bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2021‒10‒10
sixty-five papers selected by
Catalina Vasilescu
University of Helsinki

  1. Cell Metab. 2021 Oct 05. pii: S1550-4131(21)00425-3. [Epub ahead of print]33(10): 1905-1907
      Leigh syndrome, a mitochondrial disease, can be modeled in mice with a deficiency in mitochondrial complex I that results in a decreased NAD+/NADH ratio. In this issue of Cell Metabolism, Liu et al. (2021) identify glycerol-3-phosphate (Gro3P) biosynthesis as a method for regenerating cytosolic NAD+ to ameliorate pathology in this mitochondrial disease model.
  2. Mol Genet Metab. 2021 Sep 25. pii: S1096-7192(21)00781-2. [Epub ahead of print]
      Most mitochondrial proteins are synthesized in the cytosol and targeted to mitochondria via N-terminal mitochondrial targeting signals (MTS) that are proteolytically removed upon import. Sometimes, MTS removal is followed by a cleavage of an octapeptide by the mitochondrial intermediate peptidase (MIP), encoded by the MIPEP gene. Previously, MIPEP variants were linked to four cases of multisystemic disorder presenting with cardiomyopathy, developmental delay, hypotonia and infantile lethality. We report here a patient carrying compound heterozygous MIPEP variants-one was not previously linked to mitochondrial disease-who did not have cardiomyopathy and who is alive at the age of 20 years. This patient had developmental delay, global hypotonia, mild optic neuropathy and mild ataxia. Functional characterization of patient fibroblasts and HEK293FT cells carrying MIPEP hypomorphic alleles demonstrated that deficient MIP activity was linked to impaired post-import processing of subunits from four of the five OXPHOS complexes and decreased abundance and activity of some of these complexes in human cells possibly underlying the development of mitochondrial disease. Thus, our work expands the genetic and clinical spectrum of MIPEP-linked disease and establishes MIP as an important regulator of OXPHOS biogenesis and function in human cells.
    Keywords:  MIP; MIPEP; Mitochondrial disease; Mitochondrial intermediate peptidase; OXPHOS assembly defect; Protein processing
  3. J Biol Chem. 2021 Oct 05. pii: S0021-9258(21)01082-6. [Epub ahead of print] 101279
      Mitochondria are essential organelles that carry out a number of pivotal metabolic processes and maintain cellular homeostasis. Mitochondrial dysfunction caused by various stresses is associated with many diseases such as type 2 diabetes, obesity, cancer, heart failure, neurodegenerative disorders, and aging. Therefore, it is important to understand the stimuli that induce mitochondrial stress. However, broad analysis of mitochondrial stress has not been carried out to date. Here, we present a set of fluorescent tools, called mito-Pain (mitochondrial PINK1 accumulation index), which enables the labeling of stressed mitochondria. Mito-Pain utilizes PINK1 stabilization on mitochondria and quantifies mitochondrial stress levels by comparison with PINK1-GFP, which is stabilized under mitochondrial stress, and RFP-Omp25, which is constitutively localized on mitochondria. To identify compounds that induce mitochondrial stress, we screened a library of 3374 compounds using mito-Pain and identified 57 compounds as mitochondrial stress inducers. Furthermore, we classified each compound into several categories based on mitochondrial response: depolarization, mitochondrial morphology, or Parkin recruitment. Parkin recruitment to mitochondria was often associated with mitochondrial depolarization and aggregation, suggesting that Parkin is recruited to heavily damaged mitochondria. In addition, many of the compounds led to various mitochondrial morphological changes, including fragmentation, aggregation, elongation, and swelling, with or without Parkin recruitment or mitochondrial depolarization. We also found that several compounds induced an ectopic response of Parkin, leading to the formation of cytosolic puncta dependent on PINK1. Thus, mito-Pain enables the detection of stressed mitochondria under a wide variety of conditions and provide insights into mitochondrial quality control systems.
    Keywords:  PTEN‐induced putative kinase 1 (PINK1); Parkin; mitochondria; mitochondrial membrane potential; mitochondrial sensor; mitochondrial stress
  4. Biochem Biophys Rep. 2021 Dec;28 101142
      The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution.
    Keywords:  Mitochondrial DNA; Mitochondrial transcription factor A (TFAM); Nucleoids; Organellar membranes; TWNK helicase
  5. Arch Dis Child Fetal Neonatal Ed. 2021 Oct 07. pii: fetalneonatal-2021-321633. [Epub ahead of print]
      OBJECTIVE: Neonatal-onset mitochondrial disease has not been fully characterised owing to its heterogeneity. We analysed neonatal-onset mitochondrial disease in Japan to clarify its clinical features, molecular diagnosis and prognosis.DESIGN: Retrospective observational study from January 2004 to March 2020.
    SETTING: Population based.
    PATIENTS: Patients (281) with neonatal-onset mitochondrial disease diagnosed by biochemical and genetic approaches.
    MAIN OUTCOME MEASURES: Disease types, initial symptoms, biochemical findings, molecular diagnosis and prognosis.
    RESULTS: Of the 281 patients, multisystem mitochondrial disease was found in 194, Leigh syndrome in 26, cardiomyopathy in 38 and hepatopathy in 23 patients. Of the 321 initial symptoms, 236 occurred within 2 days of birth. Using biochemical approaches, 182 patients were diagnosed by mitochondrial respiratory chain enzyme activity rate and 89 by oxygen consumption rate. The remaining 10 patients were diagnosed using a genetic approach. Genetic analysis revealed 69 patients had nuclear DNA variants in 36 genes, 11 of 15 patients had mitochondrial DNA variants in five genes and four patients had single large deletion. The Cox proportional hazards regression analysis showed the effects of Leigh syndrome (HR=0.15, 95% CI 0.04 to 0.63, p=0.010) and molecular diagnosis (HR=1.87, 95% CI 1.18 to 2.96, p=0.008) on survival.
    CONCLUSIONS: Neonatal-onset mitochondrial disease has a heterogenous aetiology. The number of diagnoses can be increased, and clarity regarding prognosis can be achieved by comprehensive biochemical and molecular analyses using appropriate tissue samples.
    Keywords:  data collection; mortality; neonatology; statistics
  6. Nat Rev Mol Cell Biol. 2021 Oct 07.
      The mitochondrial oxidative phosphorylation system is central to cellular metabolism. It comprises five enzymatic complexes and two mobile electron carriers that work in a mitochondrial respiratory chain. By coupling the oxidation of reducing equivalents coming into mitochondria to the generation and subsequent dissipation of a proton gradient across the inner mitochondrial membrane, this electron transport chain drives the production of ATP, which is then used as a primary energy carrier in virtually all cellular processes. Minimal perturbations of the respiratory chain activity are linked to diseases; therefore, it is necessary to understand how these complexes are assembled and regulated and how they function. In this Review, we outline the latest assembly models for each individual complex, and we also highlight the recent discoveries indicating that the formation of larger assemblies, known as respiratory supercomplexes, originates from the association of the intermediates of individual complexes. We then discuss how recent cryo-electron microscopy structures have been key to answering open questions on the function of the electron transport chain in mitochondrial respiration and how supercomplexes and other factors, including metabolites, can regulate the activity of the single complexes. When relevant, we discuss how these mechanisms contribute to physiology and outline their deregulation in human diseases.
  7. Mol Syndromol. 2021 Aug;12(5): 294-299
      Mitochondrial DNA depletion syndromes (MDDS) are a group of rare genetic disorders caused by defects in multiple genes involved in mitochondrial DNA maintenance. Among these, FBXL4 gene variants result in encephalomyopathic mtDNA depletion syndrome 13 (MTDPS13), which commonly presents as a combination of failure to thrive, neurodevelopmental delays, encephalopathy, hypotonia, a pattern of mild facial dysmorphisms, and persistent lactic acidosis. To date, 53 pathogenic FBXL4 variants and 100 cases have been described in the literature. In the present case report, we report on a 4.5-year-old boy with MTDPS13 and a novel variant. The patient had a history of antenatal hydrocephalus, severe developmental delay and mental motor retardation with psychomotor delay, severe hypotonia, mild left ventricular hypertrophic cardiomyopathy, mild facial dysmorphism, and elevated lactate levels. Symptoms suggested mitochondrial myopathy; subsequently, whole-exome sequencing was performed and a novel homozygous variant FBXL4 (NM_012160.4): c.486T>G (p.Tyr162Ter) was identified. While most of the patients with FBLX4 gene mutation have severe clinical manifestation and die at a very young age, clinical progress of our case was milder than previously reported. MDDS are very rare and can present with many different clinical signs and symptoms. In this report, we identified a novel pathogenic variant in the FBXL4 gene. This report shows that patients with FBLX4 gene mutations may present with a milder clinical phenotype than previously reported.
    Keywords:  Encephalopathy; FBXL4; Mitochondrial DNA depletion; Myopathy, MTDPS13; mtDNA depletion syndrome
  8. Elife. 2021 10 05. pii: e68806. [Epub ahead of print]10
      Ribosome assembly is an essential and conserved process that is regulated at each step by specific factors. Using cryo-electron microscopy (cryo-EM), we visualize the formation of the conserved peptidyl transferase center (PTC) of the human mitochondrial ribosome. The conserved GTPase GTPBP7 regulates the correct folding of 16S ribosomal RNA (rRNA) helices and ensures 2'-O-methylation of the PTC base U3039. GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which sequester H68-71 of the 16S rRNA and allow biogenesis factors to access the maturing PTC. Mutations that disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activate mitochondrial stress and cause viability, development, and sterility defects. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4, but not its enzymatic activity, is indispensable for mitochondrial protein synthesis.
