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.

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.

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.

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.

Assessment and Management of Cardiotoxicity in Hematologic Malignancies.

With the increasing overall survival of cancer patients due to recent discoveries in oncology, the incidence of side effects is also rising, and along with secondary malignancies, cardiotoxicity is one of the most concerning side effects, affecting the quality of life of cancer survivors. There are two types of cardiotoxicity associated with chemotherapy; the first one is acute, life-threatening but, fortunately, in most of the cases, reversible; and the second one is with late onset and mostly irreversible. The most studied drugs associated with cardiotoxicity are anthracyclines, but many new agents have demonstrated unexpected cardiotoxic effect, including those currently used in multiple myeloma treatment (proteasome inhibitors and immunomodulatory agents), tyrosine kinase inhibitors used in the treatment of chronic myeloid leukemia and some forms of acute leukemia, and immune checkpoint inhibitors recently introduced in treatment of refractory lymphoma patients. To prevent irreversible myocardial damage, early recognition of cardiac toxicity is mandatory. Traditional methods like echocardiography and magnetic resonance imaging are capable of detecting structural and functional changings, but unable to detect early myocardial damage; therefore, more sensible biomarkers like troponins and natriuretic peptides have to be introduced into the current practice. Baseline assessment of patients allows the identification of those with high risk for cardiotoxicity, while monitoring during and after treatment is important for early detection of cardiotoxicity and prompt intervention.

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.

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.

Depletion of branched-chain aminotransferase 2 (BCAT2) enzyme impairs myoblast survival and myotube formation.

Much is known about the positive effects of branched-chain amino acids (BCAA) in regulating muscle protein metabolism. Comparatively much less is known about the effects of these amino acids and their metabolites in regulating myotube formation. Using cultured myoblasts, we showed that although leucine is required for myotube formation, this requirement is easily met by α-ketoisocaproic acid, the ketoacid of leucine. We then demonstrated increases in the expression of the first two enzymes in the catabolism of the three BCAA, branched-chain amino transferase (BCAT2) and branched-chain α-ketoacid dehydrogenase (BCKD), with ~3× increase in BCKD protein expression (p < .05) during differentiation. Furthermore, depletion of BCAT2 abolished myoblast differentiation, as indicated by reduction in the levels of myosin heavy chain-1, troponin and myogenin. Supplementation of incubation medium with branched-chain α-ketoacids or related metabolites derivable from BCAT2 functions did not rescue the defects. However, co-depletion of BCKD kinase partially rescued the defects. Collectively, our data indicate a requirement for BCAA catabolism during myotube formation and that this requirement for BCAT2 likely goes beyond the need for this enzyme to generate the α-ketoacids of the BCAA.

Protective Effects of Apocynum venetum Against Pirarubicin-Induced Cardiotoxicity.

Pirarubicin (THP) is an anthracycline antibiotic, frequently used for the treatment of various human cancers. Unfortunately, the clinical effectiveness of THP is limited by its dose-related cardiotoxicity. Apocynum leaf extract is an extract of the dried leaves of Apocynum venetum L. (a member of the Apocynaceae family, AVLE) that has many positive effects on the cardiovascular system and is widely consumed as tea in China. In this study we established a cardiactoxicity rat model, which showed that pretreatment with AVLE attenuated THP-induced myocardial histopathological injury, electrocardiogram abnormalities, and cardiac dysfunction. AVLE also significantly reduced serum levels of malondialdehyde (MDA), brain natriuretic peptide (BNP), creatine kinase (CK-MB), cardiac troponin (CTnT), and lactate dehydrogenase (LDH); and increased serum superoxide dismutase (SOD) levels. Treatment with AVLE or dexrazoxane (DZR) resulted in an increase Cytochrome C (cytc) in the mitochondria and reduced Cytc and cleaved-caspase-3 levels (p<0.05) in cytoplasm. We also found that AVLE significantly reduced voltage-dependent anion channel 1 (VDAC1), adenosine nucleotide transporter 1 (ANT1), and cyclophilin D (CYPD) mRNA expression (p<0.05). Furthermore, AVLE appeared to exert therapeutic effects in a dose-dependent manner. Our study suggests the anti-oxidant and anti-apoptotic properties of AVLE may be responsible for the observed cardioprotective effects.

Expression of troponin subunits in the rat renal afferent arteriole.

Vascular smooth muscle cells of the renal afferent arteriole are unusual in that they must be able to contract very rapidly in response to a sudden increase in systemic blood pressure in order to protect the downstream glomerular capillaries from catastrophic damage. We showed that this could be accounted for, in part, by exclusive expression, at the protein level, of the "fast" (B) isoforms of smooth muscle myosin II heavy chains in the afferent arteriole, in contrast to other vascular smooth muscle cells such as the rat aorta and efferent arteriole which express exclusively the "slow" (A) isoforms (Shiraishi et al. (2003) FASEB. J. 17, 2284-2286). As contraction of the more rapidly contracting striated (skeletal and cardiac) muscles is regulated by the thin filament-associated troponin (Tn) system, we hypothesized that Tn or a Tn-like system may exist in afferent arteriolar cells and contribute to the unusually rapid contraction of this tissue in response to increased intraluminal pressure. We examined the expression of TnC (Ca2+ -binding subunit), TnI (inhibitory subunit), and TnT (tropomyosin-binding subunit) in vascular smooth muscle cells of the rat renal afferent arteriole at the mRNA level. Fast-twitch skeletal muscle and slow-twitch skeletal muscle/cardiac TnC isoforms and slow-twitch skeletal muscle and cardiac TnI isoforms were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by cDNA sequencing. Furthermore, cardiac and slow-twitch skeletal muscle TnI isoforms, but not fast-twitch skeletal muscle TnI, were detected in isolated afferent arterioles at the protein level by proximity ligation assay. Finally, striated muscle myosin II heavy chain expression was identified in isolated rat afferent arterioles by RT-PCR. We conclude that, in addition to Ca2+ -mediated phosphorylation of myosin II regulatory light chains, contraction of the afferent arteriole may be regulated by a mechanism normally associated with the much more rapidly contracting cardiac and skeletal muscles, which involves Ca2+ binding to TnC, leading to alleviation of inhibition of the actomyosin MgATPase by TnI and tropomyosin and rapid contraction of the vessel.

Ca2+-Binding Proteins of the EF-Hand Superfamily: Diagnostic and Prognostic Biomarkers and Novel Therapeutic Targets.

A multitude of Ca2+-sensor proteins containing the specific Ca2+-binding motif (helix-loop-helix, called EF-hand) are of major clinical relevance in a many human diseases. Measurements of troponin, the first intracellular Ca-sensor protein to be discovered, is nowadays the "gold standard" in the diagnosis of patients with acute coronary syndrome (ACS). Mutations have been identified in calmodulin and linked to inherited ventricular tachycardia and in patients affected by severe cardiac arrhythmias. Parvalbumin, when introduced into the diseased heart by gene therapy to increase contraction and relaxation speed, is considered to be a novel therapeutic strategy to combat heart failure. S100 proteins, the largest subgroup with the EF-hand protein family, are closely associated with cardiovascular diseases, various types of cancer, inflammation, and autoimmune pathologies. The intention of this review is to summarize the clinical importance of this protein family and their use as biomarkers and potential drug targets, which could help to improve the diagnosis of human diseases and identification of more selective therapeutic interventions.

EPA and DHA Inhibit Myogenesis and Downregulate the Expression of Muscle-related Genes in C2C12 Myoblasts.

This study was conducted to elucidate the biological effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on cell proliferation, differentiation and gene expression in C2C12 myoblasts. C2C12 were treated with various concentrations of EPA or DHA under proliferation and differentiation conditions. Cell viability was analyzed using cell counting kit-8 assays (CCK-8). The Edu assays were performed to analyze cell proliferation. To analyze cell differentiation, the expressions of myogenic marker genes were determined at the transcriptional and translational levels by qRT-PCR, immunoblotting and immunofluorescence. Global gene expression patterns were characterized using RNA-sequencing. Phosphorylation levels of ERK and Akt were examined by immunoblotting. Cell viability and proliferation was significantly inhibited after incubation with EPA (50 and 100 µM) or DHA (100 µM). Both EPA and DHA suppressed C2C12 myoblasts differentiation. RNA-sequencing analysis revealed that some muscle-related genes were significantly downregulated following EPA or DHA (50 µM) treatment, including insulin-like growth factor 2 (IGF-2), troponin T3 (Tnnt3), myoglobin (Mb), myosin light chain phosphorylatable fast skeletal muscle (Mylpf) and myosin heavy polypeptide 3 (Myh3). IGF-2 was crucial for the growth and differentiation of skeletal muscle and could activate the PI3K/Akt and the MAPK/ERK cascade. We found that EPA and DHA (50 µM) decreased the phosphorylation levels of ERK1/2 and Akt in C2C12 myoblasts. Thus, this study suggested that EPA and DHA exerted an inhibitory effect on myoblast proliferation and differentiation and downregulated muscle-related genes expression.

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.

Isolation and characterization of three skeletal troponin genes and association with growth-related traits in Exopalaemon carinicauda.

