An mRNA assay system demonstrates proteasomal-specific degradation contributes to cardiomyopathic phospholamban null mutation

Eduarde Rohner,William E Louch,Jesper Sohlmer,Makoto Sahara,Kenneth R Chien,Erwin De Genst,Nevin Witman

doi:10.1186/s10020-021-00362-8

Abstract

BackgroundThe human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics.MethodsA panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing.ResultsOur modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes.ConclusionOur results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies.

Highlights

The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified
We hypothesized that three possible mechanisms could result in a lack of detectable L39X variant protein: (1) The mutation could induce an actively degraded or unstable mRNA; (2) The mutation could affect the translational outcome of the mRNA or (3) The mutation could be responsible for driving an actively degraded and/or highly unstable protein
L39X mutation does not critically affect mRNA stability Three different Chemically modified mRNA (modRNA) molecules were generated for both WT PLN and the mutant L39X sequences

Summary

Introduction

The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics. Given SERCA’s central role in inducing cardiac relaxation in a diastole phase by recycling calcium into the sarcoplasmic reticulum, PLN has proven to be an important piece of the puzzle to elucidate mechanisms of human calcium regulation (Marks 2013; Frank and Kranias 2000; Kranias and Hajjar 2017). Despite varying sizes and geographical distribution of these mutant PLN patient populations, there is a similar strong association between PLN mutations and cardiomyopathic phenotypes led by chronic calcium misregulation (Kranias and Hajjar 2017, 2012; Opbergen et al 2017)

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