Deficiency in nebulin repeats of sarcomeric nebulette is detrimental for cardiomyocyte tolerance to exercise and biomechanical stress.

The actin-binding sarcomeric nebulette (NEBL) protein provides efficient contractile flexibility via interaction with desmin intermediate filaments. NEBL gene mutations affecting the nebulin repeat (NR) domain are known to induce cardiomyopathy. The study aimed to explore the roles of NEBL in exercise and biomechanical stress response. We ablated exon3 encoding the first NR of Nebl and created global Neblex3-/ex3- knockout mice. Cardiac function, structure, and transcriptome were assessed before and after a 4-wk treadmill regimen. A Nebl-based exercise signaling network was constructed using systems genetics methods. H9C2 and neonatal rat cardiomyocytes (NRCs) expressing wild-type or mutant NEBL underwent cyclic mechanical strain. Neblex3-/ex3- mice demonstrated diastolic dysfunction with preserved systolic function at 6 mo of age. After treadmill running, 4-mo-old Neblex3-/ex3- mice developed concentric cardiac hypertrophy and left ventricular dilation compared with running Nebl+/+ and sedentary Neblex3-/ex3- mice. Disturbance of sarcomeric Z-disks and thin filaments architecture and disruption of intercalated disks and mitochondria were found in exercised Neblex3-/ex3- mice. A Nebl-based exercise signaling network included Csrp3, Des, Fbox32, Jup, Myh6, and Myh7. Disturbed expression of TM1, DES, JUP, ß-catenin, MLP, α-actinin2, and vinculin proteins was demonstrated. In H9C2 cells, NEBL was recruited into focal adhesions at 24-h poststrain and redistributed along with F-actin at 72-h poststrain, suggesting time-dependent redistribution of NEBL in response to strain. NEBL mutations cause desmin disorganization in NRCs upon stretch. We conclude that Nebl's NR ablation causes disturbed sarcomere, Z-disks, and desmin organization, and prevents NEBL redistribution to focal adhesions in cardiomyocytes, weakening cardiac tolerance to biomechanical stress.NEW & NOTEWORTHY We demonstrate that ablation of first nebulin-repeats of sarcomeric nebulette (Nebl) causes diastolic dysfunction in Neblex3-/ex3- mice. Exercise-induced development of diastolic dysfunction, cardiac hypertrophy and ventricular dilation in knockouts. This was associated with sarcomere disturbance, intercalated disks disruption, and mitochondrial distortion upon stress and altered expression of genes involved in Nebl-based stress network. We demonstrate that G202R and A592 mutations alter actin and desmin expression causing disorganization of desmin filaments upon cyclic strain.

Accelerated cardiac remodeling in desmoplakin transgenic mice in response to endurance exercise is associated with perturbed Wnt/ß-catenin signaling.

Arrhythmogenic ventricular cardiomyopathy (AVC) is a frequent underlying cause for arrhythmias and sudden cardiac death especially during intense exercise. The mechanisms involved remain largely unknown. The purpose of this study was to investigate how chronic endurance exercise contributes to desmoplakin (DSP) mutation-induced AVC pathogenesis. Transgenic mice with overexpression of desmoplakin, wild-type (Tg-DSP(WT)), or the R2834H mutant (Tg-DSP(R2834H)) along with control nontransgenic (NTg) littermates were kept sedentary or exposed to a daily running regimen for 12 wk. Cardiac function and morphology were analyzed using echocardiography, electrocardiography, histology, immunohistochemistry, RNA, and protein analysis. At baseline, 4-wk-old mice from all groups displayed normal cardiac function. When subjected to exercise, all mice retained normal cardiac function and left ventricular morphology; however, Tg-DSP(R2834H) mutants displayed right ventricular (RV) dilation and wall thinning, unlike NTg and Tg-DSP(WT). The Tg-DSP(R2834H) hearts demonstrated focal fat infiltrations in RV and cytoplasmic aggregations consisting of desmoplakin, plakoglobin, and connexin 43. These aggregates coincided with disruption of the intercalated disks, intermediate filaments, and microtubules. Although Tg-DSP(R2834H) mice already displayed high levels of p-GSK3-ß(Ser9) and p-AKT1(Ser473) under sedentary conditions, decrease of nuclear GSK3-ß and AKT1 levels with reduced p-GSK3-ß(Ser9), p-AKT1(Ser473), and p-AKT1(Ser308) and loss of nuclear junctional plakoglobin was apparent after exercise. In contrast, Tg-DSP(WT) showed upregulation of p-AKT1(Ser473), p-AKT1(Ser308), and p-GSK3-ß(Ser9) in response to exercise. Our data suggest that endurance exercise accelerates AVC pathogenesis in Tg-DSP(R2834H) mice and this event is associated with perturbed AKT1 and GSK3-ß signaling. Our study suggests a potential mechanism-based approach to exercise management in patients with AVC.

