Impact of intermittent fasting on the heart proteome

(a) Hierarchical clustering of proteins significantly differentially expressed in each IF group and AL group (one-way ANOVA with Dunnett’s test). The color scale denotes z-score normalized protein abundances. Red denotes higher protein abundance, and blue lower protein abundance. Gene ontology-based biological processes and pathways significantly enriched in representative protein clusters across the different groups. Selected enzymes specifically modulated within each cluster are highlighted. (b) IF-modulated kinases involved in metabolic pathways and signaling pathways. (c) Enrichment analysis of modulated processes and pathways in the different IF groups in comparison to the AL group (1-D annotation enrichment using Wilcoxon-Mann-Whitney test). Each dot represents a functional process or pathway. A positive enrichment score denotes higher protein abundance, and a negative enrichment score denotes lower protein abundance. n = 4-5 mice in each experimental group.

Differential effects of IF regimen functional processes

(a) Functional transition network that shows functional remodeling of heart proteome in response to IF12 (a), IF16 (b), and EOD (c) GSEA for biological processes and pathways performed for each IF regimen comparison is shown as an enrichment map. Functions significantly enriched (p < 0.01, nominal p-value from empirical phenotype-based permutation test in GSEA) among the upregulated and downregulated proteins for each comparison are indicated by red and blue nodes, respectively. The size of the nodes indicates the number of proteins within the node and edges link nodes that share common proteins. (d) Functional processes and pathways altered by changes in transcriptome and proteome abundances through different IF regimens (Benjamini-Hochberg FDR < 0.05, 2-D annotation enrichment using MANOVA test). Each dot on the plot represents a modulation in pathway or process. The scores indicate abundance changes of transcriptome or proteome levels over AL. Upregulation and downregulation of components are denoted by positive and negative scores, respectively. n = 4-5 mice in each experimental group.

Network mapping of IF-responsive proteins

(a) Proteins with significantly altered abundance in response to IF that are overlaid on consensus protein-protein interaction network constructed using all IF-responsive proteins. The proteins that sequentially show differential modulation are indicated with different colors as yellow (IF12), blue (IF16), and red (EOD). (b) The top densely connected functional protein clusters are shown (one-sided Mann-Whitney U test as implemented in Cluster ONE). The color of each node indicates the IF regimen for which the protein was differentially expressed. Yellow, blue and red indicate IF12, IF16 and EOD regimens, respectively. The heat maps show proteome changes for the proteins in the respective clusters. Red denotes increased expression, and blue denotes decreased expression. (c) Transcription factors significantly enriched across the different IF groups. n = 4-5 mice in each experimental group.

Phosphoproteome landscape of IF-induced changes in heart

(a) Phosphorylation sites showing significant changes in the hearts of mice in the IF12, IF16, and EOD. Phosphorylation changes observed on structural sarcomere organization proteins are highlighted. (b) Hierarchical clustering of differentially altered phosphopeptides showing increased (red) and decreased (blue) phosphosite abundance. Kinase motifs enriched (p < 0.05) in representative clusters are shown (Fisher’s exact test for enrichment). (c) Pathways significantly enriched (p-value ≤ 0.05) among the differentially altered phosphoproteins across the three IF regimens are visualized as a heatmap (Fisher’s exact test for enrichment). The color scale represents negative log10-transformed p values. (d) Temporal alteration of specific IF-responsive phosphorylation sites in AMPK and cGMP-PKG signaling. n = 3 mice in each experimental group.

Immunoblot validation of the selected proteins in IF-modulated pathways

(a) Immunoblots that show the effects of IF16 on the expression of selected proteins p-AMPKα/AMPKα, PKG-1, MYPT1, PPARγ, p-GSKαβ/GSKαβ, AKT, p-IRS1/IRS-1 and Insulin receptor β compared to AL group. Data are represented as mean ± S.E.M. n = 6 mice in each experimental group. *p < 0.05 compared with young Sham; **p < 0.01 compared with AL group.

Echocardiographic analyses of IF on cardiac stress adaptation

(a) Dobutamine stress tests for ability of the heart to adapt to stress under IF. Heart rate and ejection fraction following administration of dobutamine were measured. Values are the mean ± SD; n=10 each; *p < 0.05 (Unpaired t-test). Representative images of long axis M-Mode raw data before and after dobutamine-induced stress are shown in the right panel. (b) Contractility in hearts of IF16 mice compared with AL mice as illustrated by global longitudinal strain (GLS) (upper panel) and radial strain (lower panel). Values are the mean ± SD (n = 10 in each experimental group; **p < 0.01, ***p < 0.001) before and after dobutamine (one-way ANOVA with Tukey’s multiple comparisons test). Representative images of myocardial movement (upper panel) and radial strain (lower panel) before and after dobutamine-induced stress.