Non-ischemic or dilated cardiomyopathy (DCM) is characterized by abnormal enlargement of the left ventricle, compromising the ability of the heart to pump blood to the body. All patients with DCM are offered the same treatment regimen regardless of individual differences, with highly variable results on disease progression. Some patients fully recover cardiac function, while others continue to deteriorate, requiring heart replacement therapy or palliation. Incomplete molecular knowledge of dilated cardiomyopathy pathophysiology poses challenges for discovery of new therapeutic agents. To address this, we use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to assess individual molecular signalling and functional signatures in patients with DCM. Using blood samples from two healthy controls and two patients with DCM, we generated and validated iPSC lines, then differentiated them into cardiomyocytes. Cellular signalling was assessed in each iPSC-CM line after treatment with several disease-relevant G protein-coupled receptor (GPCR)-targeting ligands, measured using nuclear and cytosolic PKA and ERK biosensors at single cell resolution. Differences in functional properties such as calcium handling, contractility, and electrophysiology revealed additional features altered in patients with DCM. We have now established a pipeline to uncover patient-specific molecular mechanisms and disease phenotypes as a pathway to the development of personalized treatment for DCM. One Sentence Summary : Building a pipeline for bench-to-bedside study of DCM

Synchronized contractions of cardiomyocytes within the heart are tightly coupled to electrical stimulation known as excitation-contraction coupling. Calcium plays a key role in this process and dysregulated calcium handling can significantly impair cardiac function and lead to the development of cardiomyopathies and heart failure. Here, we describe a method and analytical technique to study myofilament-localized calcium signaling using the intensity-based fluorescent biosensor, RGECO-TnT. Dilated cardiomyopathy is a heart muscle disease that negatively impacts the heart’s contractile function following dilatation of the left ventricle. We demonstrate how this biosensor can be used to characterize 2D hiPSC-CMs monolayers generated from a healthy control subject compared to two patients diagnosed with dilated cardiomyopathy. Lastly, we provide a step-by-step guide for single-cell data analysis and describe a custom Transient Analysis application, specifically designed to quantify features of calcium transients. All in all, we explain how this analytical approach can be applied to phenotype hiPSC-CM behaviours and stratify patient responses to identify perturbations in calcium signaling.

ID 14979 Poster Board 301 Objective: Persistent inflammation drives the pathogenesis and progression of atrial fibrillation (AF), but pharmacological agents targeting inflammatory pathways in AF remain under-explored as therapeutics. NLRP3 inflammasome signalling is activated in atrial cardiomyocytes (CMs) from patients with AF, highlighting the importance of pro-inflammatory mechanisms in non-immune cells toward AF pathogenesis. Specialized Pro-resolving lipid Mediators (SPMs) derived from N-3 and N-6 polyunsaturated fatty acids (PUFAs) reduce inflammation by promoting non-immunosuppressive inflammatory resolution via their respective G protein-coupled receptors (GPCRs). LipoxinA4 (LxA4), an N-6 PUFA-derived SPM, signals via the formyl peptide receptor-2 (FPR2). Downstream signalling mechanisms are well-established in immune cells during acute inflammatory injury. This study investigates CM-specific inflammatory signalling in AF and the role of LxA4-FPR2 signal transduction to attenuate chronic inflammation and AF pathogenesis. Hypothesis: LxA4 attenuates cardiac electrical and structural remodeling in AF by FPR2-mediated signalling, which targets the NLRP3 inflammasome in atrial cardiomyocytes. Methods: Adult dogs underwent implantation of a pacemaker lead into the right atrial appendage and were atrial tachypaced at 600 beats per minute (cAF) or remained in sinus rhythm for 3 weeks. Hearts from a separate cohort of healthy adult dogs were enzymatically digested by Langendorff perfusion to isolate left atrial CMs. Isolated CMs were paced in vitro using the IonOptix C-Pace100-culture pacer at 1Hz or 3Hz for 24 hours in parallel. Assessment of NLRP3 inflammasome, inflammatory cytokine levels, and FPR2 expression was done by western blotting and qPCR. To investigate LxA4-FPR2 downstream signalling mechanisms, iPSCs were generated from healthy adult blood cells and differentiated into iPSC-CMs. GPCR transcript levels were assessed via bulk RNA sequencing. iPSC-CMs were transfected with the ExRai-AKAR2-NLS biosensor to detect nuclear PKA activity, subsequently treated with increasing concentrations of LxA4, and imaged using an Opera Phenix high content screening system. Results: FPR2 expression was significantly reduced in whole atrial tissue from cAF canines, while expression was unchanged in CMs paced at 3Hz. NLRP3 inflammasome levels and the inflammatory cytokine, IL-1ss, were increased in paced CMs indicating a critical role of the inflammasome pathway in arrhythmogenesis. Transcripts for FPR2 and other SPM GPCRs were not expressed in iPSC-CMs likely due to their immature phenotype. LxA4 treatment of iPSC-CMs does not influence nuclear PKA activity in the absence of FRP2, indicating a receptor-dependent activity of LxA4. Conclusions and Future Directions: CM-specific NLRP3 inflammasome signaling and FPR2 expression are disrupted in models of AF. Preliminary data suggests that LxA4 does not affect downstream PKA activity in iPSC-CMs in the absence of its receptor, FPR2. Attenuation of the NLRP3 inflammasome pathway via LxA4-FPR2 signalling mechanisms remains to be assessed in our AF models and iPSC-CMs. Funding: Canadian Institutes of Health Research and the Courtois Foundation

