Mitochondrial implication in intrauterine growth restriction and cardiovascular remodelling

Abstract

Intrauterine growth restriction (IUGR) is an obstetric complication characterized by placental insufficiency and secondary cardiovascular remodeling that may lead to cardiomyopathy in adulthood. Its etiology and potential therapeutics are poorly understood. Mitochondrial bioenergetics pathways are mainly regulated by nuclear effectors such as sirtuins and are essential for embryonic development and cardiovascular function. Members of our group developed a rabbit model of IUGR and cardiovascular remodeling, in which heart, mitochondrial alterations were observed by microscopic and transcriptomic analysis. We aimed to evaluate if such alterations are translated at a functional mitochondrial level to establish the ethiopathology and potential therapeutic targets for this obstetric complication. For that aim, heart and placenta from the rabbit model was included as well as placenta from human pregnancies together with maternal and neonatal blood. At delivery, peripheral blood and cord blood mononuclear cells (PBMC and CBMC, respectively) were isolated.

For the mitochondrial characterization, we assessed: oxygen consumption of the mitochondrial respiratory chain (MRC) by polarography using endogen cellular substrates and substrates for complex I. Also, enzymatic activity of complex I, II, IV, I+III and II+III of MRC, subunit protein expression of some of the MRC complexes (CII-SDHA, CII-SDHB and CIV-COX5A), Coenzyme Q levels, mitochondrial content (through citrate synthase activity, Tom20 expression or mitochondrial DNA (mtDNA) levels), oxidative stress (by lipid peroxidation and SOD2 activity) and ATP levels. Finally, Sirtuin 3 protein expression was measured by Western Blot.

In the IUGR offspring from the rabbit model, we found a significant decrease of MRC function: enzymatic activity of complexes II, IV and II+III in IUGR hearts (p<0.05) and complexes II and II+III in IUGR placentas (p<0.05 and p<0.01, respectively). This was occurring with a not significant reduction in CI-stimulated oxygen consumption in both tissues and a significant decrease of complex II SDHB subunit expression in placenta (p<0.001). Additionally, levels of mitochondrial content, Coenzyme Q and cellular ATP were conserved. Lipid peroxidation significantly decreased in IUGR hearts (p<0.001), but not significantly increased in IUGR placentas. Finally, Sirtuin3 protein expression significantly increased in IUGR hearts (p<0.05).

In human pregnancies, IUGR placental tissue showed an altered mitochondrial phenotype with a significant decrease of CI-stimulated oxygen consumption (p<0.05) and MRC complex I enzymatic activity (p<0.05). The enzymatic activities of the others MRC complexes and CS were preserved. In blood cells, conserved cellular oxygen consumption and trends to decrease CI-stimulated oxygen consumption was observed in maternal PBMC, but trends to decrease both cellular and CI-stimulated oxygen consumption were evidenced in neonatal CBMC, pointing out that IUGR newborns presented higher mitochondrial deficits compared to mothers. Moreover, no differences in MRC enzymatic activities in maternal PBMC or in neonatal CBMC were observed. Conserved CS activity was present in maternal PBMC but was significantly decreased in neonatal CBMC. So, in front of unaltered mtDNA levels in neonatal CBMC,

alterations in neonatal CS would be related to Krebs cycle imbalances rather than to mitochondrial content. All these changes did not affect oxidative stress or ATP production in any tissue. Finally, Sirtuin3 protein expression also showed a relevant increase in human IUGR placenta (p=0.05). The relevance of this thesis relies on the description of mitochondrial impairment in the offspring of a rabbit model of IUGR but also in newborns from pregnancies complicated by IUGR. This mitochondrial imbalance is widely present in the different studied tissues, including the heart and the placenta from the rabbit model and the placenta and neonatal blood cells from human pregnancies. The mitochondrial characterization of this obstetric complication could help to greater understand the pathophysiologic mechanisms underlying cardiac remodelling and IUGR.

Els nounats amb creixement intrauterí restringit (CIR) desenvolupen un remodelat cardiovascular fetal i idiopàtic que pot portar a cardiopatia durant l’etapa adulta. La bioenergètica mitocondrial, essencial pel desenvolupament embrionari i la funció cardíaca, està regulada per diferents proteïnes, entre elles la Sirtuina 3. Es tracta d’una proteïna deacetilasa d’alt interès terapèutic, ja que es pot modular a través de la dieta. Els cors de cries amb CIR d’un model animal de conill mostren alteracions transcriptòmiques i ultraestructurals a nivell mitocondrial. L’objectiu de l’estudi ha sigut determinar la implicació d’una possible disfunció mitocondrial i de la Sirtiuna 3 en el CIR.

Les troballes demostren una alteració mitocondrial de la cadena respiratòria en el cor i la placenta de les cries amb CIR del model animal (sobretot a nivell de l’activitat enzimàtica dels complexes II i IV; p<0.05) i també a la placenta de gestants humanes amb CIR (especialment del complex I; p<0.05). A més a més, aquesta alteració mitocondrial s’ha evidenciat en els nounats amb CIR a través de la reducció de l’activitat de l’enzim citrat sintasa (p<0.05), suggerint alteracions a nivell del cicle de Krebs. L’ATP cel·lular i el dany oxidatiu es troba preservat en tots els teixits estudiats, excepte en el cor de les cries del model animal de CIR, on el trobem disminuït significativament (p<0.001). Aquest desajust mitocondrial va acompanyat d’un augment significatiu de l‘expressió de la proteïna Sirtuina 3 en el cor de les cries del model animal de CIR i també a la placenta de les gestants humanes amb CIR (p<0.05).

Les troballes derivades d’aquest estudi permeten associar la disfunció mitocondrial al desenvolupament del CIR i el remodelat cardiovascular associat, donant lloc al disseny d’estratègies dietètiques destinades a modular l’esmentat desbalanç bioenergètic a través de la regulació de la Sirtuina 3.