Aging Science Talks: The role of miR-181a in age-related loss of muscle mass and function.

The outbreak of COVID-19 was declared a Public Health Emergency of International Concern in January 2020, scientists all over the world found new ways of progressing scientific research and maintaining or even expanding collaboration in a virtual setting. “Aging Science Talks” were one of the most successful initiatives that engaged scientists all over the world working in the field of aging and highly relevant in the context of the pandemic.

Aging is the biggest risk factor for developing chronic disorders such as heart disease, type 2 diabetes, chronic obstructive pulmonary disease (COPD), stroke, neurodegenerative diseases, chronic kidney diseases (CKDs) and cancer [1,2]. Aging is a complex process with detrimental effects on cell and tissue homeostasis which ultimately effects organ function [3]. Several mechanistic pathways have been associated with aging, such as an increase in inflammation, oxidative stress, DNA mutations, mitochondrial dysfunction, accumulation of senescence cells and disrupted proteostasis [[4], [5], [6]]. Although life expectancy has increased, this has not been accompanied by an increase in health span, which can subsequently result in a greater strain on healthcare systems [7]. The susceptibility of the aged individual suffering chronic disorders has been recently highlighted due to the Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Emerging data show that older people and those with certain underlying medical conditions, such as obesity or diabetes, are particularly affected by SARS-CoV-2 [[8], [9], [10], [11]]. Aging and COVID-19 are associated with changes in systemic inflammation and dysregulation of the immune system function [8] and it has been suggested that COVID-19 survivors, especially those requiring mechanical assisted ventilation will suffer from frailty [12]. Early studies have shown the existence of musculoskeletal deterioration in patients with COVID-19, although long-term follow-up studies are needed [13].

Frailty is largely associated with sarcopenia, aging-related loss of muscle mass and function, characterised by a progressive and degenerative loss of skeletal muscle mass, quality, and strength during aging [14]. Sarcopenia affects 5–13% of 60–70 year olds and up to 50% of people over 80 [15]. Currently, diet and exercise remain the only effective interventions, that aim to maintain muscle mass and force, however no therapeutic has been established to successfully treat sarcopenia [16,17]. The molecular mechanisms underlying sarcopenia are multifactorial [[18], [19], [20]]. Nevertheless, the transcriptome and proteome changes, as well as epigenetic changes in skeletal muscle during aging have been identified [[21], [22], [23], [24], [25]] [[21], [22], [23], [24], [25]] [[21], [22], [23], [24], [25]].

The role of microRNAs (miRNAs, miRs) as epigenetic modifiers in regulating loss of muscle mass and function has become increasingly recognised (reviewed in Ref. [17]). miRs are short, non-coding RNAs which regulate the expression of approximately 2/3 human genes [26]. miRs bind to their targets, usually within the 3′UTR region of mRNAs, via sequence complementarity. This results in gene expression regulation at the post-transcriptional level, through the degradation of the target mRNA(s) and/or translational block, ultimately resulting in decreased protein levels [26].

In skeletal muscle, miRs have been demonstrated to control multiple biological processes, including development, regeneration, and aging (reviewed in Ref. [27,28]). A number of miRs are involved in the regulation of muscle protein synthesis, that target regulators involved maintaining the balance between muscle atrophy and hypertrophy, and including regeneration of skeletal muscle [[29], [30], [31], [32]]. Early studies in humans demonstrated differential expression of miRs in skeletal muscle during aging [24,33]. We and others have demonstrated the role of miRs in aging-associated processes in skeletal muscle, such as satellite cell senescence and inflammation [30,34,35].

Bioinformatic analyses of non-coding RNAs and transcripts in human and rodent muscle during aging have identified miR-181a as a potentially key regulator of muscle mass and function during aging [25,36]. The miR-181 family of miRs includes four miRs in humans and rodents: miR-181a, b, c and d. miR-181a and b are clustered together at two genomic loci on chromosomes 1 and 9 and miR-181c and d are clustered on chromosome 19 in humans. Interestingly, the two miR-181 clusters producing two members of miR-181 family each, have been shown to have divergent roles in myocardial function [37]. The miR-181 family is predicted to regulate over 1000 genes [38]. Unsurprisingly, the miR-181 family has been shown to regulate multiple genes in many tissues, including genes associated with mitochondrial dynamics [37,[39], [40], [41], [42], [43], [44], [45], [46], [47], [48]].

In skeletal muscle, miR-181a appears to be the predominant miR-181 family member in skeletal muscle affected by aging and has been suggested as biomarker of muscular health [36,46]. miR-181 has also been demonstrated to be upregulated in muscle during exercise and predicted to regulate transcription factors and co-activators involved in the adaptive response of muscle to exercise [49]. Based on computer simulation models, miR-181a was predicted to regulate muscle atrophy and hypertrophy through its target genes: HOXA11 by inhibiting MYOD, and SIRT1, through regulating FoxO3 signalling [50] (Fig. 1 ). Indeed, we and others confirmed these as miR-181a direct targets [25,50]. More recently, miR-181 family of miRs gained more attention due to their regulation of processes associated with mitochondrial dynamics and directly targets PARK2 and p62/SQSTM1 [44,46,51] (see Fig. 1).

Fig. 1

The role of miR-181a in skeletal muscle aging.

Mitochondrial dysfunction is one of the hallmarks of aging [5]. During aging, skeletal muscle is characterised by a loss of mitochondrial content [52] and disrupted mitochondrial turnover, particularly in sedentary individuals [46,[53], [54], [55], [56]]. A number of studies to date suggest that miR-181a may be a global regulator of mitochondrial dynamics, redox homeostasis and potentially energy balance of the whole organism [34,39,44,46,51].

For example, post-stroke treatment with antagomiR-181a has been shown to reduce infarction size through regulating BCL-2 expression and NF-KB activation [57]. The regulation of Bcl-1 by miR-181 has also been demonstrated in astrocytes [40]. In this study, reduction of miR-181 levels and concomitant upregulation of BCL-2 led to a controlled increase in oxidative stress and antioxidant defence. Furthermore, inhibition of miR-181a was reported to protect the brain from stroke via regulation of GRP78 and miR-181a/b downregulation has been proposed to protect retinal neurons from cell death through regulating mitochondrial homeostasis [44,58].

The role of miR-181 in regulating the response to oxidative stress has also been studied in the context of myocardial function [37]. miR-181c levels were shown to negatively correlate to those of COX1, whereas miR-181a/b were suggested to regulate the levels of PTEN [37]. The authors proposed that decreased levels of miR-181c resulted in COX1 upregulation and dysfunction of complex IV and increased ROS production in vitro [59].

It therefore appears that miR-181 may play a critical role in regulating redox homeostasis and potentially mitochondrial dynamics in nervous and cardiac cells, with studies suggesting that inhibition of miR-181 may prevent cell death and improve antioxidant response via targeting BCL-1, BCL-2, GRP78 and PTEN. Interestingly, in other tissues miR-181 has been demonstrated to be a positive regulator of metabolism, mitochondrial function and redox homeostasis [60].

miR-181 has been proposed to function as the primary metabolic rheostat in immune cells trough targeting of PTEN and PI3K signaling [61]. Loss of miR-181a1/b1 in thymocytes was demonstrated to result in major metabolic reprogramming: a dysregulated expression of key components of the glycolytic, pentose phosphate and nucleotide biosynthetic pathways and miR-181a1/b1-deficient cells failed to reach the biosynthetic potential of proliferating thymocytes [62]. This led to suboptimal glucose uptake, reduced glycolytic rates, and impaired metabolic fitness in miR-181-deficient cells [62].

Moreover, mice lacking multiple miR-181 alleles display immune phenotypes in B-cell development and T-cell homeostatic proliferation [63]. Interestingly, miR-181a1/b1; miR-181a2/b2 double knockout mice show reduced survival when compared to littermates and a significant reduction in body size [62]. Furthermore, no data on triple knock-out mice exists, which may suggest that complete deficiency of the miR-181 family is lethal [61].

