Postprandial cardiac hypertrophy is sustained by mechanics, epigenetic, and metabolic reprogramming in pythons

Abstract

Constricting pythons, known for their ability to consume infrequent, massive meals, exhibit rapid and reversible cardiac hypertrophy following feeding. Our primary goal was to investigate how python hearts achieve this adaptive response after feeding. Isolated myofibrils increased force after feeding without changes in sarcomere ultrastructure and without increasing energy cost. Ca²⁺ transients were prolonged after feeding with no changes in myofibril Ca²⁺ sensitivity. Feeding reduced titin-based tension, resulting in decreased cardiac tissue stiffness. Feeding also reduced the activity of sirtuins, a metabolically linked class of histone deacetylases, and increased chromatin accessibility. Transcription factor enrichment analysis on transposase-accessible chromatin with sequencing revealed the prominent role of transcription factors Yin Yang1 and NRF1 in postfeeding cardiac adaptation. Gene expression also changed with the enrichment of translation and metabolism. Finally, metabolomics analysis and adenosine triphosphate production demonstrated that cardiac adaptation after feeding not only increased energy demand but also energy production. These findings have broad implications for our understanding of cardiac adaptation across species and hold promise for the development of innovative approaches to address cardiovascular diseases.

Keywords: cardiac mechanics; epigenetic; heart hypertrophy; metabolism; titin.

Fig. 1.

Sarcomere ultrastructure was unchanged after feeding but isolated myofibrils generated greater force. (A) Schematic of the experimental setup. Python snakes were fasted for 28 d and fed ~25% of their body weight 24 h prior experiments. (B) Heart mass normalized by brain mass. Data from 9 fasted and 9 24 h postfed pythons. Student t test applied. (C) Cross-section electron microscopy images of fasted (blue) and 24 h postfed (red) hearts. Fibers and myofibrils were traced manually to measure relative sizes. (D) Fiber area in μm². N = 3 hearts per group, n = 60 fasted, and n = 59 24 h postfed fibers. Each color represents a python. Student t test applied. (E) Myofibril fractional area normalized by fiber area. N = 3 hearts per group, n = 62 fasted and n = 56 24 h postfed myofibers. Each color represents a python. Student t test applied. (F) Electron microscopy images of transversal sections of fasted (blue) and 24 h postfed (red) hearts. Sarcomere length (SL) and A-band length were measured. (G) Sarcomere length in fasted (blue) and 24 h postfed (red) python hearts. N = 3. Student t test applied. (H) A-band length in fasted (blue) and 24 h postfed (red) python hearts. N = 3. Student t test applied. (I) Representative mechanics traces from fasted (blue) and 24 h postfed (red) myofibrils. On the Right, magnification of the relaxation phase after switching from pCa 4.5 to pCa 9.0. (J) Maximum tension in mN/mm² measured in fasted (blue) and 24 h postfed (red) myofibrils. N = 3 hearts per group. n = 18 fasted and n = 19 24 h postfed myofibrils. Each color represents a python. Student t test applied. (K) Myofibril linear relaxation time in ms of fasted (blue) and 24 h postfed heart (red). N = 3 hearts per group, n = 20 fasted and n = 18 24 h postfed myofibrils. Each color represents a python. Student t test applied. (L) Resting tension as a function of sarcomere stretch. Nonlinear exponential growth fit applied. Global fits compared for kinetic parameter (k). (M) Bulk shear tension measured in fasted (blue) and 24 h postfed (red) hearts with a rheometer. Data reported as Young’s modulus in kPa. N = 3. Student t test applied.

Fig. 2.

Feeding-induced prolonged Ca²⁺ transients with no change in myofibril Ca²⁺ sensitivity. (A) Example of Ca²⁺ transients measured in fasted (blue) and 24 h postfed (red) python cardiomyocytes, stimulated at 0.2 Hz. (B) Averages of Ca²⁺ time-to-peak and Ca²⁺ decay at 50% (CaT₅₀). Average of 12 fasted cardiomyocytes and 20 24 h postfed cardiomyocytes. N = 3 hearts. (C) Ca²⁺ sensitivity curve of myofibrils from fasted (blue) and 24 h postfed (red) hearts. Average of 16 fasted myofibrils and 15 24 h postfed myofibrils. N = 3 hearts. (D) Electron microscopy images of longitudinal sections of fasted (blue) and 24 h postfed (red) hearts, showing peripheral couplings (white arrows). (E) Electron microscopy images of transversal sections of fasted (blue) and 24 h postfed (red) hearts. Cytoplasmic space quantified on the Right. N = 3 hearts per group, n = 62 fasted and n = 56.

Fig. 3.

Feeding reduced myofibril passive tension and tissue stiffness. (A) Resting tension as a function of sarcomere stretch. Nonlinear exponential growth fit applied. Global fits compared for kinetic parameter (k). (B) Titin gel of fasted (blue) and 24 h postfed (red) heart tissue. Mouse (M) and Human (H) heart tissues as reference. (C) Bulk shear tension measured in fasted (blue) and 24 h postfed (red) hearts with a rheometer. Data reported as Young’s modulus in kPa. N = 3. Student t test applied.

Fig. 4.

ATACseq and RNAseq revealed changes in chromatin accessibility and gene-expression promoted by shifted HDAC activity. (A) DAPI stained nuclei of fasted (blue) and 24 h postfed (red) cardiomyocytes. CCP of fasted and 24 h postfed nuclei. Data from 13 fasted and 14 24 h postfed nuclei. Student t test applied. (B) Western blot of histone three acetylation. N = 5 hearts per group. Student t test applied. (C) HDAC activity assay without and (D) with TSA. N = 4 to 5 per group. Student t test applied. (E) Average ATAC-seq peak profile (Top) and signal heatmap (Bottom) for YY1 and NRF1. N = 3 per group. (F) GOCC enrichment analysis performed on genes that were down-regulated (blue) or up-regulated (red) in 24 h postfed hearts.

Fig. 5.

Metabolic profiling reveals increased ATP production via oxidative phosphorylation and amino acid biosynthesis. (A) Partial least squares-discriminant analysis (PLS-DA) of fasted (blue) and 24 h postfed (red) metabolomics. (B) Enrichment analysis performed using compound concentrations. P-value < 0.05. (C) Heatmap of the expression of the core enrichment genes for the GOBP term “Protein folding.” (D) Western blot and quantification of eeF1a in fasted (blue) and 24 h postfed (red) hearts. N = 4 to 5 per group. Student t test applied. (E) Normalized concentrations of GMP, AMP, and phosphocreatine detected in fasted (blue) and 24 h postfed (red) hearts via metabolomics. N = 5 to 6 hearts per group. (F) Western blot and quantification of phospho-AMPK and phospho-PFK2 in fasted (blue) and 24 h postfed (red) hearts. N = 3 to 5 per group. Student t test applied. (G) ATP production rate and percentage of metabolic pathway in fasted (blue) and 24 h postfed (red) isolated live cardiomyocytes measured with Seahorse. Two-way ANOVA with Sidak's multiple comparisons test between fasted and 24 h postfed and glycolytic ATP (GlycoATP) and mitochondria ATP (MitoATP). n = 8 wells and N = 3 animals per group.