Biology subjects

Porrello, E. R.

Publications and source records attributed to Porrello, E. R..

13 recordsLinked to original sources

bioRxiv2026

High-throughput screening in hiPSC-cardiac models reveals cardiomyocyte-specific cell cycle regulatory mechanisms

Introductory ParagraphMultiple regulatory mechanisms govern cardiomyocyte proliferation including epigenetic modifications, metabolism and mechanical load. However, it is unclear whether such mechanisms can be pharmacologically targeted to induce cardiomyocyte proliferation without affecting other cell types. Here, we develop a dual-reporter (TNNT2eGFP; PCNAmScarlet-I) and a high-throughput image-based pipeline in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, with counter screening in non-myocytes, to identify compounds that selectively promote cardiomyocyte proliferation without affecting other cell types. We identify the PIM kinase inhibitor GDC-0339 as a cardiomyocyte-selective pro-proliferative compound. GDC-0339 induced proliferation of hiPSC-derived cardiomyocytes without activity in non-myocytes, non-cardiac fibroblasts or epithelial cells. Phosphoproteomic profiling of GDC-0339 in cardiomyocytes and non-cardiac fibroblasts revealed a cardiomyocyte-specific mechanism of action involving sarcomere disassembly via remodelling of the F-actin cytoskeleton and metabolic reprogramming to anaerobic metabolism via Pyruvate Dehydrogenase Kinases (PDKs). Thus, we uncover cardiomyocyte-specific mechanisms governing the cell cycle that are potentially druggable.

cell biology↗

bioRxiv2025

Contractile function maintains cardiomyocyte differentiation and inhibits cell cycle activity

Numerous endotherm species lose cardiac regenerative capacity shortly after birth, which is in contrast to many ectotherm species who regenerate throughout life. Whether the enhanced contractile function required for endothermy contributes to the cell-cycle exit remains to be explored. Herein, we use human cardiac organoids with advanced maturation combined with direct targeting of contraction using mavacamten and aficamten to enable exquisite control of active contraction over brief time windows. We show that transient inhibition of contraction re-activates the cell cycle. Multi-omics analyses demonstrated the cell cycle response to be mediated through a dedifferentiation-like process, which was swiftly reversed upon removal of the myosin inhibitors. Together these findings reveal that active contraction maintains differentiation including cell cycle arrest in cardiomyocytes.

cell biology↗

bioRxiv2025

Alpha Protein Kinase 3 Gene Therapy Restores Heart Function in Mouse and Human Models of Cardiomyopathy

Truncating variants in the Alpha Kinase 3 (ALPK3) gene have recently emerged as an important cause of genetic cardiomyopathy globally and here we demonstrate the efficacy and safety of a viral-based gene replacement therapy for ALPK3. Given the loss-of-function nature of these variants, we reasoned that a gene replacement approach would improve heart function in this patient population. We demonstrate that the delivery of full length ALPK3 via adeno-associated virus could restore contractile function in human cardiac organoids and in vivo mouse models carrying clinically relevant mutations in ALPK3. The role of disrupted proteostasis networks in multiple forms of genetic cardiomyopathy suggest that delivery this novel AAV-ALPK3 may provide functional benefit outside of cardiomyopathy induced by ALPK3. Titin truncating variants (TTNtv) are the most common cause of dilated cardiomyopathy, and interestingly also contributes to the M-Band protein quality control network coordinated by ALPK3. Notably, in human cardiac organoids carrying a TTNtv we observed that the ALPK3 gene therapy could completely restore contractile deficits. This opens the exciting prospect for indication expansion of AAV-ALPK3 into other forms of cardiomyopathy that currently have no therapeutic options.

physiology↗

bioRxiv2024

Targeted glycophagy ATG8 therapy for diabetic heart disease

Diabetic heart disease is highly prevalent and is associated with the early development of impaired diastolic relaxation. The mechanisms of diabetic heart disease are poorly understood and it is a condition for which there are no targeted therapies. Recently, disrupted glycogen-autophagy (glycophagy) and glycogen accumulation have been identified in the diabetic heart. Glycophagy involves glycogen receptor binding and linking with an ATG8 protein to locate and degrade glycogen within an intracellular phago-lysosome. Here we show that glycogen receptor protein STBD1 (starch-binding-domain-protein-1) is mobilized early in the cardiac glycogen response to metabolic challenge in vivo, and that deficiency of a specific ATG8 family protein, Gabarapl1 ({gamma}-aminobutyric-acid-receptor-associated-protein-like-1) is associated with diastolic dysfunction in diabetes. Gabarapl1 gene delivery treatment remediated cardiomyocyte and cardiac diastolic dysfunction in type 2 diabetic mice and diastolic performance of diabetic human iPSC-derived cardiac organoids. We identify glycophagy dysregulation as a mechanism and potential treatment target for diabetic heart disease.

physiology↗

bioRxiv2024

Prenylation controls proliferation in human pluripotent stem cell-derived cardiomyocytes

Induction of cardiomyocyte proliferation to replace damaged heart tissue is a promising therapeutic approach. A recent drug screen revealed that cardiomyocytes require the mevalonate pathway for proliferation, although the specific mechanisms are unknown. In this study, we use human pluripotent stem cell-derived cardiomyocytes and cardiac organoids to further interrogate the role of the mevalonate pathway in cardiomyocyte proliferation. Chemical and genetic perturbations of the mevalonate pathway indicated that the post-translational modification, prenylation, regulates cardiomyocyte proliferation. We use prenyl probes and mass spectrometry to identify a catalogue of 40 prenylated proteins in human cardiac cells, including proteins where prenylated function had not yet been investigated. We show that multiple prenylated proteins control cardiomyocyte proliferation including RRAS2 and NAP1L4. We demonstrate that prenylation has differential effects on distinct proteins, with RRAS2 prenylation controlling membrane localization and NAP1L4 prenylation regulating cardiomyocyte mitosis and centrosome homeostasis. Together, these data show that protein prenylation is required for cardiomyocyte proliferation through multiple targets and these processes may need to be re-activated for cardiac regeneration.

cell biology↗

bioRxiv2023

Affinity purification-mass spectrometry and single fiber physiology/proteomics reveals mechanistic insights of C18ORF25

C18ORF25 was recently shown to be phosphorylated at S67 by AMP-activated protein kinase (AMPK) in skeletal muscle following acute exercise in humans. Phosphorylation was shown to improve ex vivo skeletal muscle contractile function in mice but our understanding of the molecular mechanisms is incomplete. Here, we profiled the interactome of C18ORF25 in mouse myotubes using affinity purification coupled to mass spectrometry. This analysis included an investigation of AMPK-dependent and S67-dependent protein:protein interactions. Several nucleocytoplasmic and contractile-associated proteins were identified, and revealed a subset of GTPases that associate with C18ORF25 in an AMPK- and S67 phosphorylation-dependent manner. We confirmed that C18ORF25 is localised to the nucleus and the contractile apparatus in skeletal muscle. Mice lacking C18Orf25 display defects in calcium handling specifically in fast-twitch muscle fibers. To investigate these mechanisms, we developed an integrated single fiber physiology and single fiber proteomic platform. The approach enabled detailed assessment of various steps in the excitation-contraction (EC) pathway including SR calcium handling and force generation followed by paired single fiber proteomic analysis. This enabled us to identify >700 protein:phenotype associations and 36 fiber-type specific differences following loss of C18Orf25. Taken together, our data provide unique insights into the function of C18ORF25 and its role in skeletal muscle physiology.

physiology↗

bioRxiv2023

A transient modified mRNA encoding Myc and Cyclin T1 induces cardiac regeneration and improves cardiac function after myocardial injury

Cardiac injury, such as myocardial infarction (MI), results in permanent loss of cardiomyocytes and in many cases heart failure. Transgenic expression of the pro-proliferative transcription factor Myc and Cyclin T1 can drive substantial adult cardiomyocyte proliferation to replace lost cardiomyocytes. Herein, we show that Myc and Cyclin T1 induced cardiomyocyte proliferation leads to myocardial repair and functional (long-term) recovery post-MI in mice. To provide a more translational approach, we developed modified mRNA (modRNA) encoding Myc-Ccnt1 as a transient and non-integrating strategy for regeneration. One dose of Myc-Ccnt1 modRNA is sufficient to transiently drives cardiomyocyte proliferation in human pluripotent stem cell-derived cardiomyocytes and a mouse MI model, where it leads to better heart function. Using single nuclei sequencing and proteomics, we show this was functionally mediated by transcriptional activation of cell-cycle regulating genes, which ultimately results in mitosis and cytokinesis of cardiomyocytes. Collectively, these findings indicate that Myc-Ccnt1 modRNA has the potential to be an effective regenerative therapeutic.

molecular biology↗

bioRxiv2023

Spatially Resolved Transcriptomics Mining in 3D and Virtual Reality Environments with VR-Omics

The field of spatial transcriptomics is rapidly evolving, with increasing sample complexity, resolution, and tissue size. Yet the field lacks comprehensive solutions for automated integration and analysis of multi-slice data in either stacked (3D) or co-planar (2D) formation. To address this, we developed VR-Omics, a free, platform-agnostic software that distinctively provides end-to-end automated processing of multi-slice data through a biologist-friendly interface. Benchmarking against existing methods demonstrates VR-Omics unique strengths to perform comprehensive end-to-end analysis of multi-slice stacked data. Applied to rare paediatric cardiac rhabdomyomas, VR-Omics uncovered previously undetected dysregulated metabolic networks through co-planar slice analysis, demonstrating its potential for biological discoveries.

bioinformatics↗

bioRxiv2022

Alpha kinase 3 signaling at the M-band maintains sarcomere integrity and proteostasis in striated muscle.

Pathogenic variants in alpha kinase 3 (ALPK3) cause cardiomyopathy and musculoskeletal disease. How ALPK3 mutations result in disease remains unclear because little is known about this atypical kinase. Using a suite of engineered human pluripotent stem cells (hPSCs) we show that ALPK3 localizes to the M-Band of the sarcomere. ALPK3 deficiency disrupted sarcomeric organization and calcium kinetics in hPSC-derived cardiomyocytes and reduced force generation in cardiac organoids. Phosphoproteomic profiling identified ALPK3-dependant phospho-peptides that were enriched for sarcomeric components of the M-band and the ubiquitin-binding protein SQSTM1. Analysis of the ALPK3 interactome confirmed binding to M-band proteins including SQSTM1. Importantly, in hPSC-derived cardiomyocytes modeling ALPK3 deficiency and cardiomyopathic ALPK3 mutations, sarcomeric organization and M-band localization ofSQSTM1 were abnormal. These data suggest ALPK3 has an integral role in maintaining sarcomere integrity and proteostasis in striated muscle. We propose this mechanism may underly disease pathogenesis in patients with ALPK3 variants.

cell biology↗

bioRxiv2021

propeller: testing for differences in cell type proportions in single cell data

MotivationSingle cell RNA Sequencing (scRNA-seq) has rapidly gained popularity over the last few years for profiling the transcriptomes of thousands to millions of single cells. This technology is now being used to analyse experiments with complex designs including biological replication. One question that can be asked from single cell experiments, which has been difficult to directly address with bulk RNA-seq data, is whether the cell type proportions are different between two or more experimental conditions. As well as gene expression changes, the relative depletion or enrichment of a particular cell type can be the functional consequence of disease or treatment. However, cell type proportions estimates from scRNA-seq data are variable and statistical methods that can correctly account for different sources of variability are needed to confidently identify statistically significant shifts in cell type composition between experimental conditions. ResultsWe have developed propeller, a robust and flexible method that leverages biological replication to find statistically significant differences in cell type proportions between groups. Using simulated cell type proportions data we show that propeller performs well under a variety of scenarios. We applied propeller to test for significant changes in proportions of cell types related to human heart development, ageing and COVID-19 disease severity. Availability and implementationThe propeller method is publicly available in the open source speckle R package (https://github.com/phipsonlab/speckle). All the analysis code for the paper is available at https://github.com/phipsonlab/propeller-paper-analysis/, and the associated analysis website is available at https://phipsonlab.github.io/propeller-paper-analysis/. ContactAlicia Oshlack: Alicia.Oshlack@petermac.org Belinda Phipson: phipson.b@wehi.edu.au Supplementary informationYes.

bioinformatics↗

bioRxiv2021

Protective role of the Atg8 1 homologue Gabarapl1 in regulating cardiomyocyte glycophagy in diabetic heart disease

Diabetic heart disease is highly prevalent and characterized by diastolic dysfunction. The mechanisms of diabetic heart disease are poorly understood and no targeted therapies are available. Here we show that the diabetic myocardium (type 1 and type 2) is characterized by marked glycogen elevation and ectopic cellular localization - a paradoxical metabolic pathology given suppressed cardiomyocyte glucose uptake in diabetes. We demonstrate involvement of a glycogen-selective autophagy pathway ( glycophagy) defect in mediating this pathology. Genetically manipulated deficiency of Gabarapl1, an Atg8 autophagy homologue, induces cardiac glycogen accumulation and diastolic dysfunction. Stbd1, the Gabarapl1 cognate autophagosome partner is identified as a unique component of the early glycoproteome response to hyperglycemia in cardiac, but not skeletal muscle. Cardiac-targeted in vivo Gabarapl1 gene delivery normalizes glycogen levels, diastolic function and cardiomyocyte mechanics. These findings reveal that cardiac glycophagy is a key metabolic homeostatic process perturbed in diabetes that can be remediated by Gabarapl1 intervention.

molecular biology↗

bioRxiv2021

The human FLT1 regulatory element directs vascular expression and modulates angiogenesis pathways in vitro and in vivo

There is growing evidence that mutations in non-coding cis-regulatory elements (CREs) disrupt proper development. However, little is known about human CREs that are crucial for cardiovascular development. To address this, we bioinformatically identified cardiovascular CREs based on the occupancy of the CRE by the homeodomain protein NKX2-5 and cardiac chromatin histone modifications. This search defined a highly conserved CRE within the FLT1 locus termed enFLT1. We show that the human enFLT1 is an enhancer capable of driving reporter transgene expression in vivo throughout the developing cardiovascular system of medaka. Deletion of the human enFLT1 enhancer ({Delta}enFLT1) triggered molecular perturbations in extracellular matrix organisation and blood vessel morphogenesis in vitro in endothelial cells derived from human embryonic stem cells and vascular defects in vivo in medaka. These findings highlight the crucial role of the human FLT1 enhancer and its function as a regulator and buffer of transcriptional regulation in cardiovascular development.

genomics↗

bioRxiv2020

Discovery of drugs to treat cytokine storm-induced cardiac dysfunction using human cardiac organoids

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined, but could be direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory cytokine-storm, a cocktail of interferon gamma, interleukin 1{beta} and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids and hearts of SARS-CoV-2 infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCO and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the FDA breakthrough designated drug apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.

cell biology↗