Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness.

Mitochondrial respiration is an essential component of cellular metabolism. It is a process of energy conversion through enzymatically mediated reactions, the energy of taken-up substrates transformed to the ATP production. Seahorse equipment allows to measure oxygen consumption in living cells and estimate key parameters of mitochondrial respiration in real-time mode. Four key mitochondrial respiration parameters could be measured: basal respiration, ATP-production coupled respiration, maximal respiration, and proton leak. This approach demands the application of mitochondrial inhibitors-oligomycin to inhibit ATP synthase, FCCP-to uncouple the inner mitochondrial membrane and allow maximum electron flux through the electron transport chain, rotenone, and antimycin A to inhibit complexes I and III, respectively. This chapter describes two protocols of seahorse measurements performed on iPSC-derived cardiomyocytes and TAZ knock-out C2C12 cell line.

Modulation of Notch Signaling at Early Stages of Differentiation of Human Induced Pluripotent Stem Cells to Dopaminergic Neurons.

Elaboration of protocols for differentiation of human pluripotent stem cells to dopamine neurons is an important issue for development of cell replacement therapy for Parkinson's disease. A number of protocols have been already developed; however, their efficiency and specificity still can be improved. Investigating the role of signaling cascades, important for neurogenesis, can help to solve this problem and to provide a deeper understanding of their role in neuronal development. Notch signaling plays an essential role in development and maintenance of the central nervous system after birth. In our study, we analyzed the effect of Notch activation and inhibition at the early stages of differentiation of human induced pluripotent stem cells to dopaminergic neurons. We found that, during the first seven days of differentiation, the cells were not sensitive to the Notch inhibition. On the contrary, activation of Notch signaling during the same time period led to significant changes and was associated with an increase in expression of genes, specific for caudal parts of the brain, a decrease of expression of genes, specific for forebrain, as well as a decrease of expression of genes, important for the formation of axons and dendrites and microtubule stabilizing proteins.

LMNA mutation leads to cardiac sodium channel dysfunction in the Emery-Dreifuss muscular dystrophy patient.

Pathogenic variants in the LMNA gene are known to cause laminopathies, a broad range of disorders with different clinical phenotypes. LMNA genetic variants lead to tissue-specific pathologies affecting various tissues and organs. Common manifestations of laminopathies include cardiovascular system abnormalities, in particular, cardiomyopathies and conduction disorders. In the present study, we used induced pluripotent stem cells from a patient carrying LMNA p.R249Q genetic variant to create an in vitro cardiac model of laminopathy. Induced pluripotent stem cell-derived cardiomyocytes with LMNA p.R249Q genetic variant showed a decreased sodium current density and an impaired sodium current kinetics alongside with changes in transcription levels of cardiac-specific genes. Thus, we obtained compelling in vitro evidence of an association between LMNA p.R249Q genetic variant and cardiac-related abnormalities.

Generation of iPSC line FAMRCi010-A from patient with restrictive cardiomyopathy carrying genetic variant FLNC p.Gly2011Arg.

Human iPSC cell line FAMRCi010-A was generated from a patient with restrictive cardiomyopathy carrying FLNC p.Gly2011Arg genetic variant. Patient-specific peripheral blood mononuclear cells were reprogrammed using non-integrative Sendai viruses containing OCT4, SOX2, KLF4, and CMYC. FAMRCi010-A was generated and characterized through the study. The reported iPSC line could be useful tool for in vitro modeling of FLNC-associated cardiomyopathies.

Generation of iPSC line (FAMRCi009-A) from patient with familial progressive cardiac conduction disorder carrying genetic variant FLNC p.Val2264Met.

Human iPSC cell line FAMRCi009-A was generated from a patient with restrictive cardiomyopathy and congenital myopathy carrying FLNC p.Val2264Met genetic variant. Patient-specific peripheral blood mononuclear cells were reprogrammed using non-integrative Sendai viruses. Generated iPSC lines showed normal karyotype, expressed pluripotency markers and exhibited trilineage differentiation potential in vitro. The reported iPSC lines could be used for a deeper study of filaminopathies.

