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. 2022 Oct 19;7(12):1214-1228.
doi: 10.1016/j.jacbts.2022.06.003. eCollection 2022 Dec.

Targeting the Autophagy-Lysosome Pathway in a Pathophysiologically Relevant Murine Model of Reversible Heart Failure

Sarah Evans  1 , Xiucui Ma  1 , Xiqiang Wang  1 , Yana Chen  2 , Chen Zhao  1 , Carla J Weinheimer  1 , Attila Kovacs  1 , Brian Finck  1   2 , Abhinav Diwan  1 , Douglas L Mann  1
Affiliations

Targeting the Autophagy-Lysosome Pathway in a Pathophysiologically Relevant Murine Model of Reversible Heart Failure

Sarah Evans et al. JACC Basic Transl Sci. .

Abstract

The key biological "drivers" that are responsible for reverse left ventricle (LV) remodeling are not well understood. To gain an understanding of the role of the autophagy-lysosome pathway in reverse LV remodeling, we used a pathophysiologically relevant murine model of reversible heart failure, wherein pressure overload by transaortic constriction superimposed on acute coronary artery (myocardial infarction) ligation leads to a heart failure phenotype that is reversible by hemodynamic unloading. Here we show transaortic constriction + myocardial infarction leads to decreased flux through the autophagy-lysosome pathway with the accumulation of damaged proteins and organelles in cardiac myocytes, whereas hemodynamic unloading is associated with restoration of autophagic flux to normal levels with incomplete removal of damaged proteins and organelles in myocytes and reverse LV remodeling, suggesting that restoration of flux is insufficient to completely restore myocardial proteostasis. Enhancing autophagic flux with adeno-associated virus 9-transcription factor EB resulted in more favorable reverse LV remodeling in mice that had undergone hemodynamic unloading, whereas overexpressing transcription factor EB in mice that have not undergone hemodynamic unloading leads to increased mortality, suggesting that the therapeutic outcomes of enhancing autophagic flux will depend on the conditions in which flux is being studied.

Keywords: AAV9, adeno-associated virus 9; CMV, cytomegalovirus; CQ, chloroquine; GFP, green red fluorescent protein; HF, heart failure; HF-DB, TAC + MI mice that have undergone debanding; LFEF, left ventricular ejection fraction; LV, left ventricle; MI, myocardial infarction; RFP, red fluorescent protein; TAC, transaortic constriction; TEM, transmission electron microscopic; TFEB, transcription factor EB; autophagy; dsDNA, double stranded DNA; eGFP, enhanced green fluorescent protein; mTOR, mammalian target of rapamycin; reverse left ventricle remodeling.

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Conflict of interest statement

This study was supported by research funds from the National Institutes of Health (R01HL147968, R01 HL155344), the Veterans Administration (AN # 4345132), and the Wilkinson Foundation to Dr Mann. Dr Diwan was supported by grants from the National Institutes of Health (HL107594, HL143431, and NS094692) and the Department of Veterans Affairs (I01BX004235). Dr Finck was supported by grants from the National Institutes of Health (R01 HL119225, P30 DK05634). Dr Kovacs was supported by grants from the National Institutes of Health (S10 OD028597). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Figures

None
Graphical abstract
Figure 1
Figure 1
Autophagic Flux in a Model of Reversible HF Autophagic flux was determined in sham, heart failure (HF), and heart failure-deband (HF-DB) mice at 6 weeks by measuring the accumulation of LC3-II and p62 proteins in cardiac extracts (see Methods and Supplemental Figure 1A). (A) Representative immunoblots of LC3-II and p62 levels with (+) and without (-) chloroquine pretreatment. Each pair of + and – chloroquine samples was run on adjacent lanes on the same gel and blotted for LC3-II, p62, and actin. Sham, HF, and HF-DB pairs were cut From separate blots and combined for the representative images. (B) Group data for LC3-II levels relative to actin loading controls (n = 4-6 mice/group) (t test) and (C) group data for p62 levels relative to actin loading controls (n = 4-6 mice/group) (t test for sham and HF) (Mann-Whitney test for HF-DB). Autophagosome and autolysosome abundance was determined using immunofluorescence microscopy of myocardial sections from sham, HF, and HF-DB CAG-RFP-EGFP-LC3 reporter mice at 6 weeks. (D) Representative fluorescent micrographs (scale bar = 50 µm). (E) Group data for abundance of autolysosomes (red fluorescent puncta) and autophagosomes (yellow fluorescent puncta) per 40X field (n = 3-6 mice/group) (Tukey test). ∗P < 0.05, ∗∗P < 0.01. n.s. = nonsignificant.
Figure 2
Figure 2
Accumulation of Polyubiquitinated Proteins and Damaged Organelles in a Model of Reversible HF (A) Representative immunoblots depicting levels of Lys63 polyubiquitinated proteins in sham, HF, or HF-DB mice at 6 weeks. (B) Group data for Lys63 polyubiquitinated proteins relative to actin loading control in sham, HF, or HF-DB mice (n = 4-6 mice/group) (Tukey test). (C) Representative transmission electron microscopy (TEM) images of cardiac tissue from sham, HF, or HF-DB mice at 6 weeks. Scale bar = 50 nm. Large white arrows depict double membrane-bound autophagosomes, white arrowheads denote endoplasmic reticulum, black arrows indicate mitochondria. (D) Group data for the mitochondrial aspect ratio (ratio of length/width) of sham, HF and HF-DB mice determined from TEM images (n = 9-12 images from 3-4 hearts/group) (Tukey test). ∗P < 0.05, ∗∗P < 0.01. Abbreviations as in Figure 1.
Figure 3
Figure 3
Characterization of Autophagy-Related Proteins (A) Representative immunoblots of LAMP1, LAMP2, cathepsin D, and actin loading controls at 4 weeks. Samples derived from same experiment and blots processed in parallel. (B) Representative immunoblots of phospho-mTOR, total mTOR, phospho-p70s6K, total p70s6K, phospho-4EBP1, and total 4EBP1. Samples derived from same experiment and blots processed in parallel. Group data (n = 6 mice/group) for levels of (C) LAMP1 (Tukey test), (D) LAMP2 (Tukey test), (E) Cathepsin D (pro) (Tukey test) relative to actin loading controls. Group data (n = 6 mice/group) for the ratio of (F) phospho-mTOR to total mTOR (Tukey test); (G) phospho-p70s6K to total p70s6K (Dunn test), (H) phospho-4EBP1 to total 4EBP1 (Dunn test). ∗P < 0.05, ∗∗P < 0.01. mTOR = mammalian target of rapamycin; other abbreviations as in Figure 1.
Figure 4
Figure 4
Mitochondrial Respiration and Mitophagy High-resolution respirometry was performed to measure oxygen consumption (JO2) in sham, HF, and HF-DB mice hearts (4-9 hearts/group) in the presence of (A) ADP (complex I) (Tukey test), (B) succinate (complex I + II) (Tukey test), and (C) rotenone (complex II) (Tukey test). (D) Representative fluorescent micrographs images of myocardial sections from sham, HF, and HF-DB mice depicting colocalization of PicoGreen (green) with an anti-LC3 antibody (red). White arrowheads denote colocalization of PicoGreen and LC3 (yellow fluorescence). Scale bar = 20 μm. (E) Group data for the number of yellow puncta reflecting the colocalization of PicoGreen and LC3 double-positive deposits (n = 9-12 fields from 3-4 mice /group) (Tukey test). ∗P < 0.05, ∗∗∗P < 0.001. ADP = adenosine diphosphate; other abbreviations as in Figure 1.
Figure 5
Figure 5
Effect of TFEB-Mediated Enhancement of Autophagy in a Mouse Model of Reversible HF (A) Kaplan-Meier analysis of HF and HF-DB mice injected with adeno-associated virus 9 (AAV9)-cytomegalovirus (CMV)- transcription factor EB (TFEB) or AAV9-CMV-green fluorescent protein (GFP) (control). HF mice were injected 1 day after sham debanding surgery 2 weeks after transaortic constriction (TAC) + myocardial infarction (MI). HF-DB mice were injected 1 day after hemodynamic unloading (day 0) 2 weeks after TAC + MI (n = 5 mice/group/intervention) (log-rank). (B) Representative TEM images of myocardial sections of HF-DB mice injected with AAV9-CMV-TFEB or AAV9-CMV-GFP (6 weeks). Black arrows indicate mitochondria. Scale bars, 2 μmol/L (upper) and 500 nm (lower). (C) Group data for mitochondrial aspect ratio (ratio length/width) in HF-DB mice treated with AAV9-CMV-TFEB or AAV9-CMV-GFP for 4 weeks (n = 9-12 images from 3-4 mice/group) (t test). Two-dimensional echocardiograms were performed 4 weeks after debanding in the AAV9 injected HF-DB mice and compared with the respective values at the time of debanding, before the injection of AAV9 (Supplemental Figure 1B). (D) Percent change in left ventricle (LV) end-diastolic volume in HF-DB mice treated with AAV9-CMV-TFEB or AAV9-CMV-GFP for 4 weeks (Mann-Whitney test). (E) Percent change in LV mass in HF-DB mice treated for 4 weeks with AAV9-CMV-TFEB or AAV9-CMV-GFP (n = 4-5 mice/group) (t test). ∗P < 0.05, ∗∗P < 0.01, ††P < 0.01 by log-rank. Abbreviations as in Figures 1 and 2.
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