1Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, Via Montpellier 1, Rome 00133, Italy. 2IRCCS San Raffaele, 00166, Rome, Italy. 3Department of Orthopedics and Traumatology, "Tor Vergata" University of Rome, "Policlinico Tor Vergata" Foundation, Rome, Italy. 4Neuromed IRCCS, Pozzilli (IS), Italy. 5Department of Experimental Medicine and Surgery, University “Tor Vergata”, Rome 00133, Italy.
In this study, we investigated the relationship between sarcopenia (evaluated in term of fibers atrophy), vitamin d receptor protein expression and TaqI/Cdx2/FokI VDR genotypes in an Italian cohort of osteoporosis(n=44) and osteoarthritis (n=55) patients. Muscle biopsies were fixed and investigated by both immunohistochemistry (vitamin d receptor expression) and transmission electron microscopy (satellite stem cells niches). Vitamin d receptor polymorphisms were studied on DNA extracted from muscle paraffin sections. For the first time, we reported that aging differently affects the VDR activation in OA and OP patients. In particular, while in OP patients we observed a significant reduction of VDR positive myonuclei with age, no “age effect” was observed in OA patients. The frequent activation of VDR could explain the lower number of atrophic fiber that we observed in OA patients respect to OP. From genetic point of view, we showed a putative association among polymorphisms FokI and Cdx2 of VDR gene, vitamin d receptor activation and the occurrence of sarcopenia. Altogether these data open new prospective for the prevention and cure of age-related muscle disorders.
Scimeca Manuel,Centofanti Federica,Celi Monica, et al. Vitamin D Receptor in Muscle Atrophy of Elderly Patients: A Key Element of Osteoporosis-Sarcopenia Connection[J]. Aging and disease,
2018, 9(6): 952-964.
Figure 1. Vitamin D serum level and muscle quality. (A) Graph shows mean value of vitamin D serum concentration in OA e OP patients. (B) Graph displays the percentage of atrophic fibers in OA patients. (C) Graph displays the percentage of atrophic fibers in OP patients. (D) Graph shows higher number of Pax7 positive satellite cells in OA as compared OP patients. (E) Representative image of anti-Pax7 immunoreaction of a muscle biopsies of an OA patient (scale bar represents 50µm). Arrows indicate several Pax7 positive myonuclei. (F) Representative image of anti-Pax7 immunoreaction of a muscle biopsies of an OP patient (scale bar represents 50µm) Arrow indicates a Pax7 positive cell. (G) Electron micrograph displays a well conserved satellite cell (arrow) net to myonucleus (asterisk) in a muscle biopsy of an OA patient (scale bar represents 2µm). H) Electron micrograph shows a satellite cells in degeneration (arrow) next to myonucleus (asterisk) in a muscle biopsy of an OP patient (scale bar represents 2µm).
Body Mass Index
25.84 ± 0.85
23.33 ± 3.21
T score L1-L4
-1.4 ± 1.12
-2.4 ± 1.08
Femoral neck T score
-1.2 ± 0.99
-2.99 ± 1.12
Table 2 Baseline characteristics of AO and OP patients.
Figure 2. VDR expression in muscle biopsies of OA and OP patients. (A) Representative image of anti-VDR immunoreaction of a muscle biopsies of an AO patient (scale bar represents 50µm). Arrows indicate numerous VDR positive myonuclei. (B) Representative image of anti-VDR immunoreaction of a muscle biopsies of an OP patient (scale bar represents 50µm). Arrows indicate rare VDR positive myonuclei. (C) Graph shows higher number of VDR positive myonuclei in OA patients as compared to OP. (D) There is no significant difference between cytoplasmic expression of VDR in OA and OP patients. (E) Graph displays correlation between VDR nuclear expression and the age of the patients. (F) Graph shows correlation between VDR nuclear expression and the age of OA patients. Graph displays correlation between VDR nuclear expression and the age of OP patients.
Figure 3. Genotypes distribution of the Cdx2, FokI and TaqI polymorphisms between OP and OA groups. (A) Fisher’s Exact test analysis (online VassarStats Statistical Software) showed a significant difference in genotype distribution for Cdx2 polymorphism between the OP and OA groups (p= 0.002). (B, C) There was not a significant difference in in genotype distribution for FokI and TaqI (0.50 and 0.48 respectively) polymorphisms in OP and OA patients (B-C). P-value was calculated using Fisher Exact Probability Test for a two-rows by three-columns contingency table (p< 0.05).
Table 3 Genotypes and Alleles frequencies of VDR Gene Polymorphisms in OP and OA patients. Deviations from Hardy-Weinberg equilibrium for the three SNPs were not observed for all three SNPs: Cdx2, FokI and TaqI (p > 0.05).
Figure 4. Comparison between FokI, Cdx2 and TaqI polymorphisms and the number of positive myonuclei. (A) The comparison between FokI polymorphism and the number of positive myonuclei showed a significant difference in patients with CC genotype respect to patients with CT genotype (p=0.0044). (B) No significant difference in the nuclear expression of VDR was observed in Cdx2. (C) No significant difference in the nuclear expression of VDR was observed in TaqI.
Figure 5. Comparison between FokI, Cdx2 and TaqI polymorphisms and the percentage of positive myonuclei. (A) The comparison between Cdx2 genotypes and muscle atrophy displays a significant group effect (GG =50%; GA= 38%; AA=18%; p=0,0004). Mann-Whitney test shows significant higher percentage of muscle atrophy (Type I = 17% vs Type II = 33%) in patients with GG genotype respect to patients with GA genotype (Type I = 17% vs Type II = 19%) (p=0,0035) and in patients with GA (Type I = 17% vs Type II = 19%) respect to patients with AA (Type I = 5% vs Type II = 13%) genotypes (p=0.0004). (B) The comparison between genotypes of FokI polymorphisms and muscle atrophy (CC =38%; CT= 45%; TT=46%) did not show a significant group effect (p=0,1080). Mann-Whitney test shows significant higher percentage of muscle atrophy in patients with CT genotype (Type I = 17% vs Type II = 28%) compared to patients with CC genotype (Type I = 17% vs Type II = 20%) (p= 0,0440). (C) The comparison between genotypes of TaqI polymorphisms and muscle atrophy (TT=50%; TC= 40%; CC=37%) did not reveal a significant group effect (p=0,1282). (D) Patients with GG/CT and GG/CC genotypes showed a significant higher percentage of type II atrophic fibers (Type II =35% and 25% respectively) respect to other genotypes combination (Type I =18% and 15% respectively).
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