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Supplementary Figure 2 from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

Supplementary Figure 1 from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

Supplementary Table 1 from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

Hypertrophic cardiomyopathy (HCM) affects as many as ~1 in 500 individuals, and is often typified by hyperdynamic contraction and poor cellular relaxation. HCM can be caused by mutations in a variety of key contractile proteins of the sarcomere. A large proportion of these variants are found in MYBPC3, MYH7, TNNT2, and TNNI3. These genes encode proteins that control cardiac muscle contraction at the thick (MYBPC3 and MYH7) and thin filaments (TNNT2 and TNNI3) of the sarcomere. In this study we use human induced pluripotent stem cell derived cardiomyocytes to model HCM across all of these genes. We do this to define key mechanistic differences between thick and thin filament HCM. We define sarcomeric contractility (SarcTrack) calcium transients (CalTrack) and myosin states using the mant-ATP assay. We use the parametric data from these experimental studies in iPSC-CMs to model possible disease mechanisms in silico. Our experimental analysis highlights that both thick and thin filament HCM variants cause cellular hypercontractility, with slowed cellular relaxation. We find that thick filament HCM variants drive cellular HCM phenotypes by destabilising the myosin interacting heads motif (IHM), showing a marked reduction in the super relaxed state of myosin. Counterintuitively thin filament based HCM variants show a reduction in DRX myosin. When applying Mavacamten the allosteric myosin ATPase inhibitor to our thin and thick filament HCM variant iPSC-CMs we find a dichotomy of cellular responses. The thick filament variants studied all show a clear resolution of cellular HCM. However, not all cellular phenotypes of thin filament HCM are corrected by Mavacamten treatment, although there is benefit. We conclude that causal mechanisms of thick filament HCM are well corrected at the molecular and cellular level by Mavacamten, but these causal mechanisms in thin filament based HCM are not suitably corrected. We highlight key mechanistic pharmacological targets for thin filament variants that could add cellular benefit to HCM phenotype resolution.

Supplemental Digital Content is available in the text., Rationale: Calcium transient analysis is central to understanding inherited and acquired cardiac physiology and disease. Although the development of novel calcium reporters enables assays of CRISPR/Cas-9 genome-edited induced pluripotent stem cell–derived cardiomyocytes and primary adult cardiomyocytes, existing calcium-detection technologies are often proprietary and require specialist equipment, whereas open-source workflows necessitate considerable user expertise and manual input. Objective: We aimed to develop an easy to use open-source, adaptable, and automated analysis pipeline for measuring cellular calcium transients, from image stack to data output, inclusive of cellular identification, background subtraction, photobleaching correction, calcium transient averaging, calcium transient fitting, data collation, and aberrant behavior recognition. Methods and Results: We developed CalTrack, a MatLab-based algorithm, to monitor fluorescent calcium transients in living cardiomyocytes, including isolated single cells or those contained in 3-dimensional tissues or organoids, and to analyze data acquired using photomultiplier tubes or employing line scans. CalTrack uses masks to segment cells allowing multiple cardiomyocyte transients to be measured from a single field of view. After automatically correcting for photobleaching, CalTrack averages and fits a string of transients and provides parameters that measure time to peak, time of decay, tau, peak fluorescence/baseline fluorescence (Fmax/F0), and others. We demonstrate the utility of CalTrack in primary and induced pluripotent stem cell–derived cell lines in response to pharmacological compounds and in phenotyping cells carrying hypertrophic cardiomyopathy variants. Conclusions: CalTrack, an open-source tool that runs on a local computer, provides automated high-throughput analysis of calcium transients in response to development, genetic or pharmacological manipulations, and pathological conditions. We expect that CalTrack analyses will accelerate insights into physiological and abnormal calcium homeostasis that influence diverse aspects of cardiomyocyte biology.

Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00853-z.

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.