Therapy-induced DNA methylation inactivates MCT1 and renders tumor cells vulnerable to MCT4 inhibition.

Metabolic plasticity in cancer cells makes use of metabolism-targeting agents very challenging. Drug-induced metabolic rewiring may, however, uncover vulnerabilities that can be exploited. We report that resistance to glycolysis inhibitor 3-bromopyruvate (3-BrPA) arises from DNA methylation in treated cancer cells and subsequent silencing of the monocarboxylate transporter MCT1. We observe that, unexpectedly, 3-BrPA-resistant cancer cells mostly rely on glycolysis to sustain their growth, with MCT4 as an essential player to support lactate flux. This shift makes cancer cells particularly suited to adapt to hypoxic conditions and resist OXPHOS inhibitors and anti-proliferative chemotherapy. In contrast, blockade of MCT4 activity in 3-BrPA-exposed cancer cells with diclofenac or genetic knockout, inhibits growth of derived spheroids and tumors in mice. This study supports a potential mode of collateral lethality according to which metabolic adaptation of tumor cells to a first-line therapy makes them more responsive to a second-line treatment.

Electrical signals affect the cardiomyocyte transcriptome independently of contraction.

Cardiomyocytes in vivo are continuously subjected to electrical signals that evoke contractions and instigate drastic changes in the cells' morphology and function. Studies on how electrical stimulation affects the cardiac transcriptome have remained limited to a small number of heart-specific genes. Furthermore, these studies have ignored the interplay between the electrical excitation and the subsequent contractions. We carried out a genomewide assessment of the effects of electrical signaling on gene expression, while distinguishing between the effects deriving from the electrical pulses themselves and the effects instigated by the evoked contractions. Changes in gene expression in primary cultures of neonatal ventricular cardiomyocytes from Lewis Rattus norvegicus were investigated with microarrays and RT-quantitative PCR (QPCR). A series of experiments was included in which the culture medium was supplemented with the contraction inhibitor blebbistatin to allow for electrical stimulation in the absence of contraction. Electrical stimulation was shown to directly enhance calcium handling and induce cardiomyocyte differentiation by arresting cell division and activating key cardiac transcription factors as well as additional differentiation mechanisms such as wnt signaling. Several genes involved in metabolism were also directly activated by electrical stimulation. Furthermore, our data suggest that contraction exerts negative feedback on the transcription of various genes. Together, these observations indicate that intercellular electric currents between adjacent cardiomyocytes have an important role in cardiomyocyte development. They act at least partially through a pulse-specific gene expression program that is activated independently from the evoked contractions.

Functional annotation of heart enriched mitochondrial genes GBAS and CHCHD10 through guilt by association.

Despite the mitochondria ubiquitous nature many of their components display divergences in their expression profile across different tissues. Using the bioinformatics-approach of guilt by association (GBA) we exploited these variations to predict the function of two so far poorly annotated genes: Coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) and glioblastoma amplified sequence (GBAS). We predicted both genes to be involved in oxidative phosphorylation. Through in vitro experiments using gene-knockdown we could indeed confirm this and furthermore we asserted CHCHD10 to play a role in complex IV activity.