    Keywords:  C. elegans; RNA modifications; biochemistry; chemical biology; cryo-EM; human; mitochondrial ribosome; molecular biophysics; peptidyl transferase center; structural biology
  9. Front Mol Neurosci. 2021 ;14 727552
      Charcot-Marie-Tooth (CMT) disease is one of the most common genetically inherited neurological disorders and CMT type 2A (CMT 2A) is caused by dominant mutations in the mitofusin-2 (MFN2) gene. MFN2 is located in the outer mitochondrial membrane and is a mediator of mitochondrial fusion, with an essential role in maintaining normal neuronal functions. Although loss of MFN2 induces axonal neuropathy, the detailed mechanism by which MFN2 deficiency results in axonal degeneration of human spinal motor neurons remains largely unknown. In this study, we generated MFN2-knockdown human embryonic stem cell (hESC) lines using lentivirus expressing MFN2 short hairpin RNA (shRNA). Using these hESC lines, we found that MFN2 loss did not affect spinal motor neuron differentiation from hESCs but resulted in mitochondrial fragmentation and dysfunction as determined by live-cell imaging. Notably, MFN2-knockodwn spinal motor neurons exhibited CMT2A disease-related phenotypes, including extensive perikaryal inclusions of phosphorylated neurofilament heavy chain (pNfH), frequent axonal swellings, and increased pNfH levels in long-term cultures. Importantly, MFN2 deficit impaired anterograde and retrograde mitochondrial transport within axons, and reduced the mRNA and protein levels of kinesin and dynein, indicating the interfered motor protein expression induced by MFN2 deficiency. Our results reveal that MFN2 knockdown induced axonal degeneration of spinal motor neurons and defects in mitochondrial morphology and function. The impaired mitochondrial transport in MFN2-knockdown spinal motor neurons is mediated, at least partially, by the altered motor proteins, providing potential therapeutic targets for rescuing axonal degeneration of spinal motor neurons in CMT2A disease.
    Keywords:  CMT2A; MFN2; human embryonic stem cell; mitochondrial transport; spinal motor neuron
  10. J Inherit Metab Dis. 2021 Oct 05.
      Barth syndrome is a multisystem disorder caused by an abnormal metabolism of the mitochondrial lipid cardiolipin. In this review, we discuss physical properties, biosynthesis, membrane assembly, and function of cardiolipin. We hypothesize that cardiolipin reduces packing stress in the inner mitochondrial membrane, which arises as a result of protein crowding. According to this hypothesis, patients with Barth syndrome are unable to meet peak energy demands because they fail to concentrate the proteins of oxidative phosphorylation to a high surface density in the inner mitochondrial membrane. This article is protected by copyright. All rights reserved.
  11. Biosci Rep. 2021 Oct 05. pii: BSR20211320. [Epub ahead of print]
      Mitochondria are highly specialised organelles required for cellular processes including ATP-production through cellular respiration and controlling apoptosis. Mitochondria contain their own DNA genome which encodes both protein and RNA required for cellular respiration. Each cell may contain hundreds to thousands of copies of the mitochondrial genome, which is essential for normal cellular function - deviation of mitochondrial DNA (mtDNA) copy number is associated with cellular aging and disease. Furthermore, mtDNA lesions can arise from both endogenous or exogenous sources and must either be tolerated or corrected to preserve mitochondrial function. Importantly, replication of damaged mtDNA can lead to stalling and introduction of mutations or genetic loss, mitochondria have adapted mechanisms to repair damaged DNA. These mechanisms rely on nuclear encoded DNA repair proteins that are translocated into the mitochondria. Despite the presence of many known nuclear DNA repair proteins being found in the mitochondrial proteome, it remains to be established which DNA repair mechanisms are functional in mammalian mitochondria. Here, we summarise the existing and emerging research, alongside examining proteomic evidence, demonstrating that mtDNA damage can be repaired using Base Excision Repair (BER), Homologous Recombination (HR) and Microhomology-mediated End Joining (MMEJ). Critically, these repair mechanisms do not operate in isolation and evidence for interplay between pathways and repair associated with replication is discussed. Importantly, characterising non-canonical functions of key proteins and understanding the bespoke pathways used to tolerate, repair or bypass DNA damage will be fundamental in fully understanding the causes of mitochondrial genome mutations and mitochondrial dysfunction.
    Keywords:  DNA replication and recombination; DNA synthesis and repair; genome integrity; mtDNA
  12. Cell Rep. 2021 Oct 05. pii: S2211-1247(21)01221-3. [Epub ahead of print]37(1): 109767
      Cardiac metabolism is a high-oxygen-consuming process, showing a preference for long-chain fatty acid (LCFA) as the fuel source under physiological conditions. However, a metabolic switch (favoring glucose instead of LCFA) is commonly reported in ischemic or late-stage failing hearts. The mechanism regulating this metabolic switch remains poorly understood. Here, we report that loss of PHD2/3, the cellular oxygen sensors, blocks LCFA mitochondria uptake and β-oxidation in cardiomyocytes. In high-fat-fed mice, PHD2/3 deficiency improves glucose metabolism but exacerbates the cardiac defects. Mechanistically, we find that PHD2/3 bind to CPT1B, a key enzyme of mitochondrial LCFA uptake, promoting CPT1B-P295 hydroxylation. Further, we show that CPT1B-P295 hydroxylation is indispensable for its interaction with VDAC1 and LCFA β-oxidation. Finally, we demonstrate that a CPT1B-P295A mutant constitutively binds to VDAC1 and rescues LCFA metabolism in PHD2/3-deficient cardiomyocytes. Together, our data identify an oxygen-sensitive regulatory axis involved in cardiac metabolism.
    Keywords:  cardiac metabolism switch; cardiomyocyte; carnitine O-palmitoyltransferase 1b; heart failure; hypoxia; long-chain fatty acid; myocardial infarction; prolyl hydroxylase domain protein; voltage-dependent anion channel
  13. Bio Protoc. 2021 Sep 05. 11(17): e4139
      Understanding the structure and dynamics of DNA-protein interactions during DNA replication is crucial for elucidating the origins of disorders arising from its dysfunction. In this study, we employed Atomic Force Microscopy as a single-molecule imaging tool to examine the mitochondrial DNA helicase Twinkle and its interactions with DNA. We used imaging in air and time-lapse imaging in liquids to observe the DNA binding and unwinding activities of Twinkle hexamers at the single-molecule level. These procedures helped us visualize Twinkle loading onto and unloading from the DNA in the open-ring conformation. Using traditional methods, it has been shown that Twinkle is capable of unwinding dsDNA up to 20-55 bps. We found that the addition of mitochondrial single-stranded DNA binding protein (mtSSB) facilitates a 5-fold increase in the DNA unwinding rate for the Twinkle helicase. The protocols developed in this study provide new platforms to examine DNA replication and to explore the mechanism driving DNA deletion and human diseases. Graphic abstract: Mitochondrial Twinkle Helicase Dynamics.
    Keywords:  Atomic Force Microscope; Liquid AFM imaging; Mitochondria; Mitochondrial replication; Single molecule imaging; Twinkle helicase
  14. Mitochondrion. 2021 Sep 29. pii: S1567-7249(21)00137-9. [Epub ahead of print]
      Mitofusin (MFN) 2 belongs to the large family of mitochondrial transmembrane GTPases and has a role in dynamic mitochondrial remodeling process governed by fusion and fission. MFN2 pathogenic variants classically cause Charcot-Marie-Tooth disease type 2A (CMT2A), the most common axonal form of CMT, but patients with complex and unusual phenotypes involving the central and peripheral nervous system have been described, with mitochondrial dysfunction proposed as the underlying pathogenic mechanism. Here, we report the first description of a neurochemical pattern of secondary alterations in the metabolism of biogenic amines linked to the de novo presence of the hotspot MFN2 pathogenic variant p.Arg104Trp. The infant presented a very early onset choreic movement disorder associated with severe axial hypotonia and fluctuating dystonia of limbs. The relationship between mitochondrial DNA (mtDNA) maintenance defects and dopaminergic neurotransmitter disorders, governed by MFN2, is discussed.
    Keywords:  CSF biogenic amine disorder; Charcot-Marie-Tooth disease; MFN2; mtDNA depletion syndrome
  15. Bioessays. 2021 Oct 07. e2100168
      PTEN-induced kinase 1 (PINK1) is a Parkinson's disease gene that acts as a sensor for mitochondrial damage. Its best understood role involves phosphorylating ubiquitin and the E3 ligase Parkin (PRKN) to trigger a ubiquitylation cascade that results in selective clearance of damaged mitochondria through mitophagy. Here we focus on other physiological roles of PINK1. Some of these also lie upstream of Parkin but others represent autonomous functions, for which alternative substrates have been identified. We argue that PINK1 orchestrates a multi-arm response to mitochondrial damage that impacts on mitochondrial architecture and biogenesis, calcium handling, transcription and translation. We further discuss a role for PINK1 in immune signalling co-ordinated at mitochondria and consider the significance of a freely diffusible cleavage product, that is constitutively generated and degraded under basal conditions.
    Keywords:  ISR; PINK1; Parkin; Parkinson's disease; mitochondria; mitochondrial quality control; mitophagy; stress response
  16. J Inherit Metab Dis. 2021 Oct 09.
      Cardiolipin (CL) is the signature phospholipid (PL) of mitochondria and plays a pivotal role in mitochondrial and cellular function. Disruption of the CL remodeling gene tafazzin (TAZ) causes the severe genetic disorder Barth syndrome (BTHS). Our current understanding of the function of CL and the mechanism underlying the disease has greatly benefited from studies utilizing the powerful yeast model Saccharomyces cerevisiae. In this review, we discuss important findings on the function of CL and its remodeling from yeast studies and the implications of these findings for BTHS, highlighting the potential physiological modifiers that may contribute to the disparities in clinical presentation among BTHS patients. This article is protected by copyright. All rights reserved.
