Troponin T is elevated in a relevant proportion of patients with 5q-associated spinal muscular atrophy.

Troponin T concentration (TNT) is commonly considered a marker of myocardial damage. However, elevated concentrations have been demonstrated in numerous neuromuscular disorders, pointing to the skeletal muscle as a possible extracardiac origin. The aim of this study was to determine disease-related changes of TNT in 5q-associated spinal muscular atrophy (SMA) and to screen for its biomarker potential in SMA. We therefore included 48 pediatric and 45 adult SMA patients in this retrospective cross-sequential observational study. Fluid muscle integrity and cardiac markers were analyzed in the serum of treatment-naïve patients and subsequently under disease-modifying therapies. We found a TNT elevation in 61% of SMA patients but no elevation of the cardiospecific isoform Troponin I (TNI). TNT elevation was more pronounced in children and particularly infants with aggressive phenotypes. In adults, TNT correlated to muscle destruction and decreased under therapy only in the subgroup with elevated TNT at baseline. In conclusion, TNT was elevated in a relevant proportion of patients with SMA with emphasis in infants and more aggressive phenotypes. Normal TNI levels support a likely extracardiac origin. Although its stand-alone biomarker potential seems to be limited, exploring TNT in SMA underlines the investigation of skeletal muscle integrity markers.

A rare TNNT1 gene variant causing creatine kinase elevation in nemaline myopathy: c.271_273del (p.Lys91del).

BACKGROUND: Nemaline Myopathy (NM) is a rare genetic disorder that affects muscle function and is characterized by the presence of nemaline rods in muscle fibers. These rods are abnormal structures that interfere with muscle contraction and can cause muscle weakness, respiratory distress, and other complications. NM is caused by variants in several genes, including TNNT1, which encodes the protein troponin T1. NM is inherited in an autosomal recessive pattern. The prevalence of heterozygous TNNT1 variants has been reported to be 1/152,000, indicating that the disease is relatively rare. OBJECTIVE: Investigation of TNNT1 gene variants that may cause cretin kinase elevation. METHODS: Detailed family histories and clinical data were recorded. Whole exome sequencing was performed and family segregation was done by Sanger sequencing. RESULTS: In this study, we report a 5-year-old girl with a novel variant recessive congenital TNNT1 myopathy. The patient had a novel homozygous (c.271_273del) deletion in the TNNT1 gene that is associated with creatine kinase elevation, which is a marker of muscle damage. CONCLUSION: This case expands the phenotypic spectrum of TNNT1 myopathy and highlights the importance of genetic testing and counseling for families affected by this rare disorder. In this study provides valuable insights into the genetic basis of NM and highlights the importance of early diagnosis and management for patients with this rare disorder. Further research is needed to better understand the pathophysiology of TNNT1 myopathy and to develop effective treatments for this debilitating condition.

Hypertrophic cardiomyopathy dysfunction mimicked in human engineered heart tissue and improved by sodium-glucose cotransporter 2 inhibitors.

AIMS: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy, often caused by pathogenic sarcomere mutations. Early characteristics of HCM are diastolic dysfunction and hypercontractility. Treatment to prevent mutation-induced cardiac dysfunction is lacking. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are a group of antidiabetic drugs that recently showed beneficial cardiovascular outcomes in patients with acquired forms of heart failure. We here studied if SGLT2i represent a potential therapy to correct cardiomyocyte dysfunction induced by an HCM sarcomere mutation. METHODS AND RESULTS: Contractility was measured of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) harbouring an HCM mutation cultured in 2D and in 3D engineered heart tissue (EHT). Mutations in the gene encoding ß-myosin heavy chain (MYH7-R403Q) or cardiac troponin T (TNNT2-R92Q) were investigated. In 2D, intracellular [Ca2+], action potential and ion currents were determined. HCM mutations in hiPSC-CMs impaired relaxation or increased force, mimicking early features observed in human HCM. SGLT2i enhance the relaxation of hiPSC-CMs, to a larger extent in HCM compared to control hiPSC-CMs. Moreover, SGLT2i-effects on relaxation in R403Q EHT increased with culture duration, i.e. hiPSC-CMs maturation. Canagliflozin's effects on relaxation were more pronounced than empagliflozin and dapagliflozin. SGLT2i acutely altered Ca2+ handling in HCM hiPSC-CMs. Analyses of SGLT2i-mediated mechanisms that may underlie enhanced relaxation in mutant hiPSC-CMs excluded SGLT2, Na+/H+ exchanger, peak and late Nav1.5 currents, and L-type Ca2+ current, but indicate an important role for the Na+/Ca2+ exchanger. Indeed, electrophysiological measurements in mutant hiPSC-CM indicate that SGLT2i altered Na+/Ca2+ exchange current. CONCLUSION: SGLT2i (canagliflozin > dapagliflozin > empagliflozin) acutely enhance relaxation in human EHT, especially in HCM and upon prolonged culture. SGLT2i may represent a potential therapy to correct early cardiac dysfunction in HCM.

