Mice

miR21 floxed mouse strain was engineered with lox cassettes on both sides of the mmu-miR-21 genomic locus (named miR21fl/fl) [15], and crossed with DMP1-8 kb-Cre mice [18] to obtain female and male miR21fl/fl and miR21fl/fl;DMP1-8 kb-Cre (OtmiR21Δ) mice [19]. Four-month-old female and male littermate mice were analyzed. All mice were of the C57BL/6 background, fed a regular diet and water ad libitum and maintained on a 12-h light/dark cycle. Mice were genotyped by PCR using genomic DNA extraction from mouse ear notches and genotyped using primers as previously described [15]. At 4 months of age, food was removed, and mice were euthanized by isoflurane overdose and cervical dislocation 3 h later. Calvaria bone and himdlimb skeletal muscles were collected, snapped frozen, and stored at -80 °C until used, or prepared for organ culture as detailed below. The protocols involving mice were approved by the Institutional Animal Care and Use Committee of Indiana University School of Medicine.

qPCR

Total RNA was isolated using TRIzol (Invitrogen, Grand Island, NY) [20]. Expression levels of miR21 (assay ID:000397) and the house-keeping miR135 (assay ID:001230) were evaluated using Applied Biosystem reagents, as published [15]. No differences were detected between genotypes in miR135 Ct values for either female or male mice (Ct values: female miR21fl/fl, 35.0 ± 0.5; female ΔOtmiR21, 34.3 ± 1.0; 19.6 ± 1.4; male miR21fl/fl, 33.7 ± 0.6; male ΔOtmiR21, 35.0 ± 0.7). Reverse transcription was performed using a high-capacity cDNA kit (Applied Biosystems, Foster City, CA). qPCR was performed using the Gene Expression Assay Mix TaqMan Universal Master Mix with the 7500 Real Time PCR/StepOne Plus system and software (Life Technologies). Gene expression was corrected by the levels of the house-keeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which showed Ct values that did not differ among groups as assessed by 2-way ANOVA (Ct values: female miR21fl/fl, 20.3 ± 1.6; female ΔOtmiR21, 19.6 ± 1.4; male miR21fl/fl, 19.3 ± 1.1; male ΔOtmiR21, 18.3 ± 0.8). Primers and probes were commercially available (Applied Biosystems, Foster City, CA) or were designed using the Assay Design Center (Roche Applied Science, Indianapolis, IN, USA) (Additional file 1: Table S1). Relative expression was calculated using the ∆Ct method.

Body weight and body composition by dual-energy X-ray absorptiometry (DXA)

DXA/PIXImus scans were performed in 4-month-old mice (G.E. Medical Systems, Lunar Division, Madison, WI, USA) [20] a day prior to euthanizing the mice. Body weight was measured at the time of the DXA scan. Calibration was performed using a standard control phantom before scanning, as recommended by the manufacturer. The total tissue mass (TTM) measurement was used to calculate the fat percentage (total fat body mass (g)/tissue total mass) and lean percentage (total lean body mass (g)/tissue total mass) as previously described [21].

Grip strength

The evaluation of the whole body strength in mice was assessed as previously described [22] one week before euthanizing the mice. The absolute grip strength (peak force, expressed in grams) was recorded by means of a grip strength meter (Columbus Instruments, Columbus, OH, USA) and corrected by the corresponding body weight (BW) to render normalized force. Five measurements were completed, and the top three measurements were included in the analysis.

In vivo muscle contractility

A separate cohort of mice was tested for muscle force by in vivo plantarflexion (Aurora Scientific, Aurora, ON, Canada), as described previously [23, 24]. Briefly, the left hind foot was taped to the force transducer and positioned to where the foot and tibia were aligned at 90°. The knee was then clamped at the femoral condyles, avoiding compression of the fibular nerve. Two disposable monopolar electrodes (Natus Neurology, Middleton, WI, USA) were placed subcutaneously posterior/medial to the knee in order to stimulate the tibial nerve. Peak twitch torque was first established in order to determine maximal stimulus intensity. Plantarflexion force was measured following stimulation at 100 Hz, and corrected by the weight of the corresponding mouse.

Multiplex cell-signaling assays

Cell-signaling pathway alterations induced by deletion of osteocytic miR21 were examined in miR21fl/fl and OtmiR21Δ soleus skeletal muscle lysates, prepared following the instructions from the Milliplex multi-pathway 9-plex phospho- and total protein kits (Millipore Sigma catalog # 48-680MAG and 48-681MAG, respectively), as previously reported [17]. Phospho-cAMP response element-binding protein, CREB (pS133), extracellular-regulated signal kinase, ERK1/2 (pT185/pY187), nuclear factor kappa-light-chain-enhancer of activated B cells, NFκB (pS536), c-Jun N-terminal kinase, JNK (pT183/pY185), p38 mitogen-activated protein kinase, p38 (Thr180/Tyr182), ribosomal protein S6 kinase beta-1, p70S6K (Thr412), signal transducer and activator of transcription STAT3 (pS727) and STAT5A/B (pY694/699), and protein kinase B (Akt pS473) as well as total protein levels for each kinase were measured.

Ex vivo bone organ cultures

Long bones were isolated from male and female 4-month-old miR21fl/fl and OtmiR21Δ mice. Bone-marrow cells (BMCs) were flushed out with α-MEM and osteocyte-enriched long bones were cultured ex vivo in 10% FBS and 1% penicillin/streptomycin (P/S)-α-MEM supplemented for 48 h. Conditioned media were collected and stored at -20 °C until used for the C2C12 assays.

Assessment of muscle cross-sectional area (CSA)

Ten-μm-thick cryosections of gastrocnemius muscles taken at the mid-belly were processed for immunostaining [22]. Samples were marked with a histology marking pen, blocked in phosphate buffered saline (PBS) containing 8% bovine serum albumin (BSA) for 1 h at room temperature, and incubated at 4 °C overnight with dystrophin primary antibody (1:200 in 8% BSA, Developmental Studies Hybridoma Bank, Iowa City, IA; #MANDRA1(7A10)). After the overnight incubation, samples were washed prior to incubation with a secondary antibody (1:500 in 8% BSA, ThermoFisher Scientific; AlexaFluor 555, #A-11032) for 1 h. Samples were then washed with PBS and mounted with ProLong Antifade mounting medium (ThermoFisher Scientific). For determination of the CSA, the entire muscle section was imaged and quantified by using the Lionheart XL microscope system and the Gen5 software (BioTek, Winooski, VT, USA).

C2C12 myotube differentiation

Murine C2C12 skeletal myoblasts (ATCC, Manassas, VA, USA) were grown in high glucose DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM l-glutamine, and maintained at 37 °C in 5% CO2, as previously published [25]. Myotubes were generated by exposing the myoblasts to DMEM containing 2% horse serum (i.e., differentiation medium), and replacing the medium every other day for 5 days. In order to determine the dependence of myotube size on bone-derived factors, myotubes were exposed to 5% bone conditioned medium (CM) for 48 h. Cells were fixed and stained [26, 27].

Assessment of myotube size

C2C12 cell layers were fixed in ice-cold acetone–methanol and incubated with an anti-Myosin Heavy Chain antibody (MF-20, 1:200; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) and an AlexaFluor 488-labeled secondary antibody (Invitrogen, Grand Island, NY, USA), as reported in [26, 27]. Analysis of myotube size was performed by measuring the minimum diameter of long, multi-nucleated fibers avoiding regions of clustered nuclei on a calibrated image using the Image J 1.43 software [28]. Images were taken using a Axio Observer.Z1 motorized fluorescence microscope (Zeiss, Oberchoken, Germany). Three biological replicates (n = 3) were generated for each experimental condition, and about 200–350 myotubes per replicate were measured. The results of each replicate were then averaged to obtain the final myotube size.

Statistics

All statistical analyses were performed using SigmaPlot (Systat Software Inc., San Jose, CA). Data are reported as mean ± SD and as individual values. Data were evaluated by two-way ANOVA, followed by All Pairwise Multiple Comparison Procedures (Holm–Sidak method). Statistical significance was set at p < 0.05.