    Keywords:  Barth syndrome; Cardiolipin; mitochondrial disease; taffazzin; yeast
  17. Nucleic Acids Res. 2021 Oct 06. pii: gkab901. [Epub ahead of print]
      Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational 'hot-spots' or 'cold-spots'. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.
  18. Biochim Biophys Acta Gen Subj. 2021 Oct 05. pii: S0304-4165(21)00176-8. [Epub ahead of print] 130017
      BACKGROUND: Autophagy, a highly conserved homeostatic mechanism, is essential for cell survival. The decline of autophagy function has been implicated in various diseases as well as aging. Although mitochondria play a key role in the autophagy process, whether mitochondrial-derived peptides are involved in this process has not been explored.METHODS: We developed a high through put screening method to identify potential autophagy inducers among mitochondrial-derived peptides. We used three different cell lines, mice, c.elegans, and a human cohort to validate the observation.
    RESULTS: Humanin, a mitochondrial-derived peptide, increases autophagy and maintains autophagy flux in several cell types. Humanin administration increases the expression of autophagy-related genes and lowers accumulation of harmful misfolded proteins in mice skeletal muscle, suggesting that humanin-induced autophagy potentially contributes to the improved skeletal function. Moreover, autophagy is a critical role in humanin-induced lifespan extension in C. elegans.
    CONCLUSIONS: Humanin is an autophagy inducer.
    GENERAL SIGNIFICANCE: This paper presents a significant, novel discovery regarding the role of the mitochondrial derived peptide humanin in autophagy regulation and as a possible therapeutic target for autophagy in various age-related diseases.
    Keywords:  Autophagy; Humanin; Lifespan; Mitochondrial-derived peptides
  19. iScience. 2021 Oct 22. 24(10): 103118
      The mitochondrial unfolded protein response (UPRmt) is an organellar stress signaling pathway that functions to detect and restore disruption of mitochondrial proteostasis. The UPRmt is involved in a wide range of physiological and disease conditions, including aging, stem cell maintenance, innate immunity, neurodegeneration, and cancer. Here we report that the UPRmt is integral to zebrafish fin regeneration. Taking advantage of a novel zebrafish UPRmt reporter, we observed that UPRmt activation occurs in regenerating fin tissue shortly after injury. Through chemical and genetic approaches, we discovered that the Sirt1-UPRmt pathway, best known for its role in promoting lifespan extension, is crucial for fin regeneration. The metabolism of NAD+ is an important contributor to Sirt1 activity in this context. We propose that Sirt1 activation induces mitochondrial biogenesis in injured fin tissue, which leads to UPRmt activation and promotes tissue regeneration.
    Keywords:  Cell biology; Developmental biology; Molecular biology
  20. J Am Soc Nephrol. 2021 Oct 04. pii: ASN.2021050596. [Epub ahead of print]
      Background: Gitelman syndrome (GS) is the most frequent hereditary salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. GS is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+-Cl- cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants of CLCNKB, HNF1B, FXYD2, or KCNJ10 may result in the same renal phenotype of GS, as they can lead to reduced NCC activity. For approximately 10 percent of patients with a GS phenotype, the genotype is unknown. Methods: We identified mitochondrial DNA (mtDNA) variants in three families with GS-like electrolyte abnormalities, then investigated 156 families for variants in MT-TI and MT-TF, which encode the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. Mitochondrial dysfunction was induced In NCC-expressing HEK293 cells to assess the effect on thiazide-sensitive 22Na+ transport. Results: Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT-TF) and m.4291T>C (n=4, in MT-TI). Variants were near homoplasmic in affected individuals. All variants were classified as pathogenic, except for m.643A>G, which was classified as a variant of uncertain significance. Importantly, affected members of six families with an MT-TF variant additionally suffered from progressive chronic kidney disease. Dysfunction of oxidative phosphorylation complex IV reduced maximal mitochondrial respiratory capacity in patient fibroblasts. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, inhibited NCC phosphorylation and NCC-mediated sodium uptake. Conclusion: Pathogenic mtDNA variants in MT-TF and MT-TI can cause a GS-like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained GS-like tubulopathies.
  21. Aging Cell. 2021 Oct 06. e13487
      The association between blood-based estimates of mitochondrial DNA parameters, mitochondrial DNA copy number (mtDNA-CN) and heteroplasmy load, with skeletal muscle bioenergetic capacity was evaluated in 230 participants of the Baltimore Longitudinal Study of Aging (mean age:74.7 years, 53% women). Participants in the study sample had concurrent data on muscle oxidative capacity (τPCr ) assessed by 31 P magnetic resonance spectroscopy, and mitochondrial DNA parameters estimated from whole-genome sequencing data. In multivariable linear regression models, adjusted for age, sex, extent of phosphocreatine (PCr) depletion, autosomal sequencing coverage, white blood cell total, and differential count, as well as platelet count, mtDNA-CN and heteroplasmy load were not significantly associated with τPCr (both p > 0.05). However, in models evaluating whether the association between mtDNA-CN and τPCr varied by heteroplasmy load, there was a significant interaction between mtDNA-CN and heteroplasmy load (p = 0.037). In stratified analysis, higher mtDNA-CN was significantly associated with lower τPCr among participants with high heteroplasmy load (n = 84, β (SE) = -0.236 (0.115), p-value = 0.044), but not in those with low heteroplasmy load (n = 146, β (SE) = 0.046 (0.119), p-value = 0.702). Taken together, mtDNA-CN and heteroplasmy load provide information on muscle bioenergetics. Thus, mitochondrial DNA parameters may be considered proxy measures of mitochondrial function that can be used in large epidemiological studies, especially when comparing subgroups.
    Keywords:  aging; mitochondrial DNA; skeletal muscle
  22. EMBO Rep. 2021 Oct 07. e52964
      While mitochondrial function is essential for life in all multicellular organisms, a mild impairment of mitochondrial function can extend longevity in model organisms. By understanding the molecular mechanisms involved, these pathways might be targeted to promote healthy aging. In studying two long-lived mitochondrial mutants in C. elegans, we found that disrupting subunits of the mitochondrial electron transport chain results in upregulation of genes involved in innate immunity, which is driven by the mitochondrial unfolded protein response (mitoUPR) but also dependent on the canonical p38-mediated innate immune signaling pathway. Both of these pathways are required for the increased resistance to bacterial pathogens and extended longevity of the long-lived mitochondrial mutants, as is the FOXO transcription factor DAF-16. This work demonstrates that both the p38-mediated innate immune signaling pathway and the mitoUPR act in concert on the same innate immunity genes to promote pathogen resistance and longevity and that input from the mitochondria can extend longevity by signaling through these pathways. This indicates that multiple evolutionarily conserved genetic pathways controlling innate immunity also function to modulate lifespan.
    Keywords:   C. elegans ; aging; innate immunity; mitochondria; mitochondrial unfolded protein response
  23. Elife. 2021 Oct 06. pii: e71636. [Epub ahead of print]10
      Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis and biochemically couple FASII activity to mitochondrial electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast, the mitochondrion of Plasmodium falciparum malaria parasites lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of Nfs1 and the Rieske Fe-S protein in ETC Complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other apicomplexan pathogens, including Toxoplasma and Babesia. This discovery unveils an evolutionary driving force to retain interaction of mitochondrial Fe-S cluster biogenesis with ACP independent of its eponymous function in FASII.
    Keywords:  P. falciparum; biochemistry; chemical biology; infectious disease; microbiology
  24. Mitochondrion. 2021 Oct 01. pii: S1567-7249(21)00140-9. [Epub ahead of print]
      Although alterations in cellular mitochondrial DNA (mtDNA) content have been linked to various pathological conditions, the mechanisms that govern mtDNA copy number (mtCN) control remain poorly understood. Moreover, techniques for mtDNA quantification do not allow for direct comparisons of absolute mtCNs between labs. Here we report the development of a direct droplet digital PCR technique for the determination of mtCNs in whole-cell lysates. Using this technique, we demonstrate that cellular mtDNA content can fluctuate in culture by as much as 50% and provide evidence for both cell proliferation-coupled and uncoupled mtDNA replication.
    Keywords:  Mitochondrial DNA; X-ray; cell cycle; cell proliferation; mtDNA copy number; mtDNA replication
  25. Orphanet J Rare Dis. 2021 10 02. 16(1): 407
      BACKGROUND: Mitochondrial diseases are difficult to diagnose and treat. Recent advances in genetic diagnostics and more effective treatment options can improve patient diagnosis and prognosis, but patients with mitochondrial disease typically experience delays in diagnosis and treatment. Here, we describe a unique collaborative practice model among physicians and scientists in Spain focused on identifying TK2 deficiency (TK2d), an ultra-rare mitochondrial DNA depletion and deletions syndrome.MAIN BODY: This collaboration spans research and clinical care, including laboratory scientists, adult and pediatric neuromuscular clinicians, geneticists, and pathologists, and has resulted in diagnosis and consolidation of care for patients with TK2d. The incidence of TK2d is not known; however, the first clinical cases of TK2d were reported in 2001, and only ~ 107 unique cases had been reported as of 2018. This unique collaboration in Spain has led to the diagnosis of more than 30 patients with genetically confirmed TK2d across different regions of the country. Research affiliate centers have led investigative treatment with nucleosides based on understanding of TK2d clinical manifestations and disease mechanisms, which resulted in successful treatment of a TK2d mouse model with nucleotide therapy in 2010. Only 1 year later, this collaboration enabled rapid adoption of treatment with pyrimidine nucleotides (and later, nucleosides) under compassionate use. Success in TK2d diagnosis and treatment in Spain is attributable to two important factors: Spain's fully public national healthcare system, and the designation in 2015 of major National Reference Centers for Neuromuscular Disorders (CSURs). CSUR networking and dissemination facilitated development of a collaborative care network for TK2d disease, wherein participants share information and protocols to request approval from the Ministry of Health to initiate nucleoside therapy. Data have recently been collected in a retrospective study conducted under a Good Clinical Practice-compliant protocol to support development of a new therapeutic approach for TK2d, a progressive disease with no approved therapies.