Different Expressions of Pericardial Fluid MicroRNAs in Patients With Arrhythmogenic Right Ventricular Cardiomyopathy and Ischemic Heart Disease Undergoing Ventricular Tachycardia Ablation.

Introduction: Pericardial fluid is enriched with biologically active molecules of cardiovascular origin including microRNAs. Investigation of the disease-specific extracellular microRNAs could shed light on the molecular processes underlying disease development. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease characterized by life-threatening arrhythmias and progressive heart failure development. The current data about the association between microRNAs and ARVC development are limited. Methods and Results: We performed small RNA sequence analysis of microRNAs of pericardial fluid samples obtained during transcutaneous epicardial access for ventricular tachycardia (VT) ablation of six patients with definite ARVC and three post-infarction VT patients. Disease-associated microRNAs of pericardial fluid were identified. Five microRNAs (hsa-miR-1-3p, hsa-miR-21-5p, hsa-miR-122-5p, hsa-miR-206, and hsa-miR-3679-5p) were found to be differentially expressed between patients with ARVC and patients with post-infarction VT. Enrichment analysis of differentially expressed microRNAs revealed their close linkage to cardiac diseases. Conclusion: Our data extend the knowledge of pericardial fluid microRNA composition and highlight five pericardial fluid microRNAs potentially linked to ARVC pathogenesis. Further studies are required to confirm the use of pericardial fluid RNA sequencing in differential diagnosis of ARVC.

Use of hiPSC-Derived Cardiomyocytes to Rule Out Proarrhythmic Effects of Drugs: The Case of Hydroxychloroquine in COVID-19.

In the early phases of the COVID-19 pandemic, drug repurposing was widely used to identify compounds that could improve the prognosis of symptomatic patients infected by SARS-CoV-2. Hydroxychloroquine (HCQ) was one of the first drugs used to treat COVID-19 due to its supposed capacity of inhibiting SARS-CoV-2 infection and replication in vitro. While its efficacy is debated, HCQ has been associated with QT interval prolongation and potentially Torsades de Pointes, especially in patients predisposed to developing drug-induced Long QT Syndrome (LQTS) as silent carriers of variants associated with congenital LQTS. If confirmed, these effects represent a limitation to the at-home use of HCQ for COVID-19 infection as adequate ECG monitoring is challenging. We investigated the proarrhythmic profile of HCQ with Multi-Electrode Arrays after exposure of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from two healthy donors, one asymptomatic and two symptomatic LQTS patients. We demonstrated that: I) HCQ induced a concentration-dependent Field Potential Duration (FPD) prolongation and halted the beating at high concentration due to the combined effect of HCQ on multiple ion currents. II) hiPSC-CMs from healthy or asymptomatic carriers tolerated higher concentrations of HCQ and showed lower susceptibility to HCQ-induced electrical abnormalities regardless of baseline FPD. These findings agree with the clinical safety records of HCQ and demonstrated that hiPSC-CMs potentially discriminates symptomatic vs. asymptomatic mutation carriers through pharmacological interventions. Disease-specific cohorts of hiPSC-CMs may be a valid preliminary addition to assess drug safety in vulnerable populations, offering rapid preclinical results with valuable translational relevance for precision medicine.

FLNC Expression Level Influences the Activity of TEAD-YAP/TAZ Signaling.

Filamin C (FLNC), being one of the major actin-binding proteins, is involved in the maintenance of key muscle cell functions. Inherited skeletal muscle and cardiac disorders linked to genetic variants in FLNC have attracted attention because of their high clinical importance and possibility of genotype-phenotype correlations. To further expand on the role of FLNC in muscle cells, we focused on detailed alterations of muscle cell properties developed after the loss of FLNC. Using the CRISPR/Cas9 method we generated a C2C12 murine myoblast cell line with stably suppressed Flnc expression. FLNC-deficient myoblasts have a significantly higher proliferation rate combined with an impaired cell migration capacity. The suppression of Flnc expression leads to inability to complete myogenic differentiation, diminished expression of Myh1 and Myh4, alteration of transcriptional dynamics of myogenic factors, such as Mymk and Myog, and deregulation of Hippo signaling pathway. Specifically, we identified elevated basal levels of Hippo activity in myoblasts with loss of FLNC, and ineffective reduction of Hippo signaling activity during myogenic differentiation. The latter was restored by Flnc overexpression. In summary, we confirmed the role of FLNC in muscle cell proliferation, migration and differentiation, and demonstrated for the first time the direct link between Flnc expression and activity of TEAD-YAP\TAZ signaling. These findings support a role of FLNC in regulation of essential muscle processes relying on mechanical as well as signaling mechanisms.

