Extended Data Fig. 8: GDF15 treatment and GFRAL expression in the heart.
From: Cardiac adaptation to endurance exercise training requires suppression of GDF15 via PGC-1α
a, Representative images (left) and quantification (right) of NRVMs stained with sarcomeric α-actinin after treatment with PBS or recombinant human GDF15 (Sino Biological 10936-H01Y, 100 ng/mL) for 48 hours in serum-free media (n = 3 biologically independent wells per condition, separate biological replication experiment performed once). b, Gdf15 mRNA expression (left) and GDF15 protein levels in conditioned media (right) from NRVMs transduced with Ad.GFP or Ad.Gdf15 at a multiplicity of infection (MOI) of 50 for 72 hours (n = 3 biologically independent wells per condition, separate biological replication experiment performed once). c, Representative images (left) and quantification (right) of sarcomeric α-actinin staining of NRVMs from (b) to assess cardiomyocyte size (n = 3 biologically independent wells per condition, separate biological replication experiment performed once). d, Relative mRNA expression of Gfral, Glp1r (AP/NTS marker), and Satb2 (cortical neuron marker) in mouse area postrema/nucleus tractus solitarius (AP/NTS, n = 3 independent mice), cortex (n = 4 independent mice), and heart (n = 4 independent mice). Primer sequences are listed in Supplementary Table 4. Experiment performed in one set of mice without a separate biological replication experiment. Created with BioRender.com. e, Relative GFRAL expression across individual cardiac cell types from human hearts using single-nucleus RNA sequencing (snRNA-seq) (n = 16 independent non-failing [NF] heart samples, N = 15 hypertrophic cardiomyopathy [HCM] samples, n = 11 independent dilated cardiomyopathy [DCM] samples). Experiment performed in one cohort without a separate biological replication cohort. f, Relative expression of GFRAL, GDF15, and PPARGC1A across all nuclei from the same snRNA-seq dataset in (e). Experiment performed in one cohort without a separate biological replication cohort. Data in (a)–(c) are shown as mean±s.e.m.Box plots in (e-f) display boxes extending from 25th to 75th percentile of the distribution of the values, with whiskers extending to minimum and maximum values. For (a)–(c), statistical significance was assessed using unpaired two-tailed t-tests. For (d)–(f), statistical significance was assessed using two-sided one-way ANOVA followed by Sidak’s multiple comparisons test (d), or two-sided two-way ANOVA with Sidak’s post-test (e–f). Exact P values: a, 0.038; b, Gdf15 mRNA: 0.038, GDF15 protein: 0.000013; c, 0.0051; d, Glp1r: 0.000089 (AP/NTS vs. cortex), 0.000074 (AP/NTS vs. heart); Gfral: 0.0044 (AP/NTS vs. cortex), 0.0017 (AP/NTS vs. heart); Satb2: 0.0016 (AP/NTS vs. cortex), 1 (AP/NTS vs. heart); e, GFRAL in fibroblast I: 1.1e–15 (NF vs. DCM), 0.00032 (NF vs. HCM); f, PPARGC1A: 1 (NF vs. DCM), 1 (NF vs. HCM). Significance symbols: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; N.S., not significant. n refers to biologically independent samples. Measurements in (a)–(c) represent the average value per well from 30-35 cells per well. For (d), each data point represents an individual mouse tissue sample. For (e-f), each data point reflects one biological replicate (human donor). Representative microscopy images in (a) and (c) are from experiments independently repeated twice with similar results. Mouse strain and age: data in (d) was derived from 10 week old female C57BL/6 mice obtained from The Jackson Laboratory (age relative to initiation of the experiment) of the indicated n per group.
