Paternal Resistance Training Modulates Calcaneal Tendon Proteome in the Offspring Exposed to High-Fat Diet

Abstract

The increase in high-energy dietary intakes is a well-known risk factor for many diseases, and can also negatively impact the tendon. Ancestral lifestyle can mitigate the metabolic harmful effects of offspring exposed to high-fat diet (HF). However, the influence of paternal exercise on molecular pathways associated to offspring tendon remodeling remains to be determined. We investigated the effects of 8 weeks of paternal resistance training (RT) on offspring tendon proteome exposed to standard diet or HF diet. Wistar rats were randomly divided into two groups: sedentary fathers and trained fathers (8 weeks, three times per week, with 8-12 dynamic movements per climb in a stair climbing apparatus). The offspring were obtained by mating with sedentary females. Upon weaning, male offspring were divided into four groups (five animals per group): offspring from sedentary fathers were exposed either to control diet (SFO-C), or to high-fat diet (SFO-HF); offspring from trained fathers were exposed to control diet (TFO-C) or to a high-fat diet (TFO-HF). The Nano-LC-MS/MS analysis revealed 383 regulated proteins among offspring groups. HF diet induced a decrease of abundance in tendon proteins related to extracellular matrix organization, transport, immune response and translation. On the other hand, the changes in the offspring tendon proteome in response to paternal RT were more pronounced when the offspring were exposed to HF diet, resulting in positive regulation of proteins essential for the maintenance of tendon integrity. Most of the modulated proteins are associated to biological pathways related to tendon protection and damage recovery, such as extracellular matrix organization and transport. The present study demonstrated that the father's lifestyle could be crucial for tendon homeostasis in the first generation. Our results provide important insights into the molecular mechanisms involved in paternal intergenerational effects and potential protective outcomes of paternal RT.

Keywords: exercise; intergenerational; overweight; paternal programming; tendon proteome.

FIGURE 1

Experimental design. Schematic representation of the methodological sequence followed in the study.

FIGURE 2

The results of Principal Components Analyses (PCA). Score plot of proteomic data set acquired by Nano-LC-MS/MS of the six biological groups: SF, sedentary fathers (gray); TF, trained fathers (purple); SFO-C, offspring from sedentary fathers, exposed to control diet (yellow); TFO-C, offspring from trained fathers exposed to control diet (red); SFO-HF, offspring from sedentary fathers exposed to high-fat diet (green); TFO-HF, offspring from trained fathers exposed to a high-fat diet (blue). Each animal is represented by individual points.

FIGURE 3

Heat map of the changes for each animals in the fathers (A) and offspring groups (B). Each horizontal line represents an individual protein. The top 80 proteins regarding fathers and 50 from offspring, respectively. SF, sedentary fathers; TF, trained fathers; SFO-C, offspring from sedentary fathers, exposed to control diet; TFO-C, offspring from trained fathers exposed to control diet; SFO-HF, offspring from sedentary fathers exposed to high-fat diet; TFO-HF, offspring from trained fathers exposed to a high-fat diet. Read counts for each animal has been plotted as log2. Green and red indicate a decrease and increase of protein abundance levels, respectively. Overall number of regulated (down-regulation or up-regulation) protein for training and diet as factors (C).

FIGURE 4

Effects of resistance training on father tendon proteome. Analysis of proteins from the tendons of the trained versus control animals (fathers). Histogram of protein abundance levels from intergroup analysis considering only proteins with down-regulation and up-regulation (p ≤ 0.05), with a ln(fold change) of at least (≥) 0.5. SF, sedentary fathers; TF, trained fathers. The X-axis represents the natural logarithm of the ratio between the treatments (purple: TF:SF ratio). All altered proteins are grouped according to their biologic process as noted in Gene Ontology (GO). The proteins were considered reliably identified only if presenting an FDR < 1% and at least two matching peptides. Supplementary Data 3 shows detailed information about each protein outlined in this figure.

FIGURE 5

High-fat diet effects on tendon proteome of the offspring. Histogram of protein abundance levels from intergroup analysis considering only proteins with down-regulation and up-regulation (p ≤ 0.05), with a ln(fold change) of at least (≥) 0.5. SFO-HF, offspring from sedentary fathers exposed to high-fat diet; SFO-C, offspring from sedentary fathers exposed to control diet; TFO-HF, offspring from trained fathers exposed to a high-fat diet; TFO-C, offspring from trained fathers exposed to control diet. The X-axis represents the natural logarithm of the ratio between the treatments (green: SFO-HF:SFO-C ratio; blue: TFO-HF:TFO-C ratio). All altered proteins are grouped according to their biologic process as noted in Gen Ontology (GO). The proteins were considered reliably identified only if presenting an FDR < 1% and at least two matching peptides. Supplementary Data 3 shows detailed information about each protein outlined in this figure.

