Mitochondrial Quality Control in Aging and Heart Failure: Influence of Ketone Bodies and Mitofusin-Stabilizing Peptides

Abstract

Aim: Aging and heart failure (HF) are each characterized by increased mitochondrial damage, which may contribute to further cardiac dysfunction. Mitophagy in response to mitochondrial damage can improve cardiovascular health. HF is also characterized by increased formation and consumption of ketone bodies (KBs), which may activate mitophagy and provide an endogenous mechanism to limit the adverse effects of mitochondrial damage. However, the role of KBs in activation of mitophagy in aging and HF has not been evaluated. Methods: We assessed mitophagy by measuring mitochondrial Parkin accumulation and LC3-mediated autophagosome formation in cardiomyocytes from young (2.5 months), aged (2.5 years), and aged rabbits with HF (2.5 years) induced by aortic insufficiency and stenosis. Levels of reactive oxygen species (ROS) generation and redox balance were monitored using genetically encoded sensors ORP1-roGFP2 and GRX1-roGFP2, targeted to mitochondrial or cytosolic compartments, respectively. Results: Young rabbits exhibited limited mitochondrial Parkin accumulation with small (~1 μm²) puncta. Those small Parkin puncta increased four-fold in aged rabbit hearts, accompanied by elevated LC3-mediated autophagosome formation. HF hearts exhibited fewer small puncta, but many very large Parkin-rich regions (4-5 μm²) with completely depolarized mitochondria. Parkin protein expression was barely detectable in young animals and was much higher in aged and maximal in HF hearts. Expression of mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1) was reduced by almost 50% in HF, consistent with improper fusion-fission, contributing to mitochondrial Parkin build-up. The KB β-hydroxybutyrate (β-OHB) enhanced mitophagy in young and aging myocytes, but not in HF where β-OHB further increased the number of cells with giant Parkin-rich regions. This β-OHB effect on Parkin-rich areas was prevented by cell-permeable TAT-MP1^Gly peptide (thought to promote MFN2-dependent fusion). Basal levels of mitochondrial ROS were highest in HF, while cytosolic ROS was highest in aged compared to HF myocytes, suggesting that cytosolic ROS promotes Parkin recruitment to the mitochondria. Conclusion: We conclude that elevated KB levels were beneficial for mitochondrial repair in the aging heart. However, an impaired MFN2-DRP1-mediated fusion-fission process in HF reduced this benefit, as well as Parkin degradation and mitophagic signaling cascade.

Keywords: Parkin; aging; heart failure; ketone bodies; mitochondrial quality control; mitofusin; mitophagy; β-hydroxybutyrate.

Figure 1

Parkin-mediated mitophagy flux is activated in cardiomyocytes from young rabbit hearts upon treatment with mitochondrial uncoupler FCCP. (A) Representative images of mCherry-Parkin and GFP-LC3 distribution in untreated myocytes from young rabbit hearts (left) and cardiomyocytes treated with 100 nM (middle) or 1 μM (right panels) mitochondrial uncoupler FCCP. (B) Summary of mCherry-Parkin and GFP-LC3 puncta count per cell in untreated cardiomyocytes from young rabbit and myocytes treated with 100 nM or 1 μM FCCP, respectively. (C) In separate experiments, cells were pre-treated with 100 nM Bafilomycin A, a vacuolar H⁺-ATPase inhibitor that prevents autophagosome and lysosome fusion, and then treated with 100 nM FCCP to induce mitophagy. As shown in the summary data, mCherry-Parkin and GFP-LC3 puncta count per cell was not affected by Bafilomycin A1 significantly, indicating the slow mitophagy flux in cardiomyocytes from the young hearts. (D) Representative examples of 100 nM FCCP-treated GFP-LC3-expressing control myocyte co-loaded with mitochondrial membrane potential sensitive dye TMRM (left), and (E) mCherry-Parkin-expressing myocyte co-loaded with MitoTracker Green to identify mitochondria (right). The images below represent enlarged areas from the region of interest as shown in the rectangular area. Data expressed as mean ± SEM. n = number of cells from three different animals per experimental group. *p < 0.05 in treated vs. untreated cells in each group.

Figure 2

Parkin-mediated mitophagy flux is significantly elevated in aging hearts but impaired in HF. (A) Representative images of mCherry-Parkin and GFP-LC3 puncta distribution in young, aged control and HF myocytes. (B) Average puncta size in young, aged and HF myocytes. (C) Average puncta count per cell in young, aged and HF myocytes. (D) Cell size differences in young, aged and HF myocytes. (E) Puncta count normalized per cell size in young, aged and aged HF myocytes. Data expressed as mean ± SEM. n = number of cells from four young, five aged and six HF animals. *p < 0.05 reflects the difference in aged and HF myocytes vs. young; #p < 0.05 aged vs. HF myocytes.

Figure 3

Mitophagy signaling pathway was upregulated in the aging heart but severely impaired in HF. (A) Expression of the proteins in PINK-Parkin mitophagy pathway in cardiac myocytes from young, aged and failing hearts: PINK-1, Parkin, mitofusin 2 (MFN2), dynamin-related protein 1 (DRP1), SQSTM1/p62, pro-LC3, and LC3-I/LC3-II. GAPDH staining was used to confirm equal protein loading for each experimental group. (B–H) Summary analysis of PINK-1, Parkin, mitofusin 2 (MFN2), DRP1, SQSTM1/p62, pro-LC3, LC3-I protein expression normalized to GAPDH or VDAC1 as indicated in the figure. (I) Quantification of LC3-I conversion to LC3-II protein expression presented as LC3-II/LC3-I ratio. Data expressed as mean ± SEM. n = 3–5 animals per group, *p < 0.05 reflects a difference in aging and HF hearts vs. young hearts, #p < 0.05 reflects a difference in HF hearts vs. corresponding age-matched control hearts.

