The biphasic and age-dependent impact of klotho on hallmarks of aging and skeletal muscle function

  1. Zachary Clemens
  2. Sruthi Sivakumar
  3. Abish Pius
  4. Amrita Sahu
  5. Sunita Shinde
  6. Hikaru Mamiya
  7. Nathaniel Luketich
  8. Jian Cui
  9. Purushottam Dixit
  10. Joerg D Hoeck
  11. Sebastian Kreuz
  12. Michael Franti
  13. Aaron Barchowsky
  14. Fabrisia Ambrosio  Is a corresponding author
  1. Department of Physical Medicine & Rehabilitation, University of Pittsburgh, United States
  2. Department of Environmental and Occupational Health, University of Pittsburgh, United States
  3. Department of Bioengineering, University of Pittsburgh, United States
  4. Department of Computational & Systems Biology, School of Medicine, University of Pittsburgh, United States
  5. Department of Physics, University of Florida, United States
  6. Department of Research Beyond Borders, Regenerative Medicine, Boehringer Ingelheim Pharmaceuticals, Inc, Germany
  7. McGowan Institute for Regenerative Medicine, University of Pittsburgh, United States
9 figures, 5 tables and 3 additional files

Figures

Figure 1 with 2 supplements
Declines in muscle structure are subtle until advanced age.

(A) Tibialis anterior (TA) muscle weight as a percentage of whole-body weight in young (3–6 months), middle-aged (10-14 months), old (21-24 months), and oldest-old (27-29 months) male (N = 57) and female mice (N = 78, one-way ANOVAs). (B) Average fiber cross-sectional area of uninjured male (N = 27) and female (N = 24) mouse TAs across age groups (one-way ANOVAs). (C) Representative images of TA sections stained for laminin (gray), type IIA (purple), type IIX (black/unstained), and type IIB (red) fibers (top, main scale bars = 500 μm, inset scale bars = 250 μm ); Masson’s trichrome staining (middle, 50 µm); and lipidtox staining (bottom, lipidtox = red, laminin = green, scale bars = 50 µm). (D) Percentage of IIA and IIX fibers in the whole TA cross-section of male (N = 27) and female (N = 24) mice (one-way ANOVAs). (E) Collagen area of TA sections across ages and sexes (male N = 17, female N = 19) quantified by Masson’s Trichrome staining (one-way ANOVAs). (F) Intermuscular lipid accumulation in the TA across ages and sexes (male N = 16, female N = 15) quantified by lipidtox staining (one-way ANOVA). (G) Intramuscular lipid accumulation in the TA across ages and sexes (male N = 16, female N = 15) quantified by lipidtox staining (one-way ANOVA). All data presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 1—figure supplement 1
Characterization of sarcopenic changes in male mice.

(A) Physiological TA cross-sectional area (CSA) estimated from length and mass measurements in young (3–6 months), middle-aged (10-14 months), old (21-24 months), and oldest-old (27-29 months) male (N = 26) and female (N = 26) C57BL/6J mice across ages and sexes (one-way ANOVAs). (B) Total number of TA muscle fibers in whole TA sections across ages and sexes (male N = 27, female N = 24). (C) Representative images of male uninjured TA muscles showing fiber type staining for type IIA (purple), type IIX (black/unstained), type IIB (red), and laminin (gray, main scale bars = 500 μm, inset scale bars = 250 μm), and Masson’s trichrome staining. (D) Percentage of type IIB muscle fibers in whole TA muscle sections (male N = 27, female N = 24, one-way ANOVA). (E) Type IIB fiber CSA (male N = 27, female N = 24, one-way ANOVA). (F) Type IIA fiber CSA (male N = 27, female N = 24, one-way ANOVA). (G) Type IIX fiber CSA (male N = 27, female N = 24, one-way ANOVA). All data presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 1—figure supplement 2
Denervation gene expression in mice across age groups for Musk (N = 28), Ncam1 (N = 27), and Runx1 (N = 29).

