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. 2019 Jun 26:10:758.
doi: 10.3389/fphys.2019.00758. eCollection 2019.

Highly Dynamic Changes in the Activity and Regulation of Macroautophagy in Hearts Subjected to Increased Proteotoxic Stress

Bo Pan  1 , Megan T Lewno  1 , Penglong Wu  1   2 , Xuejun Wang  1
Affiliations

Highly Dynamic Changes in the Activity and Regulation of Macroautophagy in Hearts Subjected to Increased Proteotoxic Stress

Bo Pan et al. Front Physiol. .

Abstract

Macroautophagy (referred to as autophagy hereafter) plays an important role in the quality control of cellular proteins and organelles. Transcription Factor EB (TFEB) globally activates the expression of genes in the autophagic-lysosomal pathway (ALP) to replenish lysosomes and ALP machineries. We previously reported that myocardial TFEB signaling was impaired in advanced cardiac proteinopathy; however, myocardial ALP status and TFEB activity at earlier stages of cardiac proteinopathy remain uncharacterized. Here a stable line of CryABR120G transgenic (R120G) and non-transgenic (NTG) littermate mice with cardiomyocyte-restricted overexpression of CryABR120G were used at 1, 3, and 6 months of age. At 1 month when no cardiac phenotypes other than aberrant protein aggregation are discernible, R120G mice displayed a 5-fold increase in myocardial LC3-II flux. Interestingly, the LC3-II flux increase co-existed with increases in mTOR complex 1 (mTORC1) activities as well as cytoplasmic, but not nuclear, TFEB proteins. This increase in cytoplasmic TFEB proteins occurred without any discernible alteration in TFEB activity as reflected by unchanged mRNA levels of representative TFEB target genes (Mcoln1, M6pr, Sqstm1, Vps18, and Uvrag). At 3 months of age when hypertrophy and diastolic malfunction start to develop, the LC3-II flux remained significantly increased but to a lesser degree (2-fold) than at 1 month. The LC3-II flux increase was associated with decreased mTORC1 activities and with increased nuclear TFEB proteins and TFEB activities. At 6 months of age when congestive heart failure is apparent in R120G mice, both LC3-II flux and TFEB activities were severely suppressed, while mTORC1 activity increased. We conclude that changes in both autophagy and TFEB signaling are highly dynamic during the progression of cardiac proteinopathy. Increases in autophagy occur before increases in TFEB activities but both increase in the compensatory stage of cardiac proteinopathy. Once congestive heart failure develops, both autophagy and TFEB signaling become impaired. Our results suggest that TFEB signaling is regulated by both mTORC1-dependent and -independent mechanisms in hearts subjected to increased proteotoxic stress. For therapeutic exploration, it will be important to test the effect of TFEB stimulation at the early, intermediate, and late stages of cardiac proteinopathy.

Keywords: TFEB; mTOR; macroautophagy; mice; proteinopathy; proteotoxicity.

