Integrated proteomics reveals alterations in sarcomere composition and developmental processes during postnatal swine heart development

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Abstract

The neonatal swine heart possesses an endogenous ability to regenerate injured myocardium through the proliferation of pre-existing cardiomyocyte (CM) populations. However, this regenerative capacity is lost shortly after birth. Normal postnatal developmental processes and the regenerative capacity of mammalian hearts are tightly linked, but not much is known about how the swine cardiac proteome changes throughout postnatal development. Herein, we integrated robust and quantitative targeted “top-down” and global “bottom-up” proteomic workflows to comprehensively define the dynamic landscape of the swine cardiac proteome throughout postnatal maturation. Using targeted top-down proteomics, we were able to identify significant alterations in sarcomere composition, providing new insight into the proteoform landscape of sarcomeres that can disassemble, a process necessary for productive CM proliferation. Furthermore, we quantified global changes in protein abundance using bottom-up proteomics, identified over 700 differentially expressed proteins throughout postnatal development, and mapped these proteins to changes in developmental and metabolic processes. We envision these results will help guide future investigations to comprehensively understand endogenous cardiac regeneration toward the development of novel therapeutic strategies for heart failure.

Introduction

Heart failure (HF) affects over 6 million people in the United States and is one of the leading causes of death globally [1], yet there are currently no curative therapies available for patients. The inability of the adult human heart to restore lost cardiomyocyte (CM) populations after ischemic injury can lead to systolic heart failure [2,3]. Previous studies have demonstrated that neonatal mice are able to robustly regenerate infarcted cardiac tissue through CM proliferation; however, this regenerative potential in neonatal mice rapidly diminishes after birth [4,5]. Recent studies have demonstrated that neonatal swine are also capable of completely regenerating damaged cardiac tissue after myocardial infarction (MI) [6,7]. Similar to regeneration in neonatal mice, this regenerative capacity stems from proliferation of pre-existing CM populations, as well as disassembly of the cardiac sarcomeres prior to successful mitosis and cytokinesis, but this regenerative potential is also lost shortly after birth [6,7]. Endogenous cardiac regeneration in swine has high translational potential, as the swine and human heart are anatomically and physiologically similar [8]. Understanding the molecular mechanisms involved with this regenerative process will require a comprehensive understanding of postnatal swine heart development, as lessons from cardiac development can inform studies directed at enhancing cardiac regeneration [3].

Previous studies have characterized the changes in swine hearts throughout postnatal development and regeneration using targeted western blots and immunohistochemical (IHC) analyses as well as global transcriptomics (specifically RNAseq) [[9], [10], [11]]. While these studies provide valuable information about postnatal development and regeneration, global transcriptomic analysis alone does not provide a complete understanding of biological function, nor does transcript level always reflect protein abundance [12]. Using high-resolution mass spectrometry (MS)-based proteomics, we can deeply and accurately probe the molecular alterations associated with postnatal swine heart maturation at the protein level. MS-based bottom-up proteomics, where proteins are digested into peptides prior to analysis, is a powerful tool for global analyses, providing deep proteome coverage for differential expression analysis. However, bottom-up proteomics is suboptimal to discern changes in isoform expression and post-translation modification (PTM) abundance. On the other hand, top-down proteomics, where proteins are analyzed in their intact state, is useful for the analysis of “proteoforms” [13]—all the protein products that arise from a single gene due to mutations, PTMs, and alternative splicing—but provides limited proteome coverage.

Herein, we integrated robust and quantitative top-down and bottom-up proteomics pipelines to comprehensively analyze the global cardiac proteome and the sarcomeric sub-proteome of swine hearts throughout their postnatal development. We applied top-down proteomics to quantitatively analyze the proteoform changes in the sarcomeric sub-proteome during postnatal swine heart development to decipher the composition of sarcomeres that can disassemble during CM proliferation. Furthermore, using bottom-up proteomics, we investigated the global dynamics of the cardiac proteomic landscape, quantified over 4000 protein groups, and identified over 700 differentially expressed proteins (DEPs), which defined biological processes that are altered throughout postnatal development. Overall, this study provides a comprehensive proteomic landscape of postnatal cardiac development in swine and can be used to inform future studies aimed at unraveling the complexities of cardiac regeneration.

Section snippets

Reagents and chemicals

All reagents were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA) unless otherwise noted. High performance LC-grade water, acetonitrile, and ethanol were purchased from Fischer Scientific (Fair Lawn, NJ, USA).

Sample collection

Prestage farm pigs (Prestage Farm Inc., West Point, MS, USA) were sacrificed at postnatal days 01, 07, 28 and 56 (n = 3 per group). Detailed information on sex and heart weight is included in Table S1. The hearts were excised from the organism, and the left-ventricle (LV) free-wall

Results and discussion

The neonatal swine heart can fully regenerate after cardiac injury, but this regenerative potential is lost by postnatal day (p) 3 [6,7]. Herein, we sought to determine how the cardiac proteome in swine is altered throughout postnatal development to elucidate potential molecular mechanisms that support endogenous cardiac regeneration in early postnatal swine. To do so, we sacrificed swine at p01, p07, p28, and p56 (n = 3 per group), isolated the left ventricle free-wall, and analyzed the tissue

Conclusions

In summary, we applied integrated top-down and bottom-up proteomic analyses to understand how the sarcomeric sub-proteome and global cardiac proteome change throughout postnatal swine heart development. We were able to temporally resolve how the sarcomere is reconfigured and identified key isoform switches and changes in PTMs, which provides a holistic understanding of the composition of sarcomeres that can disassemble to promote regenerative growth. We also defined how the global proteome

Author contributions

T.J.A., D.S.R., and E.F.B. performed the proteomic analyses. T.J.A., A.I.M. and Y.G. analyzed the data. W.Z. and G.W. collected the swine heart tissue. T.J.A., D.S.R., E.F.B, W.Z, G.W., A.I.M., J.Z., and Y.G. wrote the manuscript.

Disclosures

Y.G. is a co-inventor on a patent that covers the detergent Azo. Other authors have no conflict of interest to declare.

Acknowledgements

Financial support was provided by the National Institutes of Health (NIH) R01 HL096971 (to Y.G.), HL156855 (to W.Z. and Y.G.). Y.G. would also like to acknowledge R01 GM117058, GM125085, HL109810 and S10 OD018475. J.Z. would like to acknowledge R01 HL131017 and R01 HL149137. T.JA. would like to acknowledge support from the Training Program in Molecular and Cellular Pharmacology, T32 GM008688. D.S.R. acknowledges the support from the American Heart Association Predoctoral Fellowship Grant No.

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