TGFß2-induced formation of lipid droplets supports acidosis-driven EMT and the metastatic spreading of cancer cells.

Acidosis, a common characteristic of the tumor microenvironment, is associated with alterations in metabolic preferences of cancer cells and progression of the disease. Here we identify the TGF-ß2 isoform at the interface between these observations. We document that acidic pH promotes autocrine TGF-ß2 signaling, which in turn favors the formation of lipid droplets (LD) that represent energy stores readily available to support anoikis resistance and cancer cell invasiveness. We find that, in cancer cells of various origins, acidosis-induced TGF-ß2 activation promotes both partial epithelial-to-mesenchymal transition (EMT) and fatty acid metabolism, the latter supporting Smad2 acetylation. We show that upon TGF-ß2 stimulation, PKC-zeta-mediated translocation of CD36 facilitates the uptake of fatty acids that are either stored as triglycerides in LD through DGAT1 or oxidized to generate ATP to fulfill immediate cellular needs. We also address how, by preventing fatty acid mobilization from LD, distant metastatic spreading may be inhibited.

MRI Assessment of Cardiomyopathy Induced by ß1-Adrenoreceptor Autoantibodies and Protection Through ß3-Adrenoreceptor Overexpression.

The cardiopathogenic role of autoantibodies (aabs) directed against ß1-adrenoreceptors (ß1-AR) is well established. In mouse models, they cause progressive dilated cardiomyopathy (DCM) whose characterization with echocardiography requires prolonged protocols with numerous animals, complicating the evaluation of new treatments. Here, we report on the characterization of ß1-aabs-induced DCM in mice using 11.7T MRI. C57BL/6J mice (n = 10 per group) were immunized against the ß1-AR and left ventricular (LV) systolic function was assessed at 10, 18 and 27 weeks. Increase in LV mass/tibial length ratio was detected as the first modification at 10 weeks together with dilation of cavities, thereby outperforming echocardiography. Significant impairment in diastolic index was also observed in immunized animals before the onset of systolic dysfunction. Morphometric and histological measurements confirmed these observations. The same protocol performed on ß3-AR-overexpressing mice and wild-type littermates (n = 8-12 per group) showed that transgenic animals were protected with reduced LV/TL ratio compared to wild-type animals and maintenance of the diastolic index. This study demonstrates that MRI allows a precocious detection of the subtle myocardial dysfunction induced by ß1-aabs and that ß3-AR stimulation blunts the development of ß1-aabs-induced DCM, thereby paving the way for the use of ß3AR-stimulating drugs to treat this autoimmune cardiomyopathy.

Inhibition of glucose metabolism prevents glycosylation of the glutamine transporter ASCT2 and promotes compensatory LAT1 upregulation in leukemia cells.

Leukemia cells are highly dependent on glucose and glutamine as bioenergetic and biosynthetic fuels. Inhibition of the metabolism of glucose but also of glutamine is thus proposed as a therapeutic modality to block leukemia cell growth. Since glucose also supports protein glycosylation, we wondered whether part of the growth inhibitory effects resulting from glycolysis inhibition could indirectly result from a defect in glycosylation of glutamine transporters. We found that ASCT2/SLC1A5, a major glutamine transporter, was indeed deglycosylated upon glucose deprivation and 2-deoxyglucose exposure in HL-60 and K-562 leukemia cells. Inhibition of glycosylation by these modalities as well as by the bona fide glycosylation inhibitor tunicamycin however marginally influenced glutamine transport and did not impact on ASCT2 subcellular location. This work eventually unraveled the dispensability of ASCT2 to support HL-60 and K-562 leukemia cell growth and identified the upregulation of the neutral amino acid antiporter LAT1/SLC7A5 as a mechanism counteracting the inhibition of glycosylation. Pharmacological inhibition of LAT1 increased the growth inhibitory effects and the inactivation of the mTOR pathway resulting from glycosylation defects, an effect further emphasized during the regrowth period post-treatment with tunicamycin. In conclusion, this study points towards the underestimated impact of glycosylation inhibition in the interpretation of metabolic alterations resulting from glycolysis inhibition, and identifies LAT1 as a therapeutic target to prevent compensatory mechanisms induced by alterations in the glycosylating process.

Acidosis Drives the Reprogramming of Fatty Acid Metabolism in Cancer Cells through Changes in Mitochondrial and Histone Acetylation.

Bioenergetic preferences of cancer cells foster tumor acidosis that in turn leads to dramatic reduction in glycolysis and glucose-derived acetyl-coenzyme A (acetyl-CoA). Here, we show that the main source of this critical two-carbon intermediate becomes fatty acid (FA) oxidation in acidic pH-adapted cancer cells. FA-derived acetyl-CoA not only fuels the tricarboxylic acid (TCA) cycle and supports tumor cell respiration under acidosis, but also contributes to non-enzymatic mitochondrial protein hyperacetylation, thereby restraining complex I activity and ROS production. Also, while oxidative metabolism of glutamine supports the canonical TCA cycle in acidic conditions, reductive carboxylation of glutamine-derived α-ketoglutarate sustains FA synthesis. Concomitance of FA oxidation and synthesis is enabled upon sirtuin-mediated histone deacetylation and consecutive downregulation of acetyl-CoA carboxylase ACC2 making mitochondrial fatty acyl-CoA degradation compatible with cytosolic lipogenesis. Perturbations of these regulatory processes lead to tumor growth inhibitory effects further identifying FA metabolism as a critical determinant of tumor cell proliferation under acidosis.

Reducing the serine availability complements the inhibition of the glutamine metabolism to block leukemia cell growth.

Leukemia cells are described as a prototype of glucose-consuming cells with a high turnover rate. The role of glutamine in fueling the tricarboxylic acid cycle of leukemia cells was however recently identified confirming its status of major anaplerotic precursor in solid tumors. Here we examined whether glutamine metabolism could represent a therapeutic target in leukemia cells and whether resistance to this strategy could arise. We found that glutamine deprivation inhibited leukemia cell growth but also led to a glucose-independent adaptation maintaining cell survival. A proteomic study revealed that glutamine withdrawal induced the upregulation of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT), two enzymes of the serine pathway. We further documented that both exogenous and endogenous serine were critical for leukemia cell growth and contributed to cell regrowth following glutamine deprivation. Increase in oxidative stress upon inhibition of glutamine metabolism was identified as the trigger of the upregulation of PHGDH. Finally, we showed that PHGDH silencing in vitro and the use of serine-free diet in vivo inhibited leukemia cell growth, an effect further increased when glutamine metabolism was blocked. In conclusion, this study identified serine as a key pro-survival actor that needs to be handled to sensitize leukemia cells to glutamine-targeting modalities.

Paracrine nitric oxide induces expression of cardiac sarcomeric proteins in adult progenitor cells through soluble guanylyl cyclase/cyclic-guanosine monophosphate and Wnt/ß-catenin inhibition.

