The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including β-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both β-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies.

Protein phosphorylation is a major regulator of protein function and biological outcomes. This was first recognized through functional biochemical experiments, and in the past decade, major technological advances in mass spectrometry have enabled the study of protein phosphorylation on a global scale. This rapidly growing field of phosphoproteomics has revealed that more than 100,000 distinct phosphorylation events occur in human cells, which likely affect the function of every protein. Phosphoproteomics has improved the understanding of the function of even the most well-characterized protein kinases by revealing new downstream substrates and biology. However, current biochemical and bioinformatic approaches have only identified kinases for less than 5% of the phosphoproteome, and functional assignments of phosphosites are almost negligible. Notably, our understanding of the relationship between kinases and their substrates follows a power law distribution, with almost 90% of phosphorylation sites currently assigned to the top 20% of kinases. In addition, more than 150 kinases do not have a single known substrate. Despite a small group of kinases dominating biomedical research, the number of substrates assigned to a kinase does not correlate with disease relevance as determined by pathogenic human mutation prevalence and mouse model phenotypes. Improving our understanding of the substrates targeted by all kinases and functionally annotating the phosphoproteome will be broadly beneficial. Advances in phosphoproteomics technologies, combined with functional screening approaches, should make it feasible to illuminate the connectivity and functionality of the entire phosphoproteome, providing enormous opportunities for discovering new biology, therapeutic targets, and possibly diagnostics.

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Dysregulation of lipid homeostasis is a precipitating event in the pathogenesis and progression of hepatosteatosis and metabolic syndrome. These conditions are highly prevalent in developed societies and currently have limited options for diagnostic and therapeutic intervention. Here, using a proteomic and lipidomic-wide systems genetic approach, we interrogated lipid regulatory networks in 107 genetically distinct mouse strains to reveal key insights into the control and network structure of mammalian lipid metabolism. These include the identification of plasma lipid signatures that predict pathological lipid abundance in the liver of mice and humans, defining subcellular localization and functionality of lipid-related proteins, and revealing functional protein and genetic variants that are predicted to modulate lipid abundance. Trans-omic analyses using these datasets facilitated the identification and validation of PSMD9 as a previously unknown lipid regulatory protein. Collectively, our study serves as a rich resource for probing mammalian lipid metabolism and provides opportunities for the discovery of therapeutic agents and biomarkers in the setting of hepatic lipotoxicity. There is an increasingly urgent need to understand the causal factors that contribute to excess lipid accumulation in the liver known as hepatosteatosis, and an equally important need to discover biomarkers and interventions for its early diagnosis and treatment. A major proportion of current and predicted global health burden stems from conditions in which hepatosteatosis is an underlying pathology 1. Defining the mechanisms that causally influence hepatosteatosis has historically proven challenging, largely owing to an ill-defined interaction between genetic and environmental factors 2. This, together with the insufficient ability for standard genome-wide association studies to capture the effect of environment on complex traits, probably explains why only a small fraction of the estimated 30% heritability for hepatosteatosis has been assigned to specific gene variants 3. Genetic reference panels (GRPs) have become a more tractable way of studying the influence of genetics and environment on complex traits, because unlike studies in humans, GRPs allow for accurate control of environment as well as access to critical metabolic tissues. Importantly, integrating intermediate phenotypes such as transcriptomics, Parker et al.

Circulating proteins have important functions in inflammation and a broad range of diseases. To identify genetic influences on inflammation-related proteins, we conducted a genome-wide protein quantitative trait locus (pQTL) study of 91 plasma proteins measured using the Olink Target platform in 14,824 participants. We identified 180 pQTLs (59 cis, 121 trans). Integration of pQTL data with eQTL and disease genome-wide association studies provided insight into pathogenesis, implicating lymphotoxin-α in multiple sclerosis. Using Mendelian randomization (MR) to assess causality in disease etiology, we identified both shared and distinct effects of specific proteins across immune-mediated diseases, including directionally discordant effects of CD40 on risk of rheumatoid arthritis versus multiple sclerosis and inflammatory bowel disease. MR implicated CXCL5 in the etiology of ulcerative colitis (UC) and we show elevated gut CXCL5 transcript expression in patients with UC. These results identify targets of existing drugs and provide a powerful resource to facilitate future drug target prioritization.

Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry‐based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and in situ muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross‐species phosphosite responses, as well as unique model‐specific regulation. We identified > 22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK‐mediated phosphorylation of STIM1 negatively regulates store‐operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross‐species resource of exercise‐regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.

Three dimensional engineered culture systems are powerful tools to rapidly expand our knowledge of human biology and identify novel therapeutic targets for disease. Bioengineered skeletal muscle has been recently shown to recapitulate many features of native muscle biology. However, current skeletal muscle bioengineering approaches require large numbers of cells, reagents and labour, limiting their potential for high-throughput studies. Herein, we use a miniaturized 96-well micro-muscle platform to facilitate semiautomated tissue formation, culture and analysis of human skeletal micro muscles (hµMs). Utilising an iterative screening approach we define a serum-free differentiation protocol that drives rapid, directed differentiation of human myoblast to skeletal myofibres. The resulting hµMs comprised organised bundles of striated and functional myofibres, which respond appropriately to electrical stimulation. Additionally, we developed an optogenetic approach to chronically stimulate hµM to recapitulate known features of exercise training including myofibre hypertrophy and increased expression of metabolic proteins. Taken together, our miniaturized approach provides a new platform to enable high-throughput studies of human skeletal muscle biology and exercise physiology.

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