Sex Differences in Response to Diet Enriched With Glutathione Precursors in the Aging Heart

Abstract

Common features of the aging heart are dysregulated metabolism, inflammation, and fibrosis. Elevated oxidative stress is another hallmark of cardiac aging that can exacerbate each of these conditions. We hypothesize that by increasing natural antioxidant levels (glutathione), we will improve cardiac function. Twenty-one-month-old mice were fed glycine and N-acetyl cysteine (GlyNAC; glutathione precursors)-supplemented or control diets for 12 weeks. Heart function was monitored longitudinally, and the exercise performance was determined at the end of the study. We found that the GlyNAC diet was beneficial for old male but not old female mice, leading to an increase of Ndufb8 expression (a subunit of the mitochondrial respiratory chain complex), and higher enzymatic activity for CPT1b and CrAT, 2 carnitine acyltransferases that are critical to cardiomyocyte metabolism. Although no quantifiable change of collagen turnover was detected, hearts from GlyNAC-fed old males exhibited a slight but significant enrichment in Fmod, a protein that can inhibit collagen fibril formation, possibly reducing extracellular matrix stiffness and thus improving diastolic function. Cardiac diastolic function was modestly improved in males but not females, and surprisingly GlyNAC-fed female mice showed a decline in exercise performance. In summary, our work supports the concept that aged male and female hearts are phenotypically different. These basic differences may affect the response to pharmacological and diet interventions, including antioxidants.

Keywords: Biology of aging; Cardiovascular disease; Extracellular matrix; Mitochondria; Sex differences.

Graphical Abstract

Figure 1.

Mass spectrometry identifies changes in protein expression in response to GlyNAC. (A) Score plots of PC1 against PC2 of the proteomics data set. The markers indicate the sex variable (females are represented by circles and males by triangles), whereas the closed or half-closed symbol indicates treatment as the second variable) (control [closed circle or triangle] versus GlyNAC [half-closed circle or triangle]). (B) and (C) Volcano plots showing the log2 against the adjusted log10 p-value (upper panels), and the Top10 pathways significantly enriched in each group were shown as a dot plot (lower right panels) in control versus GlyNAC female mice (B), control versus GlyNAC male mice (C). (D) Heatmap showing the protein expression of mitochondrial complexes and membrane components detected by mass spectrometry that are significantly different between tested groups. The rectangular indicates changes in Ndufb8 protein expression.

Figure 1.

Mass spectrometry identifies changes in protein expression in response to GlyNAC. (A) Score plots of PC1 against PC2 of the proteomics data set. The markers indicate the sex variable (females are represented by circles and males by triangles), whereas the closed or half-closed symbol indicates treatment as the second variable) (control [closed circle or triangle] versus GlyNAC [half-closed circle or triangle]). (B) and (C) Volcano plots showing the log2 against the adjusted log10 p-value (upper panels), and the Top10 pathways significantly enriched in each group were shown as a dot plot (lower right panels) in control versus GlyNAC female mice (B), control versus GlyNAC male mice (C). (D) Heatmap showing the protein expression of mitochondrial complexes and membrane components detected by mass spectrometry that are significantly different between tested groups. The rectangular indicates changes in Ndufb8 protein expression.

Figure 2.

GlyNAC diet improves mitochondrial health in the old male mouse heart. (A) Improvement of mitochondria shape and alignment in GlyNAC male mouse heart. Left: Representative electronic transmission microscopy pictures showing the effect of GlyNAC on morphology in male cardiac mitochondria. The upper panels show lower magnification (scale bar: 2 µm), and the lower panel a higher magnification (lower panels, scale bar: 0.5 µm). Right panel: A graph representing the ratio (%) of healthy/total number of mitochondria in control versus GlyNAC male mouse hearts. An unpaired t-test was performed to test the significance, and the p-value was indicated in the graph. N = 4 hearts; for each heart, 3–4 images were analyzed. (B) Representative Western blot (left panel) of NDUFb8 in mitochondrial extracts from the hearts of GlyNAC versus control male and female mice. The level of HSP60 was used as a loading control for densitometry calculation (right panel). (C) Representative Western blot showing the changes in CPT1b protein level in cardiac mitochondrial extracts from mice fed with control or GlyNAC diet (left panel). HSP60 was used as a loading control. The right panel depicts the calculated expression of CPT1b. (D) Effect of GlyNAC on CPT1b activity estimated by the DTNB assay in mitochondrial lysates isolated from old male and old female hearts. (E) Representative Western blot depicting the level of CrAT protein in mitochondrial lysates prepared from GlyNAC or control old male and female hearts. HSP60 was used as a loading control. (F) Effect of GlyNAC on CrAT activity estimated by DTNB assay in mitochondrial lysates from control or GlyNAC old male and old female hearts. (G) Representative oxyblot (protein carbonylation, DNPH signal) in the total protein lysates of GlyNAC versus control male and female mouse hearts. M indicates the lane of the molecular marker, and IC is the interblot calibrator. Total protein staining was used as a loading control. For each diagram depicting normalized Western blot densitometry or enzymatic activity, the histogram bar shows individually plotted values and results as an average ± SEM for each group (N = 6–9). Two-way ANOVA with multiple comparisons (Tukey’s correction) was performed, and significant p-values were indicated directly on the graphs. ◊ indicates a significant difference (adjusted p < .05) among the groups, # and $ indicate a significant effect of diet (#) or sex ($).