    CONCLUSIONS: The Spanish experience in diagnosis and treatment of TK2d is a model for the diagnosis and development of new treatments for very rare diseases within an existing healthcare system.
    Keywords:  Mitochondrial disease; Mitochondrial medicine; Thymidine kinase 2 deficiency (TK2d)
  26. Heart Fail Rev. 2021 Oct 08.
      Barth syndrome is a rare and potentially fatal X-linked disease characterized by cardiomyopathy, skeletal muscle weakness, growth delays, and cyclic neutropenia. Patients with Barth syndrome are prone to high risk of mortality in infancy and the development of cardiomyopathy with severe weakening of the immune system. Elamipretide is a water-soluble, aromatic-cationic, mitochondria-targeting tetrapeptide that readily penetrates and transiently localizes to the inner mitochondrial membrane. Therapy with elamipretide facilitates cell health by improving energy production and inhibiting excessive formation of reactive oxygen species, thus alleviating oxidative stress. Elamipretide crosses the outer membrane of the mitochondrion and becomes associated with cardiolipin, a constituent phospholipid of the inner membrane. Elamipretide improves mitochondrial bioenergetics and morphology rapidly in induced pluripotent stem cells from patients with Barth syndrome and other genetically related diseases characterized by pediatric cardiomyopathy. Data with elamipretide across multiple models of disease are especially promising, with results from several studies supporting the use of elamipretide as potential therapy for patients with Barth syndrome, particularly where there is a confirmed diagnosis of cardiomyopathy. This review highlights the challenges and opportunities presented in treating Barth syndrome cardiomyopathy patients with elamipretide and addresses evidence supporting the durability of effect of elamipretide as a therapeutic agent for Barth syndrome, especially its likely durable effects on progression of cardiomyopathy following the cessation of drug treatment and the capability of elamipretide to structurally reverse remodel the failing left ventricle at the global, cellular, and molecular level in a gradual manner through specific targeting of the mitochondrial inner membrane.
    Keywords:  Barth syndrome; Cardiomyopathy; Elamipretide; Mitochondria
  27. Am J Med Genet A. 2021 Oct 08.
      D-bifunctional protein (DBP) deficiency is a rare, autosomal recessive peroxisomal enzyme deficiency resulting in a high burden of morbidity and early mortality. Patients with DBP deficiency resemble those with a severe Zellweger phenotype, with neonatal hypotonia, seizures, craniofacial dysmorphisms, psychomotor delay, deafness, blindness, and death typically within the first 2 years of life, although patients with residual enzyme function can survive longer. The clinical severity of the disease depends on the degree of enzyme deficiency. Loss-of-function variants typically result in no residual enzyme activity; however, splice variants may result in protein with residual function. We describe a full-term newborn presenting with hypotonia, seizures, and unexplained hypoglycemia, who was later found to have rickets at follow up. Rapid whole genome sequencing identified two HSD17B4 variants in trans; one likely pathogenic variant and one variant of uncertain significance (VUS) located in the polypyrimidine tract of intron 13. To determine the functional consequence of the VUS, we analyzed RNA from the patient's father with RNA-seq which showed skipping of Exon 14, resulting in a frameshift mutation three amino acids from the new reading frame. This RNA-seq analysis was correlated with virtually absent enzyme activity, elevated very-long-chain fatty acids in fibroblasts, and a clinically severe phenotype. Both variants are reclassified as pathogenic. Due to the clinical spectrum of DBP deficiency, this provides important prognostic information, including early mortality. Furthermore, we add persistent hypoglycemia to the clinical spectrum of the disease, and advocate for the early management of fat-soluble vitamin deficiencies to reduce complications.
    Keywords:  D-bifunctional protein deficiency; Zellweger spectrum disorders; peroxisomal biogenesis disorders; rapid whole genome sequencing; very-long-chain fatty acids
  28. Front Cardiovasc Med. 2021 ;8 739095
      Mitochondria are the most abundant organelles in cardiac cells, and are essential to maintain the normal cardiac function, which requires mitochondrial dynamics and mitophagy to ensure the stability of mitochondrial quantity and quality. When mitochondria are affected by continuous injury factors, the balance between mitochondrial dynamics and mitophagy is broken. Aging and damaged mitochondria cannot be completely removed in cardiac cells, resulting in energy supply disorder and accumulation of toxic substances in cardiac cells, resulting in cardiac damage and cardiotoxicity. This paper summarizes the specific underlying mechanisms by which various adverse factors interfere with mitochondrial dynamics and mitophagy to produce cardiotoxicity and emphasizes the crucial role of oxidative stress in mitophagy. This review aims to provide fresh ideas for the prevention and treatment of cardiotoxicity induced by altered mitochondrial dynamics and mitophagy.
    Keywords:  cardiotoxicity; mitochondrial dynamics; mitochondrial fission; mitochondrial fusion; mitophagy
  29. Neurotherapeutics. 2021 Oct 04.
      Inherited peripheral neuropathies are a genetically and phenotypically diverse group of disorders that lead to degeneration of peripheral neurons with resulting sensory and motor dysfunction. Genetic neuropathies that primarily cause axonal degeneration, as opposed to demyelination, are most often classified as Charcot-Marie-Tooth disease type 2 (CMT2) and are the focus of this review. Gene identification efforts over the past three decades have dramatically expanded the genetic landscape of CMT and revealed several common pathological mechanisms among various forms of the disease. In some cases, identification of the precise genetic defect and/or the downstream pathological consequences of disease mutations have yielded promising therapeutic opportunities. In this review, we discuss evidence for pathogenic overlap among multiple forms of inherited neuropathy, highlighting genetic defects in axonal transport, mitochondrial dynamics, organelle-organelle contacts, and local axonal protein translation as recurrent pathological processes in inherited axonal neuropathies. We also discuss how these insights have informed emerging treatment strategies, including specific approaches for single forms of neuropathy, as well as more general approaches that have the potential to treat multiple types of neuropathy. Such therapeutic opportunities, made possible by improved understanding of molecular and cellular pathogenesis and advances in gene therapy technologies, herald a new and exciting phase in inherited peripheral neuropathy.
    Keywords:  Axonal neuropathy; Charcot-Marie-Tooth disease; Pathogenesis; Peripheral neuropathy; Treatment
  30. Mov Disord. 2021 Oct 06.
      BACKGROUND: Parkinson's disease (PD) is a highly age-related disorder, where common genetic risk variants affect both disease risk and age at onset. A statistical approach that integrates these effects across all common variants may be clinically useful for individual risk stratification. A polygenic hazard score methodology, leveraging a time-to-event framework, has recently been successfully applied in other age-related disorders.OBJECTIVES: We aimed to develop and validate a polygenic hazard score model in sporadic PD.
    METHODS: Using a Cox regression framework, we modeled the polygenic hazard score in a training data set of 11,693 PD patients and 9841 controls. The score was then validated in an independent test data set of 5112 PD patients and 5372 controls and a small single-study sample of 360 patients and 160 controls.
    RESULTS: A polygenic hazard score predicts the onset of PD with a hazard ratio of 3.78 (95% confidence interval 3.49-4.10) when comparing the highest to the lowest risk decile. Combined with epidemiological data on incidence rate, we apply the score to estimate genetically stratified instantaneous PD risk across age groups.
    CONCLUSIONS: We demonstrate the feasibility of a polygenic hazard approach in PD, integrating the genetic effects on disease risk and age at onset in a single model. In combination with other predictive biomarkers, the approach may hold promise for risk stratification in future clinical trials of disease-modifying therapies, which aim at postponing the onset of PD. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
    Keywords:  Parkinson's disease; age at onset; genetics; polygenic score; prediction
  31. Dev Cell. 2021 Sep 29. pii: S1534-5807(21)00731-0. [Epub ahead of print]
      The metabolic coupling of Schwann cells (SCs) and peripheral axons is poorly understood. Few molecules in SCs are known to regulate axon stability. Using SC-specific Rheb knockout mice, we demonstrate that Rheb-regulated mitochondrial pyruvate metabolism is critical for SC-mediated non-cell-autonomous regulation of peripheral axon stability. Rheb knockout suppresses pyruvate dehydrogenase (PDH) activity (independently of mTORC1) and shifts pyruvate metabolism toward lactate production in SCs. The increased lactate causes age-dependent peripheral axon degeneration, affecting peripheral nerve function. Lactate, as an energy substrate and a potential signaling molecule, enhanced neuronal mitochondrial metabolism and energy production of peripheral nerves. Albeit beneficial to injured peripheral axons in the short term, we show that persistently increased lactate metabolism of neurons enhances ROS production, eventually damaging mitochondria, neuroenergetics, and axon stability. This study highlights the complex roles of lactate metabolism to peripheral axons and the importance of lactate homeostasis in preserving peripheral nerves.
    Keywords:  ROS; Rheb; Schwann cells; axon degeneration; lactate shuttle; mTORC1; metabolic coupling; oxidative stress; peripheral axons; pyruvate metabolism
  32. J Comp Neurol. 2021 Oct 05.
      In the highly dynamic metabolic landscape of a neuron, mitochondrial membrane architectures can provide critical insight into the unique energy balance of the cell. Current theoretical calculations of functional outputs like ATP and heat often represent mitochondria as idealized geometries and therefore can miscalculate the metabolic uxes. To analyze mitochondrial morphology in neurons of mouse cerebellum neuropil, 3D tracings of complete synaptic and axonal mitochondria were constructed using a database of serial TEM tomography images and converted to watertight meshes with minimal distortion of the original microscopy volumes with a granularity of 1.64 nanometer isotropic voxels. The resulting in silico representations were subsequently quanti ed by di erential geometry methods in terms of the mean and Gaussian curvatures, surface areas, volumes, and membrane motifs, all of which can alter the metabolic output of the organelle. Finally, we identify structural motifs present across this population of mitochondria, which may contribute to future modeling studies of mitochondrial physiology and metabolism in neurons. This article is protected by copyright. All rights reserved.
