Aging is associated with a decline in Atg9b-mediated autophagosome formation and appearance of enlarged mitochondria in the heart

Abstract

Advancing age is a major risk factor for developing heart disease, and the biological processes contributing to aging are currently under intense investigation. Autophagy is an important cellular quality control mechanism that is reduced in tissues with age but the molecular mechanisms underlying the age-associated defects in autophagy remain poorly characterized. Here, we have investigated how the autophagic process is altered in aged mouse hearts. We report that autophagic activity is reduced in aged hearts due to a reduction in autophagosome formation. Gene expression profile analysis to evaluate changes in autophagy regulators uncovered a reduction in Atg9b transcript and protein levels. Atg9 proteins are critical in delivering membrane to the growing autophagosome, and siRNA knockdown of Atg9b in cells confirmed a reduction in autophagosome formation. Autophagy is also the main pathway involved in eliminating dysfunctional mitochondria via a process known as mitophagy. The E3 ubiquitin ligase Parkin plays a key role in labeling mitochondria for mitophagy. We also found increased levels of Parkin-positive mitochondria in the aged hearts, an indication that they have been labeled for mitophagy. In contrast, Nrf1, a major transcriptional regulator of mitochondrial biogenesis, was significantly reduced in aged hearts. Additionally, our data showed reduced Drp1-mediated mitochondrial fission and formation of enlarged mitochondria in the aged heart. Overall, our findings suggest that cardiac aging is associated with reduced autophagosome number, decreased mitochondrial turnover, and formation of megamitochondria.

Keywords: Atg9; Parkin; aging; autophagy; heart; mitochondria; mitophagy.

FIGURE 1

Assessment of general autophagy in hearts from young and aged mice. (a) Representative Western blot and quantitation of LC3 levels from heart lysates of young and aged mice (n = 9–12). (b) Representative Western blot and quantitation of p62 levels in young and aged hearts (n = 11–12). (c) Representative images for LC3 immunostaining in heart sections (n = 3 for each). (d) Representative Western blot and quantitation of protein ubiquitination in young and aged hearts using a polyclonal ubiquitin antibody (n = 10). (e) Representative Western blot and quantitation of protein ubiquitination in young and aged hearts using a monoclonal ubiquitin antibody (n = 10–12). (f) Representative Western blot and quantitation of Parkin protein and transcript levels in young and aged hearts (n = 10–12). Scale bars are 20 μm. Data represent the mean ± SEM (*p < 0.05, **p < 0.01, ****p < 0.0001, ns = not significant)

FIGURE 2

Reduced formation of autophagosomes in aged mice. (a) Representative Western blot and quantitation of LC3II levels in young and aged hearts following administration of vehicle or rapamycin (n = 5–6). (b) Assessment of mTOR activity in young and aged hearts. Representative Western blots of p‐mTOR(Ser2448), mTOR, p‐pS6K(Ser371 and Thr389), p70S6K, p‐Ulk1(Ser757), and Ulk1. (c) Quantitation of protein levels (n = 10). Data are mean ± SEM (*p < 0.05, ns = not significant)

FIGURE 3

Atg9b transcript and protein levels are reduced in aged hearts. (a) Analysis of autophagy gene expression in young and aged hearts using the RT² Profiler PCR autophagy gene array (n = 5). (b) Scatter plot for RT² array analysis of genes with differential expression in aged compared to young mice. (c) Analysis of Atg9b, Atg10, and Atg12 mRNA levels by qPCR in young and aged hearts (n = 10). (d) Representative Western blot and quantitation of Atg9b protein levels in young and aged hearts (n = 10). (e) Representative Western blot and quantitation of ATG9B and LC3 levels following knockdown of ATG9B using siRNA in HeLa cells (n = 8). Data are shown as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant)

FIGURE 4

Reduced mitochondrial turnover in aged hearts. Representative Western blots and quantitation of (a) Parkin, LC3, p62, and (b) ubiquitin levels in the mitochondrial fraction from young and aged hearts (n = 9). Ubiquitin was detected using a polyclonal antibody. (c) Mitochondrial DNA (mtDNA) content in young and aged hearts (n = 11). (d) Representative Western blots and quantitation of proteins involved in mitochondrial oxidative phosphorylation: COX I = complex I subunit NDUFB8; COX II = complex II subunit 30 kDa; COX III = complex III subunit core 2; COX IV = complex IV subunit II; ATP synthase = ATP synthase subunit α (n = 9–10). (e) Representative Western blots and quantitation of mitochondrial proteins Tim23 and Tom20 (n = 10). (f) Analysis of PGC‐1α, Tfam, and Nrf1 mRNA levels by qPCR in young and aged hearts (n = 8–10). Data are shown as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 ns = not significant)

FIGURE 5

Megamitochondria are present in aged hearts. (a) Representative transmission electron micrographs of young and aged heart tissues reveal the presence of enlarged mitochondria in the hearts of aged mice. Scale bars are 500 nm. (b) Analysis of mean mitochondrial area (μm²) in young and aged myocytes. One hundred mitochondria per heart were scored (n = 3). Representative Western blots and quantitation of (c) Parkin, LC3II, and (d) ubiquitin levels in large and small mitochondria from aged hearts separated by differential centrifugation (n = 5). A monoclonal antibody was used to detect ubiquitin levels. (*p < 0.05, **p < 0.01, ****p < 0.0001).

FIGURE 6

Mitochondrial fission is reduced in aged hearts. (a) Representative Western blots and quantitation of mitochondrial fusion proteins Mfn1, Mfn2, and Opa1 in whole heart lysates from young and aged mice (n = 5–10). (b) Representative Western blots and quantitation of mitochondrial fission proteins Drp1 and Fis1 in whole heart lysates from young and aged mice (n = 5–10). (c) Representative Western blots and quantitation of the phosphorylation status of Drp1 at Ser637 and Ser616 in young and aged hearts (n = 9–10). Data are mean ± SEM (*p < 0.05, ns = not significant)