Sodium current abnormalities and deregulation of Wnt/ß-catenin signaling in iPSC-derived cardiomyocytes generated from patient with arrhythmogenic cardiomyopathy harboring compound genetic variants in plakophilin 2 gene.

BACKGROUND: Mutations in desmosomal genes linked to arrhythmogenic cardiomyopathy are commonly associated with Wnt/ß-catenin signaling abnormalities and reduction of the sodium current density. Inhibitors of GSK3B were reported to restore sodium current and improve heart function in various arrhythmogenic cardiomyopathy models, but mechanisms underlying this effect remain unclear. We hypothesized that there is a crosstalk between desmosomal proteins, signaling pathways, and cardiac sodium channels. METHODS AND RESULTS: To reveal molecular mechanisms of arrhythmogenic cardiomyopathy, we established human iPSC-based model of this pathology. iPSC-derived cardiomyocytes from patient carrying two genetic variants in PKP2 gene demonstrated that PKP2 haploinsufficiency due to frameshift variant, in combination with the missense variant expressed from the second allele, was associated with decreased Wnt/ß-catenin activity and reduced sodium current. Different approaches were tested to restore impaired cardiomyocytes functions, including wild type PKP2 transduction, GSK3B inhibition and Wnt/ß-catenin signaling modulation. Inhibition of GSK3B led to the restoration of both Wnt/ß-catenin signaling activity and sodium current density in patient-specific cardiomyocytes while GSK3B activation led to the reduction of sodium current density. Moreover, we found that upon inhibition GSK3B sodium current was restored through Wnt/ß-catenin-independent mechanism. CONCLUSION: We propose that alterations in GSK3B-Wnt/ß-catenin signaling pathways lead to regulation of sodium current implying its role in molecular pathogenesis of arrhythmogenic cardiomyopathy.

Generation of two iPSC lines (FAMRCi007-A and FAMRCi007-B) from patient with Emery-Dreifuss muscular dystrophy and heart rhythm abnormalities carrying genetic variant LMNA p.Arg249Gln.

Human iPSC lines were generated from peripheral blood mononuclear cells of patient carrying LMNA mutation associated with Emery-Dreifuss muscular dystrophy accompanied by atrioventricular block and paroxysmal atrial fibrillation. Reprogramming factors OCT4, KLF4, SOX2, CMYC were delivered using Sendai virus transduction. iPSCs were characterized in order to prove the pluripotency markers expression, normal karyotype, ability to differentiate into three embryonic germ layers. Generated iPSC lines would be useful model to investigate disease development associated with genetic variants in LMNA gene.

Impact of the DSP-H1684R Genetic Variant on Ion Channels Activity in iPSC-Derived Cardiomyocytes.

BACKGROUND/AIMS: Mutations of desmosomal genes are known to cause arrhythmogenic cardiomyopathy characterized by arrhythmias and sudden cardiac death. Previously, we described a novel genetic variant H1684R in desmoplakin gene (DSP), associated with a progressive cardiac conduction disease (PCCD). In the present study, we aimed to investigate an effect of the DSP-H1684R genetic variant on the activity of ion channels. METHODS: We used cardiomyocytes derived from induced pluripotent stem cells (iPSC cardiomyocytes) from a patient with DSP-H1684R genetic variant and from two healthy donors. Immunofluorescent staining and western blot analyses were used to characterize patient-specific cardiomyocytes. By the whole-cell voltage-clamp technique we estimated the activity of voltage-gated sodium, calcium, and potassium channels that are responsible for action potential generation and its shape. Action potentials' parameters were measured using whole-cell current-clamp technique. RESULTS: In patient-specific cardiomyocytes we observed both lower amplitudes of currents through sodium Nav1.5 channels and L-type calcium channels, but higher amplitude of current through transient-outward potassium channels in comparison to donor cardiomyocytes. Current-clamp measurements revealed shortening of action-potential in DSP-H1684R-carrying iPSC cardiomyocytes. Therefore, observed alterations in the channels activity might have a great impact on the properties of action potential and development of PCCD. CONCLUSION: Our results show that desmoplakin genetic variants, besides conduction slowing caused by structural heart remodeling, could affect multiple ion channel activity aggravating arrhythmia manifestation in PCCD.

