Fusion of Wild-Type Mesoangioblasts with Myotubes of mtDNA Mutation Carriers Leads to a Proportional Reduction in mtDNA Mutation Load.

In 25% of patients with mitochondrial myopathies, pathogenic mitochondrial DNA (mtDNA) mutation are the cause. For heteroplasmic mtDNA mutations, symptoms manifest when the mutation load exceeds a tissue-specific threshold. Therefore, lowering the mutation load is expected to ameliorate disease manifestations. This can be achieved by fusing wild-type mesoangioblasts with mtDNA mutant myotubes. We have tested this in vitro for female carriers of the m.3271T>C or m.3291T>C mutation (mutation load >90%) using wild-type male mesoangioblasts. Individual fused myotubes were collected by a newly-developed laser capture microdissection (LCM) protocol, visualized by immunostaining using an anti-myosin antibody. Fusion rates were determined based on male-female nuclei ratios by fluorescently labelling the Y-chromosome. Using combined 'wet' and 'air dried' LCM imaging improved fluorescence imaging quality and cell yield. Wild-type mesoangioblasts fused in different ratios with myotubes containing either the m.3271T>C or the m.3291T>C mutation. This resulted in the reduction of the mtDNA mutation load proportional to the number of fused wild-type mesoangioblasts for both mtDNA mutations. The proportional reduction in mtDNA mutation load in vitro after fusion is promising in the context of muscle stem cell therapy for mtDNA mutation carriers in vivo, in which we propose the same strategy using autologous wild-type mesoangioblasts.

Healthy, mtDNA-mutation free mesoangioblasts from mtDNA patients qualify for autologous therapy.

BACKGROUND: Myopathy and exercise intolerance are prominent clinical features in carriers of a point-mutation or large-scale deletion in the mitochondrial DNA (mtDNA). In the majority of patients, the mtDNA mutation is heteroplasmic with varying mutation loads between tissues of an individual. Exercise-induced muscle regeneration has been shown to be beneficial in some mtDNA mutation carriers, but is often not feasible for this patient group. In this study, we performed in vitro analysis of mesoangioblasts from mtDNA mutation carriers to assess their potential to be used as source for autologous myogenic cell therapy. METHODS: We assessed the heteroplasmy level of patient-derived mesoangioblasts, isolated from skeletal muscle of multiple carriers of different mtDNA point-mutations (n = 25). Mesoangioblast cultures with < 10% mtDNA mutation were further analyzed with respect to immunophenotype, proliferation capacity, in vitro myogenic differentiation potential, mitochondrial function, and mtDNA quantity. RESULTS: This study demonstrated that mesoangioblasts in half of the patients contained no or a very low mutation load (< 10%), despite a much higher mutation load in their skeletal muscle. Moreover, none of the large-scale mtDNA deletion carriers displayed the deletion in mesoangioblasts, despite high percentages in skeletal muscle. The mesoangioblasts with no or a very low mutation load (< 10%) displayed normal mitochondrial function, proliferative capacity, and myogenic differentiation capacity. CONCLUSIONS: Our data demonstrates that in half of the mtDNA mutation carriers, their mesoangioblasts are (nearly) mutation free and can potentially be used as source for autologous cell therapy for generation of new muscle fibers without mtDNA mutation and normal mitochondrial function.

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.

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.

"When looking back on my past life I regret...": retrospective regret in the second half of life.

The study investigated the frequency, themes, and attributions for significant regrets in a random probability sample of 3,917 German and Dutch nationals between the ages of 40 and 85 years. It was found that 14% did not have any regrets in spontaneous memory, and that this increased with the age of the respondents. With respect to mentioned regrets, older people, women, and those living in the former East Germany were more likely to recall externally attributed events; younger participants, men, West Germans, and the Dutch recalled more internally attributed events. Largely, memories related to 4 major themes: (a) mistakes, behavior, and bad decisions in general; (b) hard times; (c) social relationships; and (d) missed educational opportunities. The importance of these themes, however, varied according to age, gender, and regional belonging. Differences in the kind of attribution and in the centrality of themes are discussed in terms of lifespan theory, death preparation, and cultural differences.

Cognitive representations of future gains, maintenance, and losses in the second half of life.

The aim of this study is to explore three different developmental dimensions in an aging population. Based on sentence completion responses, the investigation examines personal anticipations of possible gains, maintenance, and losses. Additionally, the effects of age and other personal and situational factors are examined. The study sample consists of 2,934 participants ranging from 40 to 85 years of age, who participated in the German Aging Survey of 1996. Study findings indicate that, to a large extent, the anticipated gains include positive changes in the way of life and increased leisure projects while anticipated maintenance refer to physical and behavioral resources and to life style. Anticipated losses are related to concerns about external living conditions and physical decline. There is a strong association of anticipated gains and maintenance with age, while present health conditions are related to expectations of loss. The implications of the study results for lifespan expectations in the second half of life and for lifespan theory are discussed.