Dynamic cardiac hyperplasia and hypertrophy in Burmese Pythons

Abstract

Cardiomyocyte hyperplasia is the primary form of fetal heart growth, whereas this proliferative capacity is largely lost in adults across most mammalian species. The limited ability of adult cardiomyocytes to re-enter the cell cycle is a major cause of cardiac injury-induced morbidity and mortality. Here, we report that post-prandial Burmese python cardiomyocytes activate cell cycle re-entry to promote persistent cardiac growth. Burmese pythons normally eat large meals infrequently, resulting in reversible cardiomyocyte hypertrophy. We found that frequent feeding of large meals amplifies the modest post-prandial cardiac proliferation identified in an infrequent feeding interval. By activating E2F and Forkhead Box M1 (FoxM1) pro-proliferation transcriptional networks, frequently fed Burmese pythons potentiate cardiomyocyte proliferation in addition to transient cardiac myocyte hypertrophy. These findings identify hyperplasia as a natural means of sustained cardiac growth in Burmese pythons and demonstrate the use of pythons as a model for investigating non-injury induced proliferative cardiac remodeling.

NEW & NOTEWORTHY

This study shows that adult Burmese pythons can not only undergo reversible cardiomyocyte hypertrophy after feeding but can also activate cardiomyocyte cell-cycle re-entry and achieve hyperplasia, especially with frequent large meal consumption. Frequent feeding amplifies proliferation by engaging E2F and FoxM1 pro-proliferative signaling, resulting in sustained heart growth through hyperplasia in adult python hearts. These findings establish Burmese pythons as a new model for proliferative cardiac remodeling with potential insights into adult heart regenerative biology.

Graphical Abstract

INTRODUCTION

Adult mammalian cardiomyocytes achieve cardiac growth via hypertrophy. This cellular growth augments cardiac function and can be enhanced by exercise and other stimuli through activation of physiological growth signaling pathways in cardiomyocytes¹. Meanwhile, pathological insults such as myocardial infarction (MI) cause irreversible cardiac tissue damage, maladaptive cardiac remodeling, and dysfunction¹. After birth, most mammalian cardiomyocytes quickly mature, become terminally differentiated and lose the proliferative capacity to regenerate^{2, 3}. This cardiac maturation process involves multinucleation, polyploidization, a metabolic switch from glucose to fatty acids and hypertrophic growth³. These changes eliminate the capacity of adult mammalian cardiomyocytes to re-enter cell cycle and achieve mitotic renewal. The low levels of cardiomyocyte turnover (~1% annually) in young adult humans further declines with age and becomes too limited to repair injury compared to the early neonatal stage^{4, 5}.

The post-prandial Burmese python heart represents a rapid and robust model of physiological remodeling. Burmese pythons evolved as ambush-hunting predators and can both consume meals greater than their body mass and survive through months-long fasts during food scarcity. The python heart rapidly increases in mass by up to 40% within three days following consumption of large prey, a magnitude and rate of hypertrophy development that are unparalleled in other species^{6, 7}. This rare example of heart plasticity and rapid reversible growth offers an evolutionarily distinct model system for identifying molecular mechanisms that regulate physiological cardiac remodeling. Our earlier studies demonstrated that post-prandial cardiomyocyte hypertrophy in Burmese pythons is driven by activation of mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K)-AKT signaling⁷, while activation of Forkhead Box O1 (FoxO1) regulates the subsequent regression of hypertrophy⁸. We concluded that the cardiac growth in post-prandial Burmese pythons was achieved through cellular hypertrophy instead of hyperplasia since we did not observe a proliferative signal (BrdU) at 1-day post-feeding (DPF) whereas we observed proliferation in the intestine⁷. This is consistent with the observation in mammals that adult cardiomyocytes achieve growth by hypertrophy. Although most of the post-prandial cardiac growth in Burmese pythons is reversible, heart weight does not fully regress back to its pre-feeding size, which suggests the basal cardiac mass grows incrementally with each meal^{7, 9}. Only a few vertebrate species, including newts, zebrafish, and the recently identified spiny mouse, are known to reactivate cardiac mitotic signaling and cardiomyocyte proliferation as adults in response to cardiac injuries^10–12. A recent study demonstrated non-mammalian vertebrates such as zebrafish and newts reserve heart regenerative ability with the loss of endothermy as a metabolic trade-off¹³. This suggests there are likely large number of ectotherm species that evolved to share regeneration-competent cellular and molecular features for achieving myocardial proliferation during adulthood, a phenotype that has been observed in the leopard geico (Eublepharis macularius)^14,15.

In the current study, we measured and estimated that the majority (86.8%) of adult python cardiomyocytes are diploid, a cellular feature of proliferative cardiomyocytes. We detected modest cardiomyocyte proliferation in post-prandial Burmese python hearts during late digestion, a response that is potentiated by frequent feeding and contributes to long-term cardiac growth. We also demonstrated the proliferative effect could be translated to mammalian cardiomyocytes via circulating factor(s) in post-prandial plasma. These findings demonstrate that Burmese python cardiomyocytes engage its plasticity in both hypertrophic (early digestion) and hyperplastic (late digestion) signaling post-prandially.

MATERIALS AND METHODS

Animals and tissue collection

Burmese pythons (n=24) were obtained as hatchlings from Bob Clark Reptiles (Oklahoma City, OK) and housed individually at ~28°C on a 12/12-hour light/dark cycle. Pythons were fed a meal equivalent to 25% of their body mass on a biweekly basis until they reached 1300–1500g. Four months prior to the beginning of the study, each python was placed on a 4-week (28 days) feeding regime. Each meal was 25% of the python’s body mass. After four months, pythons were randomly assigned to either a ‘Fasted’ (2 females, 4 males), or ‘Normal Fed’ (1 females, 5 males), or ‘Frequent Fed’ (2 females, 4 males), or ‘Frequent Fed/Fasted’ (3 females, 3 males) group with n=6. After the four-month acclimation period, both groups of the Fasted and Normal Fed pythons were maintained on a 28-day feeding regime for the remainder of the study (8 weeks). The Frequent Fed and Frequent Fed/Fasted groups were fed every 4 days over the same 8-week period. The Frequent Fed/Fasted group was fasted for another 4 weeks following 8-week frequent feeding. Pythons were euthanized at the end of each prandial state: Normal Fed and Frequent Fed, 3 days after their final meal; Fasted and Frequent Fed/Fasted, 28 days after their final meal. Tissues and plasma were collected, snap-frozen, and stored at −80°C. All procedures were conducted under the approval of the University of Colorado, Boulder IACUC.

