Proteomic analysis reveals that aging rabbit vocal folds are more vulnerable to changes caused by systemic dehydration

Abstract

Background: Older adults are more prone to develop systemic dehydration. Systemic dehydration has implications for vocal fold biology by affecting gene and protein expression. The objective of this study was to quantify vocal fold protein changes between two age groups and hydration status, and to investigate the interaction of age and hydration status on protein expression, which has not been investigated in the context of vocal folds before. Comparative proteomics was used to analyze the vocal fold proteome of 6.5-month-old and > 3-year-old rabbits subjected to water ad libitum or water volume restriction protocol.

Results: Young and older adult rabbits (n = 22) were either euhydrated (water ad libitum) or dehydrated by water volume restriction. Dehydration was confirmed by body weight loss of - 5.4% and - 4.6% in young and older groups, respectively, and a 1.7-fold increase of kidney renin gene expression in the young rabbits. LC-MS/MS identified 2286 proteins in the rabbit vocal folds of young and older adult rabbits combined. Of these, 177, 169, and 81 proteins were significantly (p ≤ 0.05) affected by age, hydration status, or the interaction of both factors, respectively. Analysis of the interaction effect revealed 32 proteins with opposite change patterns after dehydration between older and young rabbit vocal folds, while 31 proteins were differentially regulated only in the older adult rabbits and ten only in the young rabbits in response to systemic dehydration. The magnitude of changes for either up or downregulated proteins was higher in the older rabbits. These proteins are predominantly related to structural components of the extracellular matrix and muscle layer, suggesting a disturbance in the viscoelastic properties of aging vocal fold tissue, especially when subjected to systemic dehydration.

Conclusions: Water restriction is a laboratory protocol to assess systemic dehydration-related changes in the vocal fold tissue that is translatable to human subjects. Our findings showed a higher number of proteins differentially regulated with a greater magnitude of change in the vocal folds of older adult rabbits in the presence of systemic dehydration compared to younger rabbits. The association of these proteins with vocal fold structure and biomechanical properties suggests that older human subjects may be more vulnerable to the effects of systemic dehydration on vocal function. The clinical implications of these protein changes warrant more investigation, but age should be taken into consideration when evaluating vocal treatment recommendations that interfere with body fluid balance.

Keywords: Aging; Dehydration; Larynx; Proteome; Vocal folds; Water restriction; Young.

Fig. 1

Experimental timeline and water restriction-dehydration protocol. The young (6.5 months old; n = 11) and older adult (3.2–3.7 years old; n = 11) male rabbits were acclimatized for one week. Then, rabbits were assigned to control (water ad libitum; n = 5 young, and n = 5 older adults) or dehydrated (water volume restriction; n = 6 young, and n = 6 older adults) groups. The experiment had 10 days: a baseline period of 5 days, where all animals received water ad libitum, and the water restriction-dehydration period of 5 days, where the dehydrated group had the water volume restricted to half of their average baseline consumption. Blood was collected on the last day of the baseline period (pre-dehydration) and at the end of dehydration for packed cell volume and total solids evaluation. At the end of the dehydration period, rabbits were euthanized and vocal fold tissue was collected for proteomic and RT-qPCR analyses. The figure was created with BioRender.com

Fig. 2

Water intake and dehydration markers. a Water intake by young and older adult rabbits during the baseline period where animals were given water ad libitum. Older adult rabbits consumed ~ 30% less water than young rabbits. b Comparison of water intake by young and older adult rabbits between the control and dehydrated groups during the experiment’s baseline and water restriction periods. During the water restriction period, both young and older adult rabbits in the control group received water ad libitum, while those in the dehydrated group received a restricted volume of water. c, d Percentage body weight loss. Both young and older adult rabbits in the dehydrated group had an average body weight loss of − 5.4% and − 4.6%, respectively, at the end of the water restriction period compared to the control rabbits. e, f Percentage change in packed cell volume (PCV) and total solids (TS) after the water restriction period. Water restriction did not alter the PCV or TS of rabbits in the dehydrated group in either young or older adult rabbits. g, h Kidney renin gene expression. The expression level of renin was increased in the young water restriction-dehydrated rabbits but not in the older-dehydrated rabbits compared to their respective controls. W-ad lib: control groups that received water ad libitum throughout the study. WR-dehy: dehydrated groups that received water restricted volume during the water restriction period of the study. All bars show average values ± SEM. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001. ns: non-significant

Fig. 3

Principal component analysis (PCA) based on the proteomic data showing the separation of groups by a age, b hydration status, c interaction of both factors, and d all significant proteins from each effect combined. PCA plots are based on the top 500 proteins for age and hydration status main factors, top 250 proteins for interaction, and all significant proteins (p ≤ 0.05) organized by ascending p-value. OC: older-control; OD: older-dehydrated; YC: young-control; YD: young-dehydrated

Fig. 4

Heat maps of significant proteins identified by a age, b hydration status, or c interaction effect following Two-way ANOVA showing the patterns of protein expression in each group. d Venn diagram of significant proteins (p ≤ 0.05) based on the Two-way ANOVA results. Heat maps were created with Morpheus software (https://software.broadinstitute.org/morpheus). OC: older-control; OD: older-dehydrated; YC: young-control; YD: young-dehydrated

Fig. 5

Enrichment gene ontology analysis and MCODE networks based on the protein-protein interaction (PPI) enrichment analysis. a,c,e Top 20 enriched terms identified based on the individual gene lists correspondent to significant proteins affected by age, hydration status or interaction, respectively. b,d,f PPI clusters identified by MCODE for individual gene lists based on the significant proteins identified by age, hydration status or interaction effects, respectively. Pathway and process enrichment analysis was applied to each MCODE cluster independently, and the three best-scoring terms by p-value are retained as the functional description of the corresponding clusters, shown in the tables adjacent to corresponding network plots. Nodes represent proteins, and edges represent the interaction between those proteins. Figures were obtained from enrichment and MCODE PPI analyses using Metascape

Fig. 6

Change in the expression pattern of proteins differentially regulated in the vocal folds of young and older adult rabbits by an interaction effect. a Number of upregulated, downregulated and unchanged proteins in the vocal folds of young and older adult rabbits in response to dehydration. Unchanged proteins are proteins with p ≤ 0.05 and |Log2(LFQ mean difference)| above the cut-off of 0.38. Plots show the effect of interaction in the pattern of expression for b muscle-related proteins, c extracellular matrix proteins, and d proteins associated with translation in young and older rabbit vocal folds due to dehydration

Fig. 7

Relative expression of tested genes based on proteins differentially regulated by hydration status. FBN1: Fibrillin 1, MFAP5: Microfibril associated protein 5, SLMAP: Sarcolemmal membrane-associated protein, LMNA: Lamin A/C, and NIF3L1: NIF3-like protein 1. Bars show relative expression mean ± SEM

Fig. 8

Relative expression of tested genes based on proteins differentially regulated by the interaction of age and hydration status factors. FBLN5: Fibulin 5, LAMC1: Laminin subunit gamma 1, COL18A1: Collagen type XVIII alpha-1chain/Endostatin domain-containing protein, MFAP4: Microfibril associated protein 4, MYL2: Myosin light chain 2, DMD: Dystrophin. Bars show relative expression mean ± SEM