    Keywords:  EM tomography; energetics; mitochondria; morphology; neuronal
  33. Cell Metab. 2021 Oct 05. pii: S1550-4131(21)00427-7. [Epub ahead of print]33(10): 2004-2020.e9
      Nonalcoholic steatohepatitis (NASH) is an advanced stage of nonalcoholic fatty liver disease (NAFLD) with serious consequences that currently lacks approved pharmacological therapies. Recent studies suggest the close relationship between the pathogenesis of NAFLD and the dysregulation of RNA splicing machinery. Here, we reveal death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2) is markedly upregulated in the livers of both NAFLD/NASH patients and NAFLD/NASH diet-fed mice. Hepatic deletion of DRAK2 suppresses the progression of hepatic steatosis to NASH. Comprehensive analyses of the phosphoproteome and transcriptome indicated a crucial role of DRAK2 in RNA splicing and identified the splicing factor SRSF6 as a direct binding protein of DRAK2. Further studies demonstrated that binding to DRAK2 inhibits SRSF6 phosphorylation by the SRSF kinase SRPK1 and regulates alternative splicing of mitochondrial function-related genes. In conclusion, our findings reveal an indispensable role of DRAK2 in NAFLD/NASH and offer a potential therapeutic target for this disease.
    Keywords:  Drak2; RNA alternative splicing; hepatic steatosis; mitochondrial function; mtDNA; nonalcoholic fatty liver disease; serine/arginine-rich splicing factor (SRSF)
  34. Org Biomol Chem. 2021 Oct 05.
      The 'powerhouses' of cell, mitochondria have seen an upsurge of interest in investigations pertaining to the imaging and mapping of physiological processes. By utilizing sterol-modified rhodamine, we have performed the live-cell imaging of mitochondria without dependence on a membrane potential. The sterol probes are highly biocompatible, and they can track the mitochondrial live-cell dynamics in a background-free manner with improved brightness and impressive contrast. This is the first attempt to study the stress response using a direct fluorescence readout with bio-conjugates of rhodamine inside mitochondria. The results pave the way for developing different sterol markers for understanding cellular responses and function.
  35. Annu Rev Physiol. 2021 Oct 06.
      Mitochondria serve numerous critical cellular functions, rapidly responding to extracellular stimuli and cellular demands while dynamically communicating with other organelles. Mitochondrial function in the gastrointestinal epithelium plays a critical role in maintaining intestinal health. Emerging studies implicate the involvement of mitochondrial dysfunction in inflammatory bowel disease (IBD). This review presents mitochondrial metabolism, function, and quality control that converge in intestinal epithelial stemness, differentiation programs, barrier integrity, and innate immunity to influence intestinal inflammation. Intestinal and disease characteristics that set the stage for mitochondrial dysfunction being a key factor in IBD, and in turn, pathogenic mitochondrial mechanisms influencing and potentiating the development of IBD, are discussed. These findings establish the basis for potential mitochondrial-targeted interventions for IBD therapy. Expected final online publication date for the Annual Review of Physiology, Volume 84 is February 2022. Please see for revised estimates.
  36. Mitochondrion. 2021 Oct 04. pii: S1567-7249(21)00136-7. [Epub ahead of print]
      The COVID-19 pandemic prompted the FDA to authorize a new nucleoside analogue, remdesivir, for emergency use in affected individuals. We examined the effects of its active metabolite, remdesivir triphosphate (RTP), on the activity of the replicative mitochondrial DNA polymerase, Pol γ. We found that while RTP is not incorporated by Pol γ into a nascent DNA strand, it remains associated with the enzyme impeding its synthetic activity and stimulating exonucleolysis. In spite of that, we found no evidence for deleterious effects of remdesivir treatment on the integrity of the mitochondrial genome in human cells in culture.
    Keywords:  Antiviral nucleoside analogues; COVID-19; DNA polymerase gamma; Mitochondrial DNA; Remdesivir
  37. J Cell Sci. 2021 Oct 01. pii: jcs240465. [Epub ahead of print]134(19):
      Mitochondria, which resemble their α-proteobacteria ancestors, are a major cellular asset, producing energy 'on the cheap' through oxidative phosphorylation. They are also a liability. Increased oxidative phosphorylation means increased oxidative stress, and damaged mitochondria incite inflammation through release of their bacteria-like macromolecules. Mitophagy (the selective macroautophagy of mitochondria) controls mitochondria quality and number to manage these risky assets. Parkin, BNIP3 and NIX were identified as being part of the first mitophagy pathways identified in mammals over a decade ago, with additional pathways, including that mediated by FUNDC1 reported more recently. Loss of Parkin or PINK1 function causes Parkinson's disease, highlighting the importance of mitophagy as a quality control mechanism in the brain. Additionally, mitophagy is induced in idiopathic Parkinson's disease and Alzheimer's disease, protects the heart and other organs against energy stress and lipotoxicity, regulates metabolism by controlling mitochondrial number in brown and beige fat, and clears mitochondria during terminal differentiation of glycolytic cells, such as red blood cells and neurons. Despite its importance in disease, mitophagy is likely dispensable under physiological conditions. This Review explores the in vivo roles of mitophagy in mammalian systems, focusing on the best studied examples - mitophagy in neurodegeneration, cardiomyopathy, metabolism, and red blood cell development - to draw out common themes.
    Keywords:  Mitochondria quality control; Neurodegeneration; PRKN; Park2; Park6
  38. Physiol Rep. 2021 Oct;9(19): e15045
      In native heart tissue, cardiac fibroblasts provide the structural framework of extracellular matrix (ECM) while also influencing the electrical and mechanical properties of cardiomyocytes. Recent advances in the field of stem cell differentiation have led to the availability of human pluripotent stem cell-derived cardiac fibroblasts (iPSC-CFs) in addition to cardiomyocytes (iPSC-CMs). Here we use a novel 2D in vitro micropatterned platform that provides control over ECM geometry and substrate stiffness. When cultured alone on soft micropatterned substrates, iPSC-CFs are confined to the micropatterned features and remodel the ECM into anisotropic fibers. Similar remodeling and ECM production occurs when cultured with iPSC-CMs in a co-culture model. In addition to modifications in the ECM, our results show that iPSC-CFs influence iPSC-CM function with accelerated Ca2+ transient rise-up time and greater contractile strains in the co-culture conditions compared to when iPSC-CMs are cultured alone. These combined observations highlight the important role cardiac fibroblasts play in vivo and the need for co-culture models like the one presented here to provide more representative in vitro cardiac constructs.
    Keywords:  cardiac fibroblast; cardiomyocyte; extracellular matrix; human pluripotent stem cell; micropattern
  39. J Pediatr. 2021 Oct 04. pii: S0022-3476(21)00954-9. [Epub ahead of print]
      OBJECTIVE: To determine changes of mitochondrial DNA (mtDNA) copy number in peripheral blood in Rett syndrome caused by methyl-CpG-binding protein-2 (MECP2) variants and explore the mechanism of mitochondrial dysfunction in RTT.STUDY DESIGN: Female patients who were diagnosed as RTT and had MECP2 variants (n = 142) were recruited in this study, compared with the same number of age- and sex-matched healthy control group. MtDNA copy number was quantified by real-time quantitative polymerase chain reaction with TaqMan probes. The differences in mtDNA copy number between the RTT group and the control group were analyzed by the independent sample t-test. Linear regression, biserial correlation analysis, and one-way analysis of variance were applied for the correlations between age, clinical severity, variant types, functional domains, hot-spot variants and mtDNA copy number.
    RESULTS: MtDNA copy number was found to increase significantly in RTT patients with MECP2 gene variants than in control subjects in this study. Age, clinical severity, variant types, functional domains, and hot-spot variants were not related to mtDNA copy number in RTT patients.
    CONCLUSIONS: The mtDNA copy number of RTT patients has increased significantly, suggesting that changes in mitochondrial function in RTT patients trigger a compensatory increase in mtDNA copy number, and providing new possibilities for RTT treatments such as mitochondria-targeted therapies.
    Keywords:  RT; qPCR
  40. J Am Soc Nephrol. 2021 Oct 04. pii: ASN.2021030333. [Epub ahead of print]
      Background Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all tubulopathy patients lack a genetic diagnosis. Methods We performed whole-exome and genome sequencings of a patient cohort with a novel inherited salt-losing tubulopathy, hypomagnesemia, and dilated cardiomyopathy. We also conducted subsequent functional analyses in vitro of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). Results In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD encoded by RRAGD plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro Conclusions Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.
  41. Bio Protoc. 2021 Sep 05. 11(17): e4140
      Missense mutations in leucine rich-repeat kinase 2 (LRRK2) cause forms of familial Parkinson's disease and have been linked to 'idiopathic' Parkinson's disease. Assessment of LRRK2 kinase activity has been very challenging due to its size, complex structure, and relatively low abundance. A standard in the field to assess LRRK2 kinase activity is to measure the level of substrate phosphorylation (pThr73-Rab10) or autophosphorylation of serine 1292 (i.e., phosphoserine 1292; pS1292). The levels of pS1292 have typically been assessed by western blotting, which limits cellular and anatomical resolution. Here, we describe the method for a novel proximity ligation assay (PLA) that can detect endogenous LRRK2 kinase activity (PLA LRRK2) in situ at cellular and subcellular resolutions. PLA is a fluorescence- or chromogen-based assay that can be used to either (1) detect protein-protein interactions or (2) detect and amplify post-translational modifications on proteins. We used PLA for in situ detection and amplification of LRRK2 autophosphorylation levels at serine 1292. Our findings demonstrate that PLA LRRK2 is a highly sensitive and specific assay that can be used for assessing kinase activity in cultured cells and postmortem tissues.
    Keywords:  Fluorescence; LRRK2; Parkinson’s disease; Proximity ligation assay
  42. Methods Mol Biol. 2021 Oct 06.
      Polysome profiling is a technique that uses sucrose density gradient ultracentrifugation to separate complexes of mRNAs associated with one or more ribosomes. Here we describe polysome profiling analysis in human pluripotent stem cells (hPSCs) using a continuous ultraviolet spectrophotometer and a gradient fractionator. We provide protocols for processing sucrose gradient fractions for isolation of RNA for RT-qPCR or large-scale sequencing analysis, used to establish the translational status of specific mRNAs and identify the role of noncoding RNA in translation.
    Keywords:  Pluripotent stem cells; Polysome profiling analysis; RNA isolation; Ribosome; Sucrose density gradient
  43. Respir Physiol Neurobiol. 2021 Oct 05. pii: S1569-9048(21)00185-3. [Epub ahead of print] 103799
      A computer model of the skeletal muscle bioenergetic system, involving the "Pi double-threshold" mechanism of muscle fatigue, was used to investigate the effect of muscle training on system kinetic properties in mitochondrial myopathies (MM) patients with inborn OXPHOS deficiencies. An increase in OXPHOS activity and decrease in peak Pi can account for the training-induced increase in V̇O2max, acceleration of the primary phase II of the V̇O2 on-kinetics, delay of muscle fatigue and prolongation of exercise at a given work intensity encountered in experimental studies. Depending on the mutation load and work intensity, training can bring the muscle from severe- to very-heavy- to moderate-exercise-like behavior, thus lessening the exertional fatigue and lengthening the physical activity of a given intensity. Training significantly increases critical power (CP) and slightly decreases the curvature constant (W') of the power-duration relationship. Generally, a mechanism underlying the training-induced changes in the skeletal muscle bioenergetic system in MM patients is proposed.
    Keywords:  OXPHOS deficiencies; V̇O(2) on-kinetics; V̇O(2max); exercise duration; mitochondrial myopathies; muscle training
  44. Br J Pharmacol. 2021 Oct 04.
      BACKGROUND AND PURPOSE: Ca2+ influx via TRPV4 triggers Ca2+ release from the IP3 -sensitive internal store to generate repetitive oscillations. While mitochondria are acknowledged regulators of IP3 -mediated Ca2+ release, how TRPV4-mediated Ca2+ signals are regulated by mitochondria is unknown. We show that depolarised mitochondria switch TRPV4 signalling from relying on Ca2+ -induced Ca2+ release at IP3 receptors, to being independent of Ca2+ influx and instead mediated by ATP release via pannexins.EXPERIMENTAL APPROACH: TRPV4 evoked Ca2+ signals were individually examined in hundreds of cells in the endothelium of rat mesenteric resistance arteries using the indicator Cal520.
    KEY RESULTS: TRPV4 activation with GSK1016790A(GSK) generated repetitive Ca2+ oscillations that required Ca2+ influx. However, when the mitochondrial membrane potential was depolarised, by the uncoupler CCCP or complex I inhibitor rotenone, TRPV4 activation generated large propagating, multicellular, Ca2+ waves in the absence of external Ca2+ . The ATP synthase inhibitor oligomycin did not potentiate TRPV4 mediated Ca2+ signals. GSK-evoked Ca2+ waves, when mitochondria were depolarised, were blocked by the TRPV4 channel blocker HC067047, the SERCA inhibitor cyclopiazonic acid, the phospholipase C (PLC) blocker U73122 and the inositol triphosphate receptor (IP3 R) blocker caffeine. The Ca2+ waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid.
    CONCLUSION AND IMPLICATIONS: These results highlight a previously unknown role of mitochondria in shaping TRPV4 mediated Ca2+ signalling by facilitating ATP release. When mitochondria are depolarised, TRPV4-mediated release of ATP via pannexin channels activates plasma membrane purinergic receptors to trigger IP3 evoked Ca2+ release.
    Keywords:  Intercellular Ca2+ waves; Intercellular communication; Mitochondria; Pannexin; Purinergic receptors; TRPV4; Vascular; endothelium; inositol 1,4,5-trisphosphate (IP3)
  45. Elife. 2021 Oct 06. pii: e67886. [Epub ahead of print]10
      Human organoid systems recapitulate key features of organs offering platforms for modelling developmental biology and disease. Tissue-derived organoids have been widely used to study the impact of extrinsic niche factors on stem cells. However, they are rarely used to study endogenous gene function due to the lack of efficient gene manipulation tools. Previously, we established a human foetal lung organoid system (Nikolić et al., 2017). Here, using this organoid system as an example we have systematically developed and optimised a complete genetic toolbox for use in tissue-derived organoids. This includes 'Organoid Easytag' our efficient workflow for targeting all types of gene loci through CRISPR-mediated homologous recombination followed by flow cytometry for enriching correctly-targeted cells. Our toolbox also incorporates conditional gene knock-down or overexpression using tightly-inducible CRISPR interference and CRISPR activation which is the first efficient application of these techniques to tissue-derived organoids. These tools will facilitate gene perturbation studies in tissue-derived organoids facilitating human disease modelling and providing a functional counterpart to many on-going descriptive studies, such as the Human Cell Atlas Project.
    Keywords:  developmental biology; human
  46. BMC Neurol. 2021 Oct 02. 21(1): 382
      BACKGROUND: The genetics of cerebellar ataxia is complex. Hundreds of causative genes have been identified, but only a few cause more than single cases. The spectrum of ataxia-causing genes differs considerably between populations. The aim of the study was to investigate the molecular epidemiology of ataxia in the Finnish population.PATIENTS AND METHODS: All patients in hospital database were reviewed for the diagnosis of unspecified ataxia. Acquired ataxias and nongenetic ataxias such as those related to infection, trauma or stroke were excluded. Sixty patients with sporadic ataxia with unknown etiology and 36 patients with familial ataxia of unknown etiology were recruited in the study. Repeat expansions in the SCA genes (ATXN1, 2, 3, 7, 8/OS, CACNA1A, TBP), FXN, and RFC1 were determined. Point mutations in POLG, SPG7 and in mitochondrial DNA (mtDNA) were investigated. In addition, DNA from 8 patients was exome sequenced.
    RESULTS: A genetic cause of ataxia was found in 33 patients (34.4%). Seven patients had a dominantly inherited repeat expansion in ATXN8/OS. Ten patients had mitochondrial ataxia resulting from mutations in nuclear mitochondrial genes POLG or RARS2, or from a point mutation m.8561C > G or a single deletion in mtDNA. Interestingly, five patients were biallelic for the recently identified pathogenic repeat expansion in RFC1. All the five patients presented with the phenotype of cerebellar ataxia, neuropathy, and vestibular areflexia (CANVAS). Moreover, screening of 54 patients with Charcot-Marie-Tooth neuropathy revealed four additional patients with biallelic repeat expansion in RFC1, but none of them had cerebellar symptoms.
    CONCLUSIONS: Expansion in ATXN8/OS results in the majority of dominant ataxias in Finland, while mutations in RFC1 and POLG are the most common cause of recessive ataxias. Our results suggest that analysis of RFC1 should be included in the routine diagnostics of idiopathic ataxia and Charcot-Marie-Tooth polyneuropathy.
    Keywords:  CANVAS; Hereditary ataxia; Molecular epidemiology; Repeat expansion
  47. J Clin Endocrinol Metab. 2021 Oct 06. pii: dgab725. [Epub ahead of print]
      BACKGROUND: Familial Partial Lipodystrophy (FPL), Dunnigan variety is characterized by skeletal muscle hypertrophy and insulin resistance besides fat loss from the extremities. The cause for the muscle hypertrophy, and its functional consequences is not known.OBJECTIVE: To compare muscle strength and endurance, besides muscle protein synthesis rate between subjects with FPL and matched controls (n = 6 in each group). In addition, we studied skeletal muscle mitochondrial function and gene expression pattern to help understand the mechanisms for the observed differences.
    METHODS: Body composition by DEXA, insulin sensitivity by minimal modelling, assessment of peak muscle strength and fatigue, skeletal muscle biopsy and calculation of muscle protein synthesis rate, mitochondrial respirometry, skeletal muscle transcriptome, proteome and gene set enrichment analysis.
    RESULTS: Despite increased muscularity, FPL subjects did not demonstrate increased muscle strength but had earlier fatigue on chest press exercise. Decreased mitochondrial state 3 respiration in the presence of fatty acid substrate was noted, concurrent to elevated muscle lactate and decreased long-chain acylcarnitine. Based on gene transcriptome, there was significant down regulation of many critical metabolic pathways involved in mitochondrial biogenesis and function. Moreover, the overall pattern of gene expression was indicative of accelerated aging in FPL subjects. A lower muscle protein synthesis and down regulation of gene transcripts involved in muscle protein catabolism was observed.
    CONCLUSION: Increased muscularity in FPL is not due to increased muscle protein synthesis and is likely due to reduced muscle protein degradation. Impaired mitochondrial function and altered gene expression likely explain the metabolic abnormalities and skeletal muscle dysfunction in FPL subjects.
    Keywords:  Lipodystrophy; Mitochondria; insulin resistance; skeletal muscle hypertrophy
  48. RNA Biol. 2021 Oct 04. 1-16
      RNA molecules are known to fold into specific structures which often play a central role in their functions and regulation. In silico folding of RNA transcripts, especially when assisted with structure profiling (SP) data, is capable of accurately elucidating relevant structural conformations. However, such methods scale poorly to the swaths of SP data generated by transcriptome-wide experiments, which are becoming more commonplace and advancing our understanding of RNA structure and its regulation at global and local levels. This has created a need for tools capable of rapidly deriving structural assessments from SP data in a scalable manner. One such tool we previously introduced that aims to process such data is patteRNA, a statistical learning algorithm capable of rapidly mining big SP datasets for structural elements. Here, we present a reformulation of patteRNA's pattern recognition scheme that sees significantly improved precision without major compromises to computational overhead. Specifically, we developed a data-driven logistic classifier which interprets patteRNA's statistical characterizations of SP data in addition to local sequence properties as measured with a nearest neighbour thermodynamic model. Application of the classifier to human structurome data reveals a marked association between detected stem-loops and RNA binding protein (RBP) footprints. The results of our application demonstrate that upwards of 30% of RBP footprints occur within loops of stable stem-loop elements. Overall, our work arrives at a rapid and accurate method for automatically detecting families of RNA structure motifs and demonstrates the functional relevance of identifying them transcriptome-wide.
    Keywords:  RNA binding proteins; RNA structure; machine learning; statistical models; transcriptome
  49. Front Cell Dev Biol. 2021 ;9 743892
      Mitochondria are double-membraned organelles that exhibit fluidity. They are the main site of cellular aerobic respiration, providing energy for cell proliferation, migration, and survival; hence, they are called "powerhouses." Mitochondria play an important role in biological processes such as cell death, cell senescence, autophagy, lipid synthesis, calcium homeostasis, and iron balance. Fission and fusion are active processes that require many specialized proteins, including mechanical enzymes that physically alter mitochondrial membranes, and interface proteins that regulate the interaction of these mechanical proteins with organelles. This review discusses the molecular mechanisms of mitochondrial fusion, fission, and physiopathology, emphasizing the biological significance of mitochondrial morphology and dynamics. In particular, the regulatory mechanisms of mitochondria-related genes and proteins in animal cells are discussed, as well as research trends in mitochondrial dynamics, providing a theoretical reference for future mitochondrial research.
    Keywords:  fission; fusion; machinery; mitochondrial dynamics; pathophysiology
  50. Ann N Y Acad Sci. 2021 Oct 03.
      Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17-19, 2021, experts in single cell biology met virtually for the Keystone eSymposium "Single Cell Biology" to discuss advances both in single cell applications and technologies.
    Keywords:  development; differentiation; lineage tracing; reprogramming; single cell sequencing; spatial transcriptomics
  51. Curr Protoc. 2021 Oct;1(10): e263
      Skeletal muscle stem cells (MuSCs) reside in a complex niche composed of the muscle fiber plasma membrane and the laminin-rich basal lamina surrounded by the microvasculature, as well as different supportive cell types such as fibro-adipogenic progenitors residing in the interstitial extracellular matrix. Within the first few hours after tissue damage, MuSCs undergo cytoskeletal rearrangements and transcriptional changes that prime the cells for activation. Due to their time-consuming nature, enzymatic methods for liberation of single muscle fibers with fully quiescent MuSCs are challenging. Moreover, during enzymatic digestion, important niche components including the microvasculature and the collagenous interstitial matrix are destroyed. Here, we provide a method for the visualization of MuSCs on muscle fibers in their intact niche. Our method relies on mechanical teasing of fiber bundles from fixed skeletal muscles. We demonstrate that teased muscle fiber bundles allow the investigator to capture a representative snapshot of the MuSC niche in skeletal muscle, and outline how stem cell morphology and different microenvironmental components can be visualized. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of fiber bundles Basic Protocol 2: Immunofluorescence staining of MuSCs on fiber bundles Support Protocol: Preparation of Sylgard dishes.
    Keywords:  MuSCs; microenvironment; quiescence; satellite cells; skeletal muscle; stem cell niche
  52. Oxid Med Cell Longev. 2021 ;2021 3259963
      The normal function of the mitochondria is crucial for most tissues especially for those that demand a high energy supply. Emerging evidence has pointed out that healthy mitochondrial function is closely associated with normal heart function. When these processes fail to repair the damaged mitochondria, cells initiate a removal process referred to as mitophagy to clear away defective mitochondria. In cardiomyocytes, mitophagy is closely associated with metabolic activity, cell differentiation, apoptosis, and other physiological processes involved in major phenotypic alterations. Mitophagy alterations may contribute to detrimental or beneficial effects in a multitude of cardiac diseases, indicating potential clinical insights after a close understanding of the mechanisms. Here, we discuss the current opinions of mitophagy in the progression of cardiac diseases, such as ischemic heart disease, diabetic cardiomyopathy, cardiac hypertrophy, heart failure, and arrhythmia, and focus on the key molecules and related pathways involved in the regulation of mitophagy. We also discuss recently reported approaches targeting mitophagy in the therapy of cardiac diseases.
  53. Elife. 2021 10 05. pii: e69207. [Epub ahead of print]10
      The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.
    Keywords:  GJA1-20k; actin dynamics; cell biology; human; ischemia/reperfusion; mitochondria; mitochondria dynamics; mouse; organ protection
  54. Cardiovasc Res. 2021 Oct 05. pii: cvab311. [Epub ahead of print]
      AIMS: Remdesivir is a prodrug of an adenosine triphosphate analogue and is currently the only drug formally approved for the treatment of hospitalised COVID-19 patients. Nucleoside/nucleotide analogues have been shown to induce mitochondrial damage and cardiotoxicity, and this may be exacerbated by hypoxia, which frequently occurs in severe COVID-19 patients. Although there have been few reports of adverse cardiovascular events associated with remdesivir, clinical data are limited. Here, we investigated whether remdesivir induced cardiotoxicity using an in vitro human cardiac model.METHODS AND RESULTS: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were exposed to remdesivir under normoxic and hypoxic conditions to simulate mild and severe COVID-19 respectively. Remdesivir induced mitochondrial fragmentation, reduced redox potential and suppressed mitochondrial respiration at levels below the estimated plasma concentration under both normoxic and hypoxic conditions. Non-mitochondrial damage such as electrophysiological alterations and sarcomere disarray were also observed. Importantly, some of these changes persisted after the cessation of treatment, culminating in increased cell death. Mechanistically, we found that inhibition of DRP1, a regulator of mitochondrial fission, ameliorated the cardiotoxic effects of remdesivir, showing that remdesivir-induced cardiotoxicity was preventable and excessive mitochondrial fission might contribute to this phenotype.
    CONCLUSIONS: Using an in vitro model, we demonstrated that remdesivir can induce cardiotoxicity in hiPSC-CMs at clinically relevant concentrations. These results reveal previously unknown potential side-effects of remdesivir and highlight the importance of further investigations with in vivo animal models and active clinical monitoring to prevent lasting cardiac damage to patients.
    TRANSLATIONAL PERSPECTIVE: Adult cardiomyocytes have limited ability to regenerate, thus treatment-induced cardiotoxicity can potentially cause irreparable harm. Remdesivir is currently the only FDA approved treatment for COVID-19 but clinical safety data are limited. Using human pluripotent stem cell-derived cardiomyocytes, we revealed that remdesivir induced persistent mitochondrial and structural abnormalities at clinically relevant concentrations. We advise confirmatory experiments in in vivo animal models, investigations of cardioprotective strategies, and closer patient monitoring such that treatment-induced cardiotoxicity does not contribute to the long term sequelae of COVID-19 patients.
    Keywords:  COVID-19; cardiotoxicity; human pluripotent stem cell derived cardiomyocytes; mitochondria; remdesivir
  55. Redox Biol. 2021 Oct 01. pii: S2213-2317(21)00313-X. [Epub ahead of print]47 102153
      Protein cysteine residues are essential for protein folding, participate in enzymatic catalysis, and coordinate the binding of metal ions to proteins. Enzymatically catalyzed and redox-dependent post-translational modifications of cysteine residues are also critical for signal transduction and regulation of protein function and localization. S-nitrosylation, the addition of a nitric oxide equivalent to a cysteine residue, is a redox-dependent modification. In this study, we curated and analyzed four different studies that employed various chemoselective platforms coupled to mass spectrometry to precisely identify S-nitrosocysteine residues in mouse heart proteins. Collectively 1974 S-nitrosocysteine residues in 761 proteins were identified and 33.4% were identified in two or more studies. A core of 75 S-nitrosocysteine residues in 44 proteins were identified in all four studies. Bioinformatic analysis of each study indicated a significant enrichment of mitochondrial proteins participating in metabolism. Regulatory proteins in glycolysis, TCA cycle, oxidative phosphorylation and ATP production, long chain fatty acid β-oxidation, and ketone and amino acid metabolism constitute the major functional pathways impacted by protein S-nitrosylation. In the cardiovascular system, nitric oxide signaling regulates vasodilation and cardiac muscle contractility. The meta-analysis of the proteomic data supports the hypothesis that nitric oxide signaling via protein S-nitrosylation is also a regulator of cardiomyocyte metabolism that coordinates fuel utilization to maximize ATP production. As such, protein cysteine S-nitrosylation represents a third functional dimension of nitric oxide signaling in the cardiovascular system to ensure optimal cardiac function.
    Keywords:  Cardiovascular system; Nitric oxide; Proteomics; S-nitrosylation
  56. Front Cell Dev Biol. 2021 ;9 736603
      Sepsis-induced cardiac injury (SIC) is one of the most common complications in the intensive care unit (ICU) with high morbidity and mortality. Mitochondrial dysfunction is one of the main reasons for SIC, and Interleukin-13 (IL-13) is a master regulator of mitochondria biogenesis. The aim of the present study was to investigate the role of IL-13 in SIC and explore the underlying mechanism. It was found that reactive oxygen species (ROS) production and apoptosis were significantly increased in lipopolysaccharide (LPS)-stimulated primary cardiomyocytes, which was accompanied with obvious mitochondria dysfunction. The results of RNA-sequencing (RNA-seq), mitochondrial membrane potential, fatty acid uptake and oxidation rate suggested that treatment with IL-13 could restore the function and morphology of mitochondria, indicating that it played an important role in protecting septic cardiomyocytes. These findings demonstrated that IL-13 alleviated sepsis-induced cardiac inflammation and apoptosis by improving mitochondrial fatty acid uptake and oxidation, suggesting that IL-13 may prove to be a potential promising target for SIC treatment.
    Keywords:  IL-13; cardiomyocyte apoptosis; fatty acid; mitochondria; sepsis
  57. Methods Mol Biol. 2021 Oct 06.
      The generation of cardiomyocytes (CMs) and endothelial cells (ECs) from human induced pluripotent stem cells (iPSCs) allows for precise modeling of cardiovascular disease using clinically relevant and patient-specific cells. Differentiation of human iPSCs into cardiomyocytes (iPSC-CMs) and endothelial cells (iPSC-ECs) is governed by small molecules that regulate the WNT signaling pathway. Here we outline the detailed steps to generate iPSC-CMs and iPSC-ECs through small molecule-mediated monolayer differentiation.
    Keywords:  Cardiomyocytes; Endothelial cells; Human induced pluripotent stem cells (iPSCs); iPSC-CMs; iPSC-ECs
  58. Nature. 2021 Oct 06.
      The enzymes of the mitochondrial electron transport chain are key players of cell metabolism. Despite being active when isolated, in vivo they associate into supercomplexes1, whose precise role is debated. Supercomplexes CIII2CIV1-2 (refs. 2,3), CICIII2 (ref. 4) and CICIII2CIV (respirasome)5-10 exist in mammals, but in contrast to CICIII2 and the respirasome, to date the only known eukaryotic structures of CIII2CIV1-2 come from Saccharomyces cerevisiae11,12 and plants13, which have different organization. Here we present the first, to our knowledge, structures of mammalian (mouse and ovine) CIII2CIV and its assembly intermediates, in different conformations. We describe the assembly of CIII2CIV from the CIII2 precursor to the final CIII2CIV conformation, driven by the insertion of the N terminus of the assembly factor SCAF1 (ref. 14) deep into CIII2, while its C terminus is integrated into CIV. Our structures (which include CICIII2 and the respirasome) also confirm that SCAF1 is exclusively required for the assembly of CIII2CIV and has no role in the assembly of the respirasome. We show that CIII2 is asymmetric due to the presence of only one copy of subunit 9, which straddles both monomers and prevents the attachment of a second copy of SCAF1 to CIII2, explaining the presence of one copy of CIV in CIII2CIV in mammals. Finally, we show that CIII2 and CIV gain catalytic advantage when assembled into the supercomplex and propose a role for CIII2CIV in fine tuning the efficiency of electron transfer in the electron transport chain.
  59. BMC Bioinformatics. 2021 Oct 02. 22(1): 474
      BACKGROUND: The Sequence Alignment/Map Format Specification (SAM) is one of the most widely adopted file formats in bioinformatics and many researchers use it daily. Several tools, including most high-throughput sequencing read aligners, use it as their primary output and many more tools have been developed to process it. However, despite its flexibility, SAM encoded files can often be difficult to query and understand even for experienced bioinformaticians. As genomic data are rapidly growing, structured, and efficient queries on data that are encoded in SAM/BAM files are becoming increasingly important. Existing tools are very limited in their query capabilities or are not efficient. Critically, new tools that address these shortcomings, should not be able to support existing large datasets but should also do so without requiring massive data transformations and file infrastructure reorganizations.RESULTS: Here we introduce SamQL, an SQL-like query language for the SAM format with intuitive syntax that supports complex and efficient queries on top of SAM/BAM files and that can replace commonly used Bash one-liners employed by many bioinformaticians. SamQL has high expressive power with no upper limit on query size and when parallelized, outperforms other substantially less expressive software.
    CONCLUSIONS: SamQL is a complete query language that we envision as a step to a structured database engine for genomics. SamQL is written in Go, and is freely available as standalone program and as an open-source library under an MIT license, .
    Keywords:  BAM; Big data; Genomics; SAM; SQL
  60. Genet Mol Biol. 2021 ;pii: S1415-47572021000600102. [Epub ahead of print]44(4): 20210061
      Next-generation sequencing (NGS) has altered clinical genetic testing by widening the access to molecular diagnosis of genetically determined rare diseases. However, physicians may face difficulties selecting the best diagnostic approach. Our goal is to estimate the rate of possible molecular diagnoses missed by different targeted gene panels using data from a cohort of patients with rare genetic diseases diagnosed with exome sequencing (ES). For this purpose, we simulated a comparison between different targeted gene panels and ES: the list of genes harboring clinically relevant variants from 158 patients was used to estimate the theoretical rate of diagnoses missed by NGS panels from 53 different NGS panels from eight different laboratories. Panels presented a mean rate of missed diagnoses of 64% (range 14%-100%) compared to ES, representing an average predicted sensitivity of 36%. Metabolic abnormalities represented the group with highest mean of missed diagnoses (86%), while seizure represented the group with lowest mean (46%). Focused gene panels are restricted in covering select sets of genes implicated in specific diseases and they may miss molecular diagnoses of rare diseases compared to ES. However, their role in genetic diagnosis remains important especially for well-known genetic diseases with established genetic locus heterogeneity.
  61. JACC CardioOncol. 2021 Sep;3(3): 441-443
    Keywords:  anthracycline; cardiomyopathy; heart failure
  62. Clin Chim Acta. 2021 Oct 05. pii: S0009-8981(21)00345-4. [Epub ahead of print]
      BACKGROUND: Neuromuscular disorders (NMDs) encompass a large group of genetic and acquired diseases affecting muscles, leading to progressive muscular weakness. These disorders are frequently inherited in an autosomal-recessive (AR) pattern with extreme heterogeneity and various clinical presentations. Consanguinity increases the likelihood of AR disorders, with high rates of cousin inbreeding in Jordan and other Arab countries. In Jordan, the implementation of genetic diagnosis is limited, with delayed or misdiagnosis of genetic disorders. Thus, the lack of genetic counselling and specialized treatment options is frequently encountered in the country.METHODS: Whole-exome sequencing (WES) was conducted for eleven probands from ten Jordanian families who have been formerly diagnosed with limb-girdle dystrophy (LGMD) and Charcot-Marie-Tooth disease (CMT). The previous diagnoses were established principally on clinical examination in the absence of genetic testing. Additionally, Sanger sequencing and segregation analysis were used to validate the resulted pathogenic variants.
    RESULTS: Multiple variants were identified using WES: For DYSF gene, a missense variant (c. 4076T>C, p.Leu1359Pro) in exon 38; a nonsense variant (c. 4321C>T, p.Gln1441Ter) in exon 39; a single-nucleotide deletion (c. 5711delG, p.Gly1904AlafsTer101) in exon 51. Other variants included a missense variant (c. 122G>A, p.Arg41Gln) in exon 3 of MPV17 gene, a single-nucleotide deletion (c. 859 delC, p.Lue287Ser fs14*) in exon 6 of SGCB gene, a missense variant (c. 311G>A, p.Gly104Asp) in exon 2 of SLC25A46 gene, a nonsense variant (c. 496C>T, p.Arg166Ter) in exon 5 of SGCG gene, and a nonsense variant (c.3202C>T, p.Gln1068Ter) in exon 13 of SH3TC2 gene.
    CONCLUSION: Utilization of WES is helpful to facilitate rapid and accurate NMDs diagnosis, complementing a thorough clinical evaluation. This approach can be invaluable to aid in the identification of genetic risks among consanguineous couples. Subsequently, well-informed genetic counselling and potential individualized treatment can be provided.
    Keywords:  Whole-exome sequencing; autosomal-recessive; consanguinity; genetic testing; movement disorders
  63. Front Pharmacol. 2021 ;12 707399
      Energic deficiency of cardiomyocytes is a dominant cause of heart failure. An antianginal agent, trimetazidine improves the myocardial energetic supply. We presumed that trimetazidine protects the cardiomyocytes from the pressure overload-induced heart failure through improving the myocardial metabolism. C57BL/6 mice were subjected to transverse aortic constriction (TAC). After 4 weeks of TAC, heart failure was observed in mice manifested by an increased left ventricular (LV) chamber dimension, an impaired LV ejection fraction evaluated by echocardiography analysis, which were significantly restrained by the treatment of trimetazidine. Trimetazidine restored the mitochondrial morphology and function tested by cardiac transmission electron microscope and mitochondrial dynamic proteins analysis. Positron emission tomography showed that trimetazidine significantly elevated the glucose uptake in TAC mouse heart. Trimetazidine restrained the impairments of the insulin signaling in TAC mice and promoted the translocation of glucose transporter type IV (GLUT4) from the storage vesicle to membrane. However, these cardioprotective effects of trimetazidine in TAC mice were notably abolished by compound C (C.C), a specific AMPK inhibitor. The enlargement of neonatal rat cardiomyocyte induced by mechanical stretch, together with the increased expression of hypertrophy-associated proteins, mitochondria deformation and dysfunction were significantly ameliorated by trimetazidine. Trimetazidine enhanced the isolated cardiomyocyte glucose uptake in vitro. These benefits brought by trimetazidine were also removed with the presence of C.C. In conclusion, trimetazidine attenuated pressure overload-induced heart failure through improving myocardial mitochondrial function and glucose uptake via AMPK.
    Keywords:  AMPK; heart failure; myocardial metabolism; pressure overload; trimetazidine