Background Aortic stenosis (AS) and hypertrophic cardiomyopathy (HCM) are highly distinct disorders leading to LVH, but whether cardiac metabolism substantially differs between these in humans remains to be elucidated. Method We undertook a detailed invasive (aortic root and coronary sinus) metabolic profiling in patients with severe AS and HCM in comparison to non-LVH controls, to investigate cardiac fuel selection and metabolic remodelling. These patients were assessed under different physiological states (at rest and during stress induced by pacing). The identified changes in the metabolome were further validated by metabolomic and orthogonal transcriptomic analysis, in separately recruited patient cohorts. We then present findings from the clinical trial using perhexiline, a metabolic modulator of long-chain fatty acid β-oxidation, in patients with severe AS, and contextualise these with the up-stream metabolomic findings. Results We identified a highly discriminant metabolomic signature in severe AS characterised by striking accumulation of long-chain acylcarnitines, intermediates of long-chain transport and fatty acid metabolism, and validated this in a separate cohort. Mechanistically, we identify a down-regulation in the PPAR-α transcriptional network, including expression of genes regulating FAO. Evaluation of the symptomatic impact of perhexiline in severe AS, in contrast to HCM, revealed no significant beneficial effect, suggesting the underlying metabolic changes are attempts at metabolic adaptation. Conclusions We present a comprehensive analysis of changes in the metabolic pathways (transcriptome to metabolome) in severe AS, and its comparison to HCM. Our results demonstrate fundamental distinctions in substrate preference between AS and HCM, highlighting insufficient long-chain FAO, and the PPAR-α signalling network as a specific metabolic therapeutic target in AS.

Spliceosome mutations are common in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but the oncogenic changes due to these mutations have not been identified. Here a global analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. Interleukin-1 receptor-associated kinase 4 (IRAK4) was the dominant alternatively spliced isoform in MDS and AML and is characterized by a longer isoform that retains exon 4, which encodes IRAK4-long (IRAK4-L), a protein that assembles with the myddosome, results in maximal activation of nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) and is essential for leukaemic cell function. Expression of IRAK4-L is mediated by mutant U2 small nuclear RNA auxiliary factor 1 (U2AF1) and is associated with oncogenic signalling in MDS and AML. Inhibition of IRAK4-L abrogates leukaemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable 'active' IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signalling in MDS and AML.

Myocardial fibrosis (MF) contributes to the pathogenesis of cardiac hypertrophy secondary to energetic perturbation. Recent evidence suggests an increase of purine signalling upon energetic deficiency and specifically a role of adenosine signalling in tissue fibrosis. The specific objective of this study was to delineate adenosine A2A receptors in the development of MF. In vitro, isolated cardiac fibroblasts demonstrated a significant increase in collagen production upon A2A receptor stimulation. This was inhibited by addition of a specific A2A receptor inhibitor. In vivo, models of cardiac hypertrophy including the transverse aortic constriction (TAC) model and cardiac actin E99K transgenic mice (E99K mice) were investigated as murine models for MF. In E99K mice a reduced phosphocreatine/ATP ratio was demonstrated using magnetic resonance spectroscopy. Interstitial adenosine levels measured by microdialysis correlated with collagen content, showing energy deficiency and a correlation with MF. Crossbreeding of E99K and Adenosine A2A receptor knock out (A2A KO) mice resulted in a significant reduction of MF in E99K heterozygous A2A KO animals. A2A KO mice undergoing TAC demonstrated significantly less fibrosis formation compared to wild type mice upon measurement of myocardial collagen and on histology. This was associated with a significant rescue of cardiac function. Finally, pharmacologic adenosine A2A receptor inhibition using the antagonist ZM241385 demonstrated a partial rescue effect on MF in both TAC and E99K animals. This data indicates that signalling of energy deficiency via adenosine A2A receptors play a crucial role in the formation of MF and that this pathway is susceptible to pharmacologic modulation.