The inaccessibility of human cardiomyocytes significantly hindered years of cardiovascular research efforts. To overcome these limitations, non-human cell sources were used as proxies to study heart function and associated diseases. Rodent models became increasingly acceptable surrogates to model the human heart either in vivo or through in vitro cultures. More recently, due to concerns regarding animal to human translation, including cross-species differences, the use of human iPSC-derived cardiomyocytes presented a renewed opportunity. Here, we conducted a comparative study, assessing cellular signaling through cardiac G protein-coupled receptors (GPCRs) in rat neonatal cardiomyocytes (RNCMs) and human induced pluripotent stem cell-derived cardiomyocytes. Genetically encoded biosensors were used to explore GPCR-mediated nuclear protein kinase A (PKA) and extracellular signal-regulated kinase 1/ 2 (ERK1/2) activities in both cardiomyocyte populations. To increase data granularity, a single-cell analytical approach was conducted. Using automated high content microscopy, our analyses of nuclear PKA and ERK1/2 signaling revealed distinct response clusters in rat and human cardiomyocytes. In line with this, bulk RNA-seq revealed key differences in the expression patterns of GPCRs, G proteins and downstream effector expression levels. Our study demonstrates that human stem cell-derived models of the cardiomyocyte offer distinct advantages for understanding cellular signaling in the heart.

Gil gamma subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gil gamma signaling, we investigated the functional role of Gil gamma signaling in the regu-lation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gil1 gamma dimer in the fibrotic response. Depletion of Gil1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gil1 colo-calized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented as-sociation of Gil gamma with transcription complexes. Together, our findings suggest that Gil1 gamma is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gil gamma signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gil gamma signaling in other cell types.

BACKGROUND AND AIMS:Inflammatory bowel diseases (IBD) result in chronic inflammation of the gastrointestinal tract. Genetic studies have shown that the GPR65 gene, as well as its missense coding variant, GPR65*Ile231Leu, is associated with IBD. We aimed to define the signalling and biological pathways downstream of GPR65 activation and evaluate the impact of GPR65*231Leu on these. METHODS:We used HEK 293 cells stably expressing GPR65 and deficient for either Gαs, Gαq/11 or Gα12/13, to define GPR65 signalling pathways, IBD patient biopsies and a panel of human tissues, primary immune cells and cell lines to determine biologic context, and genetic modulation of human THP-1-derived macrophages to examine the impact of GPR65 in bacterial phagocytosis and NLRP3 inflammasome activation. RESULTS:We confirmed that GPR65 signals via the Gαs pathway, leading to cAMP accumulation. GPR65 can also signal via the Gα12/13 pathway leading to formation of stress fibers, actin remodeling and RhoA activation; all impaired by the IBD-associated GPR65*231Leu allele. Gene expression profiling revealed greater expression of GPR65 in biopsies from inflamed compared to non-inflamed tissues from IBD patients or control individuals, potentially explained by infiltration of inflammatory immune cells. Decreased GPR65 expression in THP-1-derived macrophages leads to impaired bacterial phagocytosis, increased NLRP3 inflammasome activation and IL-1β secretion in response to an inflammatory stimulus. CONCLUSIONS:We demonstrate that GPR65 exerts its effects through Gαs- and Gα12/13-mediated pathways, that the IBD-associated GPR65*231Leu allele has compromised interactions with Gα12/13 and that KD of GPR65 leads to impaired bacterial phagocytosis and increased inflammatory signalling via the NLRP3 inflammasome. This work identifies a target for development of small molecule therapies.

Dilated cardiomyopathies (DCM) represent a diverse group of cardiovascular diseases impacting the structure and function of the myocardium. To better treat these diseases, we need to understand the impact of such cardiomyopathies on critical signalling pathways that drive disease progression downstream of receptors we often target therapeutically. Our understanding of cellular signalling events has progressed substantially in the last few years, in large part due to the design, validation and use of biosensor-based approaches to studying such events in cells, tissues and in some cases, living animals. Another transformative development has been the use of human induced pluripotent stem cells (hiPSCs) to generate disease-relevant models from individual patients. We highlight the importance of going beyond monocellular cultures to incorporate the influence of paracrine signalling mediators. Finally, we discuss the recent coalition of these approaches in the context of DCM. We discuss recent work in generating patient-derived models of cardiomyopathies and the utility of using signalling biosensors to track disease progression and test potential therapeutic strategies that can be later used to inform treatment options in patients.

Although technical prowess in screening for drugs has increased dramatically with the development of high content imaging, resonance energy transfer- and intensiometric biosensors, translation into the clinic has stagnated and not all drugs work in all patients. This is likely due to 1) our rudimentary understanding of disease mechanisms, and 2) our increasing use of generic, cell-based screens which have moved us away from biologically relevant tissues, organs, and patients. Here, we focus on emerging tools to undertake screening and evaluate drug actions in models ranging from heterologous expression systems, primary cells, patient-derived induced pluripotent stem cells and organoids to in vivo models.

Dilated cardiomyopathy (DCM) is a cardiovascular condition that develops when the left ventricle of the heart enlarges, compromising its function and diminishing its capacity to pump oxygenated blood throughout the body. After patients are diagnosed with DCM, disease progression can lead to heart failure and the need for a heart transplantation. DCM is a complex disease where underlying causes can be idiopathic, genetic, or environmental. An incomplete molecular understanding of disease progression poses challenges for drug discovery efforts as effective therapeutics strategies remain elusive. Decades of research using primary cells or animal models have increased our understanding of DCM but has been hampered due to the inaccessibility of human cardiomyocytes, to model cardiac disease, in vitro , in a dish. Here, our goal is to leverage patient-derived hiPSC-CMs and to combine them with biosensors to understand how cellular signalling is altered in DCM. With high sensitivity and versatility, optical biosensors represent the ideal tools to dissect the molecular determinants of cardiovascular disease, in an unbiased manner and in real-time at the level of single cells. By characterizing the pathobiology of dilated cardiomyopathy in a patient-specific manner using high content biosensor-based assays, we aim to uncover personalized mechanisms for the occurrence and development of DCM and as a pathway to development of personalized therapeutics.

Historically, cellular context was never an issue in drug development as the focus of such efforts was on animal models and tissue explants in relevant models of disease. The switch to heterologous systems, while simplifying drug discovery and increasing throughput, made many assumptions about its translatability to more relevant contexts such as primary cells or in vivo models of disease. To achieve personalized and precision medicine in a real sense, drug discovery and disease modeling will ultimately need to be done on a patient-specific basis. Here, we focus of induced pluripotent stem cells (iPSCs) as a means to develop 2D and 3D models to understand cardiovascular and neuronal disorders and to use these models for purposes of drug discovery. The combination of modern screening technologies for drug discovery combined with disease modeling in iPSCs forms the basis of a nascent approach to patient-based drug discovery.

In the heart, left ventricular hypertrophy is initially an adaptive mechanism that increases wall thickness to preserve normal cardiac output and function in the face of coronary artery disease or hypertension. Cardiac hypertrophy develops in response to pressure and volume overload but can also be seen in inherited cardiomyopathies. As the wall thickens, it becomes stiffer impairing the distribution of oxygenated blood to the rest of the body. With complex cellular signalling and transcriptional networks involved in the establishment of the hypertrophic state, several model systems have been developed to better understand the molecular drivers of disease. Immortalized cardiomyocyte cell lines, primary rodent and larger animal models have all helped understand the pathological mechanisms underlying cardiac hypertrophy. Induced pluripotent stem cell-derived cardiomyocytes are also used and have the additional benefit of providing access to human samples with direct disease relevance as when generated from patients suffering from hypertrophic cardiomyopathies. Here, we briefly review in vitro and in vivo model systems that have been used to model hypertrophy and provide detailed methods to isolate primary neonatal rat cardiomyocytes as well as to generate cardiomyocytes from human iPSCs. We also describe how to model hypertrophy in a "dish" using gene expression analysis and immunofluorescence combined with automated high-content imaging.

Drug development was historically started by targeting protein active sites as means to pharmacologically modulate the functional properties of the target. However, with high attrition rates, pharmacologists and medicinal chemists must begin thinking outside the box more earnestly when designing new drugs. Such thinking has created an impetus toward the discovery of “biased” or “allosteric” modulators to fine-tune activity, selecting for a desired therapeutic profile. This has become a sought-after approach for the therapeutic targeting of G protein-coupled receptors (GPCRs). Structure-based studies have greatly increased our mechanistic understanding of GPCR activation in large part due to the use of single domain antibodies (or nanobodies). Even if nanobodies were initially developed to assist in receptor stabilization for structure determination of GPCRs, they have now proven to be more than simple chaperones for crystallization. Nanobodies have helped elucidate key features of GPCR biological responses, as they also behave are allosteric regulators of GPCR pharmacology. For many years, optical biosensors relying on resonance energy transfer have conventionally been used to quantify allosteric regulation. Yet, the transformation of nanobodies into biosensors, sensing distinct receptor conformation or activation states, has generated unprecedented knowledge of GPCR biology and signal transduction. Further, peptidomimetic ligands or pepducins generated from the primary and secondary structures of GPCRs have also been used as key allosteric tools to understand and drive GPCR signaling. In this review, we discuss how intracellular allosteric modulators, such as intracellular ions, and tool compounds, such as pepducins and nanobodies, have informed our understanding of G protein-coupled receptors from structural studies, to the generation of receptor conformational and signaling signatures.

As the largest family of cell surface receptors, G protein-coupled receptors (GPCRs) represent an important strategic class of therapeutic targets. Attaining a clearer perspective of how such signaling complexes set molecular events in motion could have significant impact on our understanding and treatment of human diseases. As such, many experimental approaches have set out to better understand signaling networks associated with individual receptors to understand signaling architectures and their relationship to signaling outcomes. However, designing in vitro assays aimed at addressing signaling events downstream of single GPCRs must also take into account their propensity to form homo- and heterooligomeric complexes. In the context of GPCR oligomers, physical interactions with a partner protein can have a number of potential consequences, which we will explore in this review. We will also discuss methods used to identify putative dimer partners as well as the various techniques used to study the functional consequences of such complex formation. Since the full functional significance and physiological relevance of GPCR oligomers remains incompletely understood, owing in part to technical limitations, new tools to elucidate molecular mechanisms underlying allosteric co-regulation occurring between two GPCRs are required. Accordingly, using the example of the FP/AT1R heterodimer, we discuss the potential of the FlAsH-BRET approach as a simple tool to reveal how allosteric information is transmitted via conformational rearrangements within putative GPCR complexes and as a means to deorphanize receptors.

Cardiovascular homeostasis is tightly regulated by numerous neurohormonal mediators such as the renin-angiotensin system which plays an important role in the maintenance of blood pressure. Central to this system is the peptide hormone angiotensin II (Ang II) whose signals are transduced via the AT1 receptor (AT1R), an important member of the superfamily of G protein-coupled receptors (GPCRs). Ang II binding results in receptor activation characterized by structural re-arrangements within the receptor structure and the subsequent activation of its cognate G protein partners. GPCRs are allosteric in nature and their biological activity is highly dependent on the cell context in which they are expressed1. Changes in the cellular background such as the differential availability of G proteins and effector molecules including putative dimer partners can affect receptor conformation and function. As such, we are interested in understanding how AT1R conformation and signaling are modulated by the cell context in which it is expressed1. In the past, studies that aimed at understanding signaling downstream of GPCRs mostly relied on heterologous expression systems such as HEK 293 cells because of their ease of culture. Such studies led to a ‘one size fits all’ notion that our findings could be reasonably extrapolated to guide drug discovery platforms relevant for human disease. However, it is clear that with the high rate of drug attrition, we need more physiologically relevant cellular models for studies of molecular signal transduction events to be translatable. With this in mind, we are generating iPSCs that stably express a panel of conformation-sensitive biosensors that reliably report on the conformational changes in the AT1R1,2. Our biosensors use resonance energy transfer between a bioluminescent donor and a fluorescent acceptor (FlAsH) where agonist-mediated conformational changes can be recorded1. Here, we will investigate how the conformation of the AT1R changes when expressed in AT1R-relevant cell types such as iPSC-derived cardiomyocytes and vascular smooth muscle cells. We will investigate how our conformational profiles differ in different iPSC-derived cell types in response to AT1R-specific agonists. Our goal is to gain a better mechanistic understanding of how cells are differentially wired leading to cell-specific conformational and signaling responses. We hope our results can guide rational drug design to better target the AT1R and other GPCRs. 1Devost D., et al (2017). Journal of Biological Chemistry, jbc-M116. 2Pei Y., et al (2015). Scientific reports, 5, 9205.

Ligand-biased signaling could have a significant impact on drug discovery programs. As such, many approaches to screening now target a larger section of the signaling responses downstream of an individual G protein-coupled receptor (GPCR). Biosensor-based platforms have been developed to capture signaling signatures. Despite the ability to use such signaling signatures, they may still be particular to an individual cell type and thus such platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. We have developed a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin biarsenical dyes engineered into different positions into the receptor primary structure. Here, we discuss how these experiments can be conducted and combined with CRISPR/Cas9 genome editing to assess specific G protein-dependent and -independent events.

The signalling functions of many G protein-coupled receptors (GPCRs) expressed in the myocardium are incompletely understood. Among these are the endothelin receptor (ETR) family and α1-adrenergic receptor (α1-AR), which are thought to couple to the G protein Gαq. In this study, we used transcriptome analysis to compare the signalling networks downstream of these receptors in primary neonatal rat cardiomyocytes. This analysis indicated increased expression of target genes of cAMP responsive element modulator (CREM) after 24 h treatment with the α1-AR agonist phenylephrine, but not the ETR agonist endothelin-1, suggesting a specific role for the α1-AR in promoting cAMP production in cardiomyocytes. To validate the difference observed between these two GPCRs, we used heterologous expression of the receptors and genetically encoded biosensors in HEK 293 cell lines. We validated that both α1A- and α1B-AR subtypes were able to lead to the accumulation of cAMP in response to phenylephrine in both the nucleus and cytoplasm in a Gαs-dependent manner. However, the ETR subtype ETA did not affect cAMP levels in either compartment. All three receptors were coupled to Gαq signalling as expected. Further, we showed that activation of PKA in different compartments was α1-AR subtype specific, with α1B-AR able to activate PKA in the cytoplasm and nucleus and α1A-AR only able to in the nucleus. We provide evidence for a pathway downstream of the α1-AR, and show that distinct pools of a receptor lead to differential activation of downstream effector proteins dependent on their cellular compartment.

Functional selectivity or biased agonism describes ligand-specific activation of particular downstream signaling pathways following drug treatment. This phenomenon may be exploited for the development of drugs with increased target selectivity and consequently better safety profiles. Analyzing bias at the level of signal transduction pathways is challenging due to cell type-dependent differences in the expression or activity of downstream effector proteins. Bioluminescence resonance energy transfer (BRET) has previously been used to characterize biased agonism at the level of the receptor using fluorescent biarsenical hairpin (FlAsH) binders as energy acceptors. By walking the FlAsH binding tetracysteine tag into different positions within the intracellular loops and carboxyl terminus of the 5-HT2A receptor along with a C-terminally fused Renilla luciferase, we generated a panel of seven conformation-sensitive biosensors that were able to capture conformational information in response to agonist. These FlAsH BRET-based biosensors were expressed in HEK 293 cells but also in the more relevant N-2A cells, a neuronal-like cell line where 5-HT2A receptor biology can be captured. These biosensors were first validated for cell surface expression and normal signaling phenotypes, followed by conformational analysis in response to the full agonist 5-HT. These results demonstrate a potentially simple tool for drug discovery once conformational profiles are correlated with their downstream signaling pathways.

Drug discovery for G protein-coupled receptors (GPCRs) stands at an interesting juncture. Screening programs are slowly moving away from model heterologous cell systems such as human embryonic kidney (HEK) 293 cells to more relevant cellular, tissue and whole animal platforms. Investigators are now developing analytical approaches as means to undertake different aspects of drug discovery by scaling into increasingly more relevant models all the way down to the single cell level. Such approaches include cellular, tissue slice and whole animal models where biosensors that track signaling events and receptor conformational profiles can be used. Here, we review aspects of biosensor-based imaging approaches that might be used in inducible pluripotent stem cell (iPSC) and organoid models, and focus on how such models must be characterized in order to apply them in drug screening.

Drug discovery for G protein-coupled receptors (GPCRs) stands at an interesting juncture. Screening programs are slowly moving away from model heterologous cell systems such as human embryonic kidney (HEK) 293 cells to more relevant cellular, tissue and whole animal platforms. Investigators are now developing analytical approaches as means to undertake different aspects of drug discovery by scaling into increasingly more relevant models all the way down to the single cell level. Such approaches include cellular, tissue slice and whole animal models where biosensors that track signaling events and receptor conformational profiles can be used. Here, we review aspects of biosensor-based imaging approaches that might be used in inducible pluripotent stem cell (iPSC) and organoid models, and focus on how such models must be characterized in order to apply them in drug screening.