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Muscle contraction is driven by the molecular machinery of the sarcomere. As phosphorylation is a critical regulator of muscle function, the identification of regulatory kinases is important for understanding sarcomere biology. Pathogenic variants in alpha kinase 3 (ALPK3) cause cardiomyopathy and musculoskeletal disease, but little is known about this atypical kinase. Here we show that ALPK3 is an essential component of the M-band of the sarcomere and define the ALPK3-dependent phosphoproteome. ALPK3 deficiency impaired contractility both in human cardiac organoids and in the hearts of mice harboring a pathogenic truncating Alpk3 variant. ALPK3-dependent phosphopeptides were enriched for sarcomeric components of the M-band and the ubiquitin-binding protein sequestosome-1 (SQSTM1) (also known as p62). Analysis of the ALPK3 interactome confirmed binding to M-band proteins including SQSTM1. In human pluripotent stem cell-derived cardiomyocytes modeling cardiomyopathic ALPK3 mutations, sarcomeric organization and M-band localization of SQSTM1 were abnormal suggesting that this mechanism may underly disease pathogenesis.

Refereed/Peer-reviewed Crosstalk between cardiac cells is critical for heart performance. Here we show that vascular cells within human cardiac organoids (hCOs) enhance their maturation, force of contraction, and utility in disease modeling. Herein we optimize our protocol to generate vascular populations in addition to epicardial, fibroblast, and cardiomyocyte cells that self-organize into in-vivo-like structures in hCOs. We identify mechanisms of communication between endothelial cells, pericytes, fibroblasts, and cardiomyocytes that ultimately contribute to cardiac organoid maturation. In particular, (1) endothelial-derived LAMA5 regulates expression of mature sarcomeric proteins and contractility, and (2) paracrine platelet-derived growth factor receptor β (PDGFRβ) signaling from vascular cells upregulates matrix deposition to augment hCO contractile force. Finally, we demonstrate that vascular cells determine the magnitude of diastolic dysfunction caused by inflammatory factors and identify a paracrine role of endothelin driving dysfunction. Together this study highlights the importance and role of vascular cells in organoid models.

SUMMARYCell culture is generally considered to be hyperoxic. However, the importance of cellular oxygen consumption is often underappreciated, with rates of oxygen consumption often sufficient to cause hypoxia at cell monolayers. We initially focused on cultured adipocytes as a terminally differentiated cell-type with substantial oxygen consumption rates to support diverse cellular functions. Under standard conditions, cultured adipocytes are hypoxic and highly glycolytic. Increasing oxygen diverted glucose flux toward mitochondria and resulted in thousands of gene expression changes that pointed toward alleviated physiological transcriptional responses to hypoxia. Phenotypically, providing more oxygen increased adipokine secretion and rendered adipocytes more sensitive to insulin and lipolytic stimuli. The functional benefits of increasing pericellular oxygen were transferable to other cellular systems including hPSC-derived hepatocytes and cardiac organoids. Our findings suggest that oxygen is limiting in many terminally-differentiated cell culture systems, and that controlling oxygen availability can improve the quality and translatability of cell models.

SUMMARYPathogenic 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.

Improving muscle function has great potential to improve the quality of life. To identify novel regulators of skeletal muscle metabolism and function, we performed a proteomic analysis of gastrocnemius muscle from 73 genetically distinct inbred mouse strains, and integrated the data with genomics and >300 molecular/phenotypic traits via quantitative trait loci mapping and correlation network analysis. These data identified thousands of associations between protein abundance and phenotypes and can be accessed online (https://muscle.coffeeprot.com/) to identify regulators of muscle function. We used this resource to prioritize targets for a functional genomic screen in human bioengineered skeletal muscle. This identified several negative regulators of muscle function including UFC1, an E2 ligase for protein UFMylation. We show UFMylation is up-regulated in a mouse model of amyotrophic lateral sclerosis, a disease that involves muscle atrophy. Furthermore, in vivo knockdown of UFMylation increased contraction force, implicating its role as a negative regulator of skeletal muscle function.

SUMMARYThis study establishes the homeodomain only protein, HOPX, as a determinant controlling the molecular switch between cardiomyocyte progenitor and maturation gene programs. Time-course single-cell gene expression with genome-wide footprinting reveal that HOPX interacts with and controls core cardiac networks by regulating the activity of mutually exclusive developmental gene programs. Upstream hypertrophy and proliferation pathways compete to regulate HOPX transcription. Mitogenic signals override hypertrophic growth signals to suppress HOPX and maintain cardiomyocyte progenitor gene programs. Physiological studies show HOPX directly governs genetic control of cardiomyocyte cell stress responses, electro-mechanical coupling, proliferation, and contractility. We use human genome-wide association studies (GWAS) to show that genetic variation in the HOPX-regulome is significantly associated with complex traits affecting cardiac structure and function. Collectively, this study provides a mechanistic link situating HOPX between competing upstream pathways where HOPX acts as a molecular switch controlling gene regulatory programs underpinning metabolic, signaling, and functional maturation of cardiomyocytes.

SummaryDiabetic 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.

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 semi-automated 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.

A combination of olanzapine and samidorphan was recently approved by the US Food and Drug Administration for the treatment of patients with schizophrenia or bipolar I disorder. Population pharmacokinetic models for olanzapine and samidorphan were developed using data from 11 clinical studies in healthy subjects or patients with schizophrenia. A 2‐compartment disposition model with first‐order absorption and elimination and a lag time for absorption adequately described concentration‐time profiles of both olanzapine and samidorphan. Age, sex, race, smoking status, and body weight were identified as covariates that impacted the pharmacokinetics of olanzapine. A moderate effect of body weight on samidorphan pharmacokinetics was identified by the model but was not considered clinically meaningful. The effects of food, hepatic or renal impairment, and coadministration with rifampin on the pharmacokinetics of olanzapine and samidorphan, as estimated by the population pharmacokinetic analysis, were consistent with findings from dedicated clinical studies designed to evaluate these specific covariates of interest. Food intake did not have a clinically relevant effect on the pharmacokinetics of olanzapine or samidorphan. Consistent with the known metabolic pathways for olanzapine (primarily via uridine 5′‐diphospho‐glucuronosyltransferase–mediated direct glucuronidation and cytochrome P450 [CYP]‐mediated oxidation) and for samidorphan (predominantly mediated by CYP3A4), coadministration of olanzapine and samidorphan with rifampin, a strong inducer of CYP3A4 and an inducer of uridine 5′‐diphospho‐glucuronosyltransferase enzymes, significantly decreased the systemic exposure of both olanzapine and samidorphan. Severe renal impairment or moderate hepatic impairment resulted in a modest increase in olanzapine and samidorphan exposure.

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 1β 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., A combination of phosphoproteomics, drug screening and single-cell sequencing approaches identifies how cytokines elevated in COVID-19 patients drives cardiac dysfunction, with BET inhibitors serving as potential lead candidates decrease ACE2 cardiac expression and infection.

SUMMARYCardiac 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 1β 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.

This review summarizes the important role that metabolism plays in driving maturation of human pluripotent stem cell-derived cardiomyocytes. Human pluripotent stem cell-derived cardiomyocytes provide a model system for human cardiac biology. However, these models have been unable to fully recapitulate the maturity observed in the adult heart. By simulating the glucose to fatty acid transition observed in neonatal mammals, human pluripotent stem cell-derived cardiomyocytes undergo structural and functional maturation also accompanied by transcriptional changes and cell cycle arrest. The role of metabolism in energy production, signaling, and epigenetic modifications illustrates that metabolism and cellular phenotype are intimately linked. Further understanding of key metabolic factors driving cardiac maturation will facilitate the generation of more mature human pluripotent stem cell-derived cardiomyocyte models. This will increase our understanding of cardiac biology and potentially lead to novel therapeutics to enhance heart function.

The inability of the adult mammalian heart to regenerate represents a fundamental barrier in heart failure management. In contrast, the neonatal heart retains a transient regenerative capacity, but the underlying mechanisms for the developmental loss of cardiac regenerative capacity in mammals are not fully understood. Wnt/β-catenin signaling has been proposed as a key cardio-regenerative pathway driving cardiomyocyte proliferation. Here, we show that Wnt/β-catenin signaling potentiates neonatal mouse cardiomyocyte proliferation in vivo and immature human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) proliferation in vitro. In contrast, Wnt/β-catenin signaling in adult mice is cardioprotective but fails to induce cardiomyocyte proliferation. Transcriptional profiling and chromatin immunoprecipitation sequencing of neonatal mouse and hPSC-CM revealed a core Wnt/β-catenin-dependent transcriptional network governing cardiomyocyte proliferation. In contrast, β-catenin failed to re-engage this neonatal proliferative gene network in the adult heart despite partial transcriptional re-activation of a neonatal glycolytic gene program. These findings suggest that β-catenin may be repurposed from regenerative to protective functions in the adult heart in a developmental process dependent on the metabolic status of cardiomyocytes.

Summary Centrosome reduction and redistribution of pericentriolar material (PCM) coincides with cardiomyocyte transitions to a post-mitotic and matured state. However, it is unclear whether centrosome changes are a cause or consequence of terminal differentiation. We validated that centrosomes were intact and functional in proliferative human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), consistent with their immature phenotype. We generated acentrosomal hPSC-CMs, through pharmacological inhibition of centriole duplication, and showed that centrosome loss was sufficient to promote post-mitotic transitions and aspects of cardiomyocyte maturation. As Hippo kinases are activated during post-natal cardiac maturation, we pharmacologically activated the Hippo pathway using C19, which was sufficient to trigger centrosome disassembly and relocalization of PCM components to perinuclear membranes. This was due to specific activation of Hippo kinases, as direct inhibition of YAP-TEAD interactions with verteporfin had no effect on centrosome organization. This suggests that Hippo kinase-centrosome remodeling may play a direct role in cardiac maturation., Highlights • Centrosomes are intact and functional in immature human cardiomyocytes • Centrosome loss promotes maturation of human cardiomyocytes • Centrosomes are returned with cell cycle reinitiation in post-natal cardiomyocytes • Hippo kinases promote disassembly and redistribution of centrosomal proteins, In this article, Ng, Hudson, and colleagues demonstrated that the removal of centrosomes, through blocking centriole duplication, was sufficient to trigger post-mitotic transitions and differentiation of immature human cardiomyocytes. They also revealed that Hippo kinase activation triggered redistribution of centrosomal proteins to acentrosomal sites. This suggests that the regulated disassembly of centrosomes by Hippo kinases may promote cardiomyocyte maturation.

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 ‘cytokine-storm’ induced dysfunction. To identify mechanisms and discover cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with high throughput phosphoproteomics and single nuclei RNA sequencing. We identify that ‘cytokine-storm’ induced diastolic dysfunction can be caused by a cocktail of interferon gamma, interleukin 1β and poly(I:C) and also human COVID-19 serum. Bromodomain protein 4 (BRD4) is activated along with pathology driving fibrotic and induced nitric oxide synthase genes. BRD inhibitors fully recover function in hCO and totally prevent death in a cytokine-storm mouse model. BRD inhibition decreases transcription of multiple genes, including fibrotic, induced nitric oxide synthase and ACE2, and prevention of cardiac infection with SARS-CoV2. Thus, BRD inhibitors are promising candidates to prevent COVID-19 mediated cardiac damage. Funding: We acknowledge grant and fellowship support from the National Health and Medical Research Council of Australia (J.E.H., M.J.S., C.R.E., T.B.), Heart Foundation of Australia (J.E.H.), QIMR Berghofer Medical Research Institute (J.E.H.), The Stafford Fox Foundation (E.R.P.), the Royal Children’s Hospital Foundation (E.R.P.), Australian Research Council Strategic Initiative in Stem Cell Science (Stem Cells Australia) (E.R.P. and J.E.H.) and the Medical Research Future Fund (MRFF9200008) (J.E.H., T.B., M.J.S., K.P.A.MD., C.R.E., E.R.P.). M.J.S. is supported by Health and Medical Research Council of Australia Program (APP1132519) and Investigator (APP1173958) grants. A.S. is also supported by Investigator grant (APP1173880). The Murdoch Children’s Research Institute is supported by the Victorian Government’s Operational Infrastructure Support Program. This project received support from Dynomics Inc. J.E.H. is supported by a Snow Medical Fellowship. Conflict of Interest: R.J.M., J.E.H., G.A.Q.-R., D.M.T. and E.R.P. are listed as co-inventors on pending patents held by The University of Queensland and QIMR Berghofer Medical Research Institute that relate to cardiac organoid maturation and putative cardiac regeneration therapeutics. J.E.H. is a coinventor on licensed patents held by the University of Goettingen. R.J.M, E.R.P., D.M.T., B.G. and J.E.H. are co-founders, scientific advisors and stockholders in Dynomics Inc. D.M.T. and B.G. are employees of Dynomics Inc. /Dynomics Pty Ltd. QIMR Berghofer Medical Research Institute has filed a patent on the use of BRD inhibitors. Ethical Approval: Animal work was approved by the QIMR Berghofer Medical Research Institute Animal Ethics Committee. Ethical approval for the use of human embryonic stem cells (hESCs) was obtained from QIMR Berghofer’s Ethics Committee and was carried out in accordance with the National Health and Medical Research Council of Australia (NHMRC) regulations. Procedures complied with standards set under Australian guidelines for animal welfare and experiments were subject to Monash University animal welfare ethics review (Approval #MARP/2019/13606).

Aripiprazole lauroxil (AL), a long-acting injectable antipsychotic for the treatment of schizophrenia, requires 21 days of oral aripiprazole supplementation upon initiation (21-day initiation regimen). An alternative 1-day initiation regimen utilizing a nano-crystalline milled dispersion of AL (ALNCD) plus a single 30 mg oral aripiprazole dose achieved aripiprazole concentrations associated with therapeutic doses of aripiprazole in the same time frame as the 21-day initiation regimen when starting AL (441 or 882 mg). A population pharmacokinetic (PopPK) model was developed to describe aripiprazole pharmacokinetics following administration of ALNCD, AL and oral aripiprazole, and evaluate dosing scenarios likely to be encountered in clinical practice. In total, 12,768 plasma aripiprazole concentrations from 343 patients (from 4 clinical studies) were included in the PopPK analysis and used to construct the model. Concomitant administration of the 1-day initiation regimen with all approved AL dosing regimens (441, 662, or 882 mg monthly, 882 mg every 6 weeks, or 1064 mg every 2 months) is predicted to achieve aripiprazole concentrations associated with therapeutic doses of AL using the 21-day initiation regimen within 4 days, maintaining these concentrations until the next AL dose. Administration of the first AL injection 10 days after the 1-day initiation regimen resulted in median aripiprazole concentrations just before the second dose of AL ≥ 77% of that when coadministered on the same day. Coadministration of AL with a single ALNCD injection was predicted to be effective in rapidly re-establishing concentrations associated with therapeutic doses of AL following dose delay. Model-based simulations demonstrate that the 1-day initiation regimen is suitable for starting treatment with all AL doses, allowing a window of ≤ 10 days between initiation and AL administration. ALNCD may also be used to re-establish concentrations associated with therapeutic doses of AL in conjunction with a delayed AL dose.

Recent advances in cardiac tissue engineering and pluripotent stem cell technologies are providing unprecedented access to human heart tissue with in vivo-like structural and functional properties. Engineered heart tissue (EHT) is emerging as a powerful technology platform for regenerative medicine, as well as for in vitro modeling of human physiological and pathophysiological processes. Here, we discuss the potential of EHT for studying cellular interactions within the heart and for modeling genetic and environmental causes of cardiovascular disease. In addition, we highlight the utility of EHT for drug screening and the identification of novel therapeutic modalities.

Homozygous nonsense mutations in CEP55 are associated with several congenital malformations that lead to perinatal lethality suggesting that it plays a critical role in regulation of embryonic development. CEP55 has previously been studied as a crucial regulator of cytokinesis, predominantly in transformed cells, and its dysregulation is linked to carcinogenesis. However, its molecular functions during embryonic development in mammals require further investigation. We have generated a Cep55 knockout (Cep55-/-) mouse model which demonstrated preweaning lethality associated with a wide range of neural defects. Focusing our analysis on the neocortex, we show that Cep55-/- embryos exhibited depleted neural stem/progenitor cells in the ventricular zone as a result of significantly increased cellular apoptosis. Mechanistically, we demonstrated that Cep55-loss downregulates the pGsk3β/β-Catenin/Myc axis in an Akt-dependent manner. The elevated apoptosis of neural stem/progenitors was recapitulated using Cep55-deficient human cerebral organoids and we could rescue the phenotype by inhibiting active Gsk3β. Additionally, we show that Cep55-loss leads to a significant reduction of ciliated cells, highlighting a novel role in regulating ciliogenesis. Collectively, our findings demonstrate a critical role of Cep55 during brain development and provide mechanistic insights that may have important implications for genetic syndromes associated with Cep55-loss., Author summary Despite extensive investigation on the roles of CEP55 in tumorigenesis, its physiological role during development has remained largely uncharacterized. Recently, homozygous CEP55 mutations have been linked to two lethal fetal syndromes, MKS-like Syndrome and MARCH, demonstrating the importance of CEP55 in embryogenesis and neural development. These syndromes exhibit multiple severe clinical manifestations that lead to perinatal lethality. However, the exact molecular mechanism underlying complex Cep55-deficient developmental phenotypes remain elusive. To address this question, we have generated a Cep55-/- (KO) mouse model and to bridge the gap between the mouse model and human disease, we have used brain organoids generated from pluripotent stem cells as a promising approach to investigate the mechanism of Cep55-associated neurodevelopment phenotype. Our detailed mechanistic studies suggest that Cep55 regulates neural development through the Akt-downstream effector, Gsk3β, and its mediators β-Catenin and Myc, which are known regulators of neural proliferation and differentiation. Additionally, we discovered a critical role for Cep55 in regulating cilia formation. Together, these results illustrate an important role of Cep55 in regulating neurogenesis and ciliogenesis via regulation of the Akt pathway.

Background Aripiprazole lauroxil (AL) is a long-acting injectable medication approved for the treatment of schizophrenia. Current AL regimens are 441 mg, 662 mg, and 882 mg administered monthly (every 4 weeks [q4wk]), or 882 mg administered every 6 weeks (q6wk). Objective We examined the feasibility of a 2-month (every 8 weeks [q8wk]) dosing interval of AL in a phase I open-label pharmacokinetic study investigating AL 1064 mg administered q8wk for 24 weeks, followed by 20 weeks of safety and pharmacokinetic measurements (ClinicalTrials.gov ID: NCT02320032). Second, a population pharmacokinetic model (referred to as the 2MPopPK model) was generated using data collected from the present trial, as well as data obtained from earlier studies. Methods The phase I study included patients with schizophrenia or schizoaffective disorder maintained on an oral antipsychotic (n = 140) who were assigned to one of three groups: AL 441 mg q4wk, AL 882 mg q6wk, or AL 1064 mg q8wk, with a total of seven, five, or four injections administered, respectively. No oral aripiprazole lead-in supplementation was administered and patients continued on maintenance oral antipsychotics. Pharmacokinetic samples were collected at various time points during the 24-week study period and the 20-week follow-up period. Plasma concentrations obtained from the phase I study were analyzed using non-compartmental methods. Additionally, the data were combined with data collected from prior studies to develop the 2MPopPK model. Results Following the final injection of AL in the phase I study, maximum aripiprazole concentrations were achieved 24.4–35.2 days after the last dose and persisted for the duration of the study. The mean C avg,ss values were 125.8 ng/ml, 131.1 ng/ml, and 140.7 ng/ml for the 441 mg q4wk, 882 mg q6wk, and 1064 mg q8wk doses, respectively. The mean elimination half-life of aripiprazole following the last dose was 53.9 days for the 1064 mg dose, 55.1 days for the 882 mg dose, and 57.2 days for the 441 mg dose. The 2MPopPK dataset included 14,524 aripiprazole concentrations from 700 patients with schizophrenia. The duration of absorption of aripiprazole was estimated as 43 days (95% confidence interval [CI] 42–45 days), which was preceded by a 3.2-day lag time (95% CI 3.0–3.5 days) for a total duration of input into the systemic circulation of 46 days following intramuscular administration of AL. Multiple-dose simulations showed that the 1064 mg q8wk regimen provides aripiprazole concentrations within the concentration range associated with 441 mg and 882 mg q4wk doses previously demonstrated to be efficacious in a phase III study. Conclusion These data from the phase I study and the 2MPopPK model support the suitability of using the AL 1064 mg dose as a 2-month (q8wk) dose interval option for the treatment of schizophrenia. Electronic supplementary material The online version of this article (doi:10.1007/s40263-017-0447-7) contains supplementary material, which is available to authorized users.

BackgroundAripiprazole lauroxil is an extended-release prodrug of aripiprazole for intramuscular injection, approved for schizophrenia treatment. We developed a population pharmacokinetic (PopPK) model to characterize aripiprazole lauroxil PK and evaluate dosing scenarios likely to be encoun

Structural and functional changes following infarction in a human heart can be modelled with human cardiac organoids set in a hypoxic gradient and stimulated with the neurotransmitter noradrenaline.

The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of the 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-CM), 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, 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 key proliferation pathways including β-catenin and 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 novel therapeutic strategies.

Human pluripotent stem cells (hPSCs) have extensive applications in fundamental biology, regenerative medicine, disease modelling, and drug discovery/toxicology. Whilst large numbers of cardiomyocytes can be generated from hPSCs, extensive characterization has revealed that they have immature cardiac properties. This has raised potential concerns over their usefulness for many applications and has led to the pursuit of driving maturation of hPSC-cardiomyocytes. Currently, the best approach for driving maturity is the use of tissue engineering to generate highly functional three-dimensional heart tissue. Although we have made significant progress in this area, we have still not generated heart tissue that fully recapitulates all the properties of an adult heart. Deciphering the processes driving cardiomyocyte maturation will be instrumental in uncovering the mechanisms that govern optimal heart function and identifying new therapeutic targets for heart disease.

Congenital heart defects can be caused by mutations in genes that guide cardiac lineage formation. Here, we show deletion of NKX2-5, a critical component of the cardiac gene regulatory network, in human embryonic stem cells (hESCs), results in impaired cardiomyogenesis, failure to activate VCAM1 and to downregulate the progenitor marker PDGFRα. Furthermore, NKX2-5 null cardiomyocytes have abnormal physiology, with asynchronous contractions and altered action potentials. Molecular profiling and genetic rescue experiments demonstrate that the bHLH protein HEY2 is a key mediator of NKX2-5 function during human cardiomyogenesis. These findings identify HEY2 as a novel component of the NKX2-5 cardiac transcriptional network, providing tangible evidence that hESC models can decipher the complex pathways that regulate early stage human heart development. These data provide a human context for the evaluation of pathogenic mutations in congenital heart disease., A gene regulatory network, including the transcription factor Nkx2-5, regulates cardiac development. Here, the authors show that on deletion of NKX2-5 from human embryonic stem cells, there is impaired cardiomyogenesis and changes in action potentials, and that this is regulated via HEY2.

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.

Regenerative medicine aims to replace injured tissues to restore normal physiological function. One possibility for achieving this goal is to activate or enhance endogenous regenerative pathways. Therefore, human tissue regeneration models may be useful tools for the discovery and development of novel regenerative therapeutics. In this chapter, we describe methods for the generation of three-dimensional bioengineered striated muscle in vitro and a cryoinjury model that can be applied to these tissues. This technique enables mechanistic in vitro analysis of the endogenous regenerative response of human striated muscle to injury, which is not possible using other in vivo approaches.

Background: The Institute for Urban Indigenous Health believes that continuous quality improvement (CQI) contributes to the delivery of high-quality care, thereby improving health outcomes for Aboriginal and Torres Strait Islander people. The opening of a new health service in 2015 provided an opportunity to implement best practice CQI strategies and apply them to a regional influenza vaccination campaign. Objective: The aim of this project was to implement an evidence-based CQI process within one Aboriginal Community Controlled Health Service in South East Queensland and use staff engagement as a measure of success. Method: A CQI tool was selected from the Joanna Briggs Institute Practical Application of Clinical Evidence System (PACES) to be implemented in the study site. The study site was a newly established Aboriginal and Torres Strait Islander Community Controlled Health Service located in the northern suburbs of Brisbane. This project used the evidence-based information collected in PACES to develop a set of questions related to known variables resulting in proven CQI uptake. A pre implementation clinical audit, education and self-directed learning, using the Plan Do Study Act framework, included a total of seven staff and was conducted in April 2015. A post implementation audit was conducted in July 2015. Results: There were a total of 11 pre- and post-survey respondents which included representation from most of the clinical team and medical administration. The results of the pre implementation audit identified a number of possible areas to improve engagement with the CQI process including staff training and support, understanding CQI and its impacts on individual work areas, understanding clinical data extraction, clinical indicator benchmarking, strong internal leadership and having an external data extractor. There were improvements to all audit criteria in the post-survey, for example, knowledge regarding the importance of CQI activity, attendance at education and training sessions on CQI, active involvement with CQI activity and a multidisciplinary team approach to problem solving within the CQI process. Conclusion: The study found that the implementation of regular, formally organized CQI strategies does have an immediate impact on clinical practice, in this case, by increasing staff awareness regarding the uptake of influenza vaccination against regional targets. The Plan Do Study Act cycle is an efficient tool to record and monitor the change and to guide discussions. For the CQI process to be effective, continued education and training on data interpretation is pivotal to improve staff confidence to engage in regular data discussions, and this should be incorporated into all future CQI sessions.

The adult human heart possesses a limited regenerative potential following an ischemic event, and undergoes a number of pathological changes in response to injury. While cardiac regeneration has been documented in zebrafish and neonatal mouse hearts, it is currently unknown whether the immature human heart is capable of undergoing complete regeneration. Combined progress in pluripotent stem cell differentiation and tissue engineering has facilitated the development of human cardiac organoids (hCO), which resemble fetal heart tissue and can be used to address this important knowledge gap. This study aimed to characterise the regenerative capacity of immature human heart tissue in response to injury. Following cryoinjury with a dry ice probe, hCO exhibited an endogenous regenerative response with full functional recovery by two weeks following acute injury. Cardiac functional recovery occurred in the absence of pathological fibrosis or cardiomyocyte hypertrophy. Consistent with regenerative organisms and neonatal human hearts, there was a high basal level of cardiomyocyte proliferation, which may be responsible for the regenerative capacity of the hCO. This study suggests that immature human heart tissue has an intrinsic capacity to regenerate.

We have previously developed a high-throughput bioengineered human cardiac organoid (hCO) platform, which provides functional contractile tissue with biological properties similar to native heart tissue, including mature, cell-cycle-arrested cardiomyocytes. In this study, we perform functional screening of 105 small molecules with pro-regenerative potential. Our findings reveal surprising discordance between our hCO system and traditional 2D assays. In addition, functional analyses uncovered detrimental effects of many hit compounds. Two pro-proliferative small molecules without detrimental impacts on cardiac function were identified. High-throughput proteomics in hCO revealed synergistic activation of the mevalonate pathway and a cell-cycle network by the pro-proliferative compounds. Cell-cycle reentry in hCO and in vivo required the mevalonate pathway as inhibition of the mevalonate pathway with a statin attenuated pro-proliferative effects. This study highlights the utility of human cardiac organoids for pro-regenerative drug development, including identification of underlying biological mechanisms and minimization of adverse side effects.

Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways, however, it remains unclear as to what extent these pathways can be exploited, either individually or in combination, in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro, but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways, we developed a high-density microbioreactor array (HDMA) – a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt, Hedgehog, IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation, inducing cell cycle activity marked by Ki67, and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration.