Biology subjects

MacRae, C. A.

Publications and source records attributed to MacRae, C. A..

7 recordsLinked to original sources

bioRxiv2026

Damaging RBM20 E-rich domain variants are not rescued by gene replacement

The promise of precision therapeutics in genetic cardiomyopathies relies on linking specific therapies to variant mechanisms. Missense variants in the cardiac splice regulator RBM20 cause a highly penetrant and arrhythmogenic dilated cardiomyopathy. Disease-causing variants in RBM20s arginine-serine rich (RS) domain act via formation of toxic gain of function cytoplasmic granules, but this is not true for a small number of clinically adjudicated pathogenic variants in its glutamate(E)-rich domain. To better define the effects of E-rich domain variants, we developed a scalable screen based on induced pluripotent stem cell (iPSC) cardiomyocyte differentiation that identified several additional damaging variants. Several of these reduced RBM20 protein abundance and stability. We therefore hypothesized that, unlike RS domain variants, these E-rich variants might be rescued by RBM20 overexpression. To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with a pathogenic E-rich domain variant (p.E913K), and confirmed reduced RBM20 protein expression in these RBM20+/p.E913K cells after differentiation to iPSC-derived cardiomyocytes (iPSC-CM, vs. engineered isogenic RBM20+/+). These iPSC-CMs also displayed aberrant transcriptional splicing, reduced contractility, increased calcium-induced calcium release, and nuclear localization of RBM20 protein, often with more than the two expected RBM20-centric splice factories. AAV-based overexpression of RBM20 reversed some, but not all of the mis-splicing events identified in RBM20+/p.E913K iPSC-CMs, and did not improve their abnormal contractility, calcium handling or supernumerary RBM20 nuclear granules. In summary, our data indicate that pathogenic E-rich domain variants reduce RBM20 protein abundance, but that their mechanism is unlikely to be explained by haploinsufficiency alone.

genetics↗

bioRxiv2026

Beyond Glycogen Storage: AMPKγ2 Regulates Cardiac Hypertrophy and Electrophysiology via Myosin Interaction

IntroductionVariants in PRKAG2 cause hypertrophic cardiomyopathy (HCM) and conduction disturbances. While prior studies associated PRKAG2-related hypertrophy with increased glycogen storage, many HCM phenotypes remain unexplained. We aimed to uncover how PRKAG2 variants induce myocyte hypertrophy and electrical changes during early cardiac development. MethodsWe generated transgenic zebrafish expressing wild-type (TgWT) or pathogenic variant (TgR299Q) Prkag2 cDNA under a myocardium-specific promoter, and examined cardiac electrophysiology, contractile function, and cytoarchitecture during cardiogenesis and in adult hearts. ResultsTgR299Q fish showed hypertrophic cardiomyocytes and progressive contractile abnormalities, recapitulating human HCM phenotypes. Cardiomyocyte glycogen was elevated in adult but not embryonic hearts. Despite the absence of glycogen accumulation at 6-day post-fertilization, TgR299Q hearts showed electrical abnormalities, including reduced conduction velocity and prolonged action potential and Ca2+ transient durations. We observed decreased AMPK phosphorylation in the TgR299Q hearts. However, AMPK activation did not rescue the electrophysiological abnormalities in TgR299Q. Proximity ligation assays and co-immunoprecipitation identified a physical interaction between AMPK{gamma}2 and myosin, enhanced by the R299Q variant and accompanied by increased AMPK{gamma}2 localization to the myofilament. Na/Ca{superscript 2} exchanger (NCX) inhibition increased Ca2+ duration and diastolic Ca2+ in TgWT but not TgR299Q hearts, indicating reduced free cytosolic Ca2+ for NCX-mediated extrusion in TgR299Q. These findings suggest that enhanced AMPK{gamma}2-myosin interaction may promote myofilament Ca{superscript 2} retention, thereby prolonging Ca{superscript 2} transient duration and APD in the mutant. Notably, the myosin inhibitor mavacamten reduced AMPK{gamma}2-myosin interaction in TgR299Q hearts, and both mavacamten and vmhcl knockdown rescued the early electrophysiological abnormalities. ConclusionsThe PRKAG2 variant altered cardiac excitability, contractility, and Ca2+ handling during cardiogenesis, independent of glycogen accumulation. Enhanced interactions between AMPK{gamma}2 and myosin contributed to these early changes. Our study revealed a novel link between cellular energy sensing and contractile machinery, with therapeutic potential for modulating contractile function in cardiomyopathies.

physiology↗

bioRxiv2025

Scaled multidimensional assays of variant effect identify sequence-function relationships in hypertrophic cardiomyopathy

BackgroundAn estimated 1 in 500 people live with hypertrophic cardiomyopathy (HCM), a disease for which genetic diagnosis can identify family members at risk, and increasingly guide therapy. Mutations in the myosin binding protein C3 (MYBPC3) gene account for a significant proportion of HCM cases. However, many of these variants are classified as variants of uncertain significance (VUS), complicating clinical decision-making. Scalable methods for variant interpretation in disease-specific cell types are crucial for understanding variant impact and uncovering disease mechanisms. MethodsWe developed a scaled multidimensional mapping strategy to evaluate the functional impact of variants across a critical domain of MYBPC3. We incorporate saturation base editing at the native MYBPC3 locus, a long-read RNA sequencing-enabled assay of variant splice effects, and measurements of HCM-relevant phenotypes, including MYBPC3 abundance, hypertrophic signaling, and ubiquitin-proteasome function in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). ResultsOur multidimensional mapping strategy enabled high-resolution functional analysis of MYBPC3 variants in iPSC-CMs. Targeted transient base editing generated a comprehensive variant library at the native locus, capturing diverse variant effects on cellular HCM-relevant phenotypes. Our massively parallel splicing assay identified novel splice-disrupting variants. Integration of functional assays revealed that decreased MYBPC3 abundance is a key driver of HCM-related phenotypes. In parallel, downregulation of protein degradation was observed as a compensatory response to MYBPC3 loss of function, and novel disease mechanisms were identified for missense variants near a critical binding domain, underscoring their contribution to pathogenesis. Bayesian estimates of variant effects enable the reclassification of clinical variants. ConclusionsThis work provides a platform for extending genome engineering in iPSCs to multiplexed assays of variant effects across diverse disease-relevant cellular phenotypes, enhancing the understanding of variant pathogenicity and uncovering novel biological mechanisms that could inform therapeutic strategies.

genetics↗

bioRxiv2023

Prdm16 mutation determines sex-specific cardiac metabolism and identifies two novel cardiac metabolic regulators

BackgroundMutation of the PRDM16 gene has been associated with human cardiomyopathy. The PRDM16 protein is a transcriptional regulator affecting cardiac development via Tbx5 and Hand1 regulating myocardial structure. Biallelic Prdm16 inactivation induces severe cardiac dysfunction with postnatal lethality and hypertrophy in mice. Early pathological events upon Prdm16 inactivation have not been explored. MethodsThis study performed in depth pathophysiological and molecular analysis of male and female Prdm16csp1/wt mice carrying systemic, monoallelic Prdm16 gene inactivation. We systematically assessed early molecular changes with transcriptomics, proteomics, and metabolomics. Kinetic modelling of the cardiac metabolism was undertaken in silico with CARDIOKIN. ResultsPrdm16csp1/wt mice are viable up to 8 months, develop hypoplastic hearts, and diminished systolic performance that is more pronounced in female mice. Prdm16csp1/wt hearts demonstrate moderate alterations of specific transcripts and protein levels with consistent upregulation of pyridine nucleotide-disulphide oxidoreductase domain 2 (Pyroxd2) and the transcriptional regulator pre B-cell leukemia transcription factor interacting protein 1 (Pbxip1). The strongest concordant transcriptional upregulation was detected for Prdm16 itself probably by an autoregulatory mechanism. Prdm16csp1/wt cardiac tissue showed reduction of metabolites associated with amino acid as well as glycerol metabolism, glycolysis, and tricarboxylic acid cycle. Global lipid metabolism was also affected with accumulation of triacylglycerides detected in male Prdm16csp1/wt hearts. In addition, Prdm16csp1/wt cardiac tissue revealed diminished glutathione (GSH) and increased inosine monophosphate (IMP) levels indicating oxidative stress and a dysregulated energetics, respectively. Metabolic modelling in silico suggested lowered fatty acid utilization in male and reduced glucose utilization in female Prdm16csp1/wt cardiac tissue. ConclusionsMonoallelic Prdm16 mutation restricts cardiac performance in Prdm16csp1/wt mice. Metabolic alterations precede transcriptional dysregulation in Prdm16csp1/wt cardiac tissue. Female Prdm16csp1/wt mice develop a more pronounced phenotype indicating a sexual dimorphism at this early pathological window. This study suggests that metabolic dysregulation is an early event in PRDM16 associated cardiac pathology. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABS- Mutation of the PRDM16 gene has been associated with human cardiomyopathy. - Biallelic inactivation of Prdm16 in mice induces severe cardiac dysfunction with early postnatal lethality. - Prdm16 cooperates with transcription factors such as Tbx5 and Hand1 to activate transcriptional programs that define the development of the compacted myocardium. What New Information Does This Article Contribute?- Systemic, monoallelic inactivation in Prdm16csp1/wt mice induces cardiac dysfunction with normal survival. - Metabolic alterations are the leading pathophysiological consequences and induce cardiac hypoplasia. On the molecular level this is associated with upregulation of metabolic regulators Pyroxd2 and Pbxip1. - Metabolic response after Prdm16 inactivation occurs in a sex specific manner.

pathology↗

bioRxiv2021

Prdm16 and Notch functionally and physically interact during artery development

Proper arterial versus venous endothelial lineage identity is required to establish a hierarchical network of arteries and veins and prevent the occurrence of life-threatening arteriovenous malformations. The molecular mechanisms that govern arteriovenous lineage specification remain however incompletely understood. Here, we demonstrate that the transcription factor Prdm16 is expressed in arterial but not venous endothelial cells (ECs) from the earliest stages of development, where it actively promotes arterial EC identity by enhancing canonical Notch activity, while simultaneously suppressing the alternative venous cell fate. Concordantly, our results in zebrafish show that Prdm16 coordinates proper arterial development and arteriovenous lineage specification together with canonical Notch signaling, as combined loss of prdm16 and notch in zebrafish invariably leads to arteriovenous malformations (AVMs). Although the arterializing effect of Prdm16 in human ECs is dependent on the absolute levels of the cleaved intracellular domain of the Notch receptors (NICD), Prdm16 does not increase NICD levels per se. Rather, Prdm16 physically and functionally interacts with NICD to potentiate its effect. Prdm16 further finetunes Notch signaling and arterial development by complexing with the Notch downstream effector Hey2, which regulates arterial lineage specification and development across species. Together, our data demonstrate that Prdm16 act as a rheostat for endothelial Notch activity and suggest that Prdm16 signaling may constitute a novel therapeutic target for AVMs.

developmental biology↗

bioRxiv2021

Single-cell dissection of live human hearts in ischemic heart disease and heart failure reveals cell-type-specific driver genes and pathways

Ischemic heart disease is globally the leading cause of death. It plays a central role in the electrical and structural remodeling of the right atrium, predisposing to arrhythmias, heart failure, and sudden death. Here, we provide the first dissection of the gene expression changes in the live right atrial tissue, using single-nuclei RNA-seq and spatial transcriptomics. We investigate matched samples of the tissue and pericardial fluid and reveal substantial differences in disease- associated gene expression in all cell types, leading to inflammatory microvascular dysfunction and changes in the tissue composition. Our study demonstrates the importance of creating high- resolution cellular maps and partitioning disease signals beyond epicardial coronary arteries and ischemic left ventricle to identify candidate mechanisms leading to more severe types of human cardiovascular disease. One-Sentence SummarySingle-cell dissection of ex vivo heart biopsies and pericardial fluid in ischemic heart disease and heart failure

molecular biology↗

bioRxiv2017

Fragile dynamics enable diverse genomic determinants to influence arrhythmia propensity

Genotype-phenotype relationships are determinants of human diseases. Often, we know little about why so many genes are involved in complex common diseases. We hypothesized that this multigene effect arises from the relationship between genes and physiological dynamics. We tested this hypothesis for arrhythmias as physiological dynamics define this disease. We integrated graph theory analysis of genomic and protein-protein interaction networks with dynamical models of ion channel function to identify the physiological dynamics of genome wide variation for five different arrhythmias. Regulatory networks for the cardiac conduction system and arrhythmias were constructed from GWAS and known disease genes. Electrophysiological models of myocyte action potentials were used to conduct extensive parameter variations to identify robust and fragile kinetic parameters that were then, using regulatory networks, associated with genomic determinants. We find that genome-wide determinants of arrhythmias that represent many cellular processes are selectively associated with fragile physiological dynamics of ion channel kinetics. This association predicts disease propensity. Deep RNA sequencing from human left ventricular tissue of arrhythmia and control subjects confirmed the predictive relationship. Taken together these studies show that the varied multigene effects of arrhythmias arises because of associations with fragile kinetic parameters of cardiac electrophysiology.\n\nSignificance StatementOur understanding of the genetics of common diseases has advanced exponentially over the past decade. We now know that differences and variation in multiple genes contribute to disease susceptibility with significant heterogeneity in the phenotype. However, how genetic variation contributes to disease phenotypes remains unknown. We hypothesized that the relationships between physiological dynamics and genetic architecture is a fundamental determinant of disease susceptibility and genetic heterogeneity. To test our hypothesis, we integrated mathematical models of cardiac electrophysiology with genetic network models of cardiac arrhythmias. We found that disease related genome variants were selectively associated with fragile kinetic parameters that predict disease propensity and identified several novel cellular processes associated with arrhythmogenesis.

systems biology↗