FIGURE 6

Effects of paternal resistance training on tendon proteome in the offspring exposed to control and high-fat diet. Histogram of protein abundance levels from intergroup analysis considering only proteins with down-regulation and up-regulation (p ≤ 0.05), with a ln(fold change) of at least (≥) 0.5. TFO-C, offspring from trained fathers exposed to control diet; SFO-C, offspring from sedentary fathers exposed to control diet; TFO-HF, offspring from trained fathers exposed to a high-fat diet; SFO-HF, offspring from sedentary fathers exposed to high-fat diet; The X-axis represents the natural logarithm of the ratio between the treatments (red: TFO-C:SFO-C ratio, blue: TFO-HF:SFO-HF ratio). All altered proteins are grouped according to their biologic process as noted in Gen Ontology (GO). The proteins were considered reliably identified only if presenting an FDR < 1% and at least two matching peptides. Supplementary Data 3 shows detailed information about each protein outlined in this figure.

FIGURE 7

Protein-protein interaction analysis based on STRING network analysis with an interaction confidence score of 0.4. SF, sedentary fathers; TF, trained fathers; TFO-C, offspring from trained fathers exposed to control diet; SFO-HF, offspring from sedentary fathers exposed to high-fat diet; TFO-HF, offspring from trained fathers exposed to a high-fat diet. STRING analysis for differentially abundant proteins in the fathers (A,B) and offspring groups (C,D). All altered proteins are grouped according to their biologic process as noted in Gen Ontology (GO). Primary metabolism (yellow), adipokine (pink), oxidation-reduction process (dark gray) and muscle contraction (red), sarcomere organization (light gray), cytoskeleton organization (orange) and extracellular matrix organization (blue), transport (green).

FIGURE 8

Proteins involved in the same interaction network regarding TF and TFO-HF group. Protein-protein interaction analysis based on STRING network analysis with an interaction confidence score of 0.4. TF, trained fathers; TFO-HF, offspring from trained fathers exposed to a high-fat diet. Blue highlighted nodes are proteins responsible by cell-to-matrix interactions. Red highlighted nodes are extracellular matrix structural constituent. Green highlighted nodes are Proteins responsible by cytoskeleton organization. Upregulated proteins are grouped according to their biologic process as noted in Gen Ontology (GO).

FIGURE 9

Paternal resistance training affects offspring calcaneal tendon proteome exposed to high-fat diet. SFO-HF, offspring from sedentary fathers exposed to high-fat diet; TFO-HF, offspring from trained fathers exposed to a high-fat diet. The main upregulated (red) and downregulated (blue) proteins in the analysis (TFO-HF: SFO-HF). Adipocyte = Protein Adipoq (ADIPOQ). Cell membrane = Sodium/potassium-transporting ATPase subunit alpha-1 (ATP1A1). Cytoskeleton = Keratin, type II cytoskeletal 1 (KRT1). Extracellular space = Vitamin D-binding protein (GC), Serum albumin (MB). Extracellular matrix = Annexin A2 (ANXA2), Cartilage oligomeric matrix protein (COMP), Myocilin (MYOC), Procollagen C-endopeptidase enhancer 1 (PCOLCE), Prolargin (PRELP), Protein Col14a1 (COL14A1), Protein Col28a1 (COL28A1), Serine protease inhibitor A3M (SERPINA3M), Thrombospondin-1 (THBS1). Mitochondrion = Cytochrome c, somatic (CYCS). Myocyte = Calmodulin (CALM), Isoform 3 of Troponin T, slow skeletal muscle (TNNT3), Myosin light chain 1/3, skeletal muscle isoform (MYL1), Myozenin 2 (MYOZ2), Troponin I, slow skeletal muscle (TNNI1), Troponin T, fast skeletal muscle (TNNT1). Nucleus = Eukaryotic translation initiation factor 4H (EIF4A), Histone H2A (H2A), Protein Cipc (CIPC), Protein S100-A4 (S100A4).