RESUMEN
SMYD1, a striated muscle-specific lysine methyltransferase, was originally shown to play a key role in embryonic cardiac development but more recently we demonstrated that loss of Smyd1 in the murine adult heart leads to cardiac hypertrophy and failure. However, the effects of SMYD1 overexpression in the heart and its molecular function in the cardiomyocyte in response to ischemic stress are unknown. In this study, we show that inducible, cardiomyocyte-specific overexpression of SMYD1a in mice protects the heart from ischemic injury as seen by a > 50% reduction in infarct size and decreased myocyte cell death. We also demonstrate that attenuated pathological remodeling is a result of enhanced mitochondrial respiration efficiency, which is driven by increased mitochondrial cristae formation and stabilization of respiratory chain supercomplexes within the cristae. These morphological changes occur concomitant with increased OPA1 expression, a known driver of cristae morphology and supercomplex formation. Together, these analyses identify OPA1 as a novel downstream target of SMYD1a whereby cardiomyocytes upregulate energy efficiency to dynamically adapt to the energy demands of the cell. In addition, these findings highlight a new epigenetic mechanism by which SMYD1a regulates mitochondrial energetics and functions to protect the heart from ischemic injury.
Asunto(s)
Músculo Esquelético , Miocitos Cardíacos , Animales , Ratones , Cardiomegalia/metabolismo , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Miocitos Cardíacos/metabolismoRESUMEN
[This corrects the article DOI: 10.1021/acsomega.2c00984.].
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The clinical use of genomic analysis has expanded rapidly resulting in an increased availability and utility of genomic information in clinical care. We have developed an infrastructure utilizing informatics tools and clinical processes to facilitate the use of whole genome sequencing data for population health management across the healthcare system. Our resulting framework scaled well to multiple clinical domains in both pediatric and adult care, although there were domain specific challenges that arose. Our infrastructure was complementary to existing clinical processes and well-received by care providers and patients. Informatics solutions were critical to the successful deployment and scaling of this program. Implementation of genomics at the scale of population health utilizes complicated technologies and processes that for many health systems are not supported by current information systems or in existing clinical workflows. To scale such a system requires a substantial clinical framework backed by informatics tools to facilitate the flow and management of data. Our work represents an early model that has been successful in scaling to 29 different genes with associated genetic conditions in four clinical domains. Work is ongoing to optimize informatics tools; and to identify best practices for translation to smaller healthcare systems.
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Heart disease is the leading cause of death in the developed world, and its comorbidities such as hypertension, diabetes, and heart failure are accompanied by major transcriptomic changes in the heart. During cardiac dysfunction, which leads to heart failure, there are global epigenetic alterations to chromatin that occur concomitantly with morphological changes in the heart in response to acute and chronic stress. These epigenetic alterations include the reversible methylation of lysine residues on histone proteins. Lysine methylations on histones H3K4 and H3K9 were among the first methylated lysine residues identified and have been linked to gene activation and silencing, respectively. However, much less is known regarding other methylated histone residues, including histone H4K20. Trimethylation of histone H4K20 has been shown to repress gene expression; however, this modification has never been examined in the heart. Here, we utilized immunoblotting and mass spectrometry to quantify histone H4K20 trimethylation in three models of cardiac dysfunction. Our results show that lysine methylation at this site is differentially regulated in the cardiomyocyte, leading to increased H4K20 trimethylation during acute hypertrophic stress in cell models and decreased H4K20 trimethylation during sustained ischemic injury and cardiac dysfunction in animal models. In addition, we examined publicly available data sets to analyze enzymes that regulate H4K20 methylation and identified two demethylases (KDM7B and KDM7C) and two methyltransferases (KMT5A and SMYD5) that were all differentially expressed in heart failure patients. This is the first study to examine histone H4K20 trimethylation in the heart and to determine how this post-translational modification is differentially regulated in multiple models of cardiac disease.
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Smyd1, a muscle-specific histone methyltransferase, has established roles in skeletal and cardiac muscle development, but its role in the adult heart remains poorly understood. Our prior work demonstrated that cardiac-specific deletion of Smyd1 in adult mice (Smyd1-KO) leads to hypertrophy and heart failure. Here we show that down-regulation of mitochondrial energetics is an early event in these Smyd1-KO mice preceding the onset of structural abnormalities. This early impairment of mitochondrial energetics in Smyd1-KO mice is associated with a significant reduction in gene and protein expression of PGC-1α, PPARα, and RXRα, the master regulators of cardiac energetics. The effect of Smyd1 on PGC-1α was recapitulated in primary cultured rat ventricular myocytes, in which acute siRNA-mediated silencing of Smyd1 resulted in a greater than twofold decrease in PGC-1α expression without affecting that of PPARα or RXRα. In addition, enrichment of histone H3 lysine 4 trimethylation (a mark of gene activation) at the PGC-1α locus was markedly reduced in Smyd1-KO mice, and Smyd1-induced transcriptional activation of PGC-1α was confirmed by luciferase reporter assays. Functional confirmation of Smyd1's involvement showed an increase in mitochondrial respiration capacity induced by overexpression of Smyd1, which was abolished by siRNA-mediated PGC-1α knockdown. Conversely, overexpression of PGC-1α rescued transcript expression and mitochondrial respiration caused by silencing Smyd1 in cardiomyocytes. These findings provide functional evidence for a role of Smyd1, or any member of the Smyd family, in regulating cardiac energetics in the adult heart, which is mediated, at least in part, via modulating PGC-1α.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Metabolismo Energético/fisiología , N-Metiltransferasa de Histona-Lisina/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas Musculares/metabolismo , Miocardio/enzimología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/biosíntesis , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/genética , Proteínas Musculares/genética , PPAR alfa/biosíntesis , PPAR alfa/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Receptor alfa X Retinoide/biosíntesis , Receptor alfa X Retinoide/genética , Factores de Transcripción/genéticaRESUMEN
Protein methylation plays a pivotal role in the regulation of various cellular processes including chromatin remodeling and gene expression. SET and MYND domain-containing proteins (Smyd) are a special class of lysine methyltransferases whose catalytic SET domain is split by an MYND domain. The hallmark feature of this family was thought to be the methylation of histone H3 (on lysine 4). However, several studies suggest that the role of the Smyd family is dynamic, targeting unique histone residues associated with both transcriptional activation and repression. Smyd proteins also methylate several non-histone proteins to regulate various cellular processes. Although we are only beginning to understand their specific molecular functions and role in chromatin remodeling, recent studies have advanced our understanding of this relatively uncharacterized family, highlighting their involvement in development, cell growth and differentiation and during disease in various animal models. This review summarizes our current knowledge of the structure, function and methylation targets of the Smyd family and provides a compilation of data emphasizing their prominent role in cardiac and skeletal muscle physiology and pathology.
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All terminally differentiated organs face two challenges, maintaining their cellular identity and restricting organ size. The molecular mechanisms responsible for these decisions are of critical importance to organismal development, and perturbations in their normal balance can lead to disease. A hallmark of heart failure, a condition affecting millions of people worldwide, is hypertrophic growth of cardiomyocytes. The various forms of heart failure in human and animal models share conserved transcriptome remodeling events that lead to expression of genes normally silenced in the healthy adult heart. However, the chromatin remodeling events that maintain cell and organ size are incompletely understood; insights into these mechanisms could provide new targets for heart failure therapy. Using a quantitative proteomics approach to identify muscle-specific chromatin regulators in a mouse model of hypertrophy and heart failure, we identified upregulation of the histone methyltransferase Smyd1 during disease. Inducible loss-of-function studies in vivo demonstrate that Smyd1 is responsible for restricting growth in the adult heart, with its absence leading to cellular hypertrophy, organ remodeling, and fulminate heart failure. Molecular studies reveal Smyd1 to be a muscle-specific regulator of gene expression and indicate that Smyd1 modulates expression of gene isoforms whose expression is associated with cardiac pathology. Importantly, activation of Smyd1 can prevent pathological cell growth. These findings have basic implications for our understanding of cardiac pathologies and open new avenues to the treatment of cardiac hypertrophy and failure by modulating Smyd1.
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Cardiomegalia/genética , Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/genética , Insuficiencia Cardíaca/genética , Proteínas Musculares/genética , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Factores de Transcripción/genética , Animales , Western Blotting , Cardiomegalia/diagnóstico por imagen , Cardiomegalia/metabolismo , Aumento de la Célula , Ecocardiografía , Regulación de la Expresión Génica , Técnicas de Sustitución del Gen , Células HeLa , Insuficiencia Cardíaca/diagnóstico por imagen , Insuficiencia Cardíaca/metabolismo , Humanos , Ratones , Ratones Noqueados , Miocardio/patología , Miocitos Cardíacos/patología , Proteómica , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia Arriba , Remodelación Ventricular/genéticaRESUMEN
Heart failure (HF) is accompanied by complex alterations in myocardial energy metabolism. Up to 40% of HF patients have dyssynchronous ventricular contraction, which is an independent indicator of mortality. We hypothesized that electromechanical dyssynchrony significantly affects metabolic remodeling in the course of HF. We used a canine model of tachypacing-induced HF. Animals were paced at 200 bpm for 6 weeks either in the right atrium (synchronous HF, SHF) or in the right ventricle (dyssynchronous HF, DHF). We collected biopsies from left ventricular apex and performed comprehensive metabolic pathway analysis using multi-platform metabolomics (GC/MS; MS/MS; HPLC) and LC-MS/MS label-free proteomics. We found important differences in metabolic remodeling between SHF and DHF. As compared to Control, ATP, phosphocreatine (PCr), creatine, and PCr/ATP (prognostic indicator of mortality in HF patients) were all significantly reduced in DHF, but not SHF. In addition, the myocardial levels of carnitine (mitochondrial fatty acid carrier) and fatty acids (12:0, 14:0) were significantly reduced in DHF, but not SHF. Carnitine parmitoyltransferase I, a key regulatory enzyme of fatty acid ß-oxidation, was significantly upregulated in SHF but was not different in DHF, as compared to Control. Both SHF and DHF exhibited a reduction, but to a different degree, in creatine and the intermediates of glycolysis and the TCA cycle. In contrast to this, the enzymes of creatine kinase shuttle were upregulated, and the enzymes of glycolysis and the TCA cycle were predominantly upregulated or unchanged in both SHF and DHF. These data suggest a systemic mismatch between substrate supply and demand in pacing-induced HF. The energy deficit observed in DHF, but not in SHF, may be associated with a critical decrease in fatty acid delivery to the ß-oxidation pipeline, primarily due to a reduction in myocardial carnitine content.
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Metabolismo Energético/fisiología , Insuficiencia Cardíaca/metabolismo , Redes y Vías Metabólicas/fisiología , Metabolómica/métodos , Miocardio/metabolismo , Disfunción Ventricular/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Ciclo del Ácido Cítrico/fisiología , Creatina/metabolismo , Creatina Quinasa/metabolismo , Perros , Cromatografía de Gases y Espectrometría de Masas , Glucólisis , Proteómica/métodos , Espectrometría de Masas en Tándem , Factores de Tiempo , Disfunción Ventricular/fisiopatologíaRESUMEN
In bacteria, transfer-messenger RNA (tmRNA) and SmpB comprise the most common and effective system for rescuing stalled ribosomes. Ribosomes stall on mRNA transcripts lacking stop codons and are rescued as the defective mRNA is swapped for the tmRNA template in a process known as trans-translation. The tmRNA-SmpB complex is recruited to the ribosome independent of a codon-anticodon interaction. Given that the ribosome uses robust discriminatory mechanisms to select against non-cognate tRNAs during canonical decoding, it has been hard to explain how this can happen. Recent structural and biochemical studies show that SmpB licenses tmRNA entry through its interactions with the decoding center and mRNA channel. In particular, the C-terminal tail of SmpB promotes both EFTu activation and accommodation of tmRNA, the former through interactions with 16S rRNA nucleotide G530 and the latter through interactions with the mRNA channel downstream of the A site. Here we present a detailed model of the earliest steps in trans-translation, and in light of these mechanistic considerations, revisit the question of how tmRNA preferentially reacts with stalled, non-translating ribosomes.