Normalization of tissue weights

Organ weights were normalized to brain weight to account for overall body size. In frequently fed pythons, total body mass is highly dynamic due to adipose deposition and meal-associated mass changes including meal mass, gut contents and hydration, making it a variable denominator for assessing tissue-specific growth. Brain mass varies comparatively little over the experimental timeframe and is less influenced by feeding state; therefore, it was used as a conservative internal size reference. Non-normalized organ weights are presented in Table S3 and fig. S2.

Neonatal rat ventricular myocyte (NRVM) isolation and culture.

Cardiomyocytes were isolated from one-day-old Sprague Dawley rat pups as previously described⁷. Purified ventricular cardiomyocytes were plated at 250,000 cells per well on 12-well plates with glass coverslips. Approximately 24 hours after isolation, the wells were washed twice and then incubated in experimental media (MEM, 20 mM HEPES, 2 μg/mL vitamin B12) for 18 hours prior to the start of the experiment. NRVMs were treated with serum free experimental media, 10% Fasted python plasma, 10% Normal Fed python plasma, 10% Frequent Fed python plasma, and 10% calf serum with experimental media respectively at 0 hour and 24 hours for a total of 72 hours treatment.

Immunohistochemistry

Python hearts were collected and embedded in O.C.T. Compound (Fisher Healthcare, 23730571), and cryosectioned at 10 μm intervals. For immunofluorescent staining, cryosections were air-dried for 15 min at room temperature and fixed with cold 4% PFA for 30 min. Section slides were stained with wheat germ agglutinin (WGA) 555 (5 ug/mL in 1X PBS) for 30 min at room temperature. Section slides were then washed twice with PBS and permeabilized with 0.5% Triton X-100 (Fisher Scientific, BP151–500) in PBS for 20 min. Sections were then blocked in 2% BSA/0.25% Triton X-100/PBS blocking solution for 1 h at room temperature and stained with the indicated primary antibodies prepared in 2% BSA/0.125% Triton X-100/PBS at 4 °C overnight using the following dilutions: sarcomeric alpha-actinin (Thermo Fisher Scientific, MA1–22863, 1:250), pHH3 (Cell Signaling Technology, 9701, 1:200). Sections were subsequently washed with PBS three times (5 min for each wash), and incubated with corresponding secondary antibodies conjugated to Alexa Fluor 488 or 555 (Cell Signaling Technology) prepared in 2% BSA/0.125% Triton X-100/PBS at room temperature for 1.5 h. After secondary antibody incubation, sections were washed with PBS three times (5 min for each wash) and mounted using VECTASHIELD^® PLUS Antifade Mounting Medium with DAPI (Vector Laboratory, H-2000).

Immunocytochemistry

Wells with NRVMs were washed twice with PBS, and fixed with ice cold methanol for 1 min and then 4% PFA for 10 min. Fixed cells were washed twice with PBS and permeabilized with 0.5% Triton X-100 (Fisher Scientific, BP151–500) in PBS for 20 min followed by 0.1% Triton X-100 in PBS for another 30min. Permeabilized cells were then blocked in 0.5% BSA/0.1% Triton X-100/PBS blocking solution for 1 h at room temperature and stained with the indicated primary antibodies prepared in 0.5% BSA/0.1% Triton X-100/PBS at 4 °C overnight using the following dilutions: sarcomeric alpha-actinin (Thermo Fisher Scientific, MA1–22863, 1:250), pHH3 (Cell Signaling Technology, 9701, 1:200), and AURKB (Thermo Fisher Scientific, MA5–15321, 1:250). Cells were subsequently washed with PBS three times, and incubated with corresponding secondary antibodies conjugated to Alexa Fluor 488 or 555 (Cell Signaling Technology) prepared in 0.5% BSA/0.1% Triton X-100/PBS at room temperature for 1.5 h. After secondary antibody incubation, sections were washed with PBS three times and mounted using VECTASHIELD^® PLUS Antifade Mounting Medium with DAPI (Vector Laboratory, H-2000).

Microscopy and imaging analysis of cell proliferation

Burmese python ventricles section: pHH3 stain, three field of views were randomly selected from each stained tissue slide. Images (10X 3×3) used for quantifications and representative images (10X 1×1) were obtained using a Nikon Spinning Disk confocal microscope. 3D images (100X 1×1) were obtained using a Nikon A1R Laser Scanning confocal microscope. Neonatal rat ventricular myocytes: pHH3 and AURKB stain, images (10X 3×3) used for quantifications, representative images (20X 1×1, 40X 1×1), and 3D images (100X 1×1) were obtained using a Nikon Spinning Disk confocal microscope. The data analysis and visualization work were performed at the BioFrontiers Institute’s Advanced Light Microscopy Core using the software pack Imaris (Oxford Instrument).

