Heart specific scoX knockdown induces p53 dependent apoptosis and cardiomyopathy

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 11/07/2014La citocromo c oxidasa o complejo IV es el ultimo elemento de la cadena de transporte de electrones mitocondrial. Cataliza la oxidación del citocromo c transfiriendo sus electrones al oxígeno. El déficit de complejo IV debido a mutaciones en factores de ensamblaje es uno de los defectos más comunes de la cadena respiratoria. Estas patologías se caracterizan por su aparición a una edad temprana y cursar con un amplio espectro de manifestaciones clínicas como encefalopatía, cardiomiopatía, hepatopatía o leucodistrofia. SCO1 y SCO2 son dos metalochaperonas responsables de la formación del centro de cobre CuA en el ensamblaje del complejo IV. Mutaciones en SCO1 causan hepatoencefalomiopatía aunque se ha descrito un caso que también presenta cardiomiopatía hipertrófica. Las mutaciones en SCO2 causan cardiomiopatía hipertrófica y encefalopatía infantil. A excepción de un caso, todos los pacientes portan la mutación E140K. En esta tesis hemos utilizado Drosophila melanogaster como sistema modelo para el estudio de las bases genéticas y moleculares de la cardiopatía causada por mutaciones en las proteínas SCO. Los mecanismos genéticos que controlan la especificación de los cardiomiocitos y numerosos aspectos de la fisiología del corazón están conservados en Drosophila lo que hace de este organismo un excelente modelo para el estudio de la función cardiaca y las cardiomiopatías humanas. Además, recientemente se han desarrollado técnicas que permiten caracterizar la fisiología del corazón de Drosophila, facilitando el estudio de cómo defectos en la cadena de transporte de electrones, afectan a la función cardiaca. Drosophila presenta un solo ortólogo para los genes de mamífero Sco1 y Sco2, ScoX. Los resultados obtenidos demuestran que la interferencia de ScoX en el corazón causa una cardiomiopatía dilatada, viéndose gravemente afectada tanto la función como la estructura del corazón. Los cardiomiocitos sufren un cambio metabólico favoreciendo la glicólisis frente a la fosforilación oxidativa, causando, probablemente, acidosis láctica y emulando los síntomas clínicos observados en pacientes con mutaciones en Sco1 y Sco2. El fenotipo observado es debido a una activación, dependiente de dp53, de la muerte celular. Estos resultados sugieren que dp53 contribuye directamente en el desarrollo de la cardiomiopatía.Cytochrome c oxidase or complex IV is the terminal component of the mitochondrial electron transport chain. It catalyses the transfer of electrons from reduced cytochrome c to molecular oxygen. Complex IV deficiency due to mutations in assembly factors is one of the most frequent defects of respiratory chain in humans. These pathologies are characterized by a very early age of onset and the display of different clinical presentations, as encephalopathy, cardiomyopathy, hepatic failure and leukodystrophy. SCO1 and SCO2 are two metallochaperones playing a key role in the formation of cooper centre CuA during complex IV assembly. Mutations in SCO1 cause hepatoencephalomyopathy although one case has been reported which also presents hypertrophic cardiomyopathy. Mutations in SCO2 cause hypertrophic cardiomyopathy and infantile encephalomyophathy and with but one exception, all patients harbour the E140K mutation. In this thesis, we have used Drosophila melanogaster as model system to investigate the genetic and molecular mechanisms that underlie the cardiomyopathy associated with SCO deficiency. The genetic network controlling cardiac specification and differentiation as well as many aspects of heart function are conserved from flies to mammals. Thus, Drosophila has become a powerful model system for the study and understanding of cardiac function and human cardiomyopathies. Furthermore, the recently established heart function assays in Drosophila make it possible to characterize how mitochondrial electron transport chain defects affect heart function. In Drosophila there is a single ortholog of mammalian Sco1 and 2, ScoX. Our findings demonstrate that cardiac-specific knockdown of ScoX causes dileted cardiomyopathy severely compromising heart function and structure. Cardiomyocytes undergo a metabolic switch from oxidative phosphorylation to glycolysis probably accompanied by lactic acidosis and therefore mimicking the clinical features found in patients with mutations in Sco1 and Sco2. The observed phenotype is result of dp53- dependent cell death activation. These results strongly suggest that dp53 is directly involved in cardiomyopathy development