Serum circPRDM5 as a novel diagnostic biomarker for acute myocardial infarction.

BACKGROUND: Circular RNA (CircRNA) is known to play an important role in cardiovascular diseases, but its use as a biomarker of acute myocardial infarction (AMI) has not been studied. This study explores the feasibility of circPRDM5 as a novel biomarker of AMI. METHODS: CircPRDM5 was screened by bioinformatics, the correct circPRDM5 primers were tested by agarose gel electrophoresis (AGE) and Sanger sequencing, and the expression level of serum circPRDM5 was detected by Quantitative Reverse Transcription-Polymerase Chain Reaction. (qRT-PCR), and the diagnostic value of circPRDM5 was analyzed by the receiver operating characteristic (ROC) curve. RESULTS: The expression of circPRDM5 in serum of AMI patients was significantly decreased compared with that of healthy control group and angina group (P < 0.001). The area under ROC curve of serum circPRDM5 was 0.862 [95 % CI, 0.814-0.909]. The combined diagnosis of serum circPRDM5, cardiac troponin T (cTnT) and creatine kinase-MB (CK-MB) could improve the sensitivity of diagnosing AMI. The expression level of serum circPRDM5 increased after percutaneous coronary intervention (PCI). CONCLUSIONS: CircPRDM5 can be used as a novel biomarker for AMI, and its combination with cTnT and CK-MB can improve diagnostic value.

Meta-Analysis of Penetrance and Systematic Review on Transition to Disease in Genetic Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is characterized by unexplained left ventricular hypertrophy and is classically caused by pathogenic or likely pathogenic variants (P/LP) in genes encoding sarcomere proteins. Not all subclinical variant carriers will manifest clinically overt disease because penetrance (proportion of sarcomere or sarcomere-related P/LP variant carriers who develop disease) is variable, age dependent, and not reliably predicted. METHODS: A systematic search of the literature was performed. We used random-effects generalized linear mixed model meta-analyses to contrast the cross-sectional prevalence and penetrance of sarcomere or sarcomere-related genes in 2 different contexts: clinically-based studies on patients and families with HCM versus population or community-based studies. Longitudinal family/clinical studies were additionally analyzed to investigate the rate of phenotypic conversion from subclinical to overt HCM during follow-up. RESULTS: In total, 455 full-text manuscripts and articles were assessed. In family/clinical studies, the prevalence of sarcomere variants in patients diagnosed with HCM was 34%. The penetrance across all genes in nonproband relatives carrying P/LP variants identified during cascade screening was 57% (95% CI, 52%-63%), and the mean age at HCM diagnosis was 38 years (95% CI, 36%-40%). Penetrance varied from ≈32% for MYL3 (myosin light chain 3) to ≈55% for MYBPC3 (myosin-binding protein C3), ≈60% for TNNT2 (troponin T2) and TNNI3 (troponin I3), and ≈65% for MYH7 (myosin heavy chain 7). Population-based genetic studies demonstrate that P/LP sarcomere variants are present in the background population but at a low prevalence of <1%. The penetrance of HCM in incidentally identified P/LP variant carriers was also substantially lower at ≈11%, ranging from 0% in Atherosclerosis Risk in Communities to 18% in UK Biobank. In longitudinal family studies, the pooled phenotypic conversion across all genes was 15% over an average of ≈8 years of follow-up, starting from a mean of ≈16 years of age. However, short-term gene-specific phenotypic conversion varied between ≈12% for MYBPC3 and ≈23% for MYH7. CONCLUSIONS: The penetrance of P/LP variants is highly variable and influenced by currently undefined and context-dependent genetic and environmental factors. Additional longitudinal studies are needed to improve our understanding of true lifetime penetrance in families and in the community and to identify drivers of the transition from subclinical to overt HCM.

