Regression of postprandial cardiac hypertrophy in burmese pythons is mediated by FoxO1

Abstract

As ambush-hunting predators that consume large prey after long intervals of fasting, Burmese pythons evolved with unique adaptations for modulating organ structure and function. Among these is cardiac hypertrophy that develops within three days following a meal (Andersen et al., 2005, Secor, 2008), which we previously showed was initiated by circulating growth factors (Riquelme et al., 2011). Postprandial cardiac hypertrophy in pythons also rapidly regresses with subsequent fasting (Secor, 2008); however, the molecular mechanisms that regulate the dynamic cardiac remodeling in pythons during digestion are largely unknown. In this study, we employed a multiomics approach coupled with targeted molecular analyses to examine remodeling of the python ventricular transcriptome and proteome throughout digestion. We found that forkhead box protein O1 (FoxO1) signaling was suppressed prior to hypertrophy development and then activated during regression, which coincided with decreased and then increased expression, respectively, of FoxO1 transcriptional targets involved in proteolysis. To define the molecular mechanistic role of FoxO1 in hypertrophy regression, we used cultured mammalian cardiomyocytes treated with postfed python plasma. Hypertrophy regression both in pythons and in vitro coincided with activation of FoxO1-dependent autophagy; however, the introduction of a FoxO1-specific inhibitor prevented both regression of cell size and autophagy activation. Finally, to determine whether FoxO1 activation could induce regression, we generated an adenovirus expressing a constitutively active FoxO1. FoxO1 activation was sufficient to prevent and reverse postfed plasma-induced hypertrophy, which was partially prevented by autophagy inhibition. Our results indicate that modulation of FoxO1 activity contributes to the dynamic ventricular remodeling in postprandial Burmese pythons.

Keywords: Burmese python; FoxO1; autophagy; cardiac hypertrophy; hypertrophy regression.

Fig. 1.

Ventricular remodeling in Burmese pythons is associated with dynamic modulation of FoxO1 gene targets. (A) Heat map of differentially expressed python genes during digestion identified by bulk mRNA sequencing and partitioned into 3 clusters by K-means clustering. (B) Volcano plot for 1DPF vs. Fasted gene expression. (C) Volcano plot for 6DPF vs. 1DPF gene expression. (D and E) Reactome pathway (D) and ChEA Transcription Factor (E) enrichment for the differentially expressed genes in cluster 3. (F) Volcano plot displaying TMT-quantitative proteomics data from 6DPF vs. Fasted python ventricular tissue. (G) Gene Ontology Biological Process enrichment of the upregulated proteins at 6DPF vs. Fasted. (H) P62 (SQSTM1) protein abundance identified by quantitative proteomics; n = 4/group, one-way ANOVA. (I) SQSMT1 gene expression across the feeding groups identified by RNA-seq; n = 2/group, one-way ANOVA with Tukey’s post hoc test. FDR = false discovery rate adjusted P-value. Data in (H) and (I) are presented as the mean ± SEM.

Fig. 2.

FoxO1 activity and autophagy are suppressed during cardiac hypertrophy development in Burmese pythons and reactivated with regression. (A) Python heart weight normalized to prefed body weight throughout the feeding paradigm; F = Fasted. (B) Western blots for phosphorylated mTOR (S2448) and phosphorylated Akt (S473). (C) Normalized expression of p-mTOR and p-Akt represented as fold-change relative to fasted controls. (D) Western blots for phosphorylated FoxO1 (T24) and LC3B. (E and F) p-FoxO1 (E) and LC3-II (F) expression normalized to total protein. (G–K) qPCR analysis of the autophagy genes ATG10 (G), BNIP3 (H), CTSL (I), MAP1LC3B (J), and ULK1 (K) normalized to HPRT. For all, n = 4 pythons/group and data were analyzed by one-way ANOVA with Tukey’s post hoc test. Data are presented as the mean ± SEM.

Fig. 3.

Mammalian cardiomyocytes treated with postfed python plasma recapitulate molecular features of python cardiac remodeling. (A) Experimental paradigm. PE = phenylephrine, Fast = fasted python plasma, FPP = fed python plasma. (B) Percent change in cell volume compared with control for NRVMs treated with PE, 3% Fast, or 3% FPP during the Treatment and Chase. n = 10,000 to 30,000 cells/replicate, 8 biological replicates/group. (C) Representative immunofluorescence microscopy images of NRVMs stained for puromycin (red) and DAPI (blue); 100X magnification (Scale bars = 15 µm.) (D) Western blots for puromycin, representing protein synthesis rates. (E–G) Representative western blots (E) and normalized expression of p-mTOR (F) and p-Akt (G); n = 4/group. (H) Representative immunoblot for LC3B in NRVMs during Treatment and Chase with 50 nM BafA1 or equal volume DMSO control. (I and J). LC3-II expression normalized to GAPDH during the Treatment (I) and Chase (J) periods. n = 4/group. (K) qPCR analysis for Bnip3, Map1lc3b, and Ulk1 expression normalized to 18S during the Chase. n = 5/group. For all data, statistical analyses were performed by two-way ANOVA with Tukey’s post hoc test. Con = control. Data are presented as the mean ± SEM.

Fig. 4.

FoxO1 inhibition prevents regression of postfed plasma-induced hypertrophy and suppresses autophagy. (A) Representative western blots for T24 phosphorylated FoxO1 and total FoxO1 during Treatment (24 h) and Chase (6 h after agonist withdrawal). (B) p-FoxO1 (T24) normalized to GAPDH; n = 4/group; C = control, Tx = treatment. (C) Representative immunofluorescence microscopy images for NRVMs transduced with Ad-GFP-FOXO1_WT treated with FPP (Treatment) or 12 h after withdrawal of FPP (Chase); 100X magnification (Scale bar = 15 µm.) (D) Percent change in cell volume compared with control for NRVMs treated with FPP and 24 h after FPP removal with addition of either DMSO vehicle or 2 µM AS1842856 (AS, FoxO1-specific inhibitor); n = 10,000 to 30,000 cells/replicate, 5 biological replicates/group. (E–H) qPCR analysis of the autophagy genes Bnip3 (E), Gabarapl1 (F), Map1lc3b (G), and Ulk1 (H) normalized to Gapdh; n = 6/group. (I and J) Representative western blot (I) and normalized expression of (J) LC3B 24 h after FPP withdrawal and addition of AS1842856; BafA1 was added into the media 6 h before protein collection. (K and L) Representative immunofluorescence microscopy images (K) and quantification (L) of LC3B-positive area (Green) in NRVMs transduced with Ad-GFP-LC3B after FPP withdrawal with or without AS1842856 and ± BafA1; 100X magnification (Scale bar = 15 µm); n = 42 FPP → C/DMSO, 52 FPP → C/BafA1, 48 FPP → AS/DMSO, 49 FPP→AS/BafA1. (M) Representative western blot for p-ULK1 (S555). (N) p-ULK1 expression normalized to GAPDH; n = 4/group. The data were analyzed by two-way ANOVA for (B, J, and L) and by one-way ANOVA for (D–H and N). Tukey’s post hoc test was used in all cases. Data are presented as the mean ± SEM.

Fig. 5.

FoxO1 activation stimulates regression of postfed python plasma-induced hypertrophy. (A) Representative immunofluorescence microscopy images for GFP-FoxO1_ΔDB and GFP-FoxO1_CA counterstained with α-actinin; 100X magnification (Scale bars = 15 µm.) (B) Experimental paradigm for FoxO1 adenovirus experiments in NRVMs. (C) Percent change in cell volume compared with control; n = 10,000 to 30,000 cells/replicate, 5 biological replicates/group. (D–G) qPCR analysis for the autophagy genes Bnip3 (D), Gabarapl1 (E), Map1lc3b (F), and Ulk1 (G) normalized to Gapdh; n = 6/group. (H and I) Representative immunoblot (H) and normalized expression of (I) LC3-II with and without BafA1; n = 6/group. (J and K) Representative immunoblot (J) and normalized expression of (K) phospho-mTOR in NRVMs treated with FPP and each of the adenovirus constructs; n = 6 biological replicates per group. (L) Representative immunoblot for LC3B and GAPDH in NRVMs treated with BafA1 ± the autophagosome biogenesis inhibitor 3-methyl adenine (3-MA, 10 mM). (M) LC3-II expression normalized to GAPDH. n = 3/group, two-way ANOVA. (N) Percent change in cell volume in NRVMs treated with FPP and the different adenovirus constructs ±3-MA; n = 10,000 to 30,000 cells/replicate, 6 biological replicates/group. (O) Representative immunoblot for pan-ubiquitin in NRVMs treated with the proteasome inhibitor bortezomib (BTZ, 20 nM). (P) Percent change in cell volume compared with control for NRVMs treated with FPP and the different adenovirus constructs ±BTZ; n = 3/group. For all except K, data were analyzed by one-way ANOVA. Data are presented as the mean ± SEM.