Hallmarks of frailty and osteosarcopenia in prematurely aged PolgA(D257A/D257A) mice

Abstract Background Frailty is a geriatric syndrome characterized by increased susceptibility to adverse health outcomes. One major determinant thereof is the gradual weakening of the musculoskeletal system and the associated osteosarcopenia. To improve our understanding of the underlying pathophysiology and, more importantly, to test potential interventions aimed at counteracting frailty, suitable animal models are needed. Methods To evaluate the relevance of prematurely aged PolgA(D257A/D257A) mice as a model for frailty and osteosarcopenia, we quantified the clinical mouse frailty index in PolgA(D257A/D257A) and wild‐type littermates (PolgA(+/+), WT) with age and concertedly assessed the quantity and quality of bone and muscle tissue. Lastly, the anabolic responsiveness of skeletal muscle, muscle progenitors, and bone was assessed. Results PolgA(D257A/D257A) accumulated health deficits at a higher rate compared with WT, resulting in a higher frailty index at 40 and 46 weeks of age (+166%, +278%, P < 0.0001), respectively, with no differences between genotypes at 34 weeks. Concomitantly, PolgA(D257A/D257A) displayed progressive musculoskeletal deterioration such as reduced bone and muscle mass as well as impaired functionality thereof. In addition to lower muscle weights (−14%, P < 0.05, −23%, P < 0.0001) and fibre area (−20%, P < 0.05, −22%, P < 0.0001) at 40 and 46 weeks, respectively, PolgA(D257A/D257A) showed impairments in grip strength and concentric muscle forces (P < 0.05). PolgA(D257A/D257A) mutation altered the acute response to various anabolic stimuli in skeletal muscle and muscle progenitors. While PolgA(D257A/D257A) muscles were hypersensitive to eccentric contractions as well as leucine administration, shown by larger downstream signalling response of the mechanistic target of rapamycin complex 1, myogenic progenitors cultured in vitro showed severe anabolic resistance to leucine and robust impairments in cell proliferation. Longitudinal micro‐computed tomography analysis of the sixth caudal vertebrae showed that PolgA(D257A/D257A) had lower bone morphometric parameters (e.g. bone volume fraction, trabecular, and cortical thickness, P < 0.05) as well as reduced remodelling activities (e.g. bone formation and resorption rate, P < 0.05) compared with WT. When subjected to 4 weeks of cyclic loading, young but not aged PolgA(D257A/D257A) caudal vertebrae showed load‐induced bone adaptation, suggesting reduced mechanosensitivity with age. Conclusions PolgA(D257A/D257A) mutation leads to hallmarks of age‐related frailty and osteosarcopenia and provides a powerful model to better understand the relationship between frailty and the aging musculoskeletal system.


ram@ethz.ch
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Breeding scheme
As both heterozygous (PolgA D257A/+ ) and homozygous (PolgA D257A/D257A ) mice have progressive accumulation of mitochondrial DNA point mutations, specific breeding considerations were taken into account while expanding the colony. Specifically, as paternal mitochondrial DNA is actively eliminated following fertilization in mice, the (undesirable) accumulation of mutations in the germline was minimized by mating heterozygous male mice (PolgA D257A/+ ) with C57Bl/6J inbred females (Charles River Laboratories, Sulzfeld, Germany). The thus obtained heterozygous (PolgA D257A/+ ) females and males (originating from a wild type (WT) C57BL/6J mother) were crossed (age 7-9 weeks) to generate homozygous (PolgA D257A/D257A , referred to as PolgA) and WT littermates (PolgA +/+ , referred to as WT) with only a single generation of mutation burden.

Mouse genotyping
The presence of the PolgA knock-in mutation was confirmed by extracting DNA from ear clips

Complex IV Enzyme Activity
In order to confirm that the premature aging phenotypes were associated with mitochondrial dysfunction, we measured the activity of complex IV enzyme in m. gastrocnemius (GAS) using the Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911, Abcam). Briefly, GAS was homogenized using a tissue homogenizer (Omni THg, Omni International) and protein concentration was determined using the DC assay protein method (Bio-Rad). 50 µg of proteins in 200 µL assay solution were loaded into each well of 96 well plates coated with COX specific antibodies, and incubated at room temperature for 3 hours. The oxidative capacity of COX in the presence of cytochrome C was monitored by absorbance at 550 nm at 30°C for 120 minutes using a photospectrometer (Spark, TECAN), with a measurement interval of 1 minute.
Rates of oxidation were calculated by using absorbance values during the time when the decrease of OD values was linear. We found that COX IV activity in PolgA muscles was lower compared to WT muscles both at 40 (-19%, p<0.0001) and 46 weeks (-24%, p<0.0001), thus confirming that the mice used in this study had the same phenotype as those previously reported [36][37]40].

Muscle fiber cross-sectional area determination
For histology and morphometry of skeletal muscle, 10 µm thick cryosections of TA muscles were dried and washed for 5 min in PBS supplemented with 0.05% triton and subsequently incubated with wheat germ agglutinin Alexa Fluor 647 (1:250, Thermo Fischer Scientific) for 1h. Slides were mounted after a 3x 5min wash in PBS, sealed with glass cover slips and imaged with an epifluorescent microscope (Zeiss Axio observer Z.1) at 10x. Fiber cross-sectional area