Growth is among the most important traits for animal breeding. Muscle growth is controlled by different cellular and molecular pathways and environments, and it also relies heavily on high-quality muscle contractions. The troponin complex, composed of troponin T (TnT), troponin C (TnC) and troponin I (TnI), plays a vital role in the regulation of muscle contraction. In this study, the cDNA of EcTnT, EcTnC and EcTnI of the ridgetail white prawn Exopalaemon carinicauda were cloned and characterized. The full length cDNA of EcTnT, EcTnC and EcTnI were 1 373 bp, 692 bp, and 1 475 bp, encoding a protein of 385, 150 and 193 amino acid residues, respectively. The expression of all genes was predominantly detected in abdominal muscle, while extremely lesser expressed in gill and hepatopancreas. Higher expression level of EcTnI was observed in heavier shrimp of the same age during different developmental stages, excepted for 120 days. Eleven single nucleotide polymorphisms (SNPs) were revealed in the three skeletal troponin genes, and only c.TnI66 A>G from EcTnI was significantly associated with both body weight and body length (P < 0.05). In summary, the result of this study suggested that EcTnI is growth-related gene of the troponin complex gene and the presence of SNP suggests that it could be a candidate gene for shrimp genetic improvement research.

The Genetic and Molecular Bases for Hypertrophic Cardiomyopathy: The Role for Calcium Sensitization.

Hypertrophic cardiomyopathy (HCM) affects millions of people around the world as one of the most common genetic heart disorders and leads to cardiac ischemia, heart failure, dysfunction of other organ systems, and increased risk for sudden unexpected cardiac deaths. HCM can be caused by single-point mutations, insertion or deletion mutations, or truncation of cardiac myofilament proteins. The molecular mechanism that leads to disease progression and presentation is still poorly understood, despite decades of investigations. However, recent research has made dramatic advances in the understanding of HCM disease development. Studies have shown that increased calcium sensitivity is a universal feature in HCM. At the molecular level, increased crossbridge force (or power) generation resulting in hypercontractility is the prominent feature. Thus, calcium sensitization/hypercontractility is emerging as the primary stimulus for HCM disease development and phenotypic expression. Cross-bridge inhibition has been shown to halt HCM presentation, and myofilament desensitization appears to reduce lethal arrhythmias in animal models of HCM. These advances in basic research will continue to deepen the knowledge of HCM pathogenesis and are beginning to revolutionize the management of HCM.

A novel protocol to provide a suitable cardiac model from induced pluripotent stem cells.

Cumulative evidence has proven the safety, feasibility and efficacy of stem cell therapy for cardiomyocyte replacement in heart failure treatment. In contrast to embryonic stem cells, induced pluripotent stem cells (iPS cells) provide a route to the production of patient-specific stem cell lines with no ethical concerns. Recent studies have revealed that myogenic transcription factors activated the expression of conserved microRNAs (miRNAs), such as mir-1, that 'fine-tuned' the output of the transcriptional networks. To introduce an efficient and applicable protocol for establishment of autologous cardiac cellular models, herein we introduced a novel protocol for induction of iPS cells into cardiomyocytes using both microRNA-1 transduction and 5'-Azacitidine treatment. Quantitative evaluation of transcription and translation of cardiac markers such as MHC-α, GATA4, FLK and troponin, demonstrated that this new direct protocol led to cardiac differentiation of iPS cells. From a clinical point of view, these results raise the possibility that administration of miRNA mimic or miRNA inhibitor therapies could increase allocation of iPS cells into the cardiac lineage. Taking all the results into account, our novel protocol provides further progress in the application of patient's own cells for more effective therapies. Moreover, such cellular models could be used in personalized drug screening.

Clinically Divergent Mutation Effects on the Structure and Function of the Human Cardiac Tropomyosin Overlap.

The progression of genetically inherited cardiomyopathies from an altered protein structure to clinical presentation of disease is not well understood. One of the main roadblocks to mechanistic insight remains a lack of high-resolution structural information about multiprotein complexes within the cardiac sarcomere. One example is the tropomyosin (Tm) overlap region of the thin filament that is crucial for the function of the cardiac sarcomere. To address this central question, we devised coupled experimental and computational modalities to characterize the baseline function and structure of the Tm overlap, as well as the effects of mutations causing divergent patterns of ventricular remodeling on both structure and function. Because the Tm overlap contributes to the cooperativity of myofilament activation, we hypothesized that mutations that enhance the interactions between overlap proteins result in more cooperativity, and conversely, those that weaken interaction between these elements lower cooperativity. Our results suggest that the Tm overlap region is affected differentially by dilated cardiomyopathy-associated Tm D230N and hypertrophic cardiomyopathy-associated human cardiac troponin T (cTnT) R92L. The Tm D230N mutation compacts the Tm overlap region, increasing the cooperativity of the Tm filament, contributing to a dilated cardiomyopathy phenotype. The cTnT R92L mutation causes weakened interactions closer to the N-terminal end of the overlap, resulting in decreased cooperativity. These studies demonstrate that mutations with differential phenotypes exert opposite effects on the Tm-Tn overlap, and that these effects can be directly correlated to a molecular level understanding of the structure and dynamics of the component proteins.