Gßγ subunits colocalize with RNA polymerase II and regulate transcription in cardiac fibroblasts.

Gßγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gßγ signaling, we investigated the functional role of Gßγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gß1γ dimer in the fibrotic response. Depletion of Gß1 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 Gß1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gßγ with transcription complexes. Together, our findings suggest that Gß1γ 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 Gßγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gßγ signaling in other cell types.

Tools for drug discovery and disease modeling- the future is upon us.

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.

Measuring hypertrophy in neonatal rat primary cardiomyocytes and human iPSC-derived cardiomyocytes.

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.

Conformational biosensors reveal allosteric interactions between heterodimeric AT1 angiotensin and prostaglandin F2α receptors.

G protein-coupled receptors (GPCRs) are conformationally dynamic proteins transmitting ligand-encoded signals in multiple ways. This transmission is highly complex and achieved through induction of distinct GPCR conformations, which preferentially drive specific receptor-mediated signaling events. This conformational capacity can be further enlarged via allosteric effects between dimers, warranting further study of these effects. Using GPCR conformation-sensitive biosensors, we investigated allosterically induced conformational changes in the recently reported F prostanoid (FP)/angiotensin II type 1 receptor (AT1R) heterodimer. Ligand occupancy of the AT1R induced distinct conformational changes in FP compared with those driven by PGF2α in bioluminescence resonance energy transfer (BRET)-based FP biosensors engineered with Renilla luciferase (RLuc) as an energy donor in the C-tail and fluorescein arsenical hairpin binder (FlAsH)-labeled acceptors at different positions in the intracellular loops. We also found that this allosteric communication is mediated through Gαq and may also involve proximal (phospholipase C) but not distal (protein kinase C) signaling partners. Interestingly, ß-arrestin-biased AT1R agonists could also transmit a Gαq-dependent signal to FP without activation of downstream Gαq signaling. This transmission of information was specific to the AT1R/FP complex, as activation of Gαq by the oxytocin receptor did not recapitulate the same phenomenon. Finally, information flow was asymmetric in the sense that FP activation had negligible effects on AT1R-based conformational biosensors. The identification of partner-induced GPCR conformations may help identify novel allosteric effects when investigating multiprotein receptor signaling complexes.

Comparing the signaling and transcriptome profiling landscapes of human iPSC-derived and primary rat neonatal cardiomyocytes.

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.

Measuring Single-Cell Calcium Dynamics Using a Myofilament-Localized Optical Biosensor in hiPSC-CMs Derived from DCM Patients.

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.

Biosensor-based profiling to track cellular signalling in patient-derived models of dilated cardiomyopathy.

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.

IBD-associated G protein-coupled receptor 65 variant compromises signalling and impairs key functions involved in inflammation.

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-1ß 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.

Exploring functional consequences of GPCR oligomerization requires a different lens.

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.

Combining Conformational Profiling of GPCRs with CRISPR/Cas9 Gene Editing Approaches.

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.

Combining Optical Approaches with Human Inducible Pluripotent Stem Cells in G Protein-Coupled Receptor Drug Screening and Development.

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.

Receptor- and cellular compartment-specific activation of the cAMP/PKA pathway by α1-adrenergic and ETA endothelin receptors.

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.

Distinct Conformational Dynamics of Three G Protein-Coupled Receptors Measured Using FlAsH-BRET Biosensors.

A number of studies have profiled G protein-coupled receptor (GPCR) conformation using fluorescent biaresenical hairpin binders (FlAsH) as acceptors for BRET or FRET. These conformation-sensitive biosensors allow reporting of movements occurring on the intracellular surface of a receptor to investigate mechanisms of receptor activation and function. Here, we generated eight FlAsH-BRET-based biosensors within the sequence of the ß2-adrenergic receptor (ß2AR) and compared agonist-induced responses to the angiotensin II receptor type I (AT1R) and the prostaglandin F2α receptor (FP). Although all three receptors had FlAsH-binding sequences engineered into the third intracellular loops and carboxyl-terminal domain, both the magnitude and kinetics of the BRET responses to ligand were receptor-specific. Biosensors in ICL3 of both the AT1R and FP responded robustly when stimulated with their respective full agonists as opposed to the ß2AR where responses in the third intracellular loop were weak and transient when engaged by isoproterenol. C-tail sensors responses were more robust in the ß2AR and AT1R but not in FP. Even though GPCRs share the heptahelical topology and are expressed in the same cellular background, different receptors have unique conformational fingerprints.