CF2 transcription factor is involved in the regulation of Mef2 RNA levels, nuclei number and muscle fiber size

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. CF2 and Mef2 influence a variety of developmental muscle processes at distinct stages of development. Nevertheless, the exact nature of the CF2-Mef2 relationship and its effects on muscle building remain yet to be resolved. Here, we explored the regulatory role of CF2 in the Drosophila embryo muscle formation. To address this question and not having proper null CF2 mutants we exploited loss or gain of function strategies to study the contribution of CF2 to Mef2 transcription regulation and to muscle formation. Our data point to CF2 as a factor involved in the regulation of muscle final size and/or the number of nuclei present in each muscle. This function is independent of its role as a Mef2 collaborative factor in the transcriptional regulation of muscle-structural genes. Although Mef2 expression patterns do not change, reductions or increases in parallel in CF2 and Mef2 transcript abundance were observed in interfered and overexpressed CF2 embryos. Since CF2 expression variations yield altered Mef2 expression levels but with correct spatio-temporal Mef2 expression patterns, it can be concluded that only the mechanism controlling expression levels is de-regu-lated. Here, it is proposed that CF2 regulates Mef2 expression through a Feedforward Loop circuit.This work was supported by Ministerio de Economia y Competitividad español (MINECO) BFU2010-19551 to M

Cancer-associated fibroblasts modify lung cancer metabolism involving ROS and TGF-β signaling

Lung cancer is a major public health problem due to its high incidence and mortality rate. The altered metabolism in lung cancer is key for the diagnosis and has implications on both, the prognosis and the response to treatments. Although Cancer-associated fibroblasts (CAFs) are one of the major components of the tumor microenvironment, little is known about their role in lung cancer metabolism. We studied tumor biopsies from a cohort of 12 stage IIIA lung adenocarcinoma patients and saw a positive correlation between the grade of fibrosis and the glycolysis phenotype (Low PGC-1α and High GAPDH/MT-CO1 ratio mRNA levels). These results were confirmed and extended to other metabolism-related genes through the in silico data analysis from 73 stage IIIA lung adenocarcinoma patients available in TCGA. Interestingly, these relationships are not observed with the CAFs marker α-SMA in both cohorts. To characterize the mechanism, in vitro co-culture studies were carried out using two NSCLC cell lines (A549 and H1299 cells) and two different fibroblast cell lines. Our results confirm that a metabolic reprogramming involving ROS and TGF-β signaling occurs in lung cancer cells and fibroblasts independently of α-SMA induction. Under co-culture conditions, Cancer-Associated fibroblasts increase their glycolytic ability. On the other hand, tumor cells increase their mitochondrial function. Moreover, the differential capability among tumor cells to induce this metabolic shift and also the role of the basal fibroblasts Oxphos Phosphorylation (OXPHOS) function modifying this phenomenon could have implications on both, the diagnosis and prognosis of patients. Further knowledge in the mechanism involved may allow the development of new therapies.Work in the authors’ laboratories is supported by ‘‘Instituto de Salud Carlos III’’ PI13/01806 and PIE14/0064 to M.P. A.C-B, received a Spanish Lung Cancer Group fellowship. R.L-B, is supported by Comunidad Autónoma de Madrid “Garantía juvenil” contract

Cisplatin resistance involves a metabolic reprogramming through ROS and PGC-1α in NSCLC which can be overcome by OXPHOS inhibition

Background: Platinum-based chemotherapy remains the standard of care for most lung cancer cases. However chemoresistance is often developed during the treatment, limiting clinical utility of this drug. Recently, the ability of tumor cells to adapt their metabolism has been associated to resistance to therapies. In this study, we first described the metabolic reprogramming of Non-Small Cell Lung Cancer (NSCLC) in response to cisplatin treatment. Methods: Cisplatin-resistant versions of the A549, H1299, and H460 cell lines were generated by continuous drug exposure. The long-term metabolic changes, as well as, the early response to cisplatin treatment were analyzed in both, parental and cisplatin-resistant cell lines. In addition, four Patient-derived xenograft models treated with cisplatin along with paired pre- and post-treatment biopsies from patients were studied. Furthermore, metabolic targeting of these changes in cell lines was performed downregulating PGC-1α expression through siRNA or using OXPHOS inhibitors (metformin and rotenone). Results: Two out of three cisplatin-resistant cell lines showed a stable increase in mitochondrial function, PGC1-α and mitochondrial mass with reduced glycolisis, that did not affect the cell cycle. This phenomenon was confirmed in vivo. Post-treatment NSCLC tumors showed an increase in mitochondrial mass, PGC-1α and a decrease in the GAPDH/MT-CO1 ratio. In addition, we demonstrated how a ROS-mediated metabolism reprogramming, involving PGC-1α and increased mitochondrial mass, is induced during short-time cisplatin exposure. Moreover, we tested how cells with increased PGC-1a induced by ZLN005 treatment, showed reduced cisplatin-driven apoptosis. Remarkably, the long-term metabolic changes, as well as the metabolic reprogramming during short-time cisplatin exposure can be exploited as an Achilles’ heel of NSCLC cells, as demonstrated by the increased sensitivity to PGC-1α interference or OXPHOS inhibition using metformin or rotenone. Conclusion: These results describe a new cisplatin resistance mechanism in NSCLC based on a metabolic reprogramming that is therapeutically exploitable through PGC-1α downregulation or OXPHOS inhibitors.Work in the authors’ laboratories is supported by ‘‘Instituto de Salud Carlos III’’ PI13/01806 and PIE14/0064 to M.P. A.C-B, received a Spanish Lung Cancer Group fellowship. R.L-B, is supported by Comunidad Autónoma de Madrid “Garantía juvenil” contrac

Plasma Gelsolin Reinforces the Diagnostic Value of FGF-21 and GDF-15 for Mitochondrial Disorders

Mitochondrial disorders (MD) comprise a group of heterogeneous clinical disorders for which non-invasive diagnosis remains a challenge. Two protein biomarkers have so far emerged for MD detection, FGF-21 and GDF-15, but the identification of additional biomarkers capable of improving their diagnostic accuracy is highly relevant. Previous studies identified Gelsolin as a regulator of cell survival adaptations triggered by mitochondrial defects. Gelsolin presents a circulating plasma isoform (pGSN), whose altered levels could be a hallmark of mitochondrial dysfunction. Therefore, we investigated the diagnostic performance of pGSN for MD relative to FGF-21 and GDF-15. Using ELISA assays, we quantified plasma levels of pGSN, FGF-21, and GDF-15 in three age- and gender-matched adult cohorts: 60 genetically diagnosed MD patients, 56 healthy donors, and 41 patients with unrelated neuromuscular pathologies (non-MD). Clinical variables and biomarkers’ plasma levels were compared between groups. Discrimination ability was calculated using the area under the ROC curve (AUC). Optimal cut-offs and the following diagnostic parameters were determined: sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios, and efficiency. Comprehensive statistical analyses revealed significant discrimination ability for the three biomarkers to classify between MD and healthy individuals, with the best diagnostic performance for the GDF-15/pGSN combination. pGSN and GDF-15 preferentially discriminated between MD and non-MD patients under 50 years, whereas FGF-21 best classified older subjects. Conclusion: pGSN improves the diagnosis accuracy for MD provided by FGF-21 and GDF-15

Apoptosis-Inducing Factor Deficiency Induces Tissue-Specific Alterations in Autophagy: Insights from a Preclinical Model of Mitochondrial Disease and Exercise Training Effects

We analyzed the effects of apoptosis-inducing factor (AIF) deficiency, as well as those of an exercise training intervention on autophagy across tissues (heart, skeletal muscle, cerebellum and brain), that are primarily affected by mitochondrial diseases, using a preclinical model of these conditions, the Harlequin (Hq) mouse. Autophagy markers were analyzed in: (i) 2, 3 and 6 month-old male wild-type (WT) and Hq mice, and (ii) WT and Hq male mice that were allocated to an exercise training or sedentary group. The exercise training started upon onset of the first symptoms of ataxia in Hq mice and lasted for 8 weeks. Higher content of autophagy markers and free amino acids, and lower levels of sarcomeric proteins were found in the skeletal muscle and heart of Hq mice, suggesting increased protein catabolism. Leupeptin-treatment demonstrated normal autophagic flux in the Hq heart and the absence of mitophagy. In the cerebellum and brain, a lower abundance of Beclin 1 and ATG16L was detected, whereas higher levels of the autophagy substrate p62 and LAMP1 levels were observed in the cerebellum. The exercise intervention did not counteract the autophagy alterations found in any of the analyzed tissues. In conclusion, AIF deficiency induces tissue-specific alteration of autophagy in the Hq mouse, with accumulation of autophagy markers and free amino acids in the heart and skeletal muscle, but lower levels of autophagy-related proteins in the cerebellum and brain. Exercise intervention, at least if starting when muscle atrophy and neurological symptoms are already present, is not sufficient to mitigate autophagy perturbations

Bases fisiopatológicas de la deficiencia del factor inductor de apoptosis en corazón y músculo esquelético del ratón Harlequin; ejercicio físico como terapia

