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

BACKGROUND: 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. 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. METHODS: A 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. RESULTS: Our 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. CONCLUSION: Our 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.

STRA6 is essential for induction of vascular smooth muscle lineages in human embryonic cardiac outflow tract development.

AIMS: Retinoic acid (RA) signalling is essential for heart development, and dysregulation of the RA signalling can cause several types of cardiac outflow tract (OFT) defects, the most frequent congenital heart disease (CHD) in humans. Matthew-Wood syndrome is caused by inactivating mutations of a transmembrane protein gene STRA6 that transports vitamin A (retinol) from extracellular into intracellular spaces. This syndrome shows a broad spectrum of malformations including CHD, although murine Stra6-null neonates did not exhibit overt heart defects. Thus, the detailed mechanisms by which STRA6 mutations could lead to cardiac malformations in humans remain unclear. Here, we investigated the role of STRA6 in the context of human cardiogenesis and CHD. METHODS AND RESULTS: To gain molecular signatures in species-specific cardiac development, we first compared single-cell RNA sequencing (RNA-seq) datasets, uniquely obtained from human and murine embryonic hearts. We found that while STRA6 mRNA was much less frequently expressed in murine embryonic heart cells derived from the Mesp1+ lineage tracing mice (Mesp1Cre/+; Rosa26tdTomato), it was expressed predominantly in the OFT region-specific heart progenitors in human developing hearts. Next, we revealed that STRA6-knockout human embryonic stem cells (hESCs) could differentiate into cardiomyocytes similarly to wild-type hESCs, but could not differentiate properly into mesodermal nor neural crest cell-derived smooth muscle cells (SMCs) in vitro. This is supported by the population RNA-seq data showing down-regulation of the SMC-related genes in the STRA6-knockout hESC-derived cells. Further, through machinery assays, we identified the previously unrecognized interaction between RA nuclear receptors RARα/RXRα and TBX1, an OFT-specific cardiogenic transcription factor, which would likely act downstream to STRA6-mediated RA signalling in human cardiogenesis. CONCLUSION: Our study highlights the critical role of human-specific STRA6 progenitors for proper induction of vascular SMCs that is essential for normal OFT formation. Thus, these results shed light on novel and human-specific CHD mechanisms, driven by STRA6 mutations.

Placental growth factor exerts a dual function for cardiomyogenesis and vasculogenesis during heart development.

Cardiogenic growth factors play important roles in heart development. Placental growth factor (PLGF) has previously been reported to have angiogenic effects; however, its potential role in cardiogenesis has not yet been determined. We analyze single-cell RNA-sequencing data derived from human and primate embryonic hearts and find PLGF shows a biphasic expression pattern, as it is expressed specifically on ISL1+ second heart field progenitors at an earlier stage and on vascular smooth muscle cells (SMCs) and endothelial cells (ECs) at later stages. Using chemically modified mRNAs (modRNAs), we generate a panel of cardiogenic growth factors and test their effects on enhancing cardiomyocyte (CM) and EC induction during different stages of human embryonic stem cell (hESC) differentiations. We discover that only the application of PLGF modRNA at early time points of hESC-CM differentiation can increase both CM and EC production. Conversely, genetic deletion of PLGF reduces generation of CMs, SMCs and ECs in vitro. We also confirm in vivo beneficial effects of PLGF modRNA for development of human heart progenitor-derived cardiac muscle grafts on murine kidney capsules. Further, we identify the previously unrecognized PLGF-related transcriptional networks driven by EOMES and SOX17. These results shed light on the dual cardiomyogenic and vasculogenic effects of PLGF during heart development.

Unlocking the promise of mRNA therapeutics.

The extraordinary success of mRNA vaccines against coronavirus disease 2019 (COVID-19) has renewed interest in mRNA as a means of delivering therapeutic proteins. Early clinical trials of mRNA therapeutics include studies of paracrine vascular endothelial growth factor (VEGF) mRNA for heart failure and of CRISPR-Cas9 mRNA for a congenital liver-specific storage disease. However, a series of challenges remains to be addressed before mRNA can be established as a general therapeutic modality with broad relevance to both rare and common diseases. An array of new technologies is being developed to surmount these challenges, including approaches to optimize mRNA cargos, lipid carriers with inherent tissue tropism and in vivo percutaneous delivery systems. The judicious integration of these advances may unlock the promise of biologically targeted mRNA therapeutics, beyond vaccines and other immunostimulatory agents, for the treatment of diverse clinical indications.

Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure.

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.