Desmin mutations result in mitochondrial dysfunction regardless of their aggregation properties.

Desmin, being a major intermediate filament of muscle cells, contributes to stabilization and positioning of mitochondria. Desmin mutations have been reported in conjunction with skeletal myopathies accompanied by mitochondrial dysfunction. Depending on the ability to promote intracellular aggregates formation, mutations can be considered aggregate-prone or non-aggregate-prone. The aim of the present study was to describe how expression of different desmin mutant isoforms effects mitochondria and contributes to the development of myocyte dysfunction. To achieve this goal, two non-aggregate-prone (Des S12F and Des A213V) and four aggregate-prone (Des L345P, Des A357P, Des L370P, Des D399Y) desmin mutations were expressed in skeletal muscle cells. We showed that all evaluated mutations affected the morphology of mitochondrial network, suppressed parameters of mitochondrial respiration, diminished mitochondrial membrane potential, increased ADP/ATP ratio, and enhanced mitochondrial DNA (mtDNA) release. mtDNA was partially secreted through exosomes as demonstrated by GW4869 treatment. Dysfunction of mitochondria was observed regardless the type of mutation: aggregate-prone or non-aggregate-prone. However, expression of aggregate-prone mutations resulted in more prominent phenotype. Thus, in this comparative study of six pathogenic desmin mutations that cause skeletal myopathy development, we confirmed a role of mitochondrial dysfunction and mtDNA release in the pathogenesis of desmin myopathies, regardless of the aggregation capacity of the mutated desmin.

Generation of two iPSC lines (FAMRCi004-A and FAMRCi004-B) from patient with familial progressive cardiac conduction disorder carrying genetic variant DSP p.His1684Arg.

Human iPSC cell lines (FAMRCi004-A and FAMRCi004-B) were generated from patient with progressive cardiac conduction disease and sick sinus syndrome carrying DSP p.His1684Arg genetic variant. Patient-specific adipose tissue-derived mesenchymal multipotent stromal cells were reprogrammed using non-integrative Sendai viruses. Established iPSC lines showed normal karyotype, expressed pluripotent markers and were able to differentiate toward three germ layers in vitro. The reported iPSC lines could be useful tool for in vitro modeling of progressive cardiac conduction disease associated with mutations in desmosomal genes.

Generation of two induced pluripotent stem cell lines (FAMRCi005-A and FAMRCi005-B) from patient carrying genetic variant LMNA p.Asp357Val.

LMNA mutations are often linked to laminopathies characterized by tissue-specific disorders. We generated two induced pluripotent stem cells lines from patient carrying genetic variant LMNA p.Asp357Val associated with paroxysmal ventricular tachycardia and myopathy. Reprogramming of patient's peripheral blood mononuclear cells was performed using Sendai viruses. Characterization of the FAMRCi005-A and FAMRCi005-B lines revealed that generated iPSC lines expressed pluripotent stem cell markers, had normal karyotype and demonstrated triliniage differentiation ability. Generated cell lines can be used to investigate the molecular links between LMNA genetic variants and cardiac disorders.

Generation of two iPSC lines (FAMRCi006-A and FAMRCi006-B) from patient with dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy associated with genetic variant LMNAp.Arg527Pro.

Mutations in LMNA gene are known to cause a broad range of diseases called laminopathies. We have generated two induced pluripotent stem cell lines FAMRCi006-A and FAMRCi006-B from a patient carrying LMNA p. p.Arg527Pro mutation associated with Emery-Dreifuss muscular dystrophy and dilated cardiomyopathy. Patient-specific peripheral blood mononuclear cells were reprogrammed to iPSCs using Sendai virus reprogramming system. Characterization of iPSCs had revealed pluripotency marker expression, normal karyotype, ability to differentiate into three embryonic germ layers. The reported iPSC lines could be a useful tool for in vitro modeling of laminopathies associated with LMNA genetic variants.

Truncating Variant in Myof Gene Is Associated With Limb-Girdle Type Muscular Dystrophy and Cardiomyopathy.

Even though genetic studies of individuals with neuromuscular diseases have uncovered the molecular background of many cardiac disorders such as cardiomyopathies and inherited arrhythmic syndromes, the genetic cause of a proportion of cardiomyopathies associated with neuromuscular phenotype still remains unknown. Here, we present an individual with a combination of cardiomyopathy and limb-girdle type muscular dystrophy where whole exome sequencing identified myoferlin (MYOF)-a member of the Ferlin protein family and close homolog of DYSF-as the most likely candidate gene. The disease-causative role of the identified variant c.[2576delG; 2575G>C], p.G859QfsTer8 is supported by functional studies in vitro using the primary patient's skeletal muscle mesenchymal progenitor cells, including both RNA sequencing and morphological studies, as well as recapitulating the muscle phenotype in vivo in zebrafish. We provide the first evidence supporting a role of MYOF in human muscle disease.

Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications.

AlphoidtetO-type human artificial chromosome (HAC) has been recently synthetized as a novel class of gene delivery vectors for induced pluripotent stem cell (iPSC)-based tissue replacement therapeutic approach. This HAC vector was designed to deliver copies of genes into patients with genetic diseases caused by the loss of a particular gene function. The alphoidtetO-HAC vector has been successfully transferred into murine embryonic stem cells (ESCs) and maintained stably as an independent chromosome during the proliferation and differentiation of these cells. Human ESCs and iPSCs have significant differences in culturing conditions and pluripotency state in comparison with the murine naïve-type ESCs and iPSCs. To date, transferring alphoidtetO-HAC vector into human iPSCs (hiPSCs) remains a challenging task. In this study, we performed the microcell-mediated chromosome transfer (MMCT) of alphoidtetO-HAC expressing the green fluorescent protein into newly generated hiPSCs. We used a recently modified MMCT method that employs an envelope protein of amphotropic murine leukemia virus as a targeting cell fusion agent. Our data provide evidence that a totally artificial vector, alphoidtetO-HAC, can be transferred and maintained in human iPSCs as an independent autonomous chromosome without affecting pluripotent properties of the cells. These data also open new perspectives for implementing alphoidtetO-HAC as a gene therapy tool in future biomedical applications.

De novo mutations in FLNC leading to early-onset restrictive cardiomyopathy and congenital myopathy.

Mutations in FLNC for a long time are known in connection to neuromuscular disorders and only recently were described in association with various cardiomyopathies. Here, we report a new clinical phenotype of filaminopathy in four unrelated patients with early-onset restrictive cardiomyopathy (RCM) in combination with congenital myopathy due to FLNC mutations (NM_001458.4:c.3557C>T, p.A1186V, rs1114167361 in three probands and c.[3547G>C; 3548C>T], p.A1183L, rs1131692185 in one proband). In all cases, concurrent myopathy was confirmed by neurological examination, electromyography, and morphological studies. Three of the patients also presented with arthrogryposis. The pathogenicity of the described missense variants was verified by cellular and morphological studies and by in vivo modeling in zebrafish. Combination of in silico and experimental approaches revealed that FLNC missense variants localized in Ig-loop segments often lead to development of RCM. The described FLNC mutations associated with early-onset RCMP extend cardiac spectrum of filaminopathies and facilitate the differential diagnosis of restrictive cardiac phenotype associated with neuromuscular involvement in children.

Generation of iPSC line from desmin-related cardiomyopathy patient carrying splice site mutation of DES gene.

Human iPSC line was generated from patient-specific adipose tissue-derived mesenchymal multipotent stromal cells carrying desmin (DES) gene heterozygous splice site mutation using non-integrative reprogramming method. Reprogramming factors OCT4, KLF4, SOX2, CMYC were delivered using Sendai viruses. iPSCs were characterized by sequencing, karyotype analysis, STR analysis, immunocytochemistry, RT-PCR and teratoma formation.