AIM: Cardiac progenitor cells (CPC) from adult hearts can differentiate to several cell types composing the myocardium but the underlying molecular pathways are poorly characterized. We examined the role of paracrine nitric oxide (NO) in the specification of CPC to the cardiac lineage, particularly through its inhibition of the canonical Wnt/ß-catenin pathway, a critical step preceding cardiac differentiation. METHODS AND RESULTS: Sca1 + CPC from adult mouse hearts were isolated by magnetic-activated cell sorting and clonally expanded. Pharmacologic NO donors increased their expression of cardiac myocyte-specific sarcomeric proteins in a concentration and time-dependent manner. The optimal time window for NO efficacy coincided with up-regulation of CPC expression of Gucy1a3 (coding the alpha1 subunit of guanylyl cyclase). The effect of paracrine NO was reproduced in vitro upon co-culture of CPC with cardiac myocytes expressing a transgenic NOS3 (endothelial nitric oxide synthase) and in vivo upon injection of CPC in infarcted hearts from cardiac-specific NOS3 transgenic mice. In mono- and co-cultures, this effect was abrogated upon inhibition of soluble guanylyl cyclase or nitric oxide synthase, and was lost in CPC genetically deficient in Gucy1a3. Mechanistically, NO inhibits the constitutive activity of the canonical Wnt/ß-catenin in CPC and in cell reporter assays in a guanylyl cyclase-dependent fashion. This was paralleled with decreased expression of ß-catenin and down-regulation of Wnt target genes in CPC and abrogated in CPC with a stabilized, non-inhibitable ß-catenin. CONCLUSIONS: Exogenous or paracrine sources of NO promote the specification towards the myocyte lineage and expression of cardiac sarcomeric proteins of adult CPC. This is contingent upon the expression and activity of the alpha1 subunit of guanylyl cyclase in CPC that is necessary for NO-mediated inhibition of the canonical Wnt/ß-catenin pathway.

Disturbance in Z-disk mechanosensitive proteins induced by a persistent mutant myopalladin causes familial restrictive cardiomyopathy.

BACKGROUND: Familial restrictive cardiomyopathy (FRCM) has a poor prognosis due to diastolic dysfunction and restrictive physiology (RP). Myocardial stiffness, with or without fibrosis, underlie RP, but the mechanism(s) of restrictive remodeling is unclear. Myopalladin (MYPN) is a messenger molecule that links structural and gene regulatory molecules via translocation from the Z-disk and I-bands to the nucleus in cardiomyocytes. Expression of N-terminal MYPN peptide results in severe disruption of the sarcomere. OBJECTIVES: The aim was to study a nonsense MYPN-Q529X mutation previously identified in the FRCM family in an animal model to explore the molecular and pathogenic mechanisms of FRCM. METHODS: Functional (echocardiography, cardiac magnetic resonance [CMR] imaging, electrocardiography), morphohistological, gene expression, and molecular studies were performed in knock-in heterozygote (Mypn(WT/Q526X)) and homozygote mice harboring the human MYPN-Q529X mutation. RESULTS: Echocardiographic and CMR imaging signs of diastolic dysfunction with preserved systolic function were identified in 12-week-old Mypn(WT/Q526X) mice. Histology revealed interstitial and perivascular fibrosis without overt hypertrophic remodeling. Truncated Mypn(Q526X) protein was found to translocate to the nucleus. Levels of total and nuclear cardiac ankyrin repeat protein (Carp/Ankrd1) and phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (Erk1/2), Erk1/2, Smad2, and Akt were reduced. Up-regulation was evident for muscle LIM protein (Mlp), desmin, and heart failure (natriuretic peptide A [Nppa], Nppb, and myosin heavy chain 6) and fibrosis (transforming growth factor beta 1, alpha-smooth muscle actin, osteopontin, and periostin) markers. CONCLUSIONS: Heterozygote Mypn(WT/Q526X) knock-in mice develop RCM due to persistence of mutant Mypn(Q526X) protein in the nucleus. Down-regulation of Carp and up-regulation of Mlp and desmin appear to augment fibrotic restrictive remodeling, and reduced Erk1/2 levels blunt a hypertrophic response in Mypn(WT/Q526X) hearts.

Electrical stimulation of primary neonatal rat ventricular cardiomyocytes using pacemakers.

The study of gene regulation in cardiac myocytes requires a reliable in vitro model. However, monolayer cultures used for this purpose are typically not exposed to electrical stimulation, though this has been shown to strongly affect cardiomyocyte gene expression. Based on pacemakers for clinical use, we developed an easy-to-use portable system that allows the user to perform electro-stimulation of cardiomyocyte cultures in standard tissue incubators without the need for bulky equipment. In addition, we present a refined protocol for culturing high-purity cardiomyocyte cultures with excellent contractile properties for a wide variety of applications.