All methods were carried out in accordance with relevant guidelines and regulations.
SIRT1 inhibitor screen. The NatProd Collection library (MicroSource Discovery Systems, Inc. Gaylordsville, CT) in ten source plates was screened at two doses (5 µM and 50 µM final in the reaction mixture, duplicate for each dose). The inhibitory activity of the compounds was assessed in duplicate against 2ng/µL purified human SIRT1 using 3µM p53 as a substrate. The assay was a mobility shift assay based on charge differences before and after electrophoretic separation of product from fluorescently labeled substrate read using a Caliper EZ Reader (Perkin Elmer, Boston, MA). All reactions took place in the presence of excess nicotinamide adenine dinucleotide and the known SIRT1 inhibitor suramin was used as a control.
Box-Behnken model generation. Three of the inhibitors identified in the natural product screen were selected based on their previous use in humans and complementary chemical structures. These inhibitors were then used to create an incomplete multifactorial design Box-Behnken model using Design-Expert® software. The three-factor design with one central point required 12 animals. Five more animals received the central dose of all three natural products to determine the biological variability of the intervention.
Synergist ablation. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of California, Davis and are reported in accordance with ARRIVE guidelines. Eighteen rats were used for both the DOE and validation experiments. The animals were anesthetized using 2.5% isoflurane, the distal hindlimb shaved and prepared for aseptic surgery. The whole soleus and bottom two thirds of the gastrocnemius were removed at the Achilles tendon, leaving the plantaris (PLN) muscle and innervating nerve intact. The overlying fascia and skin were sutured closed, and the animals were moved to a temperature regulated area for recovery. The left leg served as a contralateral control. Animals were monitored daily to ensure they returned to normal activity and did not suffer any stress from the procedure. Animals were orally gavaged on a daily basis (just prior to lights out), in accordance with their respective treatment group.
Muscle Collection. Following the 14th day of treatment, animals were anesthetized, and the overloaded and contralateral PLN muscles, hearts, and livers were collected. Upon removal, PLN muscles were trimmed conservatively, weighed and then pinned at resting length on cork, snap-frozen in liquid nitrogen cooled isopentane and stored at -80˚C until further processing.
Histology. PLN muscles were blocked in a cryostat on corks using Tissue Tek O.C.T. Compound (Sakura), and 10µm sections were mounted onto slides for CSA quantification. Slides were prepped for histological analysis by being blocked in 5% normal goat serum (NGS) in PBST w/1% tween, and incubated in primary antibodies for type I, IIa, IIb fibers, and/or laminin over night at 4˚C. The next day, slides were washed with PBST w/0.1% Tween and incubated in HRP conjugated secondary antibodies for 60 minutes, washed again and mounted using Prolong Gold (no Dapi). Four random images were taken of each respective muscle section for CSA quantification using Fiji.
Validation experiment. Following synergist ablation, animals were randomly assigned to one of four treatment groups, control (n = 3), Least (n = 5), Moderate (n = 5), and Optimal (n = 5). Least, moderate, and optimal refer to the best (optimal) and worst (least) combination of the natural products relative to the predicted effect on muscle fiber CSA. The Moderate group was predicted to have an effect of fiber CSA between that of the Optimal and Least groups. The control group received phosphate buffered saline (PBS), whereas the least, moderate, and optimal groups received different combinations and concentrations of the three SIRT1 inhibitors as a single cocktail dissolved in PBS (Table 2). All treatments were administered via oral gavage just prior to lights out for 14 days; the first gavage was given at 5pm on the day the surgery took place.
mRNA Isolation, reverse transcription, and qPCR. Following blocking, PLN muscles were powdered using a hammer and pestle. Total RNA was extracted from weighed powdered muscle tissue using RNAzol in accordance with the manufacturer’s protocol. RNA was quantified using Biotek Epoch Microplate Reader via absorbance (Biotek,Winooski, VT), and the RNA/mg muscle was calculated by dividing total RNA in the sample by the mass of muscle powder used. 1.5µg of total RNA was then converted into cDNA using MultiScribe reverse transcriptase and oligo (DT) primers. cDNA was diluted 1:10 before conducting qPCR. qPCR was performed using CFX384 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA), along with Quantified Mastermix and Bio-Rad Sybr Green Mix solution and Bio-Rad 384 well PCR plates. PCR reactions were performed in accordance with the manufacturer’s instructions with the following primers: rITS-1 (fwd-TCCGTTTTCTCGCTCTTCCC- ; rev-CCGGAGAGATCACGTACCAC-), r5E1TS (fwd-ACGCACGCCTTCCCAGAGG-; rev-CGCGTCTCGCCTGGTCTCTTG-). Gene expression was calculated using a delta delta threshold cycle method 30 and GAPDH (fwd-TGGAAAGCTGTGGCGTGAT- ; rev-TGCTTCACCACCTTCTTGAT-) was used as the housekeeping gene. The absolute CT of GAPDH was unchanged by either overload or treatment.
Tissue homogenization and western blotting. Two scoops of muscle powder were incubated in 250 µL of sucrose lysis buffer [1M Tris, pH 7.5, 1M sucrose, 1mM EDTA, 1mM EGTA, 1% Triton X-100, and protease inhibitor complex]. The solution was set on a shaker for 60 minutes at 4˚C, spun down at 8,000g for 10 minutes, supernatants were transferred to new Eppendorf tubes and protein concentrations were then determined using a DC protein assay (Bio-Rad, Hercules, CA). 750 µg of protein was diluted in 4X Laemmli sample buffer (LSB) and boiled for 5 minutes. 10µL of protein sample was loaded onto a Criterion TGX Stain-Free Precast Gel and run for 45 minutes at a constant voltage of 200V. Proteins were then transferred to an Immobilon-P PVDF membrane, after it was activated in methanol and normalized in transfer buffer, at a constant voltage of 100V for 60 minutes. Membranes were blocked in 1% Fish Skin Gelatin (FSG) in TBST (Tris-buffered saline w/ 0.1% Tween) and incubated overnight at 4˚C with the appropriate primary antibody diluted in either TBST or 1% FSG at 1:1,000. The next day, membranes were washed three times with TBST for 5 minutes, and successively incubated at room temperature with peroxidase-conjugated secondary antibodies in a 0.5% Nonfat Milk TBST solution at 1:5,000. Bound antibodies were detected using a chemiluminescence HRP substrate detection solution (Millipore, Watford, UK). Band quantification was determined using BioRad Image Lab Software.
Immunoprecipitations. Muscle powder was homogenized, and protein quantified as above and 500µg protein was placed into a tube containing 25µL of antibody loaded Protein G-Dyna beads were aliquoted into an Eppendorf tube and prepped for immunoprecipitation using the instructed protocol (Thermo Scientific, Protein G-Dyna Beads). Antibodies for pulldown were used at a concentration of 1:100, and the final solution was submerged in a 30µL of 1X LSB, boiled for 5 minutes and stored at -80˚C. 6µL of sample was loaded per well onto a Criterion TGX Stain-Free Precast Gel and carried forward with the western blotting protocol above.
Antibodies. Primary antibodies for western blotting and immunoprecipitation were diluted to a concentration of 1:1000. Antibodies were from Cell Signaling Technology (Danvers, MA, United States)- total eEF2 (CS-2332S), p-53 (CS-2524S), phospho-eEF2 (CS-2331S), SIRT1 (CS-947S), Ac-Lys (CS9441S), phospho-S6 (CS-5364S), Ac-p53 (CS-252S), P-AKT (Ser473) (CS-4060S), Cytochrome-C (CS-4280S); Santa Cruz Biotechnology (Santa Cruz, CA, United States)- rps6 (SC-13007), rpL13a (SC-390131), Dystrophin (SC-465954); Abcam (Cambridge, UK)- Total OxPhos (ab110413); and Millipore- puromycin (MABE343).
Statistics. Data were analyzed using one-way or two-way ANOVA (loading x inhibition) using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA). Tukey’s post hoc analysis was used to determine differences when interactions existed. Statistical significance was set at p < 0.05. All data are presented as mean ± standard error mean (SEM).