Novel Pan-ERR Agonists Ameliorate Heart Failure Through Enhancing Cardiac Fatty Acid Metabolism and Mitochondrial Function

BACKGROUND: Cardiac metabolic dysfunction is a hallmark of heart failure (HF). Estrogen-related receptors ERRα and ERRγ are essential regulators of cardiac metabolism. Therefore, activation of ERR could be a potential therapeutic intervention for HF. However, in vivo studies demonstrating the potential usefulness of ERR agonist for HF treatment are lacking, because compounds with pharmacokinetics appropriate for in vivo use have not been available. METHODS: Using a structure-based design approach, we designed and synthesized 2 structurally distinct pan-ERR agonists, SLU-PP-332 and SLU-PP-915. We investigated the effect of ERR agonist on cardiac function in a pressure overload-induced HF model in vivo. We conducted comprehensive functional, multi-omics (RNA sequencing and metabolomics studies), and genetic dependency studies both in vivo and in vitro to dissect the molecular mechanism, ERR isoform dependency, and target specificity. RESULTS: Both SLU-PP-332 and SLU-PP-915 significantly improved ejection fraction, ameliorated fibrosis, and increased survival associated with pressure overload-induced HF without affecting cardiac hypertrophy. A broad spectrum of metabolic genes was transcriptionally activated by ERR agonists, particularly genes involved in fatty acid metabolism and mitochondrial function. Metabolomics analysis showed substantial normalization of metabolic profiles in fatty acid/lipid and tricarboxylic acid/oxidative phosphorylation metabolites in the mouse heart with 6-week pressure overload. ERR agonists increase mitochondria oxidative capacity and fatty acid use in vitro and in vivo. Using both in vitro and in vivo genetic dependency experiments, we show that ERRγ is the main mediator of ERR agonism-induced transcriptional regulation and cardioprotection and definitively demonstrated target specificity. ERR agonism also led to downregulation of cell cycle and development pathways, which was partially mediated by E2F1 in cardiomyocytes. CONCLUSIONS: ERR agonists maintain oxidative metabolism, which confers cardiac protection against pressure overload-induced HF in vivo. Our results provide direct pharmacologic evidence supporting the further development of ERR agonists as novel HF therapeutics

MiRNAs as regulators of gene expression modulate development and energy metabolism of skeletal muscle

It is important to understand the molecular networks affecting biological properties of muscle in order to improve the efficiency of meat production and meat quality in domestic animals. The discovery of miRNA represents an important breakthrough in biology in recent years. MiRNA function identification has become one of the active research fields in muscle biology addressing muscle development, growth and metabolism. This thesis aims at the identification of miRNAs differentially expressed in skeletal muscle at various developmental stages and in pig breeds differing in muscularity. Moreover, links between miRNAs and mRNAs should be shown in order to address biofunctions affected by miRNAs in muscle. Finally, miRNAs impacted on muscle metabolism should be validated exemplarity by in vitro cell culture experimants. The first approach demonstrates the comprehensive miRNA expression profiles of longissimus dorsi (LD) during muscle development and growth. A comparative study on two distinct phenotypic pigs were performed using miRNA custom designed arrays. Two different key stages 63 and 91 days post-conception (dpc), and one adult stage (180 days post-natum) were analysed in German Landrace (DL) and Pietrain (Pi) breeds. Several potential candidate miRNAs are significantly up-regulated and associated with muscular developmental stages and breed types. The Affymetrix GeneChip porcine genome microarrays were also used to obtain the differential transcriptional profile of mRNA targets of the same animals. The combination of miRNA–mRNA expression data and Ingenuity Pathway Analysis established complex miRNA–dependent regulatory networks. A number of miRNA–mRNA interactions, that were associated to cellular growth and proliferation and lipid-metabolism functions, revealed insights into their role during skeletal muscle development and growth. The second approach involves in muscle growth in post mortem pig traits (crossbred [PI×(DL×DE)] population, n = 207). The experiment integrated miRNA and mRNA expression together with network analysis by using weighted gene co-expression network analysis (WGCNA). In this part, we identified the negative miRNA-mRNA co-expression networks which revealed several biological pathways underlying the difference of meat properties and muscle traits (i.e. glucose metabolic process, mitochondrial ribosome and oxidative phosphorylation). In the last approach, C2C12 in vitro model studies revealed that miRNAs are modulated in cellular ATP production and energy metabolism processes during myogenic differentiation. Correlation analyses were performed between ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation. Among 14 significant miRNAs as representing cellular ATP regulators involved in mitochondrial energy metabolism, miR-423-3p is a novel regulator for cellular ATP/ energy metabolism via targeting the group of mitochondrial energy metabolism genes (Cox6a2, Ndufb7, and Ndufs5). In conclusion, the present study further adds a comprehensive knowledge on the systems perspective of the skeletal muscle miRNAs and their target genes regulation networks that influence on skeletal muscle starting from early muscle development to mature muscle growth.MiRNAs regulieren die Genexpression und modulieren die Entwicklung und den Energiestoffwechsel der Skelettmuskulatur Das Verständnis von molekularen Netzwerken mit Einfluss auf die biologischen Eigenschaften des Muskels ist notwendig, um die Effizienz der Fleischproduktion und die Fleischqualität in Nutztieren zu verbessern. Die Erforschung von miRNAs stellt einen entscheidenden Durchbruch in der Biologie in den letzten Jahren dar. Die Identifizierung von miRNA-Funktionen wurde seit dem eines der aufstrebenden Forschungsschwerpunkte in der Muskelbiologie mit Bezug auf Muskelentwicklung, -wachstum und -stoffwechsel. Das Ziel dieser Dissertation ist die Identifizierung von miRNAs mit differenzieller Expression in der Skelettmuskulatur im Hinblick auf verschiedene Entwicklungsstadien und Schweinerassen mit unterschiedlichem Muskelansatz. Im Weiteren soll die Verknüpfung von miRNA- und mRNA-Datensätzen helfen, durch miRNA beeinflusste Biofunktionen im Muskel zu benennen. Abschließend sollen exemplarisch einige miRNAs mit Einfluss auf den Muskelmetabolismus durch in vitro Zellkulturstudien validiert werden. Der erste Forschungsansatz lieferte umfassende miRNA-Expressionsprofile des longissimus dorsi (LD) während der Muskelentwicklung und des Wachstums. Dazu wurden Schweine mit unterschiedlicher phänotypischer Ausprägung unter der Verwendung von spezifisch gefertigten miRNA-Arrays vergleichend analysiert. Tiere der Deutschen Landrasse (DL) und der Rasse Pietrain (Pi) wurden zu zwei wesentlichen pränatalen Entwicklungszeitpunkten (am 63 und 91 Tag nach Empfängnis) sowie im adulten Stadium (180 Tage nach Geburt) untersucht. Für zahlreiche potentielle Kandidaten-miRNAs konnte gezeigt werden, dass diese signifikant hochreguliert sind und Assoziationen zu muskulären Entwicklungsstadien und der Rasse aufzeigten. Zusätzlich wurden porcine Genommikroarrays (Affymetrix GeneChip) verwendet um Profile der differentiell exprimierten mRNA-targets im gleichen Tier zu untersuchen. Durch die Kombination von miRNA- und mRNA-Expressionsdaten gekoppelt mit Ergebnissen aus der Analyse von regulierten Signalwegen (Ingenuity pathway analysis) konnte ein Komplex aus miRNA-abhängigen regulatorischen Netzwerken etabliert werden. Zahlreiche miRNA-mRNA-Interaktionen im Zusammenhang mit Funktionen des zellulären Wachstums, der Proliferation und des Fettstoffwechsels, ermöglichten Einblicke in die Funktion dieser Wechselwirkungen während der Entwicklung und des Wachstums der Skelettmuskulatur. Der zweite Forschungsansatz berücksichtigt das Muskelwachstum in relevanten post mortem Merkmalen (Kreuzungsrasse [Pi x (DLxDE), n=207). Für diesen Ansatz wurden die Expressionsdaten der miRNA- und mRNA-Analysen in einem Ko-Expressionsnetzwerk integriert. Dabei wurden die Wechselwirkungen zwischen verschiedenen Komponenten berücksichtigt und gewichtet. Negative miRNA-mRNA-Ko-Expressionsnetzwerke konnten identifiziert werden. Diese deuten auf biologisch relevante Signalwegen hin, welche mit unterschiedlichen Ausprägungen der Fleischeigenschaften und Merkmalen der Muskulatur in Zusammenhang stehen (z.B. Prozesse des Glucosemetabolismus, mitochondriale Ribosomen und oxidative Phosphorylierung). Im abschließenden Forschungsansatz konnte durch Analysen des C2C12-Muskelzellmodells gezeigt werden, dass miRNAs im Zusammenhang mit der zellulären ATP-Produktion und mit Prozessen des Energiemetabolismus im Rahmen der myogenen Differenzierung reguliert werden. Dazu wurden zum Zeitpunkt der C2C12-Zelldifferenzierung ermittelte ATP-Gehalte und miRNA- und mRNA-Mikroarray-Expressionsprofile miteinander verknüpft. Unter den 14 miRNAs, die als zelluläre ATP-Regulatoren am mitochondrialen Energiemetabolismus beteiligt sind, konnte miR-423-3p, durch den Einfluss auf Gene aus der Gruppe des mitochondrialen Energiemetabolismus (Cox6a2, Ndufb7 und Ndufs5), als neuer Regulator für zelluläres ATP bzw. den Energiemetabolismus bestätigt werden. Zusammenfassend liefern die vorliegenden Studien wesentliche Erkenntnisse zu systemischen Funktionen der miRNAs in der Skelettmuskulatur und verdeutlichen ihren Einfluss auf Gennetzwerke, welche die Prozesse von der frühen Muskelentwicklung bis hin zum Muskelwachstum beeinflussen

Regulation and functional role of the electron transport chain supercomplexes.

Mitochondria are one of the most exhaustively investigated organelles in the cell and most attention has been paid to the components of the mitochondrial electron transport chain (ETC) in the last 100 years. The ETC collects electrons from NADH or FADH2 and transfers them through a series of electron carriers within multiprotein respiratory complexes (complex I to IV) to oxygen, therefore generating an electrochemical gradient that can be used by the F1-F0-ATP synthase (also named complex V) in the mitochondrial inner membrane to synthesize ATP. The organization and function of the ETC is a continuous source of surprises. One of the latest is the discovery that the respiratory complexes can assemble to form a variety of larger structures called super-complexes (SCs). This opened an unexpected level of complexity in this well-known and fundamental biological process. This review will focus on the current evidence for the formation of different SCs and will explore how they modulate the ETC organization according to the metabolic state. Since the field is rapidly growing, we also comment on the experimental techniques used to describe these SC and hope that this overview may inspire new technologies that will help to advance the field.S

Regulation and functional role of the electron transport chain supercomplexes

Mitochondria are one of the most exhaustively investigated organelles in the cell and most attention has been paid to the components of the mitochondrial electron transport chain (ETC) in the last 100 years. The ETC collects electrons from NADH or FADH2 and transfers them through a series of electron carriers within multiprotein respiratory complexes (complex I to IV) to oxygen, therefore generating an electrochemical gradient that can be used by the F1-F0-ATP synthase (also named complex V) in the mitochondrial inner membrane to synthesize ATP. The organization and function of the ETC is a continuous source of surprises. One of the latest is the discovery that the respiratory complexes can assemble to form a variety of larger structures called super-complexes (SCs). This opened an unexpected level of complexity in this well-known and fundamental biological process. This review will focus on the current evidence for the formation of different SCs and will explore how they modulate the ETC organization according to the metabolic state. Since the field is rapidly growing, we also comment on the experimental techniques used to describe these SC and hope that this overview may inspire new technologies that will help to advance the fiel

The Role of Microrna in Cardioprotection: Ischemic Preconditioning and Mesenchymal Stem Cell Paracrine Effects

Changes in gene expression and protein levels are an important aspect of cardioprotection in which short non-coding RNA known as miRNA may play a key regulatory role. We investigated the functions of several miRNAs in the context of two cardioprotective stimuli, ischemic preconditioning (IPC) and mesenchymal stem cell (MSC) paracrine effects. We hypothesized that downregulation of a set of miRNAs (miR-148a/b, miR-30b, and let-7a*) augments expression of protective heat shock proteins during IPC, and that MSC exosomes transfer miR-21 to cardiomyocytes, resulting in downregulation of pro-apoptotic genes and reduction of infarct size. IPC increased the level of Hsp70, Hsp90, and Hsp40 family members within 6 hours as measured by qPCR and Western blot. Luciferase reporter assays and miRNA mimic transfection and knockdown were used to confirm effects of miR-148a/b, miR-30b, and let-7a* on translation. Combinations of miRNAs had more pronounced effects than single miRNAs alone. Pretreatment with wild type exosomes, but not those lacking miR-21, reduced cell death in vitro, and decreased infarct size in mice. The wild type exosomes, and miR-21 mimic, decreased protein levels of the miR-21 target genes Fas Ligand, Programmed Cell Death 4, Phosphatase and Tensin Homolog, and Pellino1. In conclusion, a small set of miRNAs may act synergistically as regulatory nodes in a heat shock protein expression network after IPC. Future studies will test whether manipulation of this set of miRNAs can induce cardioprotection. miR-21 plays a key role in pro-survival paracrine effects mediated by MSC exosomes. Future studies will test whether MSC exosomes mediate regeneration as well as cardioprotection

Modelling neuronal mitochondrial aminoacyl-tRNA synthetase defects

Mitochondrial diseases cover a broad group of disorders caused by mitochondrial dysfunction, often affecting organs with high metabolic demand, such as the brain and skeletal muscle. Mutations in mitochondrial aminoacyl-tRNA synthetase (MT-ARS) genes, which are crucial for mitochondrial protein synthesis and energy production via oxidative phosphorylation, are implicated in a variety of severe neurological and multisystemic diseases. Among these, mutations in mitochondrial alanyl-tRNA synthetase (AARS2), mitochondrial glutamyl-tRNA synthetase (EARS2), and mitochondrial arginyl-tRNA synthetase (RARS2) cause distinct clinical manifestations including combined oxidative phosphorylation deficiency type 8 (COXPD8), characterised by leukoencephalopathy with ovarian failure, or cardiomyopathy, leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL), and pontocerebellar hypoplasia type 6 (PCH6). Despite their significance, the underlying pathological mechanisms remain poorly understood due to the lack of physiologically relevant human models, as current research relies primarily on non-human models or patient-derived fibroblasts that do not recapitulate the tissue-specific complexities of the nervous system. Here, I address this unmet need by applying tissue-specific human models of the nervous system to investigate the cellular impact of AARS2, EARS2 and RARS2 defects. First, patient fibroblasts were reprogrammed into neuronal cells, which allows for a direct comparison of different MT-ARS mutations. These in vitro models exhibit the tissue-specificity of MT-ARS mutations by showing selective loss of mitochondrial respiratory chain complexes in differentiated neurons but not in proliferating neural progenitor cells. RNA sequencing, immunoblotting and mitochondrial respiratory analysis of these models demonstrate varying degrees of mitochondrial dysfunction along with the activation of distinct compensatory mechanisms and cellular stress responses, including the integrated stress response, and impairment in neuronal development. Furthermore, I investigate how AARS2 mutations associated with two distinct phenotypes impact mitochondrial protein synthesis in neurons in vitro using a non-radioactive technique involving click chemistry. In addition to developing neuronal models of MT-ARS defects, I sought to complement the in vitro findings with a viable in vivo model of EARS2 deficiency. Given the housekeeping role of MT-ARS, homozygous mutations in these genes are often embryonically lethal and in vivo models are rarely viable. Zebrafish offer a unique advantage in this regard, enabling viable MT-ARS whole-body knockouts to investigate developmental and systemic consequences of these mutations. I present new findings on the pathogenicity of EARS2 mutations in early development and test potential therapeutic strategies for MT-ARS-related disorders in vivo using a transgenic zebrafish model. Ears2 knockout zebrafish presented with abnormal development, early lethality, reduced locomotor activity, and activation of cellular stress, which was partially rescued by amino acid supplementation

Novel Diagnostic and Therapeutic Approaches for Mitochondrial Disorders

Mitochondrial disorders are among the most common inherited genetic disorders, with a combined prevalence of 1:5,000. These are genetically, biochemically, and clinically heterogeneous disorders affecting any organ or tissue in the body. A poor understanding of gene-to-phenotype relationships and pathophysiological mechanisms has resulted in sometimes years-long diagnostic odysseys and a lack of curative therapies. Consequently, outcomes are often poor with most patients dying in early childhood. The aim of this project is to improve patient outlooks by using novel tools to address both the diagnostic and therapeutic challenges associated with mitochondrial disease. The diagnostic aspect of the study involved the creation of four interactive diagnostic resources which can complement next generation sequencing (NGS) technologies to achieve more rapid diagnoses for patients. MitoEpilepsy Map, MitoCardio Map, MitoLiver Map, and MitoMedicine Map were created to aid in the diagnosis of mitochondrial epilepsy, cardiomyopathy, liver disease, and the entirety of mitochondrial disease, respectively. These, maps were accurate in identifying candidate genes from clinical vignettes of genetically confirmed cases of mitochondrial disease in 69-100% of cases. These maps will be valuable resources for interpreting NGS results, hopefully facilitating quicker and more accurate genetic diagnoses for affected patients. The therapeutic aspect of the project aimed to develop a new treatment strategy for mitochondrial disease caused by nonsense mutations. Translational read-through therapy involves pharmacological incorporation of a near-cognate amino acid in place of a premature stop codon during translation. A systematic in vitro proof-of-principle study was performed in patient fibroblasts harbouring bi-allelic nonsense mutations in ten different mitochondrial disease genes. In five patient cell cultures, translational read-through therapy was able to restore transcript, protein, and mitochondrial function, thus demonstrating in vitro efficacy and paving the way for future clinical development. Together, these approaches help improve outcomes for patients suffering from mitochondrial disease