Cardiomyocyte ploidy analysis and quantification

Stained tissue sections (ACTN2 488, WGA 555, DAPI) were imaged using Nikon Spinning Disk confocal miscroscope at a 40X objective. Images were taken from multiple fields of view that captures both non-cardiomyocytes and cardiomyocytes. A total of 842 cardiomyocytes from two male normal fed and two male frequent fed Burmese python ventricles were analyzed. Ploidy levels were quantified as the sum of DAPI fluorescence pixels per cell nucleus measured as in integrated density (ID). In each field of the view, non-cardiomyocyte nuclei were assumed to be diploid and used as the reference cell for cardiomyocyte nuclear ploidy measurement. Each scored nucleus required to longitudinally lay flat and non-overlapping. Cardiomyocyte nuclei were distinguished from non-cardiomyocyte nuclei by their localization in the ACTN2 (+) sarcomere regions. Software pack Imaris (Oxford Instrument) was used for quantifying ploidy. Background subtraction was first applied to the nuclei (DAPI) channel. Due to the disorganization of python cardiac muscle fibers, nuclei meet the standards mentioned above were manually selected from each field of the view. The final ID ratio was determined by diving cardiomyocyte nuclei ID over the average non-cardiomyocyte nuclei ID using the measurement from each field of the view. In this study, we used the classification system developed by Hirose et al¹³. Diploid (2N) cardiomyocyte nuclei were categorized for ID ratio between 0.5 to 1.5.

RNA-sequencing and analysis

Total RNA was isolated from two male python ventricles from each feeding group with Trizol reagent. The concentration of RNA was determined using Qubit RNA BR Assay Kit (Invitrogen Q10211) and Qubit Flex Fluorometer. Frozen RNA samples were sent to Novogene for sequencing and analyzed based on Burmese python (Python molurus bivittatus, Taxonomy ID: 176946) genome. Deferential gene expression analysis was performed using R package DESeq2³⁹. The gene set was then pre-ranked for Gene Ontology Biological Pathway analysis (c5.go.bp.v2023.1.Hs.symbols.gmt) using Gene Set Enrichment Analysis software with the Hallmark Pathway analysis (Molecular Signature Database, MSigDB)^{40, 41}. For those enriched pathway with 0 FDR-qval, 0 was assigned as 1.0*e-9 for graphing. RNA-seq analysis graphing profiles were generated using RStudio (v2023.12.1.402) and GraphPad Prism 10 (GraphPad Software Inc).

Proteomics and analysis

The protocol for tandem-mass-tag (TMT) quantitative proteomics of python ventricular samples was previously described⁸. In brief, python ventricular tissue (n = 4 per group from Fasted, Normal Fed, Frequent Fed, and Frequent Fed/Fasted) was pulverized while frozen and then added to a 5% SDS/0.75% sodium deoxycholate 50 mM Tris (pH 8.5) buffer and immediately boiled for 10 minutes. The samples were then centrifuged at 12,000 x g for 10 minutes and the supernatant collected. Protein concentration was determined by BCA assay. The protein samples were reduced, alkylated, and then digested using the SP3 method. Tryptic peptides were then labeled with TMT-Pro 16-plex (Thermo Scientific) according to the manufacturer’s protocol. The peptides were then fraction with high pH reversed-phase C18 UPLC using a custom-packed UChrom column (1.8 μm 120Å). 12 fractions were eluted at 20 μL/minute using 2–50% acetonitrile using an M-class UPLC (Waters). Peptides were then lyophilized by vacuum centrifugation, resuspended in 3% (vol/vol) ACN, 0.1% (vol/vol) trifluoroacetic acid (TFA). LC-MS/MS analysis was performed using an Ultimate 3000 nanoUPLC (Thermo Scientific) and Q-Exactive HF-X mass spectrometer (Thermo Scientific) as previously described⁷. The raw proteomics data files were searched against the Python bivittatus proteome database using MaxQuant (v.2.0.3.0) with cysteine carbamidomethylation as a fixed modification and methionine oxidation and N-terminal acetylation as variable modifications. Data were thresholded at 1% false discovery rate (FDR) and fold changes and p-values were calculated with limma using an R-script.

Quantitative real time PCR (RT-qPCR) analysis

Total RNA was extracted from python cardiac muscle tissue with Trizol and reversed transcribed using Super Scrip III (Invitrogen, 12574018) with random primers. mRNA levels were quantified with SYBR Green (Invitrogen, 4312704). Due the dramatic changes in gene expression profile in post-prandial Burmese python hearts, all conventional normalizers such as 18s were changing. Therefore, we determined concentration of RNA using more precise chemical reaction-based method, Qubit RNA BR Assay Kit (Invitrogen, Q10211) and Qubit Flex Fluorometer rather than wavelength-based Nanodrop. Based on the precisely measured concentration of RNA, the same amount of cDNA (5 ng) was loaded for each RT-qPCR reaction, and these experiments were performed in triplicate. RT-qPCR primers were designed using whole genomic and transcriptomic Burmese python sequence on NCBI (Taxonomy ID: 176946). RT-qPCR primers are available in Table S3.

Western blot

Burmese python ventricle was pulverized in liquid nitrogen cooled pulverizer, lysed overnight in RIPA buffer (Cell Signaling Technology, 9806S), supplemented with protease and phosphatase inhibitors cocktail (Thermo Scientific Technology, 8442), centrifuged at 15,000 g for 15 minutes at 4°C, and the supernatant was collected from each sample and stored at −80°C. The concentration of protein lysates was quantified using DC protein assay kit (Bio-Rad, 5000112). 25 μg of protein from the lysate was resolved by NuPAGE^™ 4–12%, Bis-Tris gel (Invitrogen, WG1403A), transferred on to nitrocellulose membrane (Bio-Rad, 1620115), blocked in 5% fat-free milk/0.2% TWEEN-20/TBS at room temperature for 1h and stained with the indicated primary antibodies prepared in 5% BSA/0.2% TWEEN-20/TBS at 4°C overnight using the following dilutions: LC3B (Cell Signaling Technology, 2775), 1:1000. Membranes were subsequently washed with TBS three times (5 min for each wash), and incubated with corresponding HRP-linked secondary antibodies (Anti-rabbit IgG, Cell Signaling Technology, 7074) prepared in 5% BSA/0.2% TWEEN-20/TBS at room temperature for 1.5 h. After secondary antibody incubation, membranes were washed with TBS three times (5 min for each wash). Membranes were imaged after 1-minute incubation of Western Lightning Plus-ECL (PerkinElmer, NEL104001EA). Images were then analyzed using ImageQuant.

Annual cell-cycle re-entry rate calculation

Cell cycle re-entry rate at the time of tissue collection was calculated by pHH3(3) nuclei per total nuclei. The annual rate is estimated by the following equation: annual rate = (1 + fix rate)¹².

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software Inc), with p-value < 0.05 considered significant unless otherwise indicated. All data were analyzed for outlier before running statistical tests. Detailed information on the statistical analysis is supplied in the figure legends. All data are displayed as means ± SDs; each data point indicates a biological replicate, unless otherwise indicated.

RESULTS

Hyperplastic growth in Burmese python hearts

Our recent study demonstrated increased cell cycle gene enrichment in 4DPF Burmese python hearts⁴, which implies the proliferative potential of some cardiac cell type. To verify whether mitosis was activated in cardiomyocytes, we co-immunostained python ventricular tissue sections with an antibody against phospho-histone H3 (pHH3), a late-stage cell cycle marker that is activated in mitosis, and an antibody against α-actinin (ACTN2), a sarcomere protein specifically expressed in cardiomyocytes. We found that the enhanced expression of cell cycle factors previously observed at 4DPF preceded an over 10-fold increase in proliferative activity in 6DPF python hearts (Fig. 1A, 1B). In postnatal mammalian hearts, cell cycle re-entry does not always correspond to cell division, as multinucleation or endoreduplication are common occurrences and can lead to polyploid cardiomyocytes¹⁶. Polyploidization promotes maturity of post-natal cardiomyocytes, but it also limits the potential of cardiomyocyte to proliferate. Similar to other non-mammalian vertebrates, we observed mononucleated python cardiomyocytes and estimated over 85% to be diploid, a pre-requisite feature for cardiomyocyte cell cycle re-entry and tissue repair (fig. S1A, S1B)^13,17,18.

Fig. 1. Frequent fed Burmese pythons experience sustained cardiac growth.

(A) Immunohistochemistry staining of phospho-histone H3 (pHH3, green) and alpha actinin 2 (ACTN2, magenta) in python ventricle sections. Scale bar, 100 μm. The lower panel is the corresponding gray scale image for the pHH3 channel, day post-fed (DPF). (B) Quantification of pHH3(+) nuclei normalized to tissue area and plotted as a fold-change (FC). Data are represented mean ± SD, n=2. (C) Schematic showing feeding regimen for Burmese pythons: Fasted, Normal Fed, Frequent Fed, and Frequent Fed/Fasted. Normal Fed and Frequent Fed endpoint: 3 days after their final meal; Fasted and Frequent Fed/Fasted endpoint: 28 days after their final meal. (D) Total body weight (Wt.) in the four groups over the course of the experiment. (E) Representative images of pythons in each group at the end of the study; scale bar, 10 cm. (F-G) Liver weight (D) and heart weight (E) normalized to brain weight (Supplemental Fig. 1). Data are represented mean ± SD, n=6 animals per condition. Ordinary one-way ANOVA with Tukey’s post-hoc test for multiple independent pairwise comparisons to the F group if it is not indicated; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

With increased meal frequency, we hypothesized that Burmese pythons would experience a sustained cardiac growth stimulus and an amplified proliferative response. Therefore, we designed a frequent feeding regimen. This approach highlights both a greater feeding frequency and a greater amount of food consumption. Burmese pythons were randomly assigned to either a ‘Fasted (four-week fast)’, or ‘Normal Fed (one meal after a four-week fast)’, presented in the upper panel (Fig. 1C), ‘Frequent Fed (fed every four days for eight weeks)’ or ‘Frequent Fed/Fasted (fed every four days for eight weeks and then fasted for four weeks)’, presented in the lower panel (Fig. 1C), with all meal sizes equivalent to 25% of python body mass (Fig. 1C). Both Normal Fed and Frequent Fed were euthanized and dissected 3 days after their final meal, while the Fasted and Frequent Fed/Fasted were euthanized and dissected 28 days following their final meal. Throughout the feeding regimen, all groups showed steady growth after each meal. Due to total body mass in this frequent feeding regimen reflects substantial adipose and meal-associated mass accumulation, we normalized organ mass to brain weight to provide an estimate of tissue remodeling independent of transient body mass increase. We observed moderate (~22%) heart growth in the Normal Fed (3-day post-feeding) pythons. Meanwhile, frequent Fed pythons nearly tripled their body weight and cardiac mass compared to the Normal Fed pythons after 16 consecutive feedings spanning two months (Fig. 1D, 1E, fig. S2, Table S3). While normalization to brain weight may not reflect isometric growth with the rest of the body, the non-normalized organ weights showed the same trend of significant organ growth (Table S3, fig S2). With subsequent fasting in Frequent Fed/Fasted pythons, body weight and major organ masses, including the liver and kidney, significantly regressed (Fig. 1D, 1E, fig. S2, Table S3). However, the cardiac ventricle and total heart weights remained elevated after fasting for one month (Fig. 1F, 1G, and fig. S3A, S3B). The fact that the heart did not regress after one month of fasting, as occurs with normal feeding frequency^{7, 9}, suggests that Frequent Fed python hearts activated differential cellular signaling and likely facilitated hyperplastic cardiac growth in addition to normal feeding-induced transient hypertrophy.

Frequent feeding amplifies cardiomyocyte cell cycle re-entry

To investigate how frequent feeding differentiated cardiac growth from normal feeding, we performed bulk RNA-sequencing (RNA-seq) on mRNA from python ventricular tissue. Principal-component analysis (PCA) indicated a sharp difference between the fed (Normal Fed & Frequent Fed) and the fasted (Fasted & Frequent Fed/Fasted) groups, while there was strong similarity between Normal Fed and Frequent Fed and between Fasted and Frequent Fed/Fasted (Fig. 2A). We first performed a combined analysis of the fed groups (Normal Fed & Frequent Fed) compared to the combined fasted groups (Fasted & Frequent Fed/Fasted) to determine the general feeding-specific transcriptional response. Differential gene expression analysis revealed 3019 upregulated genes and 2779 downregulated genes in response to feeding (adj p-value <0.05) (fig. S4A). Gene Ontology Biological Process (GO-BP) analysis revealed that upregulated pathways in the combined fed groups were enriched in metabolic processes, such as ATP synthesis and oxidative phosphorylation. The downregulated pathways were centered around cytoplasmic translation and ribosome biogenesis (fig. S4B). The RNA ribosomal regulation of transcriptional response was trending down in fed pythons, implying a turning point for transitioning digestion into the next stage after cardiac growth peaked at 72h post-feeding^{7, 8}.

Fig. 2. The frequent fed Burmese python cardiac transcriptome and proteome are enriched with cell cycle re-entry activity.

(A) Principal component analysis (PCA) of the RNA-seq data from each feeding condition; red arrows denote comparisons of key groups discussed. n=2 animals per condition. (B) Gene Ontology (GO) Biological Process analysis of the up-regulated (UP) and down-regulated (DOWN) genes in Frequent Fed versus Normal Fed pairwise transcriptome comparison. Normalized enrichment score (NES). (C) Top enriched and up-regulated Hallmark pathways in Frequent Fed versus Normal Fed pairwise transcriptome comparison. (D) Gene set enrichment plot of E2F targets differentially expressed between Frequent Fed and Normal Fed pythons. (E) Analysis of the cell cycle genes differentially expressed across all feeding conditions highlighting exclusive enrichment in Frequent Fed pythons. (F) Proteomic analysis of protein expression in Frequent Fed versus Normal Fed pairwise comparison. (G) Top enriched and up-regulated Hallmark pathways in Frequent Fed versus Normal Fed pairwise proteome comparison. (H) Proteomic analysis of cell cycle protein expressions.

Although the hearts from fed pythons shared transcriptional features of metabolic pathway regulation, there was also a pronounced difference between Frequent Fed and Normal Fed (Fig. 2A). Differential gene expression analysis revealed only ~50% of genes in Frequent Fed python hearts overlap with Normal Fed python hearts when individually compared to the Fasted pythons (fig. S4C). To determine how Frequent Fed python hearts are molecularly distinct from Normal Fed python hearts, we performed differential gene expression analysis in Frequent Fed versus Normal Fed ventricles followed by GO-BP analysis. Like the Fed vs Fasted comparison, Frequent Fed python hearts showed downregulation of cytoplasmic translation and ribosome assembly, demonstrating an exaggerated dampening of the translational response (Fig. 3B). Strikingly, the top upregulated pathways in the Frequent Fed python heart were associated with cell-cycle signaling, including chromosome separation and mitotic nuclear division. (Fig. 2B). Across all feeding conditions, the gene sets associated with mitotic pathways were exclusively enriched in the Frequent Fed python hearts (fig. S5A, S5B).

Fig. 3. Frequent feeding accelerates proliferative cardiac growth in Burmese python hearts.

(A) Immunohistochemistry staining of phospho-histone H3 (pHH3, green) and alpha actinin 2 (ACTN2, magenta) in python ventricle sections. Scale bar, 100 μm. The lower panel is the corresponding gray scale image for the pHH3 channel. (B) Quantification of pHH3(+) nuclei and total number of nuclei normalized to tissue area and plotted as a fold-change (FC). Data are represented mean ± SD, n=3 (Fasted, Normal Fed and Frequent Fed/Fasted), n=6 (Frequent Fed); ordinary one-way ANOVA test; *p ≤ 0.05; ***p ≤ 0.001. (C) Higher magnification image of a dividing cardiomyocyte identified in python ventricular tissue presented in 3D maximum intensity projection (MaxIP). Scale bar, 10 μm. (D) Model for post-prandial cardiac growth in Burmese python cardiomyocytes. Data are represented mean ± SD, n=6 animals per condition. Ordinary one-way ANOVA test with Tukey’s post-hoc test for multiple independent pairwise comparisons to the F group if it is not indicated; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

A recent study revealed that E2F transcriptional targets are distinctly enriched in embryonic mouse cardiomyocytes and mark their competency for entering the cell cycle¹⁸. Applying the same gene set enrichment Hallmark pathway analysis to our differentially expressed genes, E2F Targets emerged as the top enriched pathway in Frequent Fed python hearts compared the Normal Fed python hearts (Fig. 2C, 2D). In addition, we performed proteomics and identified the enrichment of mTOR Signaling and Glycolysis suggested Frequent Fed python hearts have more robust growth signaling and reprogramming to fetal-like metabolism (i.e., using glucose as a substrate) to support proliferative cardiac growth. Different from downregulation of translation and ribosome activity, this is similar to we observed in our other study, which suggests the disconnect and additional layers of molecular regulations between RNA and protein¹⁹. Notably, 48 of the top 50 differentially expressed genes in the E2F Targets pathway were exclusively enriched in the Frequent Fed python hearts (fig. S6). Similarly, cell cycle genes including E2F targets (CNA, CDT1, TOP2A, MCMs), cell cycle G2/M regulators (CDK1, CDK2, CCNA2, CCNB1/3, PLK1), and cytokinesis markers (PRC1, CIT, AURKA, ANLN, KIFs) were exclusively upregulated in the OF python hearts. We further validated E2F targets, cell cycle markers and cytokinesis gene expression changes using quantitative PCR (qPCR) (Fig. 2E and fig. S7). Additionally, expression of epithelial cell transforming 2 (ECT2), an essential gene for initiating cytokinesis¹⁷, was significantly increased in the Frequent Fed python hearts, and the GO-BP analysis of Frequent Fed versus Normal Fed exhibited significant enrichment in Mitotic Cytokinesis (fig. S8A, S8B). Accordingly, our transcriptomic analysis strongly supports that Burmese python cardiac cells enter mitosis, and the cell cycle re-entry signatures are amplified by frequent large meals.

To verify transcriptional changes in cell cycle re-entry at the protein level, we performed tandem-mass-tag (TMT) relative quantitative proteomics on Burmese python ventricular samples from each condition. We detected 322 significantly upregulated and 296 downregulated proteins in Frequent Fed versus Normal Fed pairwise comparison, including augmented expression of DNA replication markers (PCNA and PRIM1), cytokinetic kinesins (KIF1B and KIF5B), and decreased cell cycle inhibitor (CDKN2C) (Fig. 2F, 2H). Similar to the RNA-seq Hallmark pathway analysis in Frequent Fed versus Normal Fed, we identified increased expression of proteins involved in mTORC1 signaling, Mitotic spindle, and Myc target V1, indicating robust protein synthesis and cell cycle activity in the Frequent Fed python hearts (Fig. 2G). In contrast to downregulated cytoplasmic translation and RNA processing in transcriptomic analysis described above, our proteomics analysis revealed enhanced protein translation machinery in the Frequent Fed animals (fig. S9). The lag between transcription and translation suggests there are layers of molecular regulation in python hearts. Despite the lag between transcription and translation, these findings demonstrate aligned evidence at both transcriptomic and translational levels in supporting increased mitotic activity in Frequent Fed python cardiomyocytes.

Dynamic cardiomyocyte growth by hypertrophy and hyperplasia

The absence of regression in heart size in Frequent Fed/Fasted pythons suggests long-term cardiac muscle mass accumulation, which aligns with there being an increase in total cardiomyocyte number. The expression of pro-proliferation transcription factors including E2F, TOP2A, MCMs, FoxM1 and other S phase cell cycle markers, including PCNA and MKI-67, were upregulated in both Normal Fed and Frequent Fed python hearts (fig. S7A-S7E, S11). Transcriptomic clustering analysis in Normal Fed versus Fasted also revealed a cluster (Cluster 1) of upregulated genes highly enriched in S phase genes of DNA replication, and their expressions were strongly enhanced in the Frequent Fed group (fig. S10). Along with the mitotic and cytokinetic gene expression and protein enrichment exclusive to Frequent Fed python hearts, these data provide compelling evidence to support accelerated and amplified mitotic cardiac growth in Frequent Fed pythons. However, the bulk analyses of the transcriptome and proteome include multiple cell types, so we turned to high resolution microscopy experiments to specifically examine the proliferative potential of python cardiomyocytes.

Consistent with the RNA-seq mitotic pathway enrichment, the co-immunostaining of pHH3 and ACTN2 showed a ~40-fold increase in pHH3 signal in Frequent Fed python cardiomyocytes compared to the Fasted python (Fig. 3A, 3B). About 73% of pHH3 signals were scored from the ACTN2(+) sarcomere region, which indicates a myocardial proliferation rate of 0.22% at this fixed time point and 2.63% annually (Fig 2E Table S1). In addition to hallmarks of cardiomyocyte proliferation, the Frequent Fed group showed significant cardiomyocyte hypertrophy as indicated by the decreased number of nuclei per tissue area (Fig. 3B), while cell size returned to baseline in the Frequent Fed/Fasted group despite the sustained cardiac growth observed (Fig. 1E, 3B). Through 3D imaging, we captured numerous dividing Burmese python cardiomyocytes at high resolution. The representative image showed a pair of pHH3(+) nuclei embedded in the cardiac muscle tissue was dividing and separating apart from each other (Fig. 3C, 3D, Movie S1). Although the mitotic marker pHH3 has its drawbacks as it does not fully distinguish cytokinesis from karyokinesis, the finding that cardiac mass remained elevated in the Frequent Fed/Fasted group, while cardiomyocyte hypertrophy was restored to baseline (Fig. 3B), indicates that the sustained cardiac growth induced by frequent feeding must be a result of increased cell number. Taken together, these data indicate that frequent large meal consumption directed python cardiomyocytes into a dynamic new phase: they not only undergo hypertrophy, but also enhance the existing post-prandial proliferative response and achieve a higher degree of cardiac growth to meet the increasing metabolic demand of their rapidly growing bodies. This further verifies that normal feeding activates cardiomyocyte proliferation, and that hyperplasia is a natural means of cardiac growth in python hearts. Notably, this proliferation occurs during late stages of digestion (6DPF) after the peak of hypertrophy has begun to regress (Fig. 1A, 1B).

Given these findings, the current model of post-prandial hypertrophic growth of the Burmese python heart should be revised to a new dynamic model involving both cardiomyocyte hypertrophy and hyperplasia. First, the Burmese python heart exhibits rapid cardiac hypertrophy to accommodate the sudden rise in cardiac demand following a meal (1–3DPF), and this process is achieved via activation of mTOR and PI3K/AKT mediated growth⁷ and suppression of Atrogin-1/FBXO32 and LC-3B mediated protein catabolism (fig. S11, S18). While cardiac hypertrophy peaks at 3DPF in the Normal Fed paradigm, python cardiomyocytes initiate E2F and FoxM1 pro-proliferation transcriptional networks to promote DNA replication in later phases of digestion (4–6DPF). During the regression of post-prandial cardiac hypertrophy, a small population of cardiomyocytes enter M phase of cell cycle to proliferate (Fig. 1A, 1B, 3D). We expect this burst of proliferation following a meal contributes to lasting basal cardiac mass that steadily increases as the python grows. However, the length of this proliferative time window remains to be determined (indicated by dashed line section of the red curve in Fig. 3D).

Frequent fed python plasma promotes hypertrophy and cell-cycle re-entry in mammalian cardiomyocytes

Previously, we demonstrated post-prandial Burmese python plasma promoted physiological cardiac hypertrophy in mammalian cardiomyocytes⁷.To explore whether the cell cycle re-entry observed in the fed python heart was also caused by circulating factors, we took a similar approach and found both 10% normal fed and frequent fed post-prandial python plasma treatment not only significantly promoted cellular hypertrophy, indicated by decreased nuclei count per cell area, but also activated cell cycle re-entry at 72 hours in neonatal rat cardiomyocytes comparing to the 10% fasted plasma, 10% calf serum, and serum free conditions (Fig. 4A, 4B, 4C). Normal fed plasma induced a >8-fold increase in cell cycle re-entry, which was further enhanced (not significant) with frequent fed plasma (>12-fold increase) (Fig. 4B). We also captured numerous mammalian cardiomyocytes in different phases of mitosis indicated by the cytokinesis marker Aurora kinase B (AURKB) (Fig. 4D). More importantly, the robust cardiac cell cycle re-entry response is specific to post-prandial python plasma as fasted plasma and calf serum largely induced cellular hypertrophy only (Fig. 4B, 4C). Calf serum is commonly supplemented in the media to promote cell growth. Despite observing abundant pHH3(+) signals from the calf serum condition, most of these signals were found in non-cardiomyocytes. This demonstrates the cell cycle re-entry observed in the post-prandial python heart can be translated to mammalian cardiomyocytes, likely via cardiomyocyte-specific circulating factor(s) in the plasma.

Fig. 4. Post-prandial python plasma promotes hypertrophy and cell-cycle re-entry in mammalian cardiomyocyte.

(A) Immunofluorescent staining of phospho-histone H3 (pHH3, green) and alpha actinin 2 (ACTN2, red) in neonatal rat cardiomyocytes. (B-C) Quantification of pHH3(+) nuclei and total number of nuclei normalized to cell area and plotted as a fold-change (FC). (D) Immunofluorescent staining of aurora kinase B (AURKB, magenta) and alpha actinin 2 (ACTN2, green) captured neonatal rat cardiomyocytes in different mitotic phases after treated by frequent fed python plasma. Data are represented mean ± SD, n=3 per condition. Ordinary one-way ANOVA test with Tukey’s post-hoc test for multiple independent pairwise comparisons to the Serum Free group if it is not indicated; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

DISCUSSION

In this study, we introduced a novel frequent feeding paradigm in Burmese pythons to test the proliferative potential of their cardiomyocytes. In the Normal Fed python, we observed moderate ~22% cardiac growth, similar to the average 15% growth observed in the recent studies, whereas the 40% post-prandial growth at 3DPF from some previous studies remain exclusive^{20, 21, 22}. The large body mass loss observed in the Frequent Fed/Fasted group is primarily a result of digestion and clearance of undigested gut contents accumulated during the frequent feeding period, rather than true tissue catabolism. Knowing that almost all adult mammalian cardiomyocytes achieve postnatal cardiac growth via hypertrophy rather than hyperplasia, while ectotherm vertebrates are speculated to have higher capacity of proliferative cardiac growth¹³. Our results led to the discovery of myocardial proliferation in the Burmese python heart that activated during meal digestion, and the proliferative activity is significantly potentiated by increasing meal frequency. Our data, therefore, add Burmese pythons to the short but growing list of vertebrate species known to have cardiomyocytes with proliferative potential, which includes newts, zebrafish, leopard gecko and spiny mice^{10–12, 14}. While large number of python cardiomyocytes are visually mononucleated (Fig. S1A), we attempted to stain with label cell membrane with WGA (Fig. S1A) and laminin (not shown), we were not able quantify the percentage of mononucleated cardiomyocytes due to disorganization of muscle fibers in the python heart. However, like other myocardial regenerative-competent vertebrates (e.g. newts and zebrafish), Burmese pythons are also ectotherms, exhibit low baseline metabolic rates and with over 85% of cardiomyocytes being diploid^{13, 14}. However, unlike these previously studied species, Burmese python hearts exhibit a distinct feeding induced myocardial proliferation as a natural means of cardiac growth rather than injury-induced cardiac regeneration and repair. Compared to the cardiac dedifferentiation process during post-injury cardiac regeneration in zebrafish^{23, 24}, we observed similar sarcomere disassembly (fig. S12), upregulation of fetal genes (ACTA1, TNNT1) and downregulation of genes involved in sarcomere maintenance (TCAP). This indicates the feeding induced proliferation are resulting from existing cardiomyocytes. Different from cardiac apex resection models, there is a lack of injury-induced signaling enrichment such inflammation and macrophage infiltration from the RNA-seq in the Burmese python heart^25–28. Compared to another reptile species, the leopard gecko, which also activate cardiomyocyte proliferation in the absence of injury, post-prandial python showed similar percentage of diploid cardiomyocytes and proliferation rate^14,15. However, python cardiomyocyte requires feeding as the stimulus to achieve similar rate of myocardial renewal compared to the constitutive cycling in the gecko heart, suggesting Burmese python cardiomyocytes evolved to utilize a different type or additional layer of molecular regulation to govern postnatal heart growth following each meal.

Moreover, we found post-prandial cardiac proliferation in pythons is marked by increased FoxM1 expression (fig. S11), which is akin to post-injury zebrafish cardiomyocytes and proliferative neonatal rat cardiomyocytes²³. FoxM1, a proliferation-associated transcription factor, is essential for zebrafish cardiomyocytes to complete regeneration after injury²⁸. Additionally, cardiomyocyte-specific loss of FoxM1 leads to cell-cycle withdrawal and decreased neonatal rat cardiomyocyte proliferation³⁰. During this process, FoxM1 has been shown to play an antagonistic regulatory role with AKT/FoxO1 signaling during cardiac growth³⁰. Similar to mammalian cardiomyocytes, we recently reported that the activity of FoxO1 plays a central regulatory role in controlling post-prandial cardiac hypertrophy via Atrogin-1/FBXO32 and autophagy-mediated proteolysis in Burmese python hearts⁸. Here, we propose a similar molecular regulation: immediately after meal ingestion, python cardiomyocytes activate PI3K/AKT signaling, which deactivates FoxO1, downstream Atrogin-1 and LC3B mediated autophagy to promote robust cardiac hypertrophy. When cardiac demand starts to decrease, FoxO1 becomes re-activated and drives cardiac regression⁸. Meanwhile, increased E2F and FoxM1 target gene expression promotes cell cycle progression and proliferation, whereas the upstream proliferative growth signaling remains to be identified (Fig. 3D). On the other hand, cellular proliferation by dedifferentiating existing cardiomyocytes leads to sarcomere disassembly and therefore reduced contractile function. Notably, overexpression of cytokinesis regulators (PLK1 and ECT2) in adult mammalian hearts resulted in widespread postnatal cardiomyocytes mitosis, but also contractile dysfunction³¹. Frequent feeding significantly induced PLK1 and ECT2 expression in python hearts and resulted in 0.22% cell cycle re-entry rate at this fixed time, which translates to a projected rate of 2.63% per year (Fig. 2E, Table S1). In a normal feeding cycle, cell cycle progression initiates when cardiac demand decreases (6DPF). This separates the need for basal cardiac growth (hyperplasia at 6DPF) at the expense of reduced contractile function from the high cardiac demand during early digestion (hypertrophy only at 1–3DPF). During frequent feeding, we expect the sustained, robust cardiac hypertrophy can functionally compensate for the amplified cell cycle re-entry rate. Other cell types may also contribute to the growing population of cardiomyocytes, as such has been observed during cardiac regeneration in the aquatic salamander heart, epicardium-derived cells are recruited to the injured site and differentiate themselves to de novo cardiomyocytes³². Additionally, whether there is a unique population of cardiomyocytes that get activated to proliferate and become quiescent during the fasting period remained to be determined. Our current bulk RNA-seq data lacks resolution to distinguish transcriptional signals within cardiomyocytes and from individual cell type. Future work of single cell sequencing will be required to verify the specificity of transcriptional regulation and define the signaling network during this feeding induced proliferative remodeling in the python heart.

Burmese pythons (~2000g) used in this study were larger compared to previous and recent studies, where python weights were under 1000 g^7,9. Lately, the invasive species control program in the Greater Everglade Ecosystem, Florida USA characterized newly categorized size-stage classes based on data from more than 4000 wild Burmese pythons. By fitting sexual maturity with the snout-vent length (SVL, tip of snout to vent, cm), the size-stage classes are defined as 1) Juvenile: 39–150.9cm for female; 42–124.9cm for male; 2) Sub-adult: 151–205.9cm for female, 125–181.9cm for male; 3) Adult: 206cm+ for female; 182cm+ for male³³. Based on these standards, all Burmese pythons used in this study fall into the category of sub-adults (Table S2). By the end of frequent feeding, the 4 males in the frequent fed group grew rapidly and reached adult size with average SVL of 185.3cm, while the 2 females averaged 192.4cm, ~14cm shorter of reaching adult size. The proliferation rate of human cardiomyocyte declines dramatically with age⁴. Similarly, cardiomyocyte proliferation rates decline to low levels in the adult zebrafish heart, suggesting a switch to homeostatic maintenance and not growth^{34, 35}. Meanwhile, a similar frequent feeding study from Last et al. divided one standard meal (20% body mass) into 5 small meals (4% body mass) for frequent feeding. They found the intestinal grow rate remains the same regardless of the feeding frequency³⁶. Based on these findings and our data, the frequent feeding likely stimulated long lasting effect on growth rate at its peak including heart and other organs, whereas the growth rate only peak for a brief period from a single large caloric equivalent meal. We expect this feeding-induced body growth and transient peak rate of post-prandial cardiac proliferation to continue during adulthood and to steadily increase basal cardiac mass as the python grows from centimeters in length as a juvenile to upwards of 6 meters in adulthood.

Lastly, we found there are trophic factors circulating in the post-prandial python plasma that can potentiate mammalian cardiomyocytes growth both in size and number. Our previous study identified pro-hypertrophic factors in the normal fed python plasma⁷. The potentiated growth response in the Frequent Fed group reflects sustained transcriptional programming driven by repeated feeding episodes. We speculate pro-proliferative factor(s) are likely to be highly enriched in the frequent fed python plasma --- including growth-promoting hormones and nutrient-sensing signals --- collectively amplifies a higher cell-cycle re-entry rate in the mammalian cardiomyocytes in addition to hypertrophy. The identity of these trophic factors and underlying signaling remain to be identified in future studies.

Collectively, this study extends our molecular understanding of the dynamic post-prandial cardiac remodeling in Burmese pythons. Our data support that hyperplastic growth during late stages of digestion contributes to long-term increases in cardiac mass. Compared to existing models of cardiac regeneration following injury (e.g., zebrafish), the post-prandial Burmese python hearts represent a different model of natural proliferative cardiac remodeling. They are also ~100 million years evolutionarily closer to mammals than fish, which evolved more robust cardiac function capable of maintaining low pulmonary blood pressure while generating high systemic blood pressure, similar to mammals^{37, 38}. They have two atria and a partially septated ventricle, giving them a more structurally similar cardiac anatomy to mammals^{37, 38}. Lastly, we demonstrated translational potential of circulating factor(s) in the post-prandial python plasma to promote mammalian cardiomyocyte proliferation. Frequent fed python plasma potently induced cell cycle re-entry in neonatal rat cardiomyocytes; however, the specific circulating factor(s) and its origin remain to be determined. Taken together, our findings elevate the Burmese python as a platform to investigate non-injury and feeding induced molecular mechanisms that regulate cardiomyocyte cell cycle re-entry.

Supplementary Material

Supplemental Figs. S1-S18: DOI.10.6084/m9.figshare.32664465

Supplemental Tables S1-S4: DOI.10.6084/m9.figshare.32664465

Supplemental Movie S1: DOI.10.6084/m9.figshare.32664471

APPENDIX

N/A.

DATA AVAILABILITY

The raw RNA sequencing data will be deposited in the NCBI Gene Expression Omnibus repository. The raw proteomics data will be deposited in the Proteome Xchange Consortium via the PRIDE partner repository.