Time-restricted feeding improves aortic endothelial relaxation by enhancing mitochondrial function and attenuating oxidative stress in aged mice

Abstract

Age-related endothelial dysfunction is a pivotal factor in the development of cardiovascular diseases, stemming, at least in part, from mitochondrial dysfunction and a consequential increase in oxidative stress. These alterations are central to the decline in vascular health seen with aging, underscoring the urgent need for interventions capable of restoring endothelial function for preventing cardiovascular diseases. Dietary interventions, notably time-restricted feeding (TRF), have been identified for their anti-aging effects on mitochondria, offering protection against age-associated declines in skeletal muscle and other organs. Motivated by these findings, our study aimed to investigate whether TRF could similarly exert protective effects on endothelial health in the vasculature, enhancing mitochondrial function and reducing oxidative stress. To explore this, 12-month-old C57BL/6 mice were placed on a TRF diet, with food access limited to a 6-h window daily for 12 months. For comparison, we included groups of young mice and age-matched controls with unrestricted feeding. We evaluated the impact of TRF on endothelial function by measuring acetylcholine-induced vasorelaxation of the aorta. Mitochondrial health was assessed using fluororespirometry, and vascular reactive oxygen species (ROS) production was quantified with the redox-sensitive dye dihydroethidium. We also quantified 4-hydroxynonenal (4-HNE) levels, a stable marker of lipid peroxidation, in the aorta using ELISA. Our findings demonstrated that aged mice on a standard diet exhibited significant impairments in aortic endothelial relaxation and mitochondrial function, associated with elevated vascular oxidative stress. Remarkably, the TRF regimen led to substantial improvements in these parameters, indicating enhanced endothelial vasorelaxation, better mitochondrial function, and reduced oxidative stress in the aortas of aged mice. This investigation establishes a vital foundation, paving the way for subsequent clinical research aimed at exploring the cardiovascular protective benefits of intermittent fasting.

Keywords: Endothelium; Fluororespirometry; Intermittent fasting; Mitochondrial dysfunction; O2K; Oroboros; Vascular.

Fig. 1

Experimental design and outcome measures for evaluating the effects of TRF on vascular health in aging mice. (A) Study's timeline, incorporating control groups of young (10-months-old) and older (24-months-old) mice, with the experimental group beginning a 12-month TRF regimen at 12 months of age. Each group was provided unlimited access to a standard chow diet, with the TRF group switching to their specified feeding schedule at the appropriate time. (B) Following euthanasia and perfusion with ice-cold buffer, the aorta was excised and cleared of surrounding adipose tissue. Key evaluations include: assessment of ROS production and mitochondrial functionality using an Oroboros O2k respirometer, endothelium-mediated vasorelaxation determined by wire myography with a DMT Myograph system, and ROS quantification via DHE staining coupled with confocal microscopy analysis. These measures are essential for elucidating TRF's mechanistic impact on vascular aging, offering insight into the dietary modulation of vascular redox biology.

Fig. 2

Comparative impact of aging and TRF regimen on energy intake, serum fasting glucose levels, and body weight gain. (A) Circadian feeding cycles for unrestricted food access and TRF in the three experimental groups, indicating the specific hours of food availability and fasting. TRF animals had access to ad libitum food from ZT12 to ZT18 (Zeitgeber time). The unrestricted access model allows food intake throughout the light and dark phases, while TRF restricts it to a 6-h window in the dark phase. (B–C) Body weight (B) and fasting glucose (C) in young and aged control and TRF aged mice. Data are ±SEM. (n = 6–12 for each datapoint). (D) Metabolic cage assessments revealed that mice on the TRF regimen adhered strictly to their fasting schedule, consuming no calories during fasting periods. Interestingly, within their designated feeding windows, these TRF mice adjusted their intake to consume calorie amounts equivalent to those of both the unrestricted, age-matched control group and the younger cohort involved in the study. This pattern indicates that the TRF regimen successfully imposes a structured eating schedule without reducing overall caloric intake, allowing animals to compensate for fasting periods by adjusting their consumption during available feeding times.

Fig. 3

TRF enhances endothelium-mediated vasorelaxation in the aortas of aged mice. (A) Representative tracings illustrate acetylcholine (ACh)-induced vasorelaxation in aortic ring specimens precontracted with phenylephrine (PE), derived from young (10-month-old), (B) aged (24-26-month-old), and (C) aged mice subjected to 12 months of TRF. (B) Aggregate data depicting the vasorelaxation responses of aortic rings to increasing concentrations of ACh. The data demonstrate that TRF effectively prevents the age-associated decline in endothelial function. Values are presented as mean ± SEM for 6–12 animals per group. *p < 0.05 indicates statistical significance between groups, determined by Repeated Measures Analysis of Variance ANOVA with Bonferroni's post hoc test for multiple comparisons.

Fig. 4

Age-related decrease in mitochondrial respiration is ameliorated by TRF. Oxygen consumption was measured in young, aged, and aged TRF groups to determine changes in mitochondrial respiration through oxygen flux. (A) No significant differences were found in Complex I respiration, (B) Mitochondrial complex II respiration was increased in young aortas compared to aged aortas (**p < 0.01), and aortas measured after TRF intervention had significantly improved respiration in complex II compared to aged aortas (*p < 0.05). (C) Cumulatively Complex I and II showed a significant age-related decrease in oxygen flux. Hydrogen peroxide generation was measured in young, aged, and aged TRF groups simultaneously with oxygen consumption measurements to determine changes in ROS production. (D–F) After the addition of substrates for mitochondrial respiratory complexes I and II, including glutamate, malate, ADP, and succinate, aged mice had significantly higher ROS production than young mice (*p < 0.05), and the aged TRF group had significantly less ROS production than the aged mice (*p < 0.05). The overall differences noted in ETC ROS production amongst young, aged, and aged TRF mice are also observed in ROS production at the basal level (H). This demonstrates that intervention with TRF ameliorates age-related increases in ROS production resulting from both the ETC and sources of ROS production outside of the ETC. (G) Quantification of lipid peroxidation via measurement of 4-HNE showed a trend for an increase in aged aorta compared to young and aged + TRF. (I) After the addition of ETC substrates for complexes I and II, ROS production increased in all groups (n.s.), indicating that the ETC also contributes to ROS production in aorta tissue. Data are mean ± SEM (one-way ANOVA with post-hoc Tukey's test).

Fig. 5

TRF mitigates ROS production in aortas of aged mice. (A) Representative confocal microscopy images display DHE-stained aortic sections from young, aged, and aged-TRF mice, highlighting cellular superoxide (O2·−) production through red fluorescence intensity. (B) Quantitative analysis of relative DHE fluorescence intensity demonstrates a pronounced increase in ROS production in the aortas of aged mice compared to those of young mice (***p < 0.001). Notably, a 12-month TRF regimen significantly reduced ROS production in the aortas of aged mice relative to age-matched controls (*p < 0.05). Data are presented as mean ± SEM, analyzed using one-way ANOVA followed by Tukey's post-hoc test.