RESUMEN
Messenger RNAs lacking a stop codon trap ribosomes at their 3' ends, depleting the pool of ribosomes available for protein synthesis. In bacteria, a remarkable quality control system rescues and recycles stalled ribosomes in a process known as trans-translation. Acting as a tRNA, transfer-messenger RNA (tmRNA) is aminoacylated, delivered by EF-Tu to the ribosomal A site, and accepts the nascent polypeptide. Translation then resumes on a reading frame within tmRNA, encoding a short peptide tag that targets the nascent peptide for degradation by proteases. One unsolved issue in trans-translation is how tmRNA and its protein partner SmpB preferentially recognize stalled ribosomes and not actively translating ones. Here, we examine the effect of the length of the 3' extension of mRNA on each step of trans-translation by pre-steady-state kinetic methods and fluorescence polarization binding assays. Unexpectedly, EF-Tu activation and GTP hydrolysis occur rapidly regardless of the length of the mRNA, although the peptidyl transfer to tmRNA decreases as the mRNA 3' extension increases and the tmRNA·SmpB binds less tightly to the ribosome with an mRNA having a long 3' extension. From these results, we conclude that the tmRNA·SmpB complex dissociates during accommodation due to competition between the downstream mRNA and the C-terminal tail for the mRNA channel. Rejection of the tmRNA·SmpB complex during accommodation is reminiscent of the rejection of near-cognate tRNA from the ribosome in canonical translation.
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Proteínas Bacterianas/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3' , Unión Proteica , Biosíntesis de Proteínas , Ribosomas/metabolismoRESUMEN
In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon-anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB's affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA.
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Escherichia coli/genética , Factor Tu de Elongación Peptídica/química , ARN Bacteriano/química , Proteínas de Unión al ARN/química , Ribosomas/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antibacterianos/farmacología , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Guanosina Trifosfato/química , Hidrólisis , Cinética , Mutagénesis Sitio-Dirigida , Factor Tu de Elongación Peptídica/antagonistas & inhibidores , Factor Tu de Elongación Peptídica/genética , Mutación Puntual , Unión Proteica , Piridonas/farmacología , ARN Bacteriano/genética , Proteínas de Unión al ARN/genética , Ribosomas/genéticaRESUMEN
In bacteria, stalled ribosomes are recycled by a hybrid transfer-messenger RNA (tmRNA). Like tRNA, tmRNA is aminoacylated with alanine and is delivered to the ribosome by EF-Tu, where it reacts with the growing polypeptide chain. tmRNA entry into stalled ribosomes poses a challenge to our understanding of ribosome function because it occurs in the absence of a codon-anticodon interaction. Instead, tmRNA entry is licensed by the binding of its protein partner, SmpB, to the ribosomal decoding center. We analyzed a series of SmpB mutants and found that its C-terminal tail is essential for tmRNA accommodation but not for EF-Tu activation. We obtained evidence that the tail likely functions as a helix on the ribosome to promote accommodation and identified key residues in the tail essential for this step. In addition, our mutational analysis points to a role for the conserved K(131)GKK tail residues in trans-translation after peptidyl transfer to tmRNA, presumably EF-G-mediated translocation or translation of the tmRNA template. Surprisingly, analysis of A1492, A1493, and G530 mutants reveals that while these ribosomal nucleotides are essential for normal tRNA selection, they play little to no role in peptidyl transfer to tmRNA. These studies clarify how SmpB interacts with the ribosomal decoding center to license tmRNA entry into stalled ribosomes.
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ARN Bacteriano/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Proteínas de Unión al ARN/genética , Secuencia de Aminoácidos , Anticodón/genética , Dicroismo Circular , Codón/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Immunoblotting , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Factor G de Elongación Peptídica/genética , Factor G de Elongación Peptídica/metabolismo , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Aminoacilación de ARN de TransferenciaRESUMEN
BACKGROUND: tmRNA acts first as a tRNA and then as an mRNA to rescue stalled ribosomes in eubacteria. Two unanswered questions about tmRNA function remain: how does tmRNA, lacking an anticodon, bypass the decoding machinery and enter the ribosome? Secondly, how does the ribosome choose the proper codon to resume translation on tmRNA? According to the -1 triplet hypothesis, the answer to both questions lies in the unique properties of the three nucleotides upstream of the first tmRNA codon. These nucleotides assume an A-form conformation that mimics the codon-anticodon interaction, leading to recognition by the decoding center and choice of the reading frame. The -1 triplet hypothesis is important because it is the most credible model in which direct binding and recognition by the ribosome sets the reading frame on tmRNA. RESULTS: Conformational analysis predicts that 18 triplets cannot form the correct structure to function as the -1 triplet of tmRNA. We tested the tmRNA activity of all possible -1 triplet mutants using a genetic assay in Escherichia coli. While many mutants displayed reduced activity, our findings do not match the predictions of this model. Additional mutagenesis identified sequences further upstream that are required for tmRNA function. An immunoblot assay for translation of the tmRNA tag revealed that certain mutations in U85, A86, and the -1 triplet sequence result in improper selection of the first codon and translation in the wrong frame (-1 or +1) in vivo. CONCLUSION: Our findings disprove the -1 triplet hypothesis. The -1 triplet is not required for accommodation of tmRNA into the ribosome, although it plays a minor role in frame selection. Our results strongly disfavor direct ribosomal recognition of the upstream sequence, instead supporting a model in which the binding of a separate ligand to A86 is primarily responsible for frame selection.
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Escherichia coli/genética , ARN Bacteriano/genética , Sistemas de Lectura/genética , Secuencias Reguladoras de Ácido Ribonucleico/genética , Bacteriófagos/crecimiento & desarrollo , Secuencia de Bases , Codón/genética , Escherichia coli/virología , Resistencia a la Kanamicina/genética , Mutación , Conformación de Ácido Nucleico , ARN Bacteriano/químicaRESUMEN
tmRNA rescues stalled ribosomes in eubacteria by forcing the ribosome to abandon its mRNA template and resume translation with tmRNA itself as a template. Pseudoknot 1 (pk1), immediately upstream of this coding region in tmRNA, is a structural element that is considered essential for tmRNA function based on the analysis of pk1 mutants in vitro. pk1 binds near the ribosomal decoding site and may make base-specific contacts with tmRNA ligands. To study pk1 structure and function in vivo, we have developed a genetic selection that ties the life of Escherichia coli cells to tmRNA activity. Mutation of pk1 at 20% per base and selection for tmRNA activity yielded sequences that retain the same pseudoknot fold. In contrast, selection of active mutants from 10(6) completely random sequences identified hairpin structures that functionally replace pk1. Rational design of a hairpin with increased stability using an unrelated sequence yielded a tmRNA mutant with nearly wild-type activity. We conclude that the role of pk1 in tmRNA function is purely structural and that it can be replaced with a variety of hairpin structures. Our results demonstrate that in the study of functional RNAs, the inactivity of a mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure.