Rudolf J Wiesner - assoc. RG

Tissue stem cells (SC) show a multitude of mechanisms to avoid or repair damage, including low mitochondrial mass and minimal use of oxidative metabolism. However, mitochondrial mass must be dramatically expanded during SC differentiation, thus, intactness of mtDNA is essential. The aim of this project is to test (i) if mtDNA is only rarely replicated in quiescent muscle SCs or, alternatively, (ii) that it is purified during recruitment, i.e. wt mtDNA copies are kept in MuSCs while ΔmtDNA is passed to the progeny.

Introduction

Skeletal muscle contains a quiescent stem cell pool (satellite cells; MuSCs) which is only recruited after injury, but also after strenous, albeit physiological exercise. The regenerative capacity of MuScs declines with age and recent studies have suggested that progressive aging-related and cell-intrinsic alterations play a greater role in this functional decline than previously thought. Those cell-intrinsic alterations are discussed as important factors contributing to aging-induced sarcopenia, defined as a loss of muscle mass and function. Understanding the processes causing those aging-related alterations in MuSCs is thus essential to establish new strategies to delay the development of sarcopenia, e.g. by eccentric exercise regimes, which have been reported to be beneficial in humans. 

SCs in general have a multitude of mechanisms to avoid or repair damage to all cellular structures. For example, hematopoietic SCs have only few mitochondria and few copies of mitochondrial DNA (mtDNA), and are highly glycolytic, a metabolic strategy thought to minimize collateral damage by oxidative metabolism and maintain stemness. Nevertheless, both mitochondrial mass and mtDNA copy number must be expanded during differentiation of activated MuSc into mature fibers, which are largely dependent on mitochondrial metabolism. Thus, intactness of the mtDNA in MuSC  is essential for normal skeletal muscle regeneration, but little is known about the mechanisms maintaining its integrity, which is likely to be compromised during aging.

In order to study the cell specific consequences of mtDNA deletions, we have generated a mouse strain which expresses a dominant-negative mutant of the mitochondrial helicase Twinkle (Rosa26-Stop-construct, point mutation K320E), strongly enhancing the generation of ΔmtDNA species, as observed in tissues of affected patients.

Expression of  this construct in the myocardium (Ckmm-Cre) leads, as intended, to a tissue mosaic with only few COX^neg cells, which however causes severe arrhythmia, again a typical and important age-related disorder (Baris, …, Wiesner 2015). Expression of K320E-Twinkle in the epidermis (K14-Cre) causes severe inflammation with upregulation of a variety of pro-inflammatory cytokines (Weiland,…, Wiesner & Baris, 2018). Both studies therefore unequivocally showed that mitochondrial tissue mosaics are indeed of pathophysiological relevance.

Do mtDNA deletions in differentiated muscle cells cause sarcopenia?

We have expressed  K320E-Twinkle in differen-tiated muscle cells driven by Mlc1f-Cre. Those mice (K320E-Twinkle^skm) display a remarkable accumulation of COX^neg muscle fiber segments (Fig. 1 A,B), but surprisingly do not show hallmark features of sarcopenia (loss of muscle mass and function) as expected (Fig. 1C- H).  Transcriptomic and proteomic studies are underway to determine potential compensatory mechanisms in these mice.

Do mtDNA deletions in MuSCs cause sarcopenia?

Next, we have induced ΔmtDNA in muscle satellite cells (MuSCs) and study (i) possible mechanisms maintaining mtDNA integrity and (ii) whether these deteriorate during aging. For this purpose, we express K320E-Twinkle specifically in MuScs driven by Pax7-Cre^ERT. When the tibialis anterior (TA) muscle of those mice (K320E-Twinkle^MuSC) is damaged following cardiotoxin (CTX) injection, its regeneration from MuScs is dramatically impaired when compared to muscle regenerated in control mice (Fig. 2). This shows that mitochondrial dysfunction induced by mtDNA deletions is an important intrinsic factor impairing the regenerative capacity of MuSC. 

Perspectives 

The decline in regenerative capacity of MuSCs with age  contributes to the development  of sarcopenia. Sarcopenia leads to physical disability, but more importantly to an increased incidence of falls followed by hospitalization, which may result in premature death. Loss of muscle mass may also contribute to insulin resistance typical for old age. Understanding aging-related alterations in mechanisms that maintain mtDNA integrity in MuSCs is essential to establish new strategies to delay the development of sarcopenia.