Effects of ageing and genetic risk variants at 4q25 on the calcium homeostasis in cardiac myocytes
[eng] Background Ageing is a risk factor that promotes common cardiovascular diseases such as atrial fibrillation (AF) or heart failure (HF), which in turn are associated with pathological changes in intracellular calcium homeostasis. However, the effects that ageing could have on the calcium homeos...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2016 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/109019 |
| Acceso en línea: | https://hdl.handle.net/2445/109019 http://hdl.handle.net/10803/401751 |
| Access Level: | acceso abierto |
| Palabra clave: | Fibril·lació auricular Electrofisiologia Homeòstasi Atrial fibrillation Electrophysiology Homeostasis |
| Sumario: | [eng] Background Ageing is a risk factor that promotes common cardiovascular diseases such as atrial fibrillation (AF) or heart failure (HF), which in turn are associated with pathological changes in intracellular calcium homeostasis. However, the effects that ageing could have on the calcium homeostasis in human atrial cardiomyocytes are not well known. Furthermore, genetic risk variants at single nucleotide polymorphisms (SNPs) associated with a higher incidence of AF have been identified in the chromosomal region 4q25, close to the locus of the Pitx2 transcription factor that plays an important role in cardiac embryonic development. In the adult heart AF has been associated with changes in the expression of Pitx2, but findings are contradictory and the relationship between the 4q25 risk variants and Pitx2 function remain controversial. Moreover, no functional effects of the 4q25 risk variants on the calcium homeostasis have been identified so far. Therefore, in this thesis we investigated how the two risk factors, ageing and risk variants at 4q25, affect the intracellular calcium homeostasis with the intention of identifying mechanisms that underlie potentially arrhythmogenic changes in the calcium homeostasis caused by these risk factors. Hypothesis Ageing and 4q25 risk variants produce alterations in the intracellular calcium homeostasis in atrial myocytes that alone or in combination contribute to increase the propensity to atrial fibrillation. Aims of the thesis * Analyze the effects of ageing on the mechanisms that regulate the calcium homeostasis in human atrial myocytes. * Use transgenic murine models of ageing to identify molecular mechanisms underlying ageing-dependent changes in the calcium homeostasis. * Investigate how risk variants on chromosome 4q25 associated with increased AF risk, affect electrophysiological characteristics of human atrial myocytes and to identify underlying molecular mechanisms. * Investigate how ageing modulates the effects of 4q25 risk variants in human atrial myocytes. Methods Experiments were performed on isolated atrial or ventricular cells from human or murine models. Electrophysiological data were obtained using patch- clamp techniques and confocal microscopy. RT-PCR and western blot techniques were used to determine the expression levels of mRNA and the proteins studied. Results The results described in this thesis show that aging decreases the amplitude of the calcium current (ICa), an electrophysiological parameter that is also reduced by AF, the calcium content of the sarcoplasmic reticulum (SR), and the global calcium transient. These findings are corroborated by comparable changes in the expression of the proteins undertaking the corresponding calcium transport or buffering. Together, these changes likely reduce atrial contraction in the elderly. The results were reproduced in an animal model of premature aging (Zmpste24-/-) with defective lamin processing, reinforcing the notion that this mechanism may, at least partially, underlie the observed effects of aging on the calcium homeostasis in human atrial myocytes. In addition, ageing accentuated some of the effects of ageing on calcium handling in patients with a previous history of AF. Of particular interest, the ICa amplitude was further depressed by aging in patients with AF, which might contribute to maintain atrial arrhythmic episodes in these patients. The study of risk variants on chromosome 4q25 shows that the presence of the rs13143308T risk variant, alone or together with the risk variant rs2200733T, was associated with a higher frequency of spontaneous calcium release, transient inward currents (ITI) and membrane depolarizations, typical of AF. These results are the first to provide an electrophysiological mechanism that could explain a higher incidence of AF in individuals carrying risk variants at 4q25. Moreover, electrophysiological studies in right atrial myocytes from a mouse model with atrial Pitx2 insufficiency (NppaCre+Pitx2fl/-) reproduces all alterations in calcium homeostasis observed in patients with 4q25 risk variants. These results support the notion that modulation of the intracellular calcium handling by Pitx2 plays an important role in electrophysiological processes associated with AF. Analysis of potential synergies between risk variants at 4q25, ageing, and AF revealed that several effects of ageing were accentuated in patients with AF whereas aging per se does not modify the effects of the 4q25 risk variants on calcium homeostasis. However, as aging reduces the amplitude of ICa, which would reduce the atrial refractory period, this could prolong the duration of atrial arrhythmic episodes favored by the higher frequency and amplitude of spontaneous membrane depolarizations in carriers of 4q25 risk variants. Conclusions Ageing modulates calcium homeostasis in human atrial myocytes by decreasing ICa, calcium transient and SR calcium load. These changes are reproduced in a progeric mouse model, favoring a progressive decline of contractile function with age. Moreover, the observed ICa reduction is a characteristic feature of AF that may favor its maintenance in elder patients. Risk variants located on the chromosomal region 4q25, specifically the variant rs13143308T, alone or together with rs2200733T, increases spontaneous calcium release, ITIs, and membrane depolarizations. These changes are reproduced in a mouse model of Pitx2 insufficiency, and are all hallmarks of AF that could favor the initiation of arrhythmic events in carriers of the risk variants. The combined effects of 4q25 risk variants and ageing may work synergistically to promote atrial arrhythmia, with the former constituting an arrhythmogenic electrophysiological substrate that favor initiation of atrial arrhythmic episodes, and with ageing favoring their maintenance. |
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