Elevated propionate signalling drives Pde9a overexpression and contractile dysfunction through increased histone acetylation and propionylation
Authors: Park KC , Crump NT , Hulikova A , Ford KL , Louwman N , Carnicer R , Hauton D , Koschinski A , Mccullagh J , Zaccolo M , Krywawych S , Milne TA , Swietach P 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): British Heart Foundation and Propionic Acidemia Foundation

Background
In the heart, various metabolic pathways produce the three-carbon intermediate, propionate. This metabolite has been postulated to increase histone propionylation and acetylation (via deacetylase inhibition), and therefore affect transcription. Normally, propionate levels are kept low by propionyl-CoA carboxylase (PCC), but build-up has been reported in cardiometabolic diseases. The highest levels are attained in propionic acidaemia (PA; mutations in PCC), which also serves as a model for studying propionate biology [1].

Purpose
To establish the effect of propionate on cardiac gene expression and physiology using a mouse model of elevated propionate/propionyl-CoA signalling.

Methods
Experiments were performed using either wild-type (WT) neonatal ventricular myocytes (NRVMs) treated with propionate in vitro, or the hypomorphic mouse model of PA (Pcca-/- A138T) [2]. IC-MS metabolomics was performed on methanol-extracted metabolites. RNA-sequencing was carried out on an Illumina HiSeq 4000. For chromatin immunoprecipitation (ChIP), chromatin was isolated from PFA-fixed ventricular tissue. cGMP levels were measured by the FRET-based sensor, cGi500. Ca2+ transients were imaged in isolated myocytes using FuraRed. Cine-MRI was performed in a 7 tesla MR scanner.

Results
PA mice had the metabolic signature of propionate accumulation in plasma and cardiac lysates (metabolomics). RNA-seq of ventricular lysates identified differentially expressed genes (DEGs), but the effect was more pronounced in females. Thus, subsequent experiments were performed in females. To determine which DEGs are likely a direct response to propionate, RNA-seq was performed on propionate-treated NRVMs. The most significant DEGs common to both datasets were upregulated Pde9a (cGMP-selective phosphodiesterase) and Mme (degrades natriuretic peptides). ChIP-qPCR for histone acylation in PA and WT hearts demonstrated increases in H3K27ac at Pde9a, and strikingly, increases in propionylation at Pde9a and Mme, indicating a mechanism for this transcriptional induction. Propionate-treated NRVMs show greater sensitivity of cGMP to pharmacological inhibition of PDE9A (measured by FRET), consistent with Pde9a induction. Such changes are expected to result in diastolic dysfunction [3]. Indeed, ventricular myocytes from PA mice had higher diastolic Ca2+. Cine-MRI confirmed contractile dysfunction in vivo, with PA mice manifesting increased end-systolic and end-diastolic volumes.

Conclusions
We demonstrate that cardiac elevations of the metabolic intermediate, propionate, increases histone modifications that drive transcriptional changes in the heart, including those involved in cyclic nucleotide signalling. We also present evidence for histone propionylation, which has not been described previously in the heart. Thus, using a mouse model of a rare metabolic disease, we show how propionate/propionyl-CoA signalling affects cardiac function through epigenetic changes.