Participants

Healthy, sedentary young (aged 20–30 years) and elderly (aged 60–75 years) Caucasian men living within the local community in Copenhagen, Denmark, were recruited through newspaper and web advertisements. Exclusion criteria were: participation in resistance exercise during the preceding 6 months, intake of >5 μg vitamin-D one month prior to the trial period, body mass index (BMI) <18 or >30, smoking, history of serious knee injury, any medical condition or medication known to affect protein turnover, diagnosis or a family history of organic dysfunctions, cancer, illnesses that could affect the musculoskeletal system, or blood tests values outside normal range. During the trial period, the participants were told to refrain from increased exposures to UVB radiation through e.g. use of solarium or travel activity.

Setting

The study took place at Bispebjerg Hospital, Copenhagen, Denmark (latitude of 56°N). Inclusion was continuous from November 17, 2010 to December 21, 2010 and the last subject completed the study on April 25, 2011. Thus, the study was conducted in a period of low UVB irradiation from sunlight. Participants were informed about the study protocol, the risks of tests and investigations, and their rights according to the Declaration of Helsinki II. The study protocol was approved by Research Ethics Committee Region Hovedstaden Committee F (H-2-2010-100) and by the Danish Data Protection Agency (2011-41-5965). All participants gave written informed consent. The study was registered at ClinicalTrials.gov (nr. NCT01252381).

Randomization and blinding

Participants were continuously randomized by the envelope method. Young and elderly men were randomized separately. Allocation ratio was 1:1 for vitamin-D and placebo groups. The subjects were enrolled and randomized by the principal investigator. Personnel conducting the scans, analyzing CSA, measuring isometric muscle strength, taking biopsies and blood samples, analyzing muscle fiber type composition and performing RT-qPCR analyses, were all blinded with respect to allocation.

Interventions

Vitamin-D and calcium supplementation

The participants were randomly assigned (double-blinded) to a vitamin-D group receiving 48 μg (1920 IU) vitamin-D 3 (cholecalciferol)/day plus 800 mg calcium/day or a placebo group receiving 800 mg calcium/day alone for 16 weeks. All participants were instructed to take two tablets orally each day. The vitamin-D-group received one tablet containing 10 μg vitamin-D 3 + 400 mg calcium (Silver, Unikalk, Frederiksvaerk, DK) and one tablet containing 38 μg vitamin-D 3 + 400 mg calcium (Mega, Unikalk, Frederiksvaerk, DK), thus reaching the maximum advisable daily dose (50 μg, 2000 IU) by the Danish Health and Medicines Authority. The placebo-group received an equivalent dose of calcium by consuming two calcium tablets (400 mg per tablet) (Basic, Unikalk, Frederiksvaerk, DK). The two tablets were taken simultaneously at the same time each day together with a meal. Tablets containing vitamin-D plus calcium or calcium alone were indistinguishable and only labeled with the name of the manufacturer.

Training protocol

Participants were subjected to 36 training sessions (12 weeks with 3 sessions/week) consisting of 5–10 min warm-up on cycle ergometers followed by resistance training exercises of the lower extremities performed in commercial knee extension and leg press devices (Technogym, Super Executive Line, Gambettola, Italy). All sessions were supervised. Progressive loading levels were monitored continuously and adjusted throughout the entire training period to maintain muscle loading at the intended values.

Prior to commencement of training, 1-repetition maximum (1RM) strength was estimated from a 5-repetition-maximum (5RM) test [37]. The 5RM test was performed every two weeks, to ensure that the training load was relative to the strength progression throughout the training period. During the first 6 training sessions, participants completed 3 sets of 12–15 repetitions at 65–70 % of 1RM. During session 7–12, participants performed 3 sets of 10–12 repetitions at 70–75 % of 1RM, increasing to 4 sets at 70–75 % of 1RM during session 13–18. From session 19 and onwards, participants performed 5 sets with training load progressing from 8–10 repetitions at 75–80 % of 1RM in session 19–27 to 6–8 repetitions at 80–85 % of 1RM in session 28–36 [38]. The exercises were performed in a moderate slow, controlled manner with 1–2 s in the concentric- and eccentric phase with a rest of 1–3 min between sets. Exercise compliance (sets, repetitions, and load) was calculated from daily exercise records completed by the instructors at each training session. Participants were informed that a mean attendance of less than 2 training sessions per week resulted in exclusion. All adverse events associated with the training intervention were recorded.

Analyses

CSA of quadriceps muscle

CSA of quadriceps muscle was measured pre (week 0) and post (week 12) training intervention. Magnetic Resonance Imaging (MRI) (3.0 T, T1-weighted, Radiographic department, Bispebjerg Hospital, Copenhagen, Denmark) was conducted to obtain six images starting at the tibia plateau and proceeding proximally to the mid/proximal femur. Each slice was 6 mm thick and the distance between each slice was 54 mm. The slice tangential to the tibia plateau was used as an anatomical marker (first slice) and the subsequent slices were numbered slice-by-slice proximally. The fourth axial slice of the thigh was selected for further analysis. To avoid any effect of body fluid disturbances directly related to the preceding exercise session, the MRI-scans were performed no sooner than 48 h post exercise and before the biopsies were taken. CSA images of both legs were analyzed for changes from pre to post training by manually drawing margins of the quadriceps muscle (Osirix, open-source 32 bit, Pixmeo SARL, Geneva, Switzerland) and a mean of both legs was calculated. Images from one participant were analyzed in succession to ensure uniform measure technique. Analyses were performed blinded according to trial group and time point. Whether the pre- or post picture was analyzed first was randomized.

Isometric muscle strength

All participants were familiarized with the strength test at inclusion in order to reduce any learning effects. Isometric knee extension peak torque of both legs was measured pre and post 12 weeks training with the participants sitting in a custom-made rigid chair with hips and knees flexed to 90°. To avoid movement or unwanted contribution of other muscles, participants were strapped at the hip and the abdomen. A leg cuff, connected to a strain gauge (Bofors KRG-4, Bofors, Sweden) through a rigid steel rod perpendicular to the lower leg, was mounted on the lower leg just above the medial malleolus. The signal from the strain gauge was amplified (Noraxon Telemyo 2400 T G2) and sent to a computer to analyze the greatest force (N) for each participant (Myoresearch, XP Master Edition 1.07.25). Lever arm was defined as the distance from the joint line of the knee to the middle of the leg cuff. After a 5-min warm-up on a cycle ergometer and 2–3 submaximal pre-test-measurements, each leg was measured 3 times with 2 min rest between each measurement. Participants were randomized to start with either the left or the right leg.

On the basis of the quadriceps CSA and isometric strength measurement, isometric strength/CSA was calculated as an expression of muscle quality.

Serum 25(OH)D concentrations

Blood samples were obtained at baseline (week −4) and after 0, 2, 6 and 12 weeks of resistance training. Serum 25(OH)D concentrations were determined using an ELISA-kit (Elecsys, kit ref. 05894913190, Roche Diagnostics A/S, Denmark) measuring both 25(OH)D 2 and 25(OH)D 3 on a Cobas e411 (Roche Diagnostics A/S, Denmark) at the Department of Clinical Biochemistry, Bispebjerg Hospital.

Muscle biopsy

A biopsy from the lateral part of the vastus lateralis muscle from either the left or the right leg was obtained before the training started (week 0) and after the last training session (week 12). At week 12, two biopsies were taken; 4 h (TR+4h) and 48 h (TR+48h) after the last exercise session, respectively. The biopsies were obtained using a 4-mm biopsy needle (Bergström, Stockholm, Sweden). Briefly, the skin was shaved and disinfected before local anesthetization (1 % lidocaine). An incision was made through which the muscle biopsy was taken. Afterwards, the incision was strapped with SteaStrips and covered with waterproof plaster. The muscle specimen was quickly freed from any visible blood, fat or connective tissue and subsequently divided in two; one piece was mounted in Tissue-Tek and frozen in precooled isopentane, the other piece was frozen in liquid nitrogen. Both pieces were stored at −80 °C until further analysis.

Fiber type analysis

Cross sections (10 μm) were cut from the muscle specimen mounted in Tissue-Tek in a cryomicrotome (HM 560, Microm, Germany) at −20 °C and stained for myofibrillar ATPase at pH 9.4 after both alkaline (pH 10.3) and acid (pH 4.3 and 4.6) pre-incubations as previously described [39]. All samples were stained in the same batch to avoid inter-assay variations. Muscle fiber type and size were analyzed in a blinded fashion. Only true horizontal fibers were analyzed for size. Thereby, slightly fewer fibers could be analyzed for size (202 ± 7 fibers per biopsy, mean ± SD) than for type (204 ± 4 fibers per biopsy). As previously described [38], muscle fibers were characterized as type I, IIa, or IIx based on the ATPase staining pattern. Tema Image-analyses System (Scanbeam, Denmark) was used to analyze the cross sections for muscle fiber type and size. The fiber type distributions and mean fiber CSA were calculated. Muscle fiber morphology analysis included determination of fiber type % and mean CSA. Type IIa and IIx fiber results were pooled to yield the combined type II fiber area.

Measurement of mRNA

The mRNA expression of VDR, CYP27B1 and Myostatin was measured using real-time reverse transcriptase polymerase chain reaction (RT-PCR). Approximately 20 mg of muscle tissue was homogenized in 1000 μl TriReagent (Molecular Research Center, RT 118) using a FastPrep 24 (MP Biomedicals), with muscle tissue placed in a 2 ml microvial with a screw cap (BioSpec) containing one silicium-carbide crystal and five 2.3 mm steel beads. Thereafter, 1-Bromo-3-chloropropane (BCP) was added to the homogenized sample to separate the sample into an aqueous and an organic phase. The RNA was precipitated from the aqueous phase using isopropanol, washed with ethanol, and dissolved in RNase-free water. The RNA was then re-precipitated using sodium acetate (NaAc), washed in ethanol, and dissolved in RNase-free water. RNA concentrations and purity were determined by spectroscopy, and RNA quality was ensured by gel electrophoresis.

Synthesis of complementary DNA (cDNA) was performed using Omniscript reverse transcriptase (Qiagen, Hilden, Germany). The cDNA synthesis was performed on 500 ng of muscle RNA. cDNA was diluted 20 times in TE-buffer containing 1 ng/μl Salmon DNA. For each target, 5 μl of diluted cDNA was amplified (25 μl total volume) in Quantitect SYBR Green Master Mix (Qiagen) with specific primers (0.1 μM each, Table 1) on a real-time PCR machine (Stratagene MX3005P, Agilent Technologies, USA). The thermal profile was set to 95 °C for 10 min, followed by 50 cycles of amplification; 95 °C for 15 s., 58 °C for 30 s., 63 °C for 90 s. Signal intensity was measured at the 63 °C step, and the threshold cycle (C t ) values were related to a standard curve made from the cloned PCR product or corresponding synthetic oligonucleotides. Specificity of the PCR product was confirmed by a melting curve analysis after the last amplification cycle (55 °C to 95 °C).

Table 1 Primers used for real-time reverse transcription PCR Full size table

Ribosomal protein large P0 (RPLP0) was chosen as housekeeping gene. As a control, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), another often constitutively expressed mRNA, was normalized to RPLP0 (Fig. 1). Changes were seen in GAPDH mRNA expression when normalized to RPLP0. Most likely this was due to changes in GAPDH mRNA expression which possibly can occur due to 12 weeks resistance training. However, a change in mRNA expression such as that of the housekeeping genes in Fig. 1 would not affect normalization of the targets of interest (VDR, CYP27B1 and Myostatin) to a degree that would explain the differences we see in mRNA expression of these gene targets.

Fig. 1 GAPDH mRNA expression normalized to RPLP0. Shown as fold changes post 12 weeks training compared to pre training on logarithmic scale at 4 h (TR+4h) and 48 h (TR+48h) after the last exercise session. Results are shown as geometric mean ± back-transformed SEM. * different from pre training (p < 0.05) Full size image

Statistical analyzes

Based on a previously published article on the effect of heavy and light load resistance training, respectively, on muscle growth [40] where the heavy training was equivalent to the training used in this study a power calculation was carried out to estimate how many subjects should be included to be able to detect a significant difference between the desired outcome parameters. With a power of 0.8 and a significance level at 0.05 we calculated that a group size of 10 was needed to detect the expected increase in CSA and strength.

Analyzes were conducted per protocol and separately for the young and elderly men to explore the effect of vitamin-D intake vs. placebo. Furthermore, young vs. elderly was compared to test differences between age groups. Data were checked for normality and found to be normally distributed. Inclusion characteristic results were analyzed using an unpaired t-test. The results of serum 25(OH)D concentrations, muscle fiber type composition and gene expression were analyzed by a two-way analysis of variance (ANOVA) with repeated measures for time. Serum 25(OH)D concentrations and muscle fiber type composition are presented as mean ± standard error of the mean (SEM). Quadriceps muscle CSA, isometric muscle strength and isometric strength/CSA were calculated as relative changes from pre to post training and thus presented as mean percentage-changes ± SEM. Pearson correlation analyzes were conducted using Prism 6 (GraphPad Software Inc., CA, USA). Muscle fiber type data are shown as mean ± SEM. Results of mRNA data post training (TR) from biopsies taken 4 h (TR+4h) and 48 h (TR+48h) after the last exercise session are reported relative to the pre training (week 0) values. The mRNA data are shown as geometric (Geo) means ± back-transformed SEM. All mRNA data were log-transformed and normalized to RPLP0.

Unless otherwise stated, data were analyzed by a two-way ANOVA with repeated measures for time. When significant changes were found by ANOVA testing, a Student-Newman-Keuls post-hoc test was performed. Level of significance was p < 0.05. For a p-value between 0.05 and 0.1 a tendency was discussed. Statistical analyzes were conducted using SigmaPlot software version 11.0 unless otherwise stated.