Partial reprogramming by cyclic Nanog overexpression in aged mice

Introduction: Sarcopenia is the loss of muscle mass, strength and function that occurs during normal aging ultimately resulting in frailty. Impaired muscle regeneration, stemming from diminished stem cell functionality during aging, stands as a pivotal contributor to sarcopenia pathogenesis. Recent evidence underscores the potential of cellular reprogramming to pluripotency in mitigating age-associated cellular decline, owing to the inherent self-renewal capacity of less-committed precursor cells. In vivo, the completion of reprogramming can be observed through the formation of teratomas which is preventable by early intervention to halt the process. Nanog is one of the core transcription factors in maintaining pluripotency which targets genes frequently encoding transcription factors, many of which are developmentally important homeodomain proteins contributing to pluripotency and self-renewal. Our previous investigations demonstrated the ability of Nanog to enhance myotube formation in vitro and facilitate skeletal muscle regeneration in a murine model of accelerated aging (LAKI-tetO-Nanog) by increasing Pax7+ muscle progenitors and promoting expression of embryonic myosin heavy chain. However, the effects of Nanog in naturally aged mice have not been investigated. Here we report that intermittent administration of Nanog through systemic doxycycline (Dox) administration (in food and water) could maintain the function and promoted regeneration of skeletal muscle after injury in aged mice and provide insights on the potential mechanisms that mediate these rejuvenating effects.

Materials and

Methods: We developed a cyclic Nanog overexpression protocol in geriatric mice (18-24 month-old) by administering doxycycline in water (1mg/ml) and chow (200mg/kg) for 5 days in a one month period (Fig. A). Using this protocol, we were able to mitigate the negative consequences of in vivo reprogramming including weight loss and mortality. We investigated the effects of cyclic overexpression of Nanog followed by recovery on the physical performance of naturally aged mice at the end of each cycle. For physical performance assessments, we measured Tibialis Anterior (TA) muscle force in vivo, and utilized other methods including grip strength, treadmill, and inverted hang test. We performed a basal measurement prior to the 1st DOX administration. All assessments after cyclic Nanog overexpression were compared to the basal measurement for each animal individually and reported as percent change. In order to assess muscle regeneration capacity in old age, we performed cardiotoxin (CTX) injection in right leg of 24-month-old animals after one last cyclic Nanog overexpression allowing one week of recovery in muscle and measured muscle force generation at this point (Fig. B). Furthermore, we examined expression of markers for newly formed myofibers and muscle stem cells using embryonic myosin heavy chain (eMyhc) and Pax7 by immunostaining, as well as percentage of centrally nucleated myofibers, myofiber size and distribution.

Results, Conclusions, and Discussions: We report that Geriatric mice expressing Nanog (GN) showed enhanced twitch force (34.4±6.8%) compared to wild type geriatric controls (GC, -10.9±2.7%) and tetanic force (16.1±3.6% in GN vs -13.2±4% in GC) in TA muscle after just one cycle of Nanog overexpression, which was sustained at a similar level in subsequent cycles until 23 months of age (Figure C,D). Various physical performance assessments, including uphill treadmill running, and grip strength demonstrated similar improvements in the GN vs. GC groups (Figure E,F). Force measurements after cardiotoxin injury indicated higher percentage of force recovery in GN group at 1 week after injury (Figure G,H). Immunostaining for eMyhc+ myofibers showed the presence of newly formed myofibers in GN which were absent in GC TA muscle (Figure I,J). In addition, we detected higher percentage of Pax7+ cells in GN vs GC muscle, indicating increased number of satellite stem cells (Figure K,L). Immunostaining for laminin showed higher percentage of centrally nucleated fibers in GN compared to GC (Figure M). In addition, the fraction of fibers with an area of 500-1000 µm2 was higher in GN (Figure N), indicating the presence of newly formed fibers. At one week post cardiotoxin injury we detected increased percentage of eMyhc+ fibers at the site of the wound in the GN compared to GC mice (Figure O,P), which is in agreement with the increased percentage of Pax7+ cells in Nanog expressing CTX-injured muscle (GN+CTX vs GC+CTX, Figure R,S). Furthermore, the GN+CTX group exhibited higher percentage of centrally nucleated myofibers (Figure T), and higher percentage of fibers with an area of less than 500 µm2 (Figure U), indicating increased presence of regenerating fibers. In conclusion, we successfully developed a protocol for cyclic Nanog overexpression, which improved muscle function and enhanced physical performance and muscle regeneration capacity in geriatric mice, while avoiding unwanted side effects such as tumor formation. These results show that Nanog reversed the effects of aging with no need of reprogramming to the pluripotent state and no detrimental effects of animal health.

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