MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo.

RATIONALE: There are several methods to measure cardiomyocyte and muscle contraction, but these require customized hardware, expensive apparatus, and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming, and only specialist researchers can quantify data. OBJECTIVE: Here, we describe and validate an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes, and pharmacological responses. METHODS AND RESULTS: MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in (1) 1-dimensional in vitro models, such as isolated adult and human pluripotent stem cell-derived cardiomyocytes; (2) 2-dimensional in vitro models, such as beating cardiomyocyte monolayers or small clusters of human pluripotent stem cell-derived cardiomyocytes; (3) 3-dimensional multicellular in vitro or in vivo contractile tissues, such as cardiac "organoids," engineered heart tissues, and zebrafish and human hearts. MUSCLEMOTION was effective under different recording conditions (bright-field microscopy with simultaneous patch-clamp recording, phase contrast microscopy, and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement, such as optical flow, post deflection, edge-detection systems, or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. CONCLUSIONS: Using a single open-source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell, animal, and human models.

GNB5 Mutations Cause an Autosomal-Recessive Multisystem Syndrome with Sinus Bradycardia and Cognitive Disability.

GNB5 encodes the G protein ß subunit 5 and is involved in inhibitory G protein signaling. Here, we report mutations in GNB5 that are associated with heart-rate disturbance, eye disease, intellectual disability, gastric problems, hypotonia, and seizures in nine individuals from six families. We observed an association between the nature of the variants and clinical severity; individuals with loss-of-function alleles had more severe symptoms, including substantial developmental delay, speech defects, severe hypotonia, pathological gastro-esophageal reflux, retinal disease, and sinus-node dysfunction, whereas related heterozygotes harboring missense variants presented with a clinically milder phenotype. Zebrafish gnb5 knockouts recapitulated the phenotypic spectrum of affected individuals, including cardiac, neurological, and ophthalmological abnormalities, supporting a direct role of GNB5 in the control of heart rate, hypotonia, and vision.

Gß3 is required for normal light ON responses and synaptic maintenance.

Heterotrimeric G-proteins, comprising Gα and Gßγ subunits, couple metabotropic receptors to various downstream effectors and contribute to assembling and trafficking receptor-based signaling complexes. A G-protein ß subunit, Gß(3), plays a critical role in several physiological processes, as a polymorphism in its gene is associated with a risk factor for several disorders. Retinal ON bipolar cells express Gß(3), and they provide an excellent system to study its role. In the ON bipolar cells, mGluR6 inverts the photoreceptor's signal via a cascade in which glutamate released from photoreceptors closes the TRPM1 channel. This cascade is essential for vision since deficiencies in its proteins lead to complete congenital stationary night blindness. Here we report that Gß(3) participates in the G-protein heterotrimer that couples mGluR6 to TRPM1. Gß(3) deletion in mouse greatly reduces the light response under both scotopic and photopic conditions, but it does not eliminate it. In addition, Gß(3) deletion causes mislocalization and downregulation of most cascade elements and modulators. Furthermore, Gß(3) may play a role in synaptic maintenance since in its absence, the number of invaginating rod bipolar dendrites is greatly reduced, a deficit that was not observed at 3 weeks, the end of the developmental period.

Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells.

The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.

Rice transgenic plants with suppressed expression of the ß subunit of the heterotrimeric G protein.

The deficient mutant for the rice heterotrimeric G protein α subunit gene (RGA1), d1, showed dwarfism and set small seed due to a reduced cell number. Mutants for the rice heterotrimeric G protein ß subunit gene (RGB1) have not been isolated. To determine the functions of RGB1, transgenic rice plants with suppressed expression of RGB1 were studied using the RNAi method. RGB1 knock-down lines showed browning of the lamina joint regions and nodes and reduced fertility, but these abnormality were not observed in d1. Transgenic plants in which the G protein ß subunit was greatly decreased were not obtained, suggesting that the complete suppression of RGB1 mRNA may be lethal. In contrast, the d1 mutants, with complete loss of the G protein α subunit, were fertile and half the size of the WT. These studies suggest that RGB1 has different functions than RGA1.

Gß5-RGS complexes are gatekeepers of hyperactivity involved in control of multiple neurotransmitter systems.