Screening and evaluation of TBX20 and CITED2 mutations in children with congenital cardiac septal defects: Correlation with cardiac troponin T and caspase-3.

Congenital cardiac septal defect (CCSD) is the main type of congenital heart disease and owns a very high mortality rate among newborns. CCSD is controlled by specific transcription factors, including T-box transcription factor 20 (TBX20) and Cbp/P300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 2 (CITED2) which are key molecular actors in heart development. Here, we screened for mutations in TBX20 and CITED2 genes in Egyptian children with CCSD and assessed their association with CCSD susceptibility and with cardiac troponin T (cTnT) and the apoptotic marker caspase-3 as biochemical markers for CCSD. Thirty unrelated newborns and children affected with CCSD and 30 matched healthy controls with no personal history of cardiac diseases were recruited. Selection criteria were children (<18 years) with any age diagnosed with CCSD using ECHO. Mutational analysis and genotyping were done using PCR-Sanger DNA sequencing technique. Serum cTnT and caspase-3 were analyzed using ELISA. Sequencing analysis identified 2 TBX20 variants (c.766T>C and c.39T>C) in the CCSD and control groups and 2 CITED2 variants (c.12T>C and c.9C>T) in one CCSD patient, while were absent in controls. In silico analysis identified TBX20 c.766T>C (rs3999941) as a missense (F256L) pathogenic variant and the other three variants as synonymous and benign. Compared with controls, TBX20 c.766T>C TC genotype and minor C allele were candidate high-risk factors for CCSD. Besides, serum cTnT and caspase-3 were dramatically elevated in CCSD children compared to controls. TBX20 c.766T>C TC genotype was associated with high cTnT in CCSD children. Conclusively, we advocate TBX20 c.766T>C variant as a potential genetic marker for CCSD which might associate with high cTnT levels. CITED2 genetic variants might have rare incidence among Egyptian CCSD children. Serum cTnT and caspase-3 are useful markers for ascertaining CCSD in children. These data could be exploited in prenatal genetic counseling, pre-implantation genotyping, and therapy of CCSD.

Negative Charges Introduced Near the IT Helix of Cardiac Troponin T Stabilize the Active State of Actin Filaments.

The disordered and basic C-terminal 14 residues of human troponin T (TnT) are essential for full inhibition of actomyosin ATPase activity at low Ca2+ levels and for limiting activation at saturating Ca2+. In previous studies, stepwise truncation of the C-terminal region of TnT increased activity in proportion to the number of positive charges eliminated. To define key basic residues more closely, we generated phosphomimetic-like mutants of TnT. Phosphomimetic mutants were chosen because of reports that phosphorylation of TnT, including sites within the C terminal region, depressed activity, contrary to our expectations. Four constructs were made where one or more Ser and Thr residues were replaced with Asp residues. The S275D and T277D mutants, near the IT helix and adjacent to basic residues, produced the greatest activation of ATPase rates in solution; the effects of the S275D mutant were recapitulated in muscle fiber preparations with enhanced myofilament Ca2+ sensitivity. Actin filaments containing S275D TnT were also shown to be incapable of populating the inactive state at low Ca2+ levels. Actin filaments containing both S275D/T284D were not statistically different from those containing only S275D in both solution and cardiac muscle preparation studies. Finally, actin filaments containing T284D TnT, closer to the C-terminus and not adjacent to a basic residue, had the smallest effect on activity. Thus, the effects of negative charge placement in the C-terminal region of TnT were greatest near the IT helix and adjacent to a basic residue.

A perspective on Notch signalling in progression and arrhythmogenesis in familial hypertrophic and dilated cardiomyopathies.

In this perspective, we discussed emerging data indicating a role for Notch signalling in inherited disorders of the heart failure with focus on hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) linked to variants of genes encoding mutant proteins of the sarcomere. We recently reported an upregulation of elements in the Notch signalling cascade in cardiomyocytes derived from human inducible pluripotent stem cells expressing a TNNT2 variant encoding cardiac troponin T (cTnT-I79N+/-), which induces hypertrophy, remodelling, abnormalities in excitation-contraction coupling and electrical instabilities (Shafaattalab S et al. 2021 Front. Cell Dev. Biol. 9, 787581. (doi:10.3389/fcell.2021.787581)). Our search of the literature revealed the novelty of this finding and stimulated us to discuss potential connections between the Notch signalling pathway and familial cardiomyopathies. Our considerations focused on the potential role of these interactions in arrhythmias, microvascular ischaemia, and fibrosis. This finding underscored a need to consider the role of Notch signalling in familial cardiomyopathies which are trigged by sarcomere mutations engaging mechano-signalling pathways for which there is evidence of a role for Notch signalling with crosstalk with Hippo signalling. Our discussion included a role for both cardiac myocytes and non-cardiac myocytes in progression of HCM and DCM. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Exploring the possible causal effects of cardiac blood biomarkers in dementia and cognitive performance: a Mendelian randomization study.

Prospective cohort studies have implied associations between blood levels of troponin T, troponin I, NT-proBNP, GDF15, dementia, and cognitive function, without providing evidence favoring possible causality. We aimed to assess the causal associations of these cardiac blood biomarkers with dementia and cognition using two-sample Mendelian randomization (MR). Independent genetic instruments (p < 5e-7) for troponin T and I, N-terminal pro B-type natriuretic peptide (NT-proBNP) and growth-differentiation factor 15 (GDF15) were obtained from previously-performed genome-wide association studies of predominantly European ancestry. Summary statistics for gene-outcome associations in European-ancestry participants, for the two-sample MR analyses, were obtained for general cognitive performance (n = 257,842) and dementia (n = 111,326 clinically diagnosed and "proxy" AD cases, and 677,663 controls). Two-sample MR analyses were performed using inverse variance-weighted (IWV) analyses. Sensitivity analyses to evaluate horizontal pleiotropy included weighted median estimator, MR-Egger, and MR using cis-SNPs only. Using IVW, we did not find evidence for possible causal associations between genetically influenced cardiac biomarkers with cognition and dementia. For example, per standard deviation (SD) higher cardiac blood biomarker, the odds ratio for risk of dementia was 1.06 (95%CI 0.90; 1.21) for troponin T, 0.98 (95%CI 0.72; 1.23) for troponin I, 0.97 (95%CI 0.90; 1.06) for NT-proBNP and 1.07 (95%CI 0.93; 1.21) for GDF15. Sensitivity analyses showed higher GDF15 was significantly associated with higher dementia risk and worse cognitive function. We did not find strong evidence that cardiac biomarkers causally influence dementia risk. Future research should aim at elucidating the biological pathways through which cardiac blood biomarkers associate with dementia.

Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

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Hypertrophic cardiomyopathy (HCM) is the most common monogenic inherited myocardial disease in children, and mutations in sarcomere genes (such as MYH7 and MYBPC3) are the most common genetic etiology of HCM, among which mutations in the MYH7 gene are the most common and account for 30%-50%. MYH7 gene mutations have the characteristics of being affected by environmental factors, coexisting with multiple genetic variations, and age-dependent penetrance, which leads to different or overlapping clinical phenotypes in children, including various cardiomyopathies and skeletal myopathies. At present, the pathogenesis, course, and prognosis of HCM caused by MYH7 gene mutations in children remain unclear. This article summarizes the possible pathogenesis, clinical phenotype, and treatment of HCM caused by MYH7 gene mutations, in order to facilitate the accurate prognostic evaluation and individualized management and treatment of the children with this disorder.

Slow skeletal muscle troponin T acts as a potential prognostic biomarker and therapeutic target for hepatocellular carcinoma.

Slow skeletal muscle troponin T (TNNT1) as a poor prognostic indicator is upregulated in colon and breast cancers. However, the role of TNNT1 in the disease prognosis and biological functions of hepatocellular carcinoma (HCC) is still unclear. The Cancer Genome Atlas (TCGA), real-time quantitative RT-PCR (qRT-PCR), immunoblot, and immunohistochemical analyses were applied to evaluate the TNNT1 expression of human HCC. The impact of TNNT1 levels on disease progression and survival outcome was studied using TCGA analysis. Moreover, the bioinformatics analysis and HCC cell culture were used to investigate the biological functions of TNNT1. Besides, the immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) were used to detect the extracellular TNNT1 of HCC cells and circulating TNNT1 of HCC patients, respectively. The effect of TNNT1 neutralization on oncogenic behaviors and signaling was further validated in the cultured hepatoma cells. In this study, tumoral and blood TNNT1 was upregulated in HCC patients based on the analyses using bioinformatics, fresh tissues, paraffin sections, and serum. From the multiple bioinformatics tools, the TNNT1 overexpression was associated with advanced stage, high grade, metastasis, vascular invasion, recurrence, and poor survival outcome in HCC patients. By the cell culture and TCGA analyses, TNNT1 expression and release were positively correlated with epithelial-mesenchymal transition (EMT) processes in HCC tissues and cells. Moreover, TNNT1 neutralization suppressed oncogenic behaviors and EMT in hepatoma cells. In conclusion, TNNT1 may serve as a non-invasive biomarker and drug target for HCC management. This research finding may provide a new insight for HCC diagnosis and treatment.

Human induced pluripotent stem cell line YCMi007-A generated from a dilated cardiomyopathy patient with a heterozygous dominant c.613C > T (p. Arg205Trp) variant of the TNNT2 gene.

Cardiac muscle troponin T protein binds to tropomyosin and regulates the calcium-dependent actin-myosin interaction on thin filaments in cardiomyocytes. Recent genetic studies have revealed that TNNT2 mutations are strongly linked to dilated cardiomyopathy (DCM). In this study, we generated YCMi007-A, a human induced pluripotent stem cell (hiPSC) line from a DCM patient with a p. Arg205Trp mutation in the TNNT2 gene. The YCMi007-A cells show high expression of pluripotent markers, normal karyotype, and differentiation into three germ layers. Thus, YCMi007-A-an established iPSC-could be useful for the investigation of DCM.

A Revised Perspective on the Evolution of Troponin I and Troponin T Gene Families in Vertebrates.

The troponin (Tn) complex, responsible for the Ca2+ activation of striated muscle, is composed of three interacting protein subunits: TnC, TnI, and TnT, encoded by TNNC, TNNI, and TNNT genes. TNNI and TNNT are sister gene families, and in mammals the three TNNI paralogs (TNNI1, TNNI2, TNNI3), which encode proteins with tissue-specific expression, are each in close genomic proximity with one of the three TNNT paralogs (TNNT2, TNNT3, TNNT1, respectively). It has been widely presumed that all vertebrates broadly possess genes of these same three classes, although earlier work has overlooked jawless fishes (cyclostomes) and cartilaginous fishes (chimeras, rays, and sharks), which are distantly related to other jawed vertebrates. With a new phylogenetic and synteny analysis of a diverse array of vertebrates including these taxonomic groups, we define five distinct TNNI classes (TNNI1-5), with TNNI4 and TNNI5 being only present in non-amniote vertebrates and typically found in tandem, and four classes of TNNT (TNNT1-4). These genes are located in four genomic loci that were generated by the 2R whole-genome duplications. TNNI3, encoding "cardiac TnI" in tetrapods, was independently lost in cartilaginous and ray-finned fishes. Instead, ray-finned fishes predominantly express TNNI1 in the heart. TNNI5 is highly expressed in shark hearts and contains a N-terminal extension similar to that of TNNI3 found in tetrapod hearts. Given that TNNI3 and TNNI5 are distantly related, this supports the hypothesis that the N-terminal extension may be an ancestral feature of vertebrate TNNI and not an innovation unique to TNNI3, as has been commonly believed.

Recent research on childhood hypertrophic cardiomyopathy caused by MYH7 gene mutations / 中国当代儿科杂志

Hypertrophic cardiomyopathy (HCM) is the most common monogenic inherited myocardial disease in children, and mutations in sarcomere genes (such as MYH7 and MYBPC3) are the most common genetic etiology of HCM, among which mutations in the MYH7 gene are the most common and account for 30%-50%. MYH7 gene mutations have the characteristics of being affected by environmental factors, coexisting with multiple genetic variations, and age-dependent penetrance, which leads to different or overlapping clinical phenotypes in children, including various cardiomyopathies and skeletal myopathies. At present, the pathogenesis, course, and prognosis of HCM caused by MYH7 gene mutations in children remain unclear. This article summarizes the possible pathogenesis, clinical phenotype, and treatment of HCM caused by MYH7 gene mutations, in order to facilitate the accurate prognostic evaluation and individualized management and treatment of the children with this disorder.

Application of Long-Read Nanopore Sequencing to the Search for Mutations in Hypertrophic Cardiomyopathy.

Increasing evidence suggests that both coding and non-coding regions of sarcomeric protein genes can contribute to hypertrophic cardiomyopathy (HCM). Here, we introduce an experimental workflow (tested on four patients) for complete sequencing of the most common HCM genes (MYBPC3, MYH7, TPM1, TNNT2, and TNNI3) via long-range PCR, Oxford Nanopore Technology (ONT) sequencing, and bioinformatic analysis. We applied Illumina and Sanger sequencing to validate the results, FastQC, Qualimap, and MultiQC for quality evaluations, MiniMap2 to align data, Clair3 to call and phase variants, and Annovar's tools and CADD to assess pathogenicity of variants. We could not amplify the region encompassing exons 6-12 of MYBPC3. A higher sequencing error rate was observed with ONT (6.86-6.92%) than with Illumina technology (1.14-1.35%), mostly for small indels. Pathogenic variant p.Gln1233Ter and benign polymorphism p.Arg326Gln in MYBPC3 in a heterozygous state were found in one patient. We demonstrated the ability of ONT to phase single-nucleotide variants, enabling direct haplotype determination for genes TNNT2 and TPM1. These findings highlight the importance of long-range PCR efficiency, as well as lower accuracy of variant calling by ONT than by Illumina technology; these differences should be clarified prior to clinical application of the ONT method.

Early Divergence of the C-Terminal Variable Region of Troponin T Via a Pair of Mutually Exclusive Alternatively Spliced Exons Followed by a Selective Fixation in Vertebrate Heart.

Troponin T (TnT) is the thin filament anchoring subunit of troponin complex and plays an organizer role in the Ca2+-regulation of striated muscle contraction. From an ancestral gene emerged ~ 700 million years ago in Bilateria, three homologous genes have evolved in vertebrates to encode muscle type-specific isoforms of TnT. Alternative splicing variants of TnT are present in vertebrate and invertebrate muscles to add functional diversity. While the C-terminal region of TnT is largely conserved, it contains an alternatively spliced segment emerged early in C. elegans, which has evolved into a pair of mutually exclusive exons in arthropods (10A and 10B of Drosophila TpnT gene) and vertebrates (16 and 17 of fast skeletal muscle Tnnt3 gene). The C-terminal alternatively spliced segment of TnT interfaces with the other two subunits of troponin with functional significance. The vertebrate cardiac TnT gene that emerged from duplication of the fast TnT gene has eliminated this alternative splicing by the fixation of an exon 17-like constitutive exon, indicating a functional value in slower and rhythmic contractions. The vertebrate slow skeletal muscle TnT gene that emerged from duplication of the cardiac TnT gene has the exon 17-like structure conserved, indicating its further function in sustained and fatigue resistant contractions. This functionality-based evolution is consistent with the finding that exon 10B-encoded segment of Drosophila TnT homologous to the exon 17-encoded segment of vertebrate fast TnT is selectively expressed in insect heart and leg muscles. The evolution of the C-terminal variable region of TnT demonstrates a submolecular mechanism in modifying striated muscle contractility and for the treatment of muscle and heart diseases.