Figure 4

Formation of Parkin-rich areas in HF myocytes was associated with severely depolarized mitochondria. (A) Shown are Parkin-rich areas and GFP-LC3 puncta distribution in HF myocytes under the higher magnification. (B) Shown are representative images of healthy control aged and HF myocytes loaded with mitochondrial membrane potential sensitive dye TMRM (red), Mitotracker Green (to identify mitochondria), DRAQ5 (to identify nuclei) and overlay of all (merged). (C) Magnified images from the rectangular area shown in B in HF myocytes reflect the areas with completely depolarized mitochondria. (D) Summary of mean mitochondrial membrane potential in cardiac myocytes from young, aged, and HF myocytes. n = number of cells from four young, five aged, and six HF animals. *p < 0.05 in aged and HF vs. young; #p < 0.05 reflects a difference in HF hearts vs. corresponding age-matched control hearts. (E) Plot profile summaries of TMRM fluorescence obtained from the lines placed across the cells in aged and HF myocytes.

Figure 5

Effect of ketone body (KB) β-hydroxybutyrate (β-OHB) on mitochondrial and cytosolic oxidative stress and redox balance in young, aging and failing hearts. (A) Shown are representative images of untreated young rabbit cardiomyocytes expressing mito-roGFP2-ORP1 (green, upper left panel) or cyto-roGFP2-ORP1 (green, lower left panel) and same cells loaded with mitochondrial membrane potential sensitive dye TMRM to identify mitochondria (red, middle panels), and overlay of both channels (merged, right panels). (B) Mitochondrial basal H₂O₂ levels were the highest in HF myocytes compared to both young and aged cardiomyocytes. Cell treatment with 2 mM β-OHB significantly decreased H₂O₂ levels in all experimental groups. (C) Cytosolic H₂O₂ levels were the highest in aged cardiomyocytes compared to both young and HF myocytes. Cell treatment with 2 mM β-OHB decreased cytosolic H₂O₂ levels in young hearts, but did not change the oxidative stress in aged and HF myocytes. (D) Levels of the mitochondrial oxidized to reduced glutathione (GSSG/GSH) were the highest in the aging heart and elevated in HF compared to the young hearts. 2 mM β-OHB shifted the mitochondrial GSSG/GSH ratio toward reduced glutathione in both young and aging myocytes but not in HF myocytes. (E) The level of the cytosolic GSSG/GSH was progressively declining during aging and HF, and partially increased by 2 mM β-OHB treatment in all experimental groups. Data expressed as mean ± SEM. n = number of cells from three different animals per each group. Red bars represent untreated cells; green bars represent cells treated with 2 mM β-OHB for 24 h. *p < 0.05 reflects a significance in aged and HF rabbits vs. young rabbits; **p < 0.05 between HF and HF + OHB vs. aged and aged + OHB groups, #p < 0.05 reflects a significance in 2 mM β-OHB-treated cells vs. corresponding untreated groups.

Figure 6

Ketone body β-hydroxybutyrate (β-OHB) enhances mitophagy in young and aging hearts but not in heart failure (HF). (A) Shown are representative images of untreated control aged myocytes and myocytes treated with 2 mM β-OHB for 24 h. (B) Shown are representative images of untreated HF myocytes and HF myocytes treated with 2 mM β-OHB for 24 h. (C) Summary analysis of β-OHB effect on mitochondrial Parkin puncta accumulation and LC3-mediated autophagosome formation in young, aged, and HF myocytes. Data expressed as mean ± SEM. (C) Summary analysis of β-OHB effect on mitochondrial Parkin accumulation and LC3-mediated autophagosome formation in young, aged, and HF myocytes. Data expressed as mean ± SEM. n = number of cells from four young, five aged, and six HF animals. *p < 0.05 reflects a significance in aged and HF rabbits vs. young rabbits; **p < 0.05 reflects a significance in aged vs. HF rabbits, #p < 0.05 reflects a significance in 2 mM β-OHB-treated cells vs. corresponding untreated groups.

Figure 7

Stabilizing Mfn2 conformation prevents giant fusion formation and improves mitophagy coupling in HF myocytes. (A) Representative images showing mCherry-Parkin accumulation and LC3-GFP-mediated autophagosome formation in aged myocytes treated with 1 μM of either TAT-MP1^Gly (left) or TAT-MP2^Gly (right) peptides. (B) Representative images showing mCherry-Parkin accumulation and LC3-GFP-mediated autophagosome formation in HF myocytes treated with 1 μM of either TAT-MP1^Gly (left) or TAT-MP2^Gly (right) peptides. (C) Cell treatment TAT-MP1^Gly (MP1) increased Parkin puncta accumulation in aged myocytes and HF cardiomyocytes, while 1 μM TAT-MP2^Gly (MP2) decreased mCherry-Parkin puncta accumulation in both aged and HF myocytes. β-OHB did not increase the percentage of cells with Parkin-rich areas in cells treated with MP1. (D) Cell treatment TAT-MP1^Gly (MP1) did not affect LC3-mediated autophagosome formation in aged myocytes but increased it HF cardiomyocytes. Cell treatment with 1 μM TAT-MP2^Gly (MP2) decreased mCherry-Parkin puncta accumulation in both aged and HF myocytes. Data expressed as mean ± SEM. n = number of cells from three different animals per each group. *p < 0.05 reflects a significance in HF rabbits vs. aged rabbits; #p < 0.05 reflects a significance in peptide-treated cells vs. corresponding untreated groups.