Figure 3.

GlyNAC diet was associated with minor changes in ECM composition and deposition. (A) Heatmap from the proteomics data set, focusing on the changes in ECM-related proteins (composition and remodeling) in GlyNAC versus control male and female hearts. The rectangular indicates the change in Fmod protein expression among the groups. (B) Total collagen staining in cardiac sections of GlyNAC-fed versus control mouse hearts. Representative pictures of Picrosirius Red staining on cardiac cross sections from control versus GlyNAC old male and female mice. Upper panels: lower magnification (scale bar: 100 µm) showing the widespread interstitial and perivascular collagen; lower panels: higher magnification (scale bar: 50 µm) showing interstitial accumulations of collagen. (C) Representative immunostaining of collagen type I α1 (red) and degraded collagen (green), with nucleus counterstaining (DAPI, blue) (upper panel: scale bar: 50 µm). Lower panels right and left: diagrams showing the quantification of each staining. (D) Representative Western blot for mature collagen type I (70–90 kDa) performed from total heart lysates. (E) Evaluation of Fmod expression by Western blot performed from total heart lysates. Each histogram bar depicts the group average ± SEM and all the sample values are individually plotted (N = 6–9). Two-way ANOVA with multiple comparisons (Tukey’s correction) was performed, and significant p-values were indicated on the histograms. ◊ indicates a significant difference (adjusted p < .05) among the groups, # and $ indicate a significant effect of diet (#) or sex ($). M indicates the lane for the molecular marker, and IC denotes an interblot calibrator.

Figure 3.

GlyNAC diet was associated with minor changes in ECM composition and deposition. (A) Heatmap from the proteomics data set, focusing on the changes in ECM-related proteins (composition and remodeling) in GlyNAC versus control male and female hearts. The rectangular indicates the change in Fmod protein expression among the groups. (B) Total collagen staining in cardiac sections of GlyNAC-fed versus control mouse hearts. Representative pictures of Picrosirius Red staining on cardiac cross sections from control versus GlyNAC old male and female mice. Upper panels: lower magnification (scale bar: 100 µm) showing the widespread interstitial and perivascular collagen; lower panels: higher magnification (scale bar: 50 µm) showing interstitial accumulations of collagen. (C) Representative immunostaining of collagen type I α1 (red) and degraded collagen (green), with nucleus counterstaining (DAPI, blue) (upper panel: scale bar: 50 µm). Lower panels right and left: diagrams showing the quantification of each staining. (D) Representative Western blot for mature collagen type I (70–90 kDa) performed from total heart lysates. (E) Evaluation of Fmod expression by Western blot performed from total heart lysates. Each histogram bar depicts the group average ± SEM and all the sample values are individually plotted (N = 6–9). Two-way ANOVA with multiple comparisons (Tukey’s correction) was performed, and significant p-values were indicated on the histograms. ◊ indicates a significant difference (adjusted p < .05) among the groups, # and $ indicate a significant effect of diet (#) or sex ($). M indicates the lane for the molecular marker, and IC denotes an interblot calibrator.

Figure 4.

Effects of GlyNAC supplementation on cardiac function and exercise endurance in old male and old female mice. (A) Graphs representing the changes of the cardiovascular parameters in GlyNAC versus control old male and female mice: change with time for left atrial volume to body surface area ratio (LAV/BSA—upper left) and ejection fraction (upper right); measurement of E/eʹ ratio and eʹ values at the endpoint of the study (lower left and lower right, respectively). Data were analyzed using repeated measurements 3-way ANOVA for LAV/BSA and ejection fraction, and 2-way ANOVA for E/eʹ ratio and eʹ. Significant differences were shown directly on the graphs (N = 21 per group). (B) Treadmill performances in old male and old female mice fed with control or GlyNAC diet. Each histogram bar shows group average ± SEM and individually plotted values (N = 6–13 per group). Two-way ANOVA with multiple comparison (Tukey’s correction) was performed. P-value was indicated on the graphs when significant differences among the groups were identified. ◊ indicates a significant difference (adjusted p < .05) among the groups, # and $ indicate a significant effect of diet (#) or sex ($) among the groups.