Consistently, the role of miR-181 as a positive regulator of metabolism though regulation of PTEN, overexpression of the miR-181a/b1 cluster was also demonstrated to enhance osteogenesis through protein synthesis and mitochondrial metabolism [51]. Moreover, oxygen consumption rate and ATP-linked respiration were increased by miR-181a/b1 through its regulation of the PTEN/PI3K signalling [51]. In the context of cancer, miR-181a has been shown to have pro- but also anti-oncogenic role [39,41,[64], [65], [66]]. In CD8+ T cells, interferon-γ is regulated by miR-181a during differentiation [67].

In our study, using miR gain- and loss-of-function approaches, we have demonstrated that miR-181 regulates skeletal muscle size and function during aging through regulating mitochondrial dynamics [46]. Specifically, we demonstrated that miR-181a prevents the accumulation of Park2, DJ-1 and p62 in muscle during aging. This accumulation could be associated with disrupted mitochondrial turnover observed during aging [46]. Using the mitoQC reporter construct for the investigation of mitophagic flux [68], we demonstrated that miR-181a overexpression promotes mitophagy. The increased mitophagic flux may be associated with activation of alternative pathways than Pink/Parkin pathway, such as receptor-mediated mitophagy via BNIP3. Mitochondrial turnover is regulated by a number of different pathways and the exact regulatory mechanisms have not yet been fully determined [69,70]. Interestingly, we also observed a parallel increase in markers of mitochondrial biogenesis, e.g. increased PGC1a expression, following miR-181a overexpression in skeletal muscle [46]. A coupling of mitochondrial biogenesis and mitophagy has been previously observed in C. elegans and proposed to be mediated via NRF2 pathway [71]. Ultimately the overexpression of miR-181a resulted in increased mitochondrial content and increased myofibre size and muscle force in skeletal muscle from old mice [46]. Interestingly, the positive effects of miR-181 on mitochondrial function have been demonstrated in other tissues such as chondrocytes [72].

Whilst most data agree on the importance of miR-181 in regulation of mitochondrial dynamics, redox homeostasis and energy balance, it appears that the role of miR-181a could be context-dependent as has been reported for other miRs [30,73]. Different members of miR-181 family were shown to have divergent function in myocardial function [37,38,42,43]. miR-181 family members are ubiquitously expressed and the dose dependent reduction in organism size and viability following reduction of levels of the miR-181 family members suggests the critical role of this miR family in metabolic fitness and proliferation [61]. Moreover, miR-181 target genes such as Parkin or PTEN are conserved among species and it has been suggested that this family of miRNAs has evolved to provide anabolic robustness during development [61]. This may also suggest the important role of miR-181 family in a multitude of disorders associated with changes in mitochondrial dynamics and oxidative stress, such as sarcopenia or aging.

The potential of miRs as novel therapeutics for various disorders has been proposed due to their implication in regulating multiple pathways and involvement in many disease states including aging and sarcopenia [25,27,35]. Despite multiple miR-based therapies being in clinical trials, no miR-based drug has been yet approved for use [[74], [75], [76], [77], [78], [79]]. Since the recent approval of the first RNA drug for disease (2018) with no other treatment options, an interest in RNA therapeutics has increased. Onpattro (patisiran) infusion has been used to treat peripheral nerve disease (polyneuropathy) caused by hereditary transthyretin-mediated amyloidosis (hATTR) in adult patients [80]. As miRs regulate the expression of multiple genes simultaneously, they are attractive therapeutic targets for disorders with a complex molecular background, such as sarcopenia. miRs are small, charged, and water-soluble molecules and can be injected intravascularly or subcutaneously [81]. Moreover, synthetic miRs and their inhibitors can be efficiently delivered, into muscle without a systemic immune response [74,82]. Furthermore, various miR-based therapies are undergoing clinical trials, miR mimics and inhibitors (antagomiRs, antimiRs) [74]. Some of the remaining issues that remain to be resolved before miRs can enter the drug market are potential off-target effects and tissue-specific delivery. These would be of significance when designing a miR-based therapeutic for muscle disorders, as delivery would likely be via IV injection which could result in systemic expression. Nevertheless, miR-based approaches have shown efficacy in diseases such as cancer, hepatitis C and heart abnormalities [75,83]. Emerging data for miRs as therapeutic is encouraging and with emerging technologies, miR-based approaches may provide functional interventions for preventing aging- and disease-related loss of muscle mass and strength.

Declaration of competing interest

The authors declare no conflict of interest.