RATIONALE AND OBJECTIVES: Our knowledge about genes involved in the control of basal motor activity that may contribute to the pathology of the hyperactivity disorders, e.g., attention deficit hyperactivity disorder (ADHD), is limited. Disruption of monoamine neurotransmitter signaling through G protein-coupled receptors (GPCR) is considered to be a major contributing factor to the etiology of the ADHD. Genetic association evidence and functional data suggest that regulators of G protein signaling proteins of the R7 family (R7 RGS) that form obligatory complexes with type 5 G protein beta subunit (Gß5) and negatively regulate signaling downstream from monoamine GPCRs may play a role in controlling hyperactivity. METHODS: To test this hypothesis, we conducted behavioral, pharmacological, and neurochemical studies using a genetic mouse model that lacked Gß5, a subunit essential for the expression of the entire R7 RGS family. RESULTS: Elimination of Gß5-RGS complexes led to a striking level of hyperactivity that far exceeds activity levels previously observed in animal models. This hyperactivity was accompanied by motor learning deficits and paradoxical behavioral sensitization to a novel environment. Neurochemical studies indicated that Gß5-RGS-deficient mice had higher sensitivity of inhibitory GPCR signaling and deficits in basal levels, release, and reuptake of dopamine. Surprisingly, pharmacological treatment with monoamine reuptake inhibitors failed to alter hyperactivity. In contrast, blockade of NMDA receptors reversed the expression of hyperactivity in Gß5-RGS-deficient mice. CONCLUSIONS: These findings establish that Gß5-RGS complexes are critical regulators of monoamine-NMDA receptor signaling cross-talk and link these complexes to disorders that manifest as hyperactivity, impaired learning, and motor dysfunctions.

Knockout of G protein ß5 impairs brain development and causes multiple neurologic abnormalities in mice.

Gß5 is a divergent member of the signal-transducing G protein ß subunit family encoded by GNB5 and expressed principally in brain and neuronal tissue. Among heterotrimeric Gß isoforms, Gß5 is unique in its ability to heterodimerize with members of the R7 subfamily of the regulator of G protein signaling proteins that contain G protein-γ like domains. Previous studies employing Gnb5 knockout (KO) mice have shown that Gß5 is an essential stabilizer of such regulator of G protein signaling proteins and regulates the deactivation of retinal phototransduction and the proper functioning of retinal bipolar cells. However, little is known of the function of Gß5 in the brain outside the visual system. We show here that mice lacking Gß5 have a markedly abnormal neurologic phenotype that includes impaired development, tiptoe-walking, motor learning and coordination deficiencies, and hyperactivity. We further show that Gß5-deficient mice have abnormalities of neuronal development in cerebellum and hippocampus. We find that the expression of both mRNA and protein from multiple neuronal genes is dysregulated in Gnb5 KO mice. Taken together with previous observations from Gnb5 KO mice, our findings suggest a model in which Gß5 regulates dendritic arborization and/or synapse formation during development, in part by effects on gene expression.

A Dictyostelium chalone uses G proteins to regulate proliferation.

BACKGROUND: Several studies have shown that organ size, and the proliferation of tumor metastases, may be regulated by negative feedback loops in which autocrine secreted factors called chalones inhibit proliferation. However, very little is known about chalones, and how cells sense them. We previously identified two secreted proteins, AprA and CfaD, which act as chalones in Dictyostelium. Cells lacking AprA or CfaD proliferate faster than wild-type cells, and adding recombinant AprA or CfaD to cells slows their proliferation. RESULTS: We show here that cells lacking the G protein components Galpha8, Galpha9, and Gbeta proliferate faster than wild-type cells despite secreting normal or high levels of AprA and CfaD. Compared with wild-type cells, the proliferation of galpha8-, galpha9- and gbeta- cells are only weakly inhibited by recombinant AprA (rAprA). Like AprA and CfaD, Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate of increase in mass and protein per nucleus), whereas Galpha9 inhibits both proliferation and growth. galpha8- cells show normal cell-surface binding of rAprA, whereas galpha9- and gbeta- cells have fewer cell-surface rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the synthesis or processing of the AprA receptor. Like other ligands that activate G proteins, rAprA induces the binding of [3H]GTP to membranes, and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced [3H]GTP binding and the GTPgammaS inhibition of rAprA binding require Galpha8 and Gbeta but not Galpha9. Like aprA- cells, galpha8- cells have reduced spore viability. CONCLUSION: This study shows that Galpha8 and Gbeta are part of the signal transduction pathway used by AprA to inhibit proliferation but not growth in Dictyostelium, whereas Galpha9 is part of a differealnt pathway that regulates both proliferation and growth, and that a chalone signal transduction pathway uses G proteins.

Targeting of RGS7/Gbeta5 to the dendritic tips of ON-bipolar cells is independent of its association with membrane anchor R7BP.

Complexes of regulator of G-protein signaling (RGS) proteins with G-protein beta5 (Gbeta5) subunits are essential components of signaling pathways that regulate the temporal characteristics of light-evoked responses in vertebrate retinal photoreceptors and ON-bipolar cells. Recent studies have found that RGS/Gbeta5 complexes bind to a new family of adapter proteins, R9AP (RGS9 anchor protein) and R7 family binding protein (R7BP), that in case of the RGS9/Gbeta5 complex were shown to determine its precise subcellular targeting to either the outer segment of photoreceptors or postsynaptic structures of striatal neurons, respectively. In this study, we establish that another trimeric complex consisting of RGS7, Gbeta5, and R7BP subunits is specifically targeted to the dendritic tips of retinal bipolar cells. However, examination of the mechanisms of complex targeting in vivo surprisingly revealed that the delivery of RGS7/Gbeta5 to the dendrites of ON-bipolar cells occurs independently of its association with R7BP. These findings provide a new mechanism for adapter-independent targeting of RGS/Gbeta5 complexes.

Postnatal induction and localization of R7BP, a membrane-anchoring protein for regulator of G protein signaling 7 family-Gbeta5 complexes in brain.

Members of the regulator of G protein signaling 7 (RGS7) (R7) family and Gbeta5 form obligate heterodimers that are expressed predominantly in the nervous system. R7-Gbeta5 heterodimers are GTPase-activating proteins (GAPs) specific for Gi/o-class Galpha subunits, which mediate phototransduction in retina and the action of many modulatory G protein-coupled receptors (GPCRs) in brain. Here we have focused on the R7-family binding protein (R7BP), a recently identified palmitoylated protein that can bind R7-Gbeta5 complexes and is hypothesized to control the intracellular localization and function of the resultant heterotrimeric complexes. We show that: 1) R7-Gbeta5 complexes are obligate binding partners for R7BP in brain because they co-immunoprecipitate and exhibit similar expression patterns. Furthermore, R7BP and R7 protein accumulation in vivo requires Gbeta5. 2) Expression of R7BP in Neuro2A cells at levels approximating those in brain recruits endogenous RGS7-Gbeta5 complexes to the plasma membrane. 3) R7BP immunoreactivity in brain concentrates in neuronal soma, dendrites, spines or unmyelinated axons, and is absent or low in glia, myelinated axons, or axon terminals. 4) RGS7-Gbeta5-R7BP complexes in brain extracts associate inefficiently with detergent-resistant lipid raft fractions with or without G protein activation. 5) R7BP and Gbeta5 protein levels are upregulated strikingly during the first 2-3 weeks of postnatal brain development. Accordingly, we suggest that R7-Gbeta5-R7BP complexes in the mouse or rat could regulate signaling by modulatory Gi/o-coupled GPCRs in the developing and adult nervous systems.

Gbeta5 is required for normal light responses and morphology of retinal ON-bipolar cells.

Gbeta5 exists as two splice variants, Gbeta5-S and Gbeta5-L, which interact with and stabilize the R7 members of the regulators of G-protein signaling (RGSs): RGS6, RGS7, RGS9, and RGS11. Although the role of Gbeta5-L and RGS9-1 is established in photoreceptors, the physiological functions of Gbeta5-S and other R7 RGS proteins remain unclear. We found that the electroretinogram of Gbeta5-/- mice lacks the b-wave component and that Gbeta5-S and RGS11 colocalize with Go alpha at the tips of the ON-bipolar cell dendrites. Unexpectedly, we found a significant reduction in the number of synaptic triads in the outer plexiform layer (OPL) of the Gbeta5-/- mice, which is evident at postnatal day 14. Transgenic expression of Gbeta5-L in rods failed to rescue the b-wave or the OPL defects. These results indicate that Gbeta5-S is indispensable for OPL integrity and normal light responses of the retina.

Over-expression of a truncated Arabidopsis thaliana heterotrimeric G protein gamma subunit results in a phenotype similar to alpha and beta subunit knockouts.

Heterotrimeric G proteins (G-proteins) are a diverse class of signal transducing proteins which have been implicated in a variety of important roles in plants. When G-proteins are activated, they dissociate into two functional subunits (alpha and the betagamma dimer) that effectively relay the signal to a multitude of effectors. In animal systems, the betagamma dimer is anchored to the plasma membrane by a prenyl group present in the gamma subunit and membrane localization has proven vital for heterotrimer function. A semi-dominant negative strategy was designed aiming to disrupt heterotrimer function in Arabidopsis thaliana (ecotype Columbia) plants by over-expressing a truncated gamma subunit lacking the isoprenylation motif (gamma()). Northern analysis shows that the levels of expression of the mutant gamma subunit in several transgenic lines (35S-gamma()) are orders of magnitude higher than that of the native subunits. In-depth characterization of the 35S-gamma() lines has been carried out, specifically focusing on a number of developmental characteristics and responses to several stimuli previously shown to be affected in alpha- and beta-deficient mutants. In all cases, the transgenic lines expressing the mutant gamma subunit behave in the same way as the alpha- and/or the beta-deficient mutants, albeit with reduced severity of the phenotype. Our data indicates that signaling from both functional subunits, alpha and the beta/gamma dimer, is disrupted in the transgenic plants. Even though physical association of the subunits has been previously reported, our research provides evidence of the functional association of alpha and beta with the gamma subunits in Arabidopsis, while also suggesting that plasma membrane localization may be critical for function of plant heterotrimeric G proteins.