Human-Induced Pluripotent Stem Cell-Derived Cardiomyocyte Model for TNNT2 Δ160E-Induced Cardiomyopathy.

BACKGROUND: The Δ160E mutation in TNNT2, which encodes troponin T, is a rare pathogenic variant identified in patients with hypertrophic cardiomyopathy and is associated with poor prognosis. Thus, a convenient human model recapitulating the pathological phenotype caused by TNNT2 Δ160E is required for therapeutic development. METHODS: We identified a heterozygous in-frame deletion mutation (c.478_480del, p.Δ160E) in TNNT2 in a patient with familial hypertrophic cardiomyopathy showing progressive left ventricular systolic dysfunction, leading to advanced heart failure. To investigate the pathological phenotype caused by Δ160E, we generated a set of isogenic induced pluripotent stem cells carrying the heterozygous Δ160E, homozygously corrected or homozygously introduced Δ160E using genome editing and differentiated them into cardiomyocytes (Hetero-Δ160E-, wild type-, and Homo-Δ160E-induced pluripotent stem cells [iPSC]-derived cardiomyocytes [iPSC-CMs]). RESULTS: Hetero-Δ160E-iPSC-CMs exhibited prolonged calcium decay, relaxation impairment, and hypertrophy compared to wild type-iPSC-CMs. Notably, these phenotypes were further exacerbated in Homo-Δ160E-iPSC-CMs. Overexpression of R-GECO-fused Δ160E mutant troponin T prolonged decay time and time to peak of the myofilament-localized calcium transient in iPSC-CMs, indicating that sarcomeric calcium retention with Δ160E may affect intracellular calcium concentration. High-content imaging analysis detected remarkable nuclear translocation of NFATc1, especially in Homo-Δ160E-iPSC-CMs, indicating that the Δ160E mutation promotes hypertrophic signaling pathway in a dose-dependent manner. Increased phosphorylation of CaMKIIδ (calcium/calmodulin-dependent protein kinase IIδ) and phospholamban at Thr17 was observed in Homo- and Hetero-Δ160E-iPSC-CMs. Epigallocatechin-3-gallate, a calcium desensitizing compound, shortened prolonged calcium decay and relaxation duration in Δ160E-iPSC-CMs. CONCLUSIONS: Isogenic iPSC-CMs recapitulate the prolonged calcium decay, relaxation impairment, and subsequent calcium-regulated signaling pathways caused by the TNNT2 Δ160E mutation and can serve as a human model for therapeutic development to prevent hypertrophic cardiomyopathy pathology.

Appropriate Exogenous Expression Stoichiometry of GATA4 as an Important Factor for Cardiac Reprogramming of Human Dermal Fibroblasts.

Reprogramming of human dermal fibroblasts (HDFs) into induced cardiomyocyte-like cells (iCMs) represents a promising strategy for human cardiac regeneration. Different cocktails of cardiac transcription factors can convert HDFs into iCMs, although with low efficiency and immature phenotype. Here, GATA4, MEF2C, TBX5, MESP1, and MYOCD (GMTMeMy for short) were used to reprogram HDFs by retrovirus infection. We found that the exogenous expression stoichiometry of GATA4 (GATA4 stoichiometry) significantly affected reprogramming efficiency. When 1/8 dosage of GATA4 virus (GATA4 dosage) plus MTMeMy was used, the reprogramming efficiency was obviously improved compared with average pooled virus encoding each factor, which measured, by the expression level of cardiac genes, the percentage of cardiac troponin T and alpha-cardiac myosin heavy-chain immunopositive cells and the numbers of iCMs showing calcium oscillation or beating synchronously in co-culture with mouse CMs. In addition, we prepared conditioned maintenance medium (CMM) by CM differentiation of H9 human embryonic stem cell line. We found that compared with traditional maintenance medium (TMM), CMM made iCMs show well-organized sarcomere formation and characteristic calcium oscillation wave earlier. These findings demonstrated that appropriate GATA4 stoichiometry was essential for cardiac reprogramming and some components in CMM were important for maturation of iCMs.