Familial risk of vasospastic angina: a nationwide family study in Sweden.

OBJECTIVES: Vasospastic angina (VSA) is a complex coronary vasomotor disorder associated with an increased risk of myocardial infarction and sudden death. Despite considerable advances in understanding VSA pathophysiology, the interplay between genetic and environmental factors remains elusive. Accordingly, we aimed to determine the familial VSA risk among first-degree relatives of affected individuals. METHODS: A population-based multigenerational cohort study was conducted, including full-sibling pairs born to Swedish parents between 1932 and 2018. Register-based diagnoses were ascertained through linkage to the Swedish Multigeneration Register and National Patient Register. Incidence rate ratios (IRRs) and adjusted HRs were calculated for relatives of individuals with VSA compared with relatives of individuals without VSA. RESULTS: The total study population included 5 764 770 individuals. Overall, 3461 (0.06%) individuals (median age at disease onset 59 years, IQR: 63-76) were diagnosed with VSA. Of these, 2236 (64.61%) were women. The incidence rate of VSA for individuals with an affected sibling was 0.31 (95% CI: 0.24 to 0.42) per 1000 person-years compared with 0.04 (95% CI: 0.04 to 0.04) per 1000 person-years for those without an affected sibling, yielding an IRR of 7.58 (95% CI: 5.71 to 10.07). The risk of VSA for siblings with an affected sibling was significantly increased in the fully adjusted model (HR: 2.56; 95% CI: 1.73 to 3.79). No increased risk of VSA was observed in spouses of affected individuals (HR: 0.63; 95% CI: 0.19 to 2.09). CONCLUSIONS: In this nationwide family study, we identified high familial risk for VSA independent of shared environmental risk factors. Our findings indicate that VSA tends to cluster in families, emphasising the need to explore genetic and non-genetic factors that may contribute.

Familial risk of dilated and hypertrophic cardiomyopathy: a national family study in Sweden.

AIMS: This study aims to determine the familial incidence of dilated (DCM) and hypertrophic cardiomyopathy (HCM) in first-degree, second-degree, and third-degree relatives of affected individuals. METHODS AND RESULTS: In this population-based multigenerational cohort study, full-siblings, half-siblings, and cousin pairs born to Swedish parents between 1932 and 2015 were included, and register-based DCM and HCM diagnoses among relatives were ascertained. Adjusted odds ratios (ORs) for DCM and HCM were calculated for relatives of individuals with DCM and HCM compared with relatives of individuals without DCM and HCM for reference. Total study population included 6 334 979 subjects and consisted of 5 577 449 full-siblings, 1 321 414 half-siblings, and 3 952 137 cousins. Overall, 10 272 (0.16%) unique individuals were diagnosed with DCM and 3769 (0.06%) with HCM. Of these, 7716 (75.12%) and 2375 (63.01%) were males, respectively. Familial risk ORs for DCM were 5.35 [95% confidence intervals (CI): 4.85-5.90] for full-siblings, 2.68 (95% CI:1.86-3.87) for half-siblings, and 1.72 (95% CI:1.12-2.64) for cousins of affected individuals. The ORs for HCM were 42.44 (95% CI:37.66-47.82) for full-siblings, 32.70 (95% CI:21.32-50.15) for half-siblings, and 36.96 (95% CