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Gene expression microarray profiling in mice hearts with pathological and physiological cardiac hypertrophy

ABSTRACT: Compelling evidence suggests that mitochondrial dysfunction contributes to the pathogenesis of heart failure, including defects in the substrate oxidation, and the electron transport chain (ETC) and oxidative phosphorylation (OXPHOS). However, whether such changes occur early in the development of heart failure, and are potentially involved in the pathologic events that lead to cardiac dysfunction is unknown. To address this question, we conducted transcriptomic/metabolomics profiling in hearts of mice with two progressive stages of pressure overload-induced cardiac hypetrophy: i) cardiac hypertrophy with preserved ventricular function achieved via transverse aortic constriction for 4 weeks (TAC) and ii) decompensated cardiac hypertrophy or heart failure (HF) caused by combining 4 wk TAC with a small apical myocardial infarction. Transcriptomic analyses revealed, as shown previously, downregulated expression of genes involved in mitochondrial fatty acid oxidation in both TAC and HF hearts compared to sham-operated control hearts. Surprisingly, however, there were very few changes in expression of genes involved in other mitochondrial energy transduction pathways, ETC, or OXPHOS. Metabolomic analyses demonstrated significant alterations in pathway metabolite levels in HF (but not in TAC), including elevations in acylcarnitines, a subset of amino acids, and the lactate/pyruvate ratio. In contrast, the majority of organic acids were lower than controls. This metabolite profile suggests “bottlenecks” in the carbon substrate input to the TCA cycle. This transcriptomic/metabolomic profile was markedly different from that of mice PGC-1a/b deficiency in which a global downregulation of genes involved in mitochondrial ETC and OXPHOS was noted. In addition, the transcriptomic/metabolomic signatures of HF differed markedly from that of the exercise-trained mouse heart. We conclude that in contrast to current dogma, alterations in mitochondrial metabolism that occur early in the development of heart failure reflect largely post-transcriptional mechanisms resulting in impedance to substrate flux into the TCA cycle, reflected by alterations in the metabolome.

ORGANISM(S): Mus musculus

PROVIDER: GSE56348 | GEO | 2014/10/01

SECONDARY ACCESSION(S): PRJNA242995

REPOSITORIES: GEO

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Project description:Compelling evidence suggests that mitochondrial dysfunction contributes to the pathogenesis of heart failure, including defects in the substrate oxidation, and the electron transport chain (ETC) and oxidative phosphorylation (OXPHOS). However, whether such changes occur early in the development of heart failure, and are potentially involved in the pathologic events that lead to cardiac dysfunction is unknown. To address this question, we conducted transcriptomic/metabolomics profiling in hearts of mice with two progressive stages of pressure overload-induced cardiac hypetrophy: i) cardiac hypertrophy with preserved ventricular function achieved via transverse aortic constriction for 4 weeks (TAC) and ii) decompensated cardiac hypertrophy or heart failure (HF) caused by combining 4 wk TAC with a small apical myocardial infarction. Transcriptomic analyses revealed, as shown previously, downregulated expression of genes involved in mitochondrial fatty acid oxidation in both TAC and HF hearts compared to sham-operated control hearts. Surprisingly, however, there were very few changes in expression of genes involved in other mitochondrial energy transduction pathways, ETC, or OXPHOS. Metabolomic analyses demonstrated significant alterations in pathway metabolite levels in HF (but not in TAC), including elevations in acylcarnitines, a subset of amino acids, and the lactate/pyruvate ratio. In contrast, the majority of organic acids were lower than controls. This metabolite profile suggests “bottlenecks” in the carbon substrate input to the TCA cycle. This transcriptomic/metabolomic profile was markedly different from that of mice PGC-1a/b deficiency in which a global downregulation of genes involved in mitochondrial ETC and OXPHOS was noted. In addition, the transcriptomic/metabolomic signatures of HF differed markedly from that of the exercise-trained mouse heart. We conclude that in contrast to current dogma, alterations in mitochondrial metabolism that occur early in the development of heart failure reflect largely post-transcriptional mechanisms resulting in impedance to substrate flux into the TCA cycle, reflected by alterations in the metabolome. Microarray gene expression profiling was performed with bi-ventricle RNA isolated from (1) 3 month-old female C57Bl6/J mice with transverse aortic constriction (CH), vs sham-operated control (Sham-CH), (2) 3 month-old female C57Bl6/J mice with heart failure (HF), vs. sham-operated control (Sham-HF); (3) 2 month-old female C57Bl6/J mice with voluntary wheel training for 2 months (Run), vs. Sedentary control (Sed). Five biological replicates are included for each group.

Project description:Muscle ring finger-1 (MuRF1) is a muscle-specific protein implicated in the regulation of cardiac myocyte size and contractility. MuRF2, a closely related family member, redundantly interacts with protein substrates, and hetero-dimerizes with MuRF1. Mice lacking either MuRF1 or MuRF2 are phenotypically normal whereas mice lacking both proteins develop a spontaneous cardiac and skeletal muscle hypertrophy indicating cooperative control of muscle mass by MuRF1 and MuRF2. In order to identify the role that MuRF1 plays in regulating cardiac hypertrophy in vivo, we created transgenic mice expressing increased amounts of cardiac MuRF1. Adult MuRF1 transgenic (Tg+) hearts exhibited a non-progressive thinning of the left ventricular wall and a concomitant decrease in cardiac function. Experimental induction of cardiac hypertrophy by trans-aortic constriction (TAC) induced rapid failure of MuRF1 Tg+ hearts. Microarray analysis identified that the levels of genes associated with metabolism (and in particular mitochondrial processes) were significantly altered in MuRF1 Tg+ hearts, both at baseline and during the development of cardiac hypertrophy. Surprisingly, ATP levels in MuRF1 Tg+ mice did not differ from wild type mice despite the depressed contractility following TAC. To explain this discrepancy between the ongoing heart failure and maintained ATP levels in MuRF1 Tg+ hearts, we compared the level and activity of creatine kinase (CK) between wild type and MuRF1 Tg+ hearts. Although mCK and CK-M/B protein levels were unaffected in MuRF1 Tg+ hearts, total CK activity was significantly inhibited. We conclude that MuRF1’s inhibition of CK activity leads to increased susceptibility to heart failure following TAC, demonstrating for the first time that MuRF1 regulates cardiac energetics in vivo. Keywords: Genetic modification, physiological manipulation. Three-condition experiment, MuRF1 Tg+ vs. WT mice. Biological replicates: 4 WT baseline, 3 MuRF1 Tg+ baseline, cardiac overpressure hypertrophy induced by trans-aortic banding at 1 week (3 WT, 3 MurF Tg) and 4 weeks (3 WT, 3 MurF Tg), hearts harvested. One replicate per array.

Project description:Accurate and comprehensive de novo transcriptome profiling in heart is a central issue to better understand cardiac physiology and diseases. Although significant progress has been made in genome-wide profiling for quantitative changes in cardiac gene expression, current knowledge offers limited insights to the total complexity in cardiac transcriptome at individual exon level. To develop more robust bioinformatic approaches to analyze high-throughput RNA sequencing (RNA-Seq) data, with the focus on the investigation of transcriptome complexity at individual exon and transcript levels. In addition to overall gene expression analysis, the methods developed in this study were used to analyze RNA-Seq data with respect to individual transcript isoforms, novel spliced exons, novel alternative terminal exons, novel transcript clusters (i.e., novel genes) and long non-coding RNA genes. We applied these approaches to RNA-Seq data obtained from mouse hearts following pressure-overload induced by trans-aortic constriction. Based on experimental validations, analyses of the features of the identified exons/transcripts, and expression analyses including previously published RNA-Seq data, we demonstrate that the methods are highly effective in detecting and quantifying individual exons and transcripts. Novel insights inferred from the examined aspects of the cardiac transcriptome open ways to further experimental investigations. Our work provided a comprehensive set of methods to analyze mouse cardiac transcriptome complexity at individual exon and transcript levels. Applications of the methods may infer important new insights to gene regulation in normal and disease hearts in terms of exon utilization and potential involvement of novel components of cardiac transcriptome. Left ventricular tissues were collected from C57BL/6 mice after 1 week (hypertrophy stage, HY) and 8 weeks post trans-aortic constriction (TAC) procedure (heart failure stage, HF), respectively, and their corresponding Sham controls (Sham-HY, Sham-HF). To conduct RNA-Seq analysis, total RNAs from six TAC and Sham-operated mice at the HY stage and four TAC and corresponding Sham mice at the HF stage were obtained.

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Gene expression microarray profiling in mice hearts with pathological and physiological cardiac hypertrophy

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