Proteostatic Imbalance Drives the Pathogenesis and Age-Related Exacerbation of Heart Failure With Preserved Ejection Fraction

Figures

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  Figure 1. Aging Exacerbates 2-Hit Stress–Induced HFpEF Development and Has More Pronounced Effects on Cardiac Proteome Than 5 Weeks of 2-Hit Stress (A) Study design to investigate proteomic changes during early heart failure with preserved ejection fraction (HFpEF) development in young and old mice. (B) Probability of survival in young and old mice subjected to 2-hit stress for 5 weeks. n = 5 to 8 per group. (C) Ratio of heart weight to tibia length (HW/TL). n = 5 to 6 per group. (D) Fractional shortening (FS). n = 4 to 8 per group. (E) E/E′ ratio. n = 4 to 8 mice per group. (F) E/A′ ratio. n = 4 to 8 mice per group. (G) Isovolumic relaxation time (IVRT). n = 4 to 8 mice per group. (H) Ratio of wet lung water to tibia length (lung water/TL). n = 5 to 6 per group. (I) Two-dimensional principal component analysis scores plot showing proteomes of young control (YCL), young HFpEF (YHFpEF), old control (OCL), and old HFpEF (OHFpEF) hearts (n = 3-5 per group). (J) Volcano plots of proteomic changes between YHFpEF vs YCL, OHFpEF vs OCL, OCL vs YCL, and OHFpEF vs YHFpEF mice. (K, L) Venn diagrams presenting overlap of proteins with (K) heightened and (L) lowered expression across experimental…

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  Figure 2. Proteins Involved in Proteostasis Network Are Altered by Both 2-Hit Stress and Cardiac Aging (A, B) Differentially expressed proteins in the young and old mice subjected to 2-hit stress compared with age-matched control mice (bolded proteins are involved in Proteostasis Network). (C) Heat map of proteins differentially expressed with 2-hit stress in either young or old animals, annotated into the proteostasis network. (D) Annotation of differentially expressed proteins in specific proteostatic branches and classes. (E, F) Venn diagrams presenting overlap of proteins (E) up-regulated and (F) down-regulated by aging in control (OCL vs YCL) and HFpEF (OHFpEF vs YHFpEF) conditions. (G-M) Heat maps of proteins annotated into proteostasis network that are significantly affected by age in either control or HFpEF conditions. (N, O) Selected (N) up-regulated and (O) down-regulated Gene Ontology (GO) biological processes by age in control or HFpEF conditions. Proteins with adjusted P < 0.05 and |log2(FC)| >0.58 were subjected into proteostasis network annotation and GO analysis. FDR = false discovery rate; other abbreviations as in Figure 1.

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  Figure 3. 2-Hit Stress Induces Mortality in Old Mice and Up-Regulates Protein Synthesis in the Hearts of Both Young and Old Animals (A) Deuterium oxide (D2O) labeling study design to investigate effects of 15-week 2-hit stress on protein synthesis and ribosomal biogenesis. (B) Probability of survival in young and old mice subjected to 2-hit stress for 15 weeks. n = 19 to 30 per group. (C) Body weight change after 15 weeks of the experiment. n = 15 to 19 per group. (D) HW/TL ratio. n = 19 to 27 per group. (E) Lung water/TL ratio. n = 5 to 8 per group. (F) Ratio of tibialis anterior muscle weight to tibia length (TA/TL). n = 10 to 15 per group. (G) Ratio of gastrocnemius muscle weight to tibia length (GAS/TL). n = 10 to 15 per group. (H, I) Synthesis of new myofibrillar proteins over time and calculated protein synthesis rates (k). n = 14 to 18 per group. (J, K) Synthesis of new mitochondrial proteins over time and calculated protein synthesis rates (k). n = 14 to 18 per group. (L, M) Synthesis of new collagen over time and calculated protein synthesis rates (k). n = 13 to 18 per group. (N, O) Synthesis of new RNA in ventricular tissue over time and calculated rate of ribosomal biogenesis…

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  Figure 4. Kinetic Proteomics Revels HFpEF- and Age-Related Changes in Individual Protein Synthesis (A) Table showing coverage of proteins identified in specific experimental groups. (B to D) Number of (B) total, (C) mitochondrial, and (D) myofibrillar proteins with significantly higher (red) and lower (blue) synthesis rates in each comparison. (E) Individual mitochondrial proteins with significantly different synthesis rates. (F) Individual myofibrillar proteins with significantly different synthesis rates. (G to J) Volcano plots showing changes in protein synthesis rates between YHFpEF vs YCL, OHFpEF vs OCL, OCL vs YCL, and OHFpEF vs YHFpEF mice. (K, L) Venn diagrams presenting the overlap of proteins with higher synthesis and lower synthesis between experimental groups. n = 12 to 14 per group. Proteins with adjusted P < 0.05 were subjected to analysis. Abbreviations as in Figure 1.

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  Figure 5. 2-Hit Stress and Aging Have Opposing Effects on the Synthesis Rates of Proteins Involved in Mitochondrial Functioning, Glucose Metabolism, and Branched Amino Acid Metabolism (A) Selected enriched pathways affected by changes in individual protein synthesis within all 4 comparisons: YHFpEF/YCL, OHFpEF/OCL, OCL/YCL, and OHFpEF/YHFpEF mice. (B, C) Venn diagrams showing overlap of opposite effects of 2-hit stress and age on synthesis rates such as higher synthesis with HFpEF vs lower synthesis with age and lower synthesis with HFpEF vs higher synthesis with age. (D) Network of proteins and pathways with opposite effects of 2-hit stress (higher) and aging (lower) on their synthesis rates. (E to I) Heatmap representations of concentrations of (E) myofibrillar; (F) tricarboxylic acid cycle (TCA) cycle, electron transport chain (ETC), and other mitochondrial; (G) beta-oxidation and lipid metabolism; (H) glucose metabolism; and (I) stress response proteins measured in heart homogenates of all 4 for experimental groups acquired using high resolution accurate mass. n = 12 to 14 per group. Significance determined by 2-way ANOVA. CL = control; O = old; Y = young; other abbreviations as in…

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  Figure 6. Both 2-Hit Stress and Aging Impair Protein Degradation Pathways and Promote Accumulation of Protein Aggregates (A to D) Western blot analysis of protein degradation pathways: (A) LC3A/B II/I ratio, (B) p62, (C) Beclin-1, and (D) protein ubiquitination. n = 6 per group. (E) Representative immunoblotting images and total protein staining. (F-H) Proteolytic activity of (F) β1, (G) β2, and (H) β5 subunits of 20S proteasome. n = 6 per group. (I) Western blot analysis of protein ClpP expression levels. n = 6 per group. (J) Isolation of aggregate-enriched insoluble protein fraction (K) Ratio of insoluble to soluble protein content. n = 6 per group. (L to O) Western blot analysis of the levels of (L) protein ubiquitination and (M) p62 in the detergent-insoluble fraction. n = 6 per group. (N, O) Representative immunoblotting images and total protein staining. Data are presented as mean ± SD; P values were determined by 2-way ANOVA followed by Tukey post hoc test. Numbers in the 2-way ANOVA tables indicate P values. Symbols indicate statistical significance: ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Abbreviations as in Figure 1.

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  Figure 7. mTORC1 Is Activated by Aging and 2-Hit Stress, and Its Cardiac-Specific Inhibition Protects Against HFpEF Development (A) Effects of aging and 2-hit stress on mammalian target of rapamycin complex 1(mTORC1)–related mechanisms of proteostatic maintenance. (B to E) Western blot analysis of the phosphorylation of mTORC1 downstream targets (B) S6, (C) 1eIF4E-binding protein 1(4EBP1), (D) Unc-51-like autophagy activating kinase 1(ULK1), and (E) representative immunoblotting images. n = 6 per group. (F) Experimental design to test the effects of cardiac-specific heterozygous knockout of Raptor (c-Raptor-Het KO) on the development and progression of 2-hit stress–induced HFpEF phenotype. (G) Raptor messenger RNA (mRNA) level at baseline and after 15 weeks of 2-hit stress. n = 3 at baseline; n = 6 per group after 15 weeks. (H) HW/TL ratio at baseline and after 15 weeks of 2-hit stress. n = 3 at baseline; n = 12 to 13 per group after 15 weeks. (I) E/A′ ratio at baseline and after 5 and 15 weeks of 2-hit stress. n = 4 to 8 mice per group. (J) E/E′ ratio at baseline and after 5 and 15 weeks of 2-hit stress. n = 4 to 8 mice per group. (K) FS at baseline and after 5 and 15 weeks of 2-hit…