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 02-12-2021Esta tesis tiene embargado el acceso al texto completo hasta el 02-06-2023Las enfermedades mitocondriales son un grupo de desórdenes genéticos caracterizados por la presencia de mitocondrias cuyo sistema de fosforilación oxidativa (OXPHOS) se encuentra alterado, afectando principalmente a los órganos con mayores requerimientos energéticos, como el corazón y el músculo esquelético. El factor inductor de apoptosis (AIF) es una proteína mitocondrial con funciones relevantes tanto en la fisiología celular —entre las cuales se encuentra el mantenimiento de la función OXPHOS— como en la inducción de la muerte celular tras migrar al núcleo. Actualmente se desconocen muchos de los efectos que, a nivel molecular, la deficiencia de AIF produce en el corazón y el músculo esquelético. Por otro lado, el ejercicio físico moderado ha demostrado numerosos beneficios en los pacientes con enfermedad mitocondrial. Sin embargo, todavía se desconocen gran parte de los mecanismos moleculares que explican dichas mejoras en los músculos estriados. Los objetivos principales de la presente tesis doctoral fueron: estudiar las bases fisiopatológicas que subyacen al fenotipo causado por la deficiencia de AIF a distintas edades (2, 3 y 6 meses de edad); y estudiar los efectos de un programa de entrenamiento combinado de resistencia y fuerza de una duración de 8 semanas. Ambos objetivos se llevaron a cabo en corazón y en músculo esquelético del ratón Harlequin (Hq), caracterizado por presentar un déficit ubicuo de los niveles de AIF. Los resultados mostraron alteraciones en numerosos procesos celulares en el corazón y el músculo esquelético de los ratones Hq. Concretamente, se observó un fallo en ensamblaje de algunos complejos del sistema OXPHOS, que únicamente se tradujo en un descenso de actividad del complejo I en músculo esquelético. Además, la ultraestructura mitocondrial resultó alterada en ambos tejidos, con unas mitocondrias de mayor área y más alargadas, posiblemente debido a un aumento en la fusión mitocondrial. Tanto el corazón como el músculo esquelético mostraron un aumento del estrés oxidativo; una distribución alterada de los lípidos, con una mayor cantidad de gotas lipídicas; y un mayor contenido de aminoácidos libres. También se detectaron cambios en el proceso de autofagia, con una acumulación de distintas proteínas de la ruta, así como una mayor activación de mTOR. Además, se observó un posible fallo en la biosíntesis del grupo hemo, disminuyendo los niveles de hemoglobina y mioglobina en corazón. Además, se detectaron alteraciones en el mecanismo de excitación-contracción de los cardiomiocitos, consistentes en una mayor fuga de Ca2+ desde el retículo sarcoplásmico (RS) mediada por sparks, y una menor capacidad de recaptación del Ca2+ hacia el RS durante la diástole. Por último, pese a que el programa de entrenamiento produjo mejoras en la capacidad aeróbica y la fuerza muscular de los ratones Hq, no fue capaz atenuar las alteraciones observadas a nivel molecular. La presente tesis ha permitido descubrir importantes alteraciones moleculares en los músculos estriados del ratón Hq, que abren la puerta a futuros estudios, así como a la búsqueda de nuevas dianas terapéuticas, en el contexto de las enfermedades mitocondriales y de las patologías asociadas a una deficiencia de AIFEsta tesis doctoral ha sido realizada con cargo al proyecto de investigación FIS PI17/0009

List of Drosophila lines used in this study.

<p>List of Drosophila lines used in this study.</p

Nuclei number in Dorsal Acute 1 muscle in interfered and overexpressed stage 17 embryos.

<p>Statistical significance: **p<0.005; ***p<0.001. ns–no significant.</p

Proposed model for a mechanism of regulation in that CF2 regulates Mef2 expression through a Feedforward Loop (FFL) circuit.

<p>At stage 11, twi activates <i>Mef2</i> transcription which in turn activates its own transcription in a twi independent manner. At mid stage 12, Mef2 inducts CF2 transcription, which in turn increases Mef2 expression. Both transcription factors, Mef2 and CF2, cooperate to maintain high levels of Mef2 transcription and influence the fusion process. In the differentiated fiber, both factors collaborate in the regulation of sarcomeric genes expression (panel B). In the absence of CF2 (panel A), the feedback loop is lost and Mef2 expression is not increased by the action of CF2. Therefore, Mef2 expression relays only in the self-activation circuit, which renders low Mef2 expression levels with the concomitant impact on muscle fiber terminal differentiation, and in the regulation of sarcomeric genes expression.</p