Mutations in splicing factor genes (SF3B1, SRSF2, U2AF1 and ZRSR2) are frequently found in patients with myelodysplastic syndromes (MDS), suggesting that aberrant spliceosome function plays a key role in the pathogenesis of MDS. Splicing factor mutations have been shown to result in aberrant splicing of many downstream target genes. Recent functional studies have begun to characterize the splicing dysfunction in MDS, identifying some key aberrantly spliced genes that are implicated in disease pathophysiology. These findings have led to the development of therapeutic strategies using splicing-modulating agents and rapid progress is being made in this field. Splicing inhibitors are promising agents that exploit the preferential sensitivity of splicing factor-mutant cells to these compounds. Here, we review the known target genes associated with splicing factor mutations in MDS, and discuss the potential of splicing-modulating therapies for these disorders.

Hypoxia inducible factor-1α (HIF-1α) plays a critical role in the oxygen homeostasis of all metazoans. HIF-1α is a master transcriptional regulator which coordinates the adaptive response to low oxygen tension. Through activation of a plethora of downstream target genes, HIF-1α facilitates oxygenation by promoting angiogenesis and blood vessel dilation, in addition to modulating metabolic pathways to inhibit oxidative phosphorylation and promote glycolytic energy production. Given the critical roles of hypoxia, insufficient blood supply and perturbed energetics in the pathogenesis of cardiovascular disorders, notably ischaemic heart disease, therapeutic modulation of HIF-1α is of significant clinical interest. Previous studies have demonstrated an acute cardioprotective role for both endogenous and supraphysiological HIF-1α signalling in the context of myocardial ischaemia. In contrast, chronic supraphysiological HIF-1α activation in the unstressed heart has been shown to induce cardiac dysfunction. To address the effect of chronic endogenous HIF-1α activation post-myocardial infarction (MI), the present work employed a murine coronary artery ligation (CAL) model in conjunction with temporally-inducible, cardiac-specific deletion of Hif-1α. While CAL surgery successfully modelled myocardial infarction – eliciting substantial adverse cardiac remodelling and contractile dysfunction – there was no evidence of chronic HIF-1α activation by CAL in HIF knockout or control left ventricular samples. In keeping with this, chronic ablation of Hif-1α (from 2 weeks post-CAL) had no discernible additional effect upon cardiac function. Overall, these findings do not support a potential therapeutic role for inhibition of HIF-1α signalling in the chronic phase post-MI. The fundamental tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) converts fumarate to malate. FH deficiency is associated with smooth muscle and kidney tumours which exhibit normoxic HIF signalling due to fumarate accumulation. To investigate the potential for fumarate accumulation to elicit protective HIF signalling, a cardiac-specific Fh1 null mouse was developed through Cre-loxP recombination. Strikingly, despite interruption of the TCA cycle in a highly metabolically demanding organ, cardiac Fh1 null mice were viable, fertile and survived into adulthood, demonstrating the remarkable metabolic plasticity of the heart. However, by 3-4 months Fh1 null mice develop a lethal cardiomyopathy characterised by cardiac hypertrophy, ventricular dilatation and contractile dysfunction. Despite lack of a pseudohypoxic response, Fh1 null hearts did exhibit another phenomenon observed in FH-deficient cancers and also attributed to fumarate accumulation – activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) antioxidant pathway. Heterozygous, but not homozygous, somatic deletion of Nrf2 extended the life expectancy of cardiac Fh1 null mice. Exploration of redox status revealed a more reductive environment in Fh1 null hearts than controls. As a corollary, inhibition of the rate limiting enzyme of the pentose phosphate pathway – a major source of cellular reducing equivalents – with dehydroepiandrosterone conferred striking amelioration of the Fh1 null cardiomyopathy, suggesting a possible pathogenic role for reductive stress. While loss of mitochondrial Fh1 activity and subsequent TCA cycle dysfunction likely contribute to the Fh1 null phenotype, the importance of cytosolic FH was unclear. To clarify this, FH was expressed specifically in the cytosol in vivo. This was sufficient to substantially rescue the Fh1 null cardiomyopathy, supporting a role for cytosolic FH disruption in its pathogenesis. Taken together, these findings highlight the potential for reductive stress to contribute to cardiac dysfunction and suggest a function for cytosolic FH in cardiac metabolic homeostasis.

Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease., Author Summary Heart disease is very common. Some cases of heart disease are strongly influenced by lifestyle and diet, whereas others have a strong genetic component. A certain form of heart failure, known as dilated cardiomyopathy (DCM) quite often runs in families suggesting that a defective gene or genes underlie this disease. We describe a new mouse mutant called “Python” which suffers from a heart disease similar to DCM. We were able to pinpoint the defective gene responsible for the disease. This gene is normally involved in the division of mitochondria, the “power plants” of the cell that generate one of the main energy supplies for the cell. This is a unique model that implicates a new gene and mechanism of disease for further investigation.

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3' splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3' splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.Leukemia advance online publication, 17 June 2016; doi:10.1038/leu.2016.149.

We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure.

Over the last decade, there has been a concerted clinical effort to deliver on the laboratory promise that a variety of maneuvers can profoundly increase cardiac tolerance to ischemia and/or reduce additional damage consequent upon reperfusion. Here we will review the proximity of the metabolic approach to clinical practice. Specifically, we will focus on how the citric acid cycle is involved in cardioprotection. Inspired by cross-fertilization between fundamental cancer biology and cardiovascular medicine, a set of metabolic observations have identified novel metabolic pathways, easily manipulable in man, which can harness metabolism to robustly combat ischemia-reperfusion injury.

AIMS: This work investigates the role of myoglobin in mediating the vascular relaxation induced by nitrite. Nitrite, previously considered an inert by-product of nitric oxide metabolism, is now believed to play an important role in several areas of pharmacology and physiology. Myoglobin can act as a nitrite reductase in the heart, where it is plentiful, but it is present at a far lower level in vascular smooth muscle-indeed, its existence in the vessel wall is controversial. Haem proteins have been postulated to be important in nitrite-induced vasodilation, but the specific role of myoglobin is unknown. The current study was designed to confirm the presence of myoglobin in murine aortic tissue and to test the hypothesis that vascular wall myoglobin is important for nitrite-induced vasodilation. METHODS AND RESULTS: Aortic rings from wild-type and myoglobin knockout mice were challenged with nitrite, before and after exposure to the haem-protein inhibitor carbon monoxide (CO). CO inhibited vasodilation in wild-type rings but not in myoglobin-deficient rings. Restitution of myoglobin using a genetically modified adenovirus both increased vasodilation to nitrite and reinstated the wild-type pattern of response to CO. CONCLUSION: Myoglobin is present in the murine vasculature and contributes significantly to nitrite-induced vasodilation.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by mutations in the Krebs cycle enzyme fumarate hydratase (FH). It has been proposed that “pseudohypoxic” stabilization of hypoxia-inducible factor-α (HIF-α) by fumarate accumulation contributes to tumorigenesis in HLRCC. We hypothesized that an additional direct consequence of FH deficiency is the establishment of a biosynthetic milieu. To investigate this hypothesis, we isolated primary mouse embryonic fibroblast (MEF) lines from Fh1-deficient mice. As predicted, these MEFs upregulated Hif-1α and HIF target genes directly as a result of FH deficiency. In addition, detailed metabolic assessment of these MEFs confirmed their dependence on glycolysis, and an elevated rate of lactate efflux, associated with the upregulation of glycolytic enzymes known to be associated with tumorigenesis. Correspondingly, Fh1-deficient benign murine renal cysts and an advanced human HLRCC-related renal cell carcinoma manifested a prominent and progressive increase in the expression of HIF-α target genes and in genes known to be relevant to tumorigenesis and metastasis. In accord with our hypothesis, in a variety of different FH-deficient tissues, including a novel murine model of Fh1-deficient smooth muscle, we show a striking and progressive upregulation of a tumorigenic metabolic profile, as manifested by increased PKM2 and LDHA protein. Based on the models assessed herein, we infer that that FH deficiency compels cells to adopt an early, reversible, and progressive protumorigenic metabolic milieu that is reminiscent of that driving the Warburg effect. Targets identified in these novel and diverse FH-deficient models represent excellent potential candidates for further mechanistic investigation and therapeutic metabolic manipulation in tumors. Cancer Res; 70(22); 9153–65. ©2010 AACR.