susceptibility. Further research is needed to understand underlying mechanisms and therapeutic implications based on these markers. TRIAL REGISTRATION: 15092021-9-12. Registered 15 September 2021.

Troponin and a Myopathy-Linked Mutation in TPM3 Inhibit Cofilin-2-Induced Thin Filament Depolymerization.

Uniform actin filament length is required for synchronized contraction of skeletal muscle. In myopathies linked to mutations in tropomyosin (Tpm) genes, irregular thin filaments are a common feature, which may result from defects in length maintenance mechanisms. The current work investigated the effects of the myopathy-causing p.R91C variant in Tpm3.12, a tropomyosin isoform expressed in slow-twitch muscle fibers, on the regulation of actin severing and depolymerization by cofilin-2. The affinity of cofilin-2 for F-actin was not significantly changed by either Tpm3.12 or Tpm3.12-R91C, though it increased two-fold in the presence of troponin (without Ca2+). Saturation of the filament with cofilin-2 removed both Tpm variants from the filament, although Tpm3.12-R91C was more resistant. In the presence of troponin (±Ca2+), Tpm remained on the filament, even at high cofilin-2 concentrations. Both Tpm3.12 variants inhibited filament severing and depolymerization by cofilin-2. However, the inhibition was more efficient in the presence of Tpm3.12-R91C, indicating that the pathogenic variant impaired cofilin-2-dependent actin filament turnover. Troponin (±Ca2+) further inhibited but did not completely stop cofilin-2-dependent actin severing and depolymerization.

A Novel Variant in TPM3 Causing Muscle Weakness and Concomitant Hypercontractile Phenotype.

A novel variant of unknown significance c.8A > G (p.Glu3Gly) in TPM3 was detected in two unrelated families. TPM3 encodes the transcript variant Tpm3.12 (NM_152263.4), the tropomyosin isoform specifically expressed in slow skeletal muscle fibers. The patients presented with slowly progressive muscle weakness associated with Achilles tendon contractures of early childhood onset. Histopathology revealed features consistent with a nemaline rod myopathy. Biochemical in vitro assays performed with reconstituted thin filaments revealed defects in the assembly of the thin filament and regulation of actin-myosin interactions. The substitution p.Glu3Gly increased polymerization of Tpm3.12, but did not significantly change its affinity to actin alone. Affinity of Tpm3.12 to actin in the presence of troponin ± Ca2+ was decreased by the mutation, which was due to reduced interactions with troponin. Altered molecular interactions affected Ca2+-dependent regulation of the thin filament interactions with myosin, resulting in increased Ca2+ sensitivity and decreased relaxation of the actin-activated myosin ATPase activity. The hypercontractile molecular phenotype probably explains the distal joint contractions observed in the patients, but additional research is needed to explain the relatively mild severity of the contractures. The slowly progressive muscle weakness is most likely caused by the lack of relaxation and prolonged contractions which cause muscle wasting. This work provides evidence for the pathogenicity of the TPM3 c.8A > G variant, which allows for its classification as (likely) pathogenic.

Molecular Mechanisms of Deregulation of Muscle Contractility Caused by the R168H Mutation in TPM3 and Its Attenuation by Therapeutic Agents.

The substitution for Arg168His (R168H) in γ-tropomyosin (TPM3 gene, Tpm3.12 isoform) is associated with congenital muscle fiber type disproportion (CFTD) and muscle weakness. It is still unclear what molecular mechanisms underlie the muscle dysfunction seen in CFTD. The aim of this work was to study the effect of the R168H mutation in Tpm3.12 on the critical conformational changes that myosin, actin, troponin, and tropomyosin undergo during the ATPase cycle. We used polarized fluorescence microscopy and ghost muscle fibers containing regulated thin filaments and myosin heads (myosin subfragment-1) modified with the 1,5-IAEDANS fluorescent probe. Analysis of the data obtained revealed that a sequential interdependent conformational-functional rearrangement of tropomyosin, actin and myosin heads takes place when modeling the ATPase cycle in the presence of wild-type tropomyosin. A multistep shift of the tropomyosin strands from the outer to the inner domain of actin occurs during the transition from weak to strong binding of myosin to actin. Each tropomyosin position determines the corresponding balance between switched-on and switched-off actin monomers and between the strongly and weakly bound myosin heads. At low Ca2+, the R168H mutation was shown to switch some extra actin monomers on and increase the persistence length of tropomyosin, demonstrating the freezing of the R168HTpm strands close to the open position and disruption of the regulatory function of troponin. Instead of reducing the formation of strong bonds between myosin heads and F-actin, troponin activated it. However, at high Ca2+, troponin decreased the amount of strongly bound myosin heads instead of promoting their formation. Abnormally high sensitivity of thin filaments to Ca2+, inhibition of muscle fiber relaxation due to the appearance of the myosin heads strongly associated with F-actin, and distinct activation of the contractile system at submaximal concentrations of Ca2+ can lead to muscle inefficiency and weakness. Modulators of troponin (tirasemtiv and epigallocatechin-3-gallate) and myosin (omecamtiv mecarbil and 2,3-butanedione monoxime) have been shown to more or less attenuate the negative effects of the tropomyosin R168H mutant. Tirasemtiv and epigallocatechin-3-gallate may be used to prevent muscle dysfunction.

Impact of Troponin in Cardiomyopathy Development Caused by Mutations in Tropomyosin.

Tropomyosin (Tpm) mutations cause inherited cardiac diseases such as hypertrophic and dilated cardiomyopathies. We applied various approaches to investigate the role of cardiac troponin (Tn) and especially the troponin T (TnT) in the pathogenic effects of Tpm cardiomyopathy-associated mutations M8R, K15N, A277V, M281T, and I284V located in the overlap junction of neighboring Tpm dimers. Using co-sedimentation assay and viscosity measurements, we showed that TnT1 (fragment of TnT) stabilizes the overlap junction of Tpm WT and all Tpm mutants studied except Tpm M8R. However, isothermal titration calorimetry (ITC) indicated that TnT1 binds Tpm WT and all Tpm mutants similarly. By using ITC, we measured the direct KD of the Tpm overlap region, N-end, and C-end binding to TnT1. The ITC data revealed that the Tpm C-end binds to TnT1 independently from the N-end, while N-end does not bind. Therefore, we suppose that Tpm M8R binds to TnT1 without forming the overlap junction. We also demonstrated the possible role of Tn isoform composition in the cardiomyopathy development caused by M8R mutation. TnT1 dose-dependently reduced the velocity of F-actin-Tpm filaments containing Tpm WT, Tpm A277V, and Tpm M281T mutants in an in vitro motility assay. All mutations impaired the calcium regulation of the actin-myosin interaction. The M281T and I284V mutations increased the calcium sensitivity, while the K15N and A277V mutations reduced it. The Tpm M8R, M281T, and I284V mutations under-inhibited the velocity at low calcium concentrations. Our results demonstrate that Tpm mutations likely implement their pathogenic effects through Tpm interaction with Tn, cardiac myosin, or other protein partners.

Troponin Variants in Congenital Myopathies: How They Affect Skeletal Muscle Mechanics.

The troponin complex is a key regulator of muscle contraction. Multiple variants in skeletal troponin encoding genes result in congenital myopathies. TNNC2 has been implicated in a novel congenital myopathy, TNNI2 and TNNT3 in distal arthrogryposis (DA), and TNNT1 and TNNT3 in nemaline myopathy (NEM). Variants in skeletal troponin encoding genes compromise sarcomere function, e.g., by altering the Ca2+ sensitivity of force or by inducing atrophy. Several potential therapeutic strategies are available to counter the effects of variants, such as troponin activators, introduction of wild-type protein through AAV gene therapy, and myosin modulation to improve muscle contraction. The mechanisms underlying the pathophysiological effects of the variants in skeletal troponin encoding genes are incompletely understood. Furthermore, limited knowledge is available on the structure of skeletal troponin. This review focusses on the physiology of slow and fast skeletal troponin and the pathophysiology of reported variants in skeletal troponin encoding genes. A better understanding of the pathophysiological effects of these variants, together with enhanced knowledge regarding the structure of slow and fast skeletal troponin, will direct the development of treatment strategies.

Meta-analysis of cardiomyopathy-associated variants in troponin genes identifies loci and intragenic hot spots that are associated with worse clinical outcomes.

INTRODUCTION: Troponin (TNN)-encoded cardiac troponins (Tn) are critical for sensing calcium and triggering myofilament contraction. TNN variants are associated with development of cardiomyopathy; however, recent advances in genetic analysis have identified rare population variants. It is unclear how certain variants are associated with disease while others are tolerated. OBJECTIVE: To compare probands with TNNT2, TNNI3, and TNNC1 variants and utilize high-resolution variant comparison mapping of pathologic and rare population variants to identify loci associated with disease pathogenesis. METHODS: Cardiomyopathy-associated TNN variants were identified in the literature and topology mapping conducted. Clinical features were compiled and compared. Rare population variants were obtained from the gnomAD database. Signal-to-noise (S:N) normalized pathologic variant frequency against population variant frequency. Abstract review of clinical phenotypes was applied to "significant" hot spots. RESULTS: Probands were compiled (N = 70 studies, 224 probands) as were rare variants (N = 125,748 exomes; 15,708 genomes, MAF <0.001). TNNC1-positive probands demonstrated the youngest age of presentation (20.0 years; P = .016 vs TNNT2; P = .004 vs TNNI3) and the highest death, transplant, or ventricular fibrillation events (P = .093 vs TNNT2; P = .024 vs TNNI3; Kaplan Meir: P = .025). S:N analysis yielded hot spots of diagnostic significance within the tropomyosin-binding domains, α-helix 1, and the N-Terminus in TNNT2 with increased sudden cardiac death and ventricular fibrillation (P = .004). The inhibitory region and C-terminal region in TNNI3 exhibited increased restrictive cardiomyopathy (P =.008). HCM and RCM models tended to have increased calcium sensitivity and DCM decreased sensitivity (P < .001). DCM and HCM studies typically showed no differences in Hill coefficient which was decreased in RCM models (P < .001). CM models typically demonstrated no changes to Fmax (P = .239). CONCLUSION: TNNC1-positive probands had younger ages of diagnosis and poorer clinical outcomes. Mapping of TNN variants identified locations in TNNT2 and TNNI3 associated with heightened pathogenicity, RCM diagnosis, and increased risk of sudden death.

Identification and characterization of troponin genes in Locusta migratoria.

Troponin complex comprises three subunits, namely troponin C (TpnC), troponin I (TpnI) and troponin T (TpnT), and regulates the contraction of striated muscle. We found that the locust Locusta migratoria genome has one TpnT gene (LmTpnT), one TpnI gene (LmTpnI) and three TpnC genes (LmTpnC1, LmTpnC2 and LmTpnC3). Through alternative splicing, LmTpnT and LmTpnI potentially encode two and eight isoforms, respectively. The flight muscle and the jump muscle of L. migratoria express an identical LmTpnT isoform, but different LmTpnC isoforms and LmTpnI isoforms. LmTpnC2 and LmTpnC3 both contain highly conserved residues essential for calcium binding in the EF-hand II and IV, thus belonging two-site isoform. LmTpnC1 contains non-conserved substitutions in the EF-hand II and all highly conserved residues for calcium binding in the EF-hand IV. Mutagenesis and tyrosine fluorescence spectroscopic analysis show that both the EF-hand II and IV of LmTpnC1 can serve as calcium-binding site. Therefore, all three LmTpnC isoforms belong to two-site isoform. This is in contrast to the situation in the insect with asynchronous flight muscle, which expresses both one-site isoform and two-site isoform of TpnC. Those results suggest that the origination of insect asynchronous flight muscle is associated with the emergence of one-site isoform of TpnC.

The Dark Side of Actin: Cardiac actin variants highlight the role of allostery in disease development.

Mutations in the α-cardiac actin ACTC1 gene cause dilated or hypertrophic cardiomyopathy. These diseases are the result of changes in protein interactions between ACTC protein and force-generating ß-myosin or the calcium-dependent cardiac-tropomyosin (cTm) and cardiac troponin (cTn) regulatory complex, altering the overall contractile force. The T126I and S271F ACTC variants possess amino acid substitutions on the other side of actin relative to the myosin or regulatory protein binding sites on what we call the "dark side" of actin. The T126I change results in hyposensitivity to calcium, in accordance with the calcium sensitivity pathway of cardiomyopathy development while the S271F change alters the maximum in vitro motility sliding speed, reflecting a change in maximum force. These results demonstrate the role of actin allostery in the cardiac disease development.

Effects of the cardiomyopathy-causing E244D mutation of troponin T on the structures of cardiac thin filaments studied by small-angle X-ray scattering.

Small-angle X-ray scattering experiments were carried out to investigate the structural changes of cardiac thin filaments induced by the cardiomyopathy-causing E244D mutation in troponin T (TnT). We examined native thin filaments (NTF) from a bovine heart, reconstituted thin filaments containing human cardiac wild-type Tn (WTF), and filaments containing the E244D mutant of Tn (DTF), in the absence and presence of Ca2+. Analysis by model calculation showed that upon Ca2+-activation, tropomyosin (Tm) and Tn in the WTF and NTF moved together in a direction to expose myosin-binding sites on actin. On the other hand, Tm and Tn of the DTF moved in the opposite directions to each other upon Ca2+-activation. These movements caused Tm to expose more myosin-binding sites on actin than the WTF, suggesting that the affinity of myosin for actin is higher for the DTF. Thus, the mutation-induced structural changes in thin filaments would increase the number of myosin molecules bound to actin compared with the WTF, resulting in the force enhancement observed for the E244D mutation.