Proline restores mitochondrial function and reverses aging hallmarks in senescent cells.

Mitochondrial dysfunction is a hallmark of cellular senescence, with the loss of mitochondrial function identified as a potential causal factor contributing to senescence-associated decline in cellular functions. Our recent findings revealed that ectopic expression of the pluripotency transcription factor NANOG rejuvenates dysfunctional mitochondria of senescent cells by rewiring metabolic pathways. In this study, we report that NANOG restores the expression of key enzymes, PYCR1 and PYCR2, in the proline biosynthesis pathway. Additionally, senescent mesenchymal stem cells manifest severe mitochondrial respiratory impairment, which is alleviated through proline supplementation. Proline induces mitophagy by activating AMP-activated protein kinase α and upregulating Parkin expression, enhancing mitochondrial clearance and ultimately restoring cell metabolism. Notably, proline treatment also mitigates several aging hallmarks, including DNA damage, senescence-associated ß-galactosidase, inflammatory cytokine expressions, and impaired myogenic differentiation capacity. Overall, this study highlights the role of proline in mitophagy and its potential in reversing senescence-associated mitochondrial dysfunction and aging hallmarks.

Methionine adenosyltransferase2A inhibition restores metabolism to improve regenerative capacity and strength of aged skeletal muscle.

We investigate the age-related metabolic changes that occur in aged and rejuvenated myoblasts using in vitro and in vivo models of aging. Metabolic and signaling experiments reveal that human senescent myoblasts and myoblasts from a mouse model of premature aging suffer from impaired glycolysis, insulin resistance, and generate Adenosine triphosphate by catabolizing methionine via a methionine adenosyl-transferase 2A-dependant mechanism, producing significant levels of ammonium that may further contribute to cellular senescence. Expression of the pluripotency factor NANOG downregulates methionine adenosyltransferase 2 A, decreases ammonium, restores insulin sensitivity, increases glucose uptake, and enhances muscle regeneration post-injury. Similarly, selective inhibition of methionine adenosyltransferase 2 A activates Akt2 signaling, repairs pyruvate kinase, restores glycolysis, and enhances regeneration, which leads to significant enhancement of muscle strength in a mouse model of premature aging. Collectively, our investigation indicates that inhibiting methionine metabolism may restore age-associated impairments with significant gain in muscle function.

Reversine ameliorates hallmarks of cellular senescence in human skeletal myoblasts via reactivation of autophagy.

Cellular senescence leads to the depletion of myogenic progenitors and decreased regenerative capacity. We show that the small molecule 2,6-disubstituted purine, reversine, can improve some well-known hallmarks of cellular aging in senescent myoblast cells. Reversine reactivated autophagy and insulin signaling pathway via upregulation of Adenosine Monophosphate-activated protein kinase (AMPK) and Akt2, restoring insulin sensitivity and glucose uptake in senescent cells. Reversine also restored the loss of connectivity of glycolysis to the TCA cycle, thus restoring dysfunctional mitochondria and the impaired myogenic differentiation potential of senescent myoblasts. Altogether, our data suggest that cellular senescence can be reversed by treatment with a single small molecule without employing genetic reprogramming technologies.

Inhibition of glutaminolysis restores mitochondrial function in senescent stem cells.

Mitochondrial dysfunction, a hallmark of aging, has been associated with the onset of aging phenotypes and age-related diseases. Here, we report that impaired mitochondrial function is associated with increased glutamine catabolism in senescent human mesenchymal stem cells (MSCs) and myofibroblasts derived from patients suffering from Hutchinson-Gilford progeria syndrome. Increased glutaminase (GLS1) activity accompanied by loss of urea transporter SLC14A1 induces urea accumulation, mitochondrial dysfunction, and DNA damage. Conversely, blocking GLS1 activity restores mitochondrial function and leads to amelioration of aging hallmarks. Interestingly, GLS1 expression is regulated through the JNK pathway, as demonstrated by chemical and genetic inhibition. In agreement with our in vitro findings, tissues isolated from aged or progeria mice display increased urea accumulation and GLS1 activity, concomitant with declined mitochondrial function. Inhibition of glutaminolysis in progeria mice improves mitochondrial respiratory chain activity, suggesting that targeting glutaminolysis may be a promising strategy for restoring age-associated loss of mitochondrial function.

Ameliorating the hallmarks of cellular senescence in skeletal muscle myogenic progenitors in vitro and in vivo.

Senescence of myogenic progenitors impedes skeletal muscle regeneration. Here, we show that overexpression of the transcription factor NANOG in senescent myoblasts can overcome the effects of cellular senescence and confer a youthful phenotype to senescent cells. NANOG ameliorated primary hallmarks of cellular senescence including genomic instability, loss of proteostasis, and mitochondrial dysfunction. The rejuvenating effects of NANOG included restoration of DNA damage response via up-regulation of DNA repair proteins, recovery of heterochromatin marks via up-regulation of histones, and reactivation of autophagy and mitochondrial energetics via up-regulation of AMP-activated protein kinase (AMPK). Expression of NANOG in the skeletal muscle of a mouse model of premature aging restored the number of myogenic progenitors and induced formation of eMyHC+ myofibers. This work demonstrates the feasibility of reversing the effects of cellular senescence in vitro and in vivo, with no need for reprogramming to the pluripotent state.

Bioengineered Skeletal Muscle as a Model of Muscle Aging and Regeneration.

With age, adult skeletal muscle (SkM) is known to decrease in muscle mass, strength, and functional capacity, a state known as sarcopenia. Here we developed an in vitro three-dimensional (3D) bioengineered senescent SkM tissue using primary human myoblasts. These tissues exhibited the characteristics of atrophied muscle, including expression of senescent genes, decreased number of satellite cells, reduced number and size of myofibers, and compromised metabolism and calcium flux. As a result, senescent SkM tissues showed impaired ability to generate force in response to electrical stimulation compared with young tissues. Furthermore, in contrast to young SkM tissues, senescent tissues failed to regenerate in response to injury, possibly as a result of persistent apoptosis and failure to initiate a proliferation program. Our findings suggest that 3D senescent SkM may provide a powerful model for studying aging and a platform for drug testing and discovery of therapeutic compounds to improve the function of sarcopenic muscle. Impact statement Skeletal muscle (SkM) plays important physiological roles and has significant regenerative capacity. However, aged SkM lose their functionality and regeneration ability. In this article, we present a senescent human bioengineering SkM tissue model that can be used to investigate senescence, metabolic or genetic diseases that inflict SkM, and to test various strategies including novel small molecules that restore muscle function and promote regeneration. One key limitation of two-dimensional cell culture system is the detachment of contractile myotubes from the surface over time, thereby limiting the evaluation of myogenic function. Here we use primary human myoblasts, which exhibit all major hallmarks of aging to mimic the organization and function of native muscle. Using this system, we were able to measure the contractile function, calcium transients, and regeneration capacity of SkM tissues. We also evaluated the response of senescent SkM tissues to injury and their ability to regenerate and recover, compared with "young" tissues. Our results suggest that three-dimensional constructs enable organization of contractile units including myosin and actin filaments, thereby providing a powerful platform for the quantitative assessment of muscle myotubes in response to injury, genetic or metabolic disorders, or pharmacological testing.

Efficient and high yield isolation of myoblasts from skeletal muscle.

Skeletal muscle (SkM) regeneration relies on the activity of myogenic progenitors that reside beneath the basal lamina of myofibers. Here, we describe a protocol for the isolation of the SkM progenitors from young and old mice by exploiting their outgrowth potential from SkM explants on matrigel coated dishes in the presence of high serum, chicken embryo extract and basic fibroblast growth factor. Compared to other protocols, this method yields a higher number of myoblasts (10-20 million) by enabling the outgrowth of these cells from tissue fragments. The majority of outgrowth cells (~90%) were positive for myogenic markers such as α7-integrin, MyoD, and Desmin. The myogenic cell population could be purified to 98% with one round of pre-plating on collagen coated dishes, where differential attachment of fibroblasts and other non-myogenic progenitors separates them from myoblasts. Moreover, the combination of high serum medium and matrigel coating provided a proliferation advantage to myogenic cells, which expanded rapidly (~24 h population doubling), while non-myogenic cells diminished over time, thereby eliminating the need for further purification steps such as FACS sorting. Finally, myogenic progenitors gave rise to multinucleated myotubes that exhibited sarcomeres and spontaneous beating in the culture dish.

NANOG restores the impaired myogenic differentiation potential of skeletal myoblasts after multiple population doublings.

Adult skeletal muscle regeneration relies on the activity of satellite cells residing in the skeletal muscle niche. However, systemic and intrinsic factors decrease the myogenic differentiation potential of satellite cells thereby impairing muscle regeneration. Here we present data showing that late passage C2C12 myoblasts exhibited significantly impaired myogenic differentiation potential that was accompanied by impaired expression of myogenic regulatory factors (Myf5, MyoD, Myogenin, and MRF4) and members of myocyte enhancer factor 2 family. Notably, ectopic expression of NANOG preserved the morphology and restored the myogenic differentiation capacity of late passage myoblasts, possibly by restoring the expression level of these myogenic factors. Muscle regeneration was effective in 2D cultures and in 3D skeletal microtissues mimicking the skeletal muscle niche. The presence of NANOG was required for at least 15days to reverse the impaired differentiation potential of myoblasts. However, it was critical to remove NANOG during the process of maturation, as it inhibited myotube formation. Finally, myoblasts that were primed by NANOG maintained their differentiation capacity for 20days after NANOG withdrawal, suggesting potential epigenetic changes. In conclusion, these results shed light on the potential of NANOG to restore the myogenic differentiation potential of myoblasts, which is impaired after multiple rounds of cellular division, and to reverse the loss of muscle regeneration.