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

Cardiac deficiency of single cytochrome oxidase assembly factor scox induces p53-dependent apoptosis in a Drosophila cardiomyopathy model

et al.The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease produced by defects in the oxidative phosphorylation (OXPHOS) system are susceptible to cardiac involvement. The presentation of mitochondrial cardiomyopathy includes hypertrophic, dilated and left ventricular noncompaction, but the molecular mechanisms involved in cardiac impairment are unknown. One of the most frequent OXPHOS defects in humans frequently associated with cardiomyopathy is cytochrome c oxidase (COX) deficiency caused by mutations in COX assembly factors such as Sco1 and Sco2. To investigate the molecular mechanisms that underlie the cardiomyopathy associated with Sco deficiency, we have heart specifically interfered scox expression, the single Drosophila Sco orthologue. Cardiac-specific knockdown of scox reduces fly lifespan, and it severely compromises heart function and structure, producing dilated cardiomyopathy. Cardiomyocytes with low levels of scox have a significant reduction in COX activity and they undergo a metabolic switch from OXPHOS to glycolysis, mimicking the clinical features found in patients harbouring Sco mutations. The major cardiac defects observed are produced by a significant increase in apoptosis, which is dp53-dependent. Genetic and molecular evidence strongly suggest that dp53 is directly involved in the development of the cardiomyopathy induced by scox deficiency. Remarkably, apoptosis is enhanced in the muscle and liver of Sco2 knock-out mice, clearly suggesting that cell death is a key feature of the COX deficiencies produced by mutations in Sco genes in humans.This work was supported by grants Direccion General de Investigacion Ciencia y Tecnologia (BFU2007-61711BMC and BFU2010-19551 to M.C.), American Heart Association (Grant in Aid #14GRNT20490239 to K.O.), NASA (NRA NNH12ZTT001N to K.O. and NRA NNH12ZTT001N to R.B.), National Institute of Health (R01 HL054732, P01 AG033461, P01 HL098053 to R.B.), Centre for Biomedical Research on Rare Diseases, Instituto de Salud Carlos III (PI10/0703 and PI13/00556 to R.G.), Comunidad de Madrid (S2010/BMD-2402 to R.G.), Muscular Dystrophy Association to E.A.S., the U.S. Department of Defence (W911F-12-1-0159 to E.A.S.) and J. Willard and Alice S. Marriott Foundation to E.A.S.Peer Reviewe

List of Drosophila lines used in this study.

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

Genotype identification of an individual embryo.

<p>The presence or absence of the balancer chromosome was assayed by conventional PCR in individual embryos carrying the Dfγ²⁷ deficiency, UAS-CF2 or Gal4 insertions. Homozygous embryos are indicated with arrows. For all three fly lines the upper part of the panels show a positive control PCR against a single copy CG9650 unrelated gene. (A). Dfγ²⁷ embryos. The middle part of the panel shows the result obtained when the balancer marker gene LacZ was amplified. The lower part of the panel presents the results for the <i>CF2</i> gene. Only those embryos showing no amplification of both genes were considered homozygous for the Dfγ²⁷ deficiency. (B). UAS-CF2. The middle part of the panel shows the results obtained when the balancer marker gene GFP was amplified. The lower part of the panel shows the result for UAS sequence from the UAS-CF2 insertion. Only those embryos showing no GFP amplification and the presence of the UAS region were considered to carry the UAS-CF2 insertion in both chromosomes. (C). <i>Gal4</i> driver lines. The middle part of the panel shows the results obtained when the balancer marker gene GFP was amplified. The lower part of the panel shows the result for <i>Gal4</i> sequence in the driver. Only those embryos showing no GFP amplification and the presence of the <i>Gal4</i> region were considered to carry the two copies of the driver, one in each chromosome.</p

LT1-4 muscle size in μm² from control, homozygous Df2g27, CF2i and CF2 OE embryos.

<p>LT1 to LT4 muscle size is given as mean +/- standard deviation. Statistical significance:. * p < 0.05; ** p < 0.005.</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