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Figures

Figure 1
Figure 1
Change in myocardial LC3-II flux in CryABR120G transgenic mice at 1, 3 and 6 months of age. CryABR120G transgenic (R120G) and non-transgenic (NTG) littermate mice at 1, 3 or 6 months of age were treated with bafilomycin A1 (BFA, 3 μmol/kg) or DMSO (vehicle control), and their ventricular myocardial samples were collected for extraction of total proteins. The extracted proteins were used for western blot analyses for LC3 and GAPDH, a loading control. Shown are the representative images (A,D,G), a summary of LC3-II densitometry data (B,E,H), and the LC3-II flux (C,F,I) derived from the data presented in (B), (E) and (H), respectively. *p < 0.05, **p < 0.01 vs. NTG + DMSO; n = 3 mice per group; two-way (B,E,H) or one-way (C,F,I) ANOVA followed by Tukey test for pairwise comparison.
Figure 2
Figure 2
Western blot analyses for the indicated proteins of the mTORC1 and TFEB signaling pathways in the ventricular myocardium from the R120G and NTG littermate mice at 1 month of age. Shown are representative images of western blot for total mTOR (mTOR) and Ser2481-phosphorylated mTOR (p-mTOR), total p70 S6 kinase (p70 S6K) and Thr389-phosphorylated p70 S6K (p-p70 S6K), total 4E-BP1 and Thr37/46-phosphorylated 4E-BP1 (p-4E-BP1), TFEB as well as GAPDH (a loading control) (A), a representative stain-free total protein image on a PVDF membrane that was used for immunoblotting (B), and the pooled densitometry data (C,D). **p < 0.01 vs. NTG; n = 4 mice per group; two-tailed unpaired t-test.
Figure 3
Figure 3
Western blot analyses for subcellular distribution of myocardial TFEB proteins and RT-PCR analyses of representative target genes of TFEB in the R120G and NTG littermate mice at 1 month of age. (A–D) Cytoplasmic and nuclear protein extracts were subjected to western blot analyses for the indicated proteins. GAPDH and Histone H3 were probed as a cytoplasmic and nuclear loading control, respectively. Shown are the representative images (A), a representative stain-free total protein image of a PVDF membrane used for protein normalization (B), pooled densitometry data of subcellular distribution of TFEB proteins (C,D). (E) and (F), representative PCR images of mRNA levels of TFEB and the indicated representative target genes of TFEB (E) and the pooled densitometry data (F). **p < 0.01 vs. NTG; n = 3 mice per group; two-tailed unpaired t-test.
Figure 4
Figure 4
Western blot analyses for the indicated proteins of the mTORC1 and TFEB signaling pathways in the ventricular myocardium from the R120G and NTG littermate mice at 3 months of age. Shown are representative images of western blot for total mTOR (mTOR) and Ser2481-phosphorylated mTOR (p-mTOR), total p70 S6 kinase (p70 S6K) and Thr389-phosphorylated p70 S6K (p-p70 S6K), total 4E-BP1 and Thr37/46-phosphorylated 4E-BP1 (p-4E-BP1), TFEB as well as GAPDH (a loading control) (A), a representative stain-free total protein image on a PVDF membrane that was used for immunoblotting (B), and the pooled densitometry data (C–E). *p < 0.05, **p < 0.01 vs. NTG; n = 4 mice per group; two-tailed unpaired t-test.
Figure 5
Figure 5
Western blot analyses for subcellular distribution of myocardial TFEB proteins and RT-PCR analyses of representative target genes of TFEB in the R120G and NTG littermate mice at 3 months of age. (A–D) Cytoplasmic and nuclear protein extracts were subjected to western blot analyses for the indicated proteins. GAPDH and Histone H3 were probed as a cytoplasmic and nuclear loading control, respectively. Shown are the representative images (A), a representative stain-free total protein image of a PVDF membrane used for protein normalization (B), pooled densitometry data of subcellular distribution of TFEB proteins (C,D). (E) and (F), representative PCR images of mRNA levels of TFEB and the indicated representative target genes of TFEB (E) and the pooled densitometry data (F). **p < 0.01 vs. NTG; n = 3 mice per group; two-tailed unpaired t-test.
Figure 6
Figure 6
Western blot analyses for the indicated proteins of the mTORC1 and TFEB signaling pathways in the ventricular myocardium from the R120G and NTG littermate mice at 6 months of age. Shown are representative images of western blot for total mTOR (mTOR) and Ser2481-phosphorylated mTOR (p-mTOR), total p70 S6 kinase (p70 S6K) and Thr389-phosphorylated p70 S6K (p-p70 S6K), total 4E-BP1 and Thr37/46-phosphorylated 4E-BP1 (p-4E-BP1), TFEB as well as GAPDH (a loading control) (A), a representative stain-free total protein image on a PVDF membrane that was used for immunoblotting (B), and the pooled densitometry data (C,D). **p < 0.01 vs. NTG; n = 4 mice per group; two-tailed unpaired t-test.
Figure 7
Figure 7
Western blot analyses for subcellular distribution of myocardial TFEB proteins and RT-PCR analyses of representative target genes of TFEB in the R120G and NTG littermate mice at 6 months of age. (A–D) Cytoplasmic and nuclear protein extracts were subjected to western blot analyses for the indicated proteins. GAPDH and Histone H3 were probed as a cytoplasmic and nuclear loading control, respectively. Shown are the representative images (A), a representative stain-free total protein image of a PVDF membrane used for protein normalization (B), pooled densitometry data of subcellular distribution of TFEB proteins (C,D). (E) and (F), representative PCR images of mRNA levels of TFEB and the indicated representative target genes of TFEB (E) and the pooled densitometry data (F). **p < 0.01 vs. NTG; n = 3 mice per group; two-tailed unpaired t-test.
Figure 8
Figure 8
A schematic summery of the dynamic changes in myocardial autophagic flux as well as in mTORC1 and TFEB signaling during DRC progression in the CryABR120G transgenic mice (R120G) compared with the NTG littermates. The numerical value of mTORC1 activity is the average fold changes of p-mTOR, p-p70S16K, and p-4E-BP1. The numerical value of TFEB targets is the average fold changes of all representative target genes examined. HFpEF, heart failure with preserved ejection fraction; HFrEF, heart failure with reduced ejection fraction.
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