Data collected from the Tabula Muris Senis database. Blue dots represent male data. Red dots represent female data (one-way ANOVAs). All data presented as mean ± SD (*p<0.05, **p<0.01).

Male and female mice display a progressive loss of muscle function over time.

(A) Whole-body endurance of young (3–6 months), middle-aged (10-14 months), old (21-24 months), and oldest-old (27-29 months) male (N = 67) and female (N = 77) mice measured by the four-limb hang test (one-way ANOVA). 'g'= gravity and 's'=seconds. (B) Peak specific tetanic force production in male (N = 25) and female (N = 24) TA muscles (one-way ANOVAs). (C) Force frequency curves for TA stimulation in male mice (N = 25). (D) Force frequency curves for TA stimulation for female (N = 24) mice. (E) Half relaxation time of the TA muscle following single twitch stimulation across ages and sexes (male N = 25, female N = 24, Kruskal-Wallis tests). (F) Time to maximum force following single twitch stimulation of the TA muscle (male N = 25, female N = 24, Kruskal-Wallis tests). All data presented as mean ± SD (*p<0.05, **p<0.01, ****p<0.0001).

Figure 3 with 4 supplements
Network entropy increases from young to old mice, after which time it plateaus.

(A) RNA-seq analysis workflow. (B) Principle component analysis (PCA) showing overall gene expression patterns in young (3–6 months), old (21-24 months), and oldest-old (27-29 months) mice (N = 12). (C) Heatmap showing genes associated with aging progression derived from the 100 genes with the highest PC2 loadings. The two highlighted sections show 15/100 genes that have an increasing trend and 23/100 genes that have a decreasing trend. (D) Schematic description of network entropy computation and interpretation. PPI networks were generated based on RNA-seq data. We capture the degree sequence and the edge weights from the network obtained from experimental data in the form of constraints. The ensemble of networks that follow these constraints have similar network features. If the probability distribution is skewed, then it has a low network entropy and, if not, then it is has a high network entropy. (E) Protein-protein interaction (PPI) network entropy computed from transcriptomic data indicates an increase in molecular disorder of hallmarks of aging genes. A non-parametric Kruskal Wallis test (p=0.0741) and Dunn’s post-hoc test were performed. Entropy of young to old changed with p=0.07. Blue shaded portion indicates standard deviation (n = 4). (F) Venn Diagram showing the number of differentially expressed (DE) genes between groups old vs. young, and oldest-old vs. young mice. (G) Network plot denotes the total number of DE genes per hallmark and the corresponding proportion of DE genes that are unique to old and oldest-old, when compared to young counterparts for each hallmark of aging. Edge weights denote the number of genes that are common between the two hallmarks the edge connects. The node sizes are proportional to the number of genes that fall into each hallmark.

Figure 3—figure supplement 1
Transcriptomic changes in the context of hallmarks of aging genes.

(A) Gene Ontology enrichment of top 100 genes PC2 loadings that characterize aging progression. The majority of these level 3 GO terms fall under altered intercellular communication and nutrient-sensing deregulation. (B) Full heatmap showing genes associated with aging progression derived from the top 100 genes with the highest PC2 loadings.

Figure 3—figure supplement 2
Network entropy with all genes.

Network entropy trend with all genes considered in the PPI network. A non-parametric Kruskal Wallis test (p=0.056) with Dunn’s post-hoc test was performed. Blue shaded portion indicates standard deviation (n = 4).

Figure 3—figure supplement 3
Histogram of all gene counts across young, old, and oldest-old age groups.

(A) Histogram of all gene counts (concatenated values from four animals from each age group). ​(B) Histogram that shows the difference between age groups, with the green (Old - Young) histogram right-shifted compared to red (Oldest-old – Young).

Figure 3—figure supplement 4
Validation of network entropy trends in other samples and species.

(A) ​PPI network entropy obtained from all genes in male mice with ages ranging from young (3–6 months), middle-age (12–15 months), old (21–24 months), and oldest-old (27 months). The blue shaded region denotes standard deviation (n = 6, 7, 6, and 4, respectively). One 21-month-old male sample was excluded since the number of nodes was 33% lower than the mean of rest of the samples. (B) ​PPI network entropy obtained from all genes in male rats with ages ranging from young to old (6, 9, 12, 18, 21, 24, and 27 months). The blue shaded region denotes standard deviation (n = 7, 7, 8, 7, 7, 8, and 8, respectively ). (C) PPI network entropy obtained from all genes in human males with ages ranging from young to middle-aged to old to oldest-old, grouped as decades, namely, 20–29 years, 30–39 years, and so on to >80 years old. The blue shaded region denotes standard deviation (n = 4, 7, 4, 5, 4, 7, and 4, respectively).

Figure 4 with 1 supplement
Development and validation of an AAV approach for systemic delivery of Klotho.

(A) Changes in circulating Klotho levels measured via ELISA in young (3–6 months), middle-aged (10-14 months), old (21-24 months), and oldest-old (27-29 months) male (N = 41), and female (N = 47) mice (one-way ANOVAs). (B) Changes in circulating FGF23 levels in male (N = 20) and female (N = 39) mice. Red symbols represent undetectable levels and were set to zero (Kruskal-Wallis tests, KO values were excluded from statistical analysis). (C) Schematic of the AAV-Klotho plasmid design. (D) Liver expression of AAV vector genomes quantified via qPCR (N = 33, Kruskal-Wallis test). (E) Circulating Klotho levels measured via MSD-ELISA in young female (N = 33) mice injected with AAV-Klotho at varying doses (Kruskal-Wallis test). (F) Gene count normalized to library size for Klotho in the gastrocnemius muscle of female mice treated with GFP and AAV-Kl (N = 20, one-way ANOVA). (G,H,I) Serum concentration levels for insulin (N = 29), cholesterol (N = 35), and glucose (N = 20) in GFP- and Kl-treated female mice (one-way ANOVA). All data presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 4—figure supplement 1
Quantification of circulating lipid metabolites in mice receiving AAV-Kl treatment versus controls.

Circulating phospholipids (N = 20), NEFA (N = 20), and triglycerides (N = 20) in young (3–6 months), old (21-24 months), and oldest-old (27-29 months) female mice receiving AAV-GFP or AAV-Kl​.

Figure 5 with 1 supplement
Gene delivery of Klotho enhances functional muscle regeneration following an acute injury.

(A) Experimental design using old (21–24 months) male mice. (B) Quantification of TA average myofiber cross-sectional area (N = 13). (C) Collagen IV expression in the TA muscle of GFP- versus KL-treated mice (one-tailed Mann-Whitney test, N = 13). (D) Top: Representative images of injured TA muscles stained for collagen IV (green) and DAPI (blue, scale bars = 50 µm). Bottom: Masson’s Trichrome staining of the TA (scale bars = 50 µm). (E) Collagen area percentage in the TA quantified from Masson’s Trichrome staining (one-tailed student’s t-test, N = 16). (F) FAPs to MuSCs ratio in injured TA muscles, as determined by flow cytometry (N = 6, one-tailed student’s t-test). (G) Representative TEM images showing mitochondria in the TA muscle fibers of AAV-GFP vs. KL-treated mice. Aberrant and empty mitochondria show abnormal shape and high proportion of white space respectively (scale bars = 1μm). (H) Quantification of the quality of mitochondria (two-way ANOVA, N = 13). (I) TA specific twitch force produced 14 days post-injury (dpi) (one-tailed Student’s t-test, N = 20). (J) TA maximum specific tetanic force 14 dpi (one-tailed Student’s t-test, N = 20). (K) Change in force production of the TA over time as mice underwent a fatigue protocol consisting of repeated TA stimulation for a total of 7 min, followed by recovery over two 5-min intervals (two-way ANOVA, N = 19). (L) Fold change in whole body endurance compared to one day post injury hang impulse score (Mixed-effects analysis, N = 16). (M) TA peak tetanic specific force for mice 7 days after an eccentric injury treadmill protocol (N = 19). All data presented as mean ± SD (*p<0.05, **p<0.01).

Figure 5—figure supplement 1
Validation of the eccentric exercise injury model.

(A) Experimental design for the eccentric exercise injury model using old (21–24 months) male mice. (B) Summary of parameters for the acclimation period and injury protocol. (C) Assessment of adherence to the exercise protocol showing similar performance between mice receiving AAV-GFP and AAV-Kl (N = 24). Red symbols represent underperforming animals that were removed from further analysis (N = 2 per group). (D) Representative images in the TA showing the extent of injury induced by the exercise protocol, scale bars = 100 microns. (E) Percent of fibers with central nuclei in control vs. mice completing the treadmill protocol (N = 19, green = GFP group, blue = KL group, student’s one-tailed t-test). All data presented as mean ± SD (*p<0.05).

Figure 6 with 1 supplement
AAV-Klotho enhances muscle function in old, but not oldest-old, mice.

(A) Experimental design and timeline using old (21–24 months) and oldest-old (27-29 months) female mice. (B) Animal inclusion flow chart. Mortality describes mice that died over the course of the experiment. Morbidity describes mice in whom pathology was found at the time of euthanasia . These mice were subsequently excluded from analyses. (C) Representative images showing TA myofiber area (Laminin; green), lipid (red), and DAPI (blue) of the TA 14 dpi in old and oldest-old female mice treated with GFP or Kl. Scale bars = 50 µm. (D) TA wet weight (as a percent of total body weight) of old female mice treated with AAV-GFP or AAV-Kl (N = 10). (E) Quantification of TA muscle-fiber cross-sectional area (CSA) for old female mice (N = 10). (F) Percentage of type IIA and IIX muscle fibers in whole TA cross-sections of old female mice (N = 10). (G) Inter- and intramuscular lipid intensity in TA cross-sections of old female TAs (two-way ANOVA, N = 10). (H) Old female TA maximum specific tetanic force production (one-tailed Student’s t-test, N = 12). (I) Hang-test performance 14 days after injection of AAV-Kl or AAV-GFP, calculated relative to baseline performance (one-tailed Student’s t-test, N = 15). (J) TA wet weight (as a percent of total body weight) of oldest-old female mice treated with AAV-GFP or AAV-Kl (N = 16). (K) Quantification of TA muscle-fiber CSA for oldest-old female mice (N = 12). (L) Percentage of type IIA and IIX muscle fibers in whole TA cross-sections of oldest-old female mice (N = 12). (M) Inter- and intramuscular lipid intensity in TA cross-sections of oldest-old female TAs (two-way ANOVA, N = 8). (N) Oldest-old female TA maximum specific tetanic force production (N = 15). (I) Hang-test performance 14 days after injection, calculated relative to baseline performance (N = 14). All data presented as mean ± SD (*p<0.05, **p<0.01).

Figure 6—figure supplement 1
AAV-Administration in uninjured female Mice.

(A) The ratio of whole-body endurance of old (21–24 months) female mice 14 days after receiving GFP or AAV-Kl at three different doses (3 × 108 vg/mouse (low dose), 1 × 109 vg/mouse (mid dose), and 3 × 109 vg/mouse (high dose), N = 17, one-way ANOVA). (B) Total number of muscle fibers in the TA cross-sections of old and oldest-oldest (27–29 months) female mice treated with GFP or AAV-Kl (old N = 10, oldest-old N = 12). (C) Transcript reads of denervation-related genes in old mice (N = 8). (D) Transcript reads of denervation-related genes in oldest-old mice (N = 8). Data presented as mean ± SD error bars (**p<0.01).

Figure 7 with 2 supplements
The effect of AAV-Kl administration on genes associated with hallmarks of aging is age-dependent.

(A) Venn Diagram showing the number of differentially expressed (DE) genes between groups treated with AAV-Kl (n = 4) vs AAV-GFP (n = 4) mice. (B) Network plot with each node as a pie chart that denotes the total number of DE genes in that hallmark, and the wedges denote the proportion of DE genes between groups treated with AAV-Kl vs AAV-GFP for each hallmark of aging. The edge weights denote the number of genes that are common between the two connected hallmarks. The node sizes are proportional to the number of genes that fall into each hallmark. (C) Barplots showing GO terms associated with old vs old klotho (green), and oldest-old vs oldest-old klotho (yellow) DE genes. (D) Bar plot showing the top 20 KEGG pathways that change oppositely between old and oldest-old groups after AAV-Kl treatment ranked by largest absolute difference in total accumulation. Total accumulation is a measure of gene perturbation.

Figure 7—figure supplement 1
Investigation of the Klotho-FGF23 interaction in uninjured female mice with AAV-Kl treatment.

(A) Circulating FGF23 in the serum of old (21–24 months, N = 9) and oldest-old (27-29 months N = 10) female mice treated with GFP or AAV-Kl. (B) Gene count normalized to library size for FGF23 in the gastroc of old and oldest-old female mice treated with GFP or AAV-Kl compared to young (3–6 months) female mice treated with GFP (N = 20). (C) Gene count normalized to library size for primary interactors of FGF23 (N = 20, one-way ANOVA). All data presented as mean ± SD (*p<0.05, **p<0.01).

Figure 7—figure supplement 2
Dot plot of GO terms showing age-dependency of calcium ion transport and signaling with Klotho intervention.

These are GO terms associated with top three pathways differently perturbed between old and oldest-old mice with AAV-Kl intervention. The size of the circle represents the number of genes that are associated with each of the GO term.

Graphical abstract.

Entropy increases with increasing age before plateauing at old age (21–24 months) in mice (top panel). This is concomitant with a decline in skeletal muscle function, which continues to progress into oldest-old age (second panel). We show that AAV-Kl administration can rescue muscle functional declines when administered to old mice (third panel), but this effect is lost when AAV-Klotho is delivered to oldest-old (27–29 months) mice (bottom panel).

Author response image 1

Tables

Table 1
The top 25 GO terms associated with DE genes from old vs. old AAV-KL treated mice.
TermP-valueAdjusted P-valueOdds RatioCombined Score
regulation of endothelial cell chemotaxis to fibroblast growth factor (GO:2000544)8.17E-101.51332E-08266.6318185579.371
sodium ion export from cell (GO:0036376)7.41E-152.92305E-13149.4724774863.183
sodium ion export (GO:0071436)7.41E-152.92305E-13149.4724774863.183
cGMP biosynthetic process (GO:0006182)1.79E-211.98668E-1990.91162794343.159
cGMP metabolic process (GO:0046068)1.41E-232.15989E-2182.19439254324.53
vascular endothelial growth factor receptor signaling pathway (GO:0048010)2.24E-392.73359E-3647.49148424226.557
cyclic nucleotide biosynthetic process (GO:0009190)6.81E-184.38219E-1697.81524253866.443
adenylate cyclase-inhibiting G-protein coupled acetylcholine receptor signaling pathway (GO:0007197)3.18E-084.17609E-07221.6893423827.416
phospholipase C-activating G-protein coupled acetylcholine receptor signaling pathway (GO:0007207)3.18E-084.17609E-07221.6893423827.416
calcineurin-NFAT signaling cascade (GO:0033173)8.94E-122.35047E-10119.0319633028.225
cellular potassium ion homeostasis (GO:0030007)8.32E-132.71081E-11100.6578952799.85
cellular sodium ion homeostasis (GO:0006883)7.48E-142.64978E-1289.67431192710.338
cellular monovalent inorganic cation homeostasis (GO:0030004)6.58E-152.68262E-1382.38620692690.261
cellular response to forskolin (GO:1904322)3.21E-095.4068E-08133.3090912607.271
response to forskolin (GO:1904321)3.21E-095.4068E-08133.3090912607.271
transmembrane receptor protein tyrosine kinase signaling pathway (GO:0007169)1.92E-674.69774E-6416.45666072528.075
ceramide metabolic process (GO:0006672)4.51E-247.3577E-2245.962470.574
regulation of cardiac conduction (GO:1903779)1.84E-304.49925E-2835.71652152445.428
peptidyl-serine dephosphorylation (GO:0070262)2.96E-106.14116E-09103.9157182279.845
cyclic purine nucleotide metabolic process (GO:0052652)3.13E-192.38927E-1751.93355482212.838
calcineurin-mediated signaling (GO:0097720)2.63E-116.62923E-1089.26940642174.732
cAMP biosynthetic process (GO:0006171)2.28E-126.89002E-1180.52219682158.455
positive regulation of protein kinase B signaling (GO:0051897)1.97E-361.20529E-3322.9166811884.074
regulation of myosin-light-chain-phosphatase activity (GO:0035507)1.09E-071.28925E-06110.8390021776.808
cyclic nucleotide metabolic process (GO:0009187)1.16E-155.76736E-1448.89260971681.61
Table 2
The top 25 GO terms associated with DE genes from old vs. old AAV-Kl treated mice.
TermP-valueAdjusted P-valueOdds RatioCombined Score
negative regulation of transcription from RNA polymerase II promoter in response to stress (GO:0097201)0.0003310.0942424113.1472045105.3348
regulation of protein modification by small protein conjugation or removal (GO:1903320)0.000230.076587599.865793282.66108
response to unfolded protein (GO:0006986)5.3E-060.008962275.7218505369.50717
response to chemokine (GO:1990868)0.0060440.30782999613.130035367.07703
response to granulocyte macrophage colony-stimulating factor (GO:0097012)0.0060440.30782999613.130035367.07703
regulation of histone deacetylase activity (GO:1901725)0.0060440.30782999613.130035367.07703
negative regulation of vasculature development (GO:1901343)0.0060440.30782999613.130035367.07703
regulation of respiratory system process (GO:0044065)0.0060440.30782999613.130035367.07703
negative regulation of myeloid cell apoptotic process (GO:0033033)0.0060440.30782999613.130035367.07703
cellular response to granulocyte macrophage colony-stimulating factor stimulus (GO:0097011)0.0060440.30782999613.130035367.07703
regulation of microtubule nucleation (GO:0010968)0.0060440.30782999613.130035367.07703
cellular response to chemokine (GO:1990869)0.0060440.30782999613.130035367.07703
RNA stabilization (GO:0043489)0.0007870.1697492447.1739634351.27482
chaperone-mediated protein complex assembly (GO:0051131)0.0007870.1697492447.1739634351.27482
mitochondrial translation (GO:0032543)1.36E-060.0049707033.6307731749.05655
3'-UTR-mediated mRNA destabilization (GO:0061158)0.0037350.291113278.7581329648.95746
primary miRNA processing (GO:0031053)0.0037350.291113278.7581329648.95746
cellular response to leucine starvation (GO:1990253)0.0037350.291113278.7581329648.95746
endothelial tube morphogenesis (GO:0061154)0.0037350.291113278.7581329648.95746
negative regulation of inclusion body assembly (GO:0090084)0.0037350.291113278.7581329648.95746
mitochondrial gene expression (GO:0140053)4.92E-050.0257763034.7973619647.58752
regulation of transcription from RNA polymerase II promoter involved in heart development (GO:1901213)0.0100220.412099559.8469964745.32527
mitochondrial electron transport, ubiquinol to cytochrome c (GO:0006122)0.0020720.2813627297.3024298245.12396
mitochondrial translational elongation (GO:0070125)8.63E-060.008962273.697662243.11427
mitochondrial translational termination (GO:0070126)1.22E-050.008962273.5916266740.63
Table 3
Top 25 GO terms associated with DE genes from oldest-old vs. oldest-old AAV-Kl-treated mice.
Klotho mediated Bi-phasic KEGG Pathways Gene Expression
Old + log2FCOldest-Old + log2FC
Regulation of actin cytoskeletonF2-1.92627Bcarl0.40772Rras0.30578Rhoa0.08657
Kng2-1.09411Arpc30.22899Pik3rl-0.94428Itga50.30051
Diaph20.25901Brkl0.17587Rafl-0.19865Hras0.22869
Itga90.39371Arhgap35-0.24801Pfn10.19127
Pdgfrb-0.35413Pip4k2c-0.16982Pfn2-0.20196
ltgax1.6131Apc2-0.52877
Arpc20.1243Cdc420.11714
cGMP-PKG signalingF2-1.9263Dgke-0.3491Pik3rl-0.94428Cyth10.13784
Lpar31.82228Pdgfrb-0.35413Rafl-0.19865Rhoa0.08657
Adcy9-0.20643Dnm3-0.54357Hras0.22869
Rras0.30578Pla224e-0.25837
Sphingolipid signaling pathwayKng2-1.0941Sgppl-0.1274Pik3rl-0.94428Hras0.22869
Map3k50.27371Rafl-0.19865
Ctsd0.17911Rhoa0.08657
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Mus musculus)klotho (Kl)MGIMGI:1101771
Strain, strain background (mouse)C57BL/6JNIARRID:IMSR_JAX:000664
Cell line (Homo sapiens)HEK-293HThermo FisherCat#: 11631017
RRID:CVCL_6643
For vector construction
AntibodyAnti-Laminin
(Rabbit polyclonal)
AbcamCat#: ab11575,
RRID:AB_298179
IF(1:500)
AntibodyAnti-Type IIA Fibers (mouse monoclonal)DSHBCat#: SC-71,
RRID:AB_2147165
IF(1:100)
AntibodyAnti-Type IIB Fibers (mouse monoclonal)DSHBCat#: BF-F3,
RRID:AB_2266724
IF(1:100)
AntibodyAnti-Collagen IV (rabbit polyclonal)AbcamCat#: ab6586,
RRID:AB_305584
IF(1:500)
AntibodyAnti-Klotho capture antibody
(goat polyclonal)
R and D SystemsCat#: AF1819,
RRID:AB_2296612
MSD-ELISA(4 µg/mL)
AntibodyAnti-Klotho detection antibody
(goat polyclonal)
R and D SystemsCat#: BAF1819,
RRID:AB_2131927
MSD-ELISA(1 µg/mL)
AntibodyAnti-CD31 (rat monoclonal)Thermo FisherCat#:1 11–0311081,
RRID:AB_465011
FACS(1:500)
AntibodyAnti-CD45 (mouse monoclonal)Thermo FisherCat#: 11-0451-81,
RRID:AB_465049
FACS(1:500)
AntibodyAnti-Sca1 (rat monoclonal)Thermo FisherCat#: 25-5981-82,
RRID:AB_469669
FACS(1:33)
AntibodyAnti-α−7 (rat monoclonal)Thermo FisherCat#: MA5-23555,
RRID:AB_2607368
FACS(1:200)
Peptide, recombinant proteinKlothoR and D SystemsCat#: 1819 KL-050MSD-ELISA
OtherLipidtox stainThermo FisherCat#: H34476IF(1:500)
OtherDAPI stainInvitrogenCat#: D1306
RRID:AB_2629482
IF(1:1000)
OtherTrichrome Stain SolutionSigmaCat#: HT10516
OtherWeigarts’s HaemotoxylinPoly Scientific R and DCat#: S216B
OtherBouin’s solutionSigmaCat#: HT101128
Commercial assay or kitMouse Klotho ELISACloud-Clone Corp.Cat#: SEH757MuLot: L180223640
Commercial assay or kitFGF23 ELISAAbcamCat#: ab213863Lot: GR3326863
Commercial assay or kitAllPrep DNA/RNA 96 kitQiagenCat#: 80311
Chemical compound, drugPoly/Bed 812PolysciencesCat#: 08792–1
Chemical compound, drugCardiotoxinSigmaCat#: 217503
Recombinant DNA reagentAAV-GFPStrobel et al., 2019AAV8-LP1-eGFP
Recombinant DNA reagentAAV-KlothoThis paperAAV8-LP1-mKlotho
Mouse Klotho version of AAV vector
Sequence-based reagentLP-1 promoter (forward)This paperPCR primersGACCCCCTAAAATGGGCAAA
Sequence-based reagentLP-1 promoter (reverse)This paperPCR primersTGCCCCAGCTCCAAGGT
Biological sample (M. musculus)Mouse gastrocnemius muscleNIAFreshly dissected from C57BL/6J mice
Biological sample (M. musculus)Mouse serumNIAFreshly dissected from C57BL/6J mice
Biological sample (M. musculus)Mouse tibialis anterior muscleNIACollected fresh from C57BL/6J via cardiac puncture
Software, algorithmSTAR_2.7.0aDobin et al., 2013RRID:SCR_004463
Software, algorithmRR Project for Statistical ComputingRRID:SCR_001905
Software, algorithmggplot2R Project for Statistical ComputingRRID:SCR_014601R package
Software, algorithmclusterProfilerBioconductorRRID:SCR_016884R package
Software, algorithmVennDiagramR Project for Statistical ComputingRRID:SCR_002414R package
Software, algorithmigraphR Project for Statistical ComputingRRID:SCR_019225R package
Software, algorithmBioMartBioMart ProjectRRID:SCR_002987R package
Software, algorithmFiji-ImageJNIHRRID:SCR_002285
Software, algorithmMuscleJ MacroMayeuf-Louchart et al., 2018RRID:SCR_020995
Software, algorithmGraphPad Prism v9.0GraphPadRRID:SCR_002798
Software, algorithmR ShinyApp resourceThis paperhttps://sruthisivakumar.shinyapps.io/HallmarksAgingGenes/Gene classification based on hallmarks of aging. Building details are provided in ‘Hallmarks of aging genes classification' (Materials and methods).
Software, algorithmPython StreamlitThis paperhttps://network-entropy-calculator.herokuapp.com/Network entropy calculatorapp hosted on Heroku
Software, algorithmGitHubThis paperhttps://github.com/sruthi-hub/sarcopenia-network-entropyCode for RNA -seq network entropy provided with explanation
OtherSTRINGSTRING ConsortiumRRID:SCR_005223Protein interaction database
Table 4
Antibody/stain list.

Primary antibodies and corresponding dilutions used for immunofluorescence imaging.

Antibody/stainSourceDilution
Rabbit anti-LamininAbcam ab115751:500
Lipidtox Red StainInvitrogen H344761:200
Collagen IVAbcam ab65861:500
Type IIA Muscle FibersDSHB SC-711:100
Type IIB Muscle FibersDSHB BF-F31:100
DAPI StainInvitrogen D13061:1000

Additional files

Supplementary file 1

Full list of KEGG pathways affected by Klotho treatment in old and oldest-old animals.

https://cdn.elifesciences.org/articles/61138/elife-61138-supp1-v2.pdf
Supplementary file 2

Search terms and network properties for hallmarks of aging genes.

https://cdn.elifesciences.org/articles/61138/elife-61138-supp2-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/61138/elife-61138-transrepform-v2.docx

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  1. Zachary Clemens
  2. Sruthi Sivakumar
  3. Abish Pius
  4. Amrita Sahu
  5. Sunita Shinde
  6. Hikaru Mamiya
  7. Nathaniel Luketich
  8. Jian Cui
  9. Purushottam Dixit
  10. Joerg D Hoeck
  11. Sebastian Kreuz
  12. Michael Franti
  13. Aaron Barchowsky
  14. Fabrisia Ambrosio
(2021)
The biphasic and age-dependent impact of klotho on hallmarks of aging and skeletal muscle function
eLife 10:e61138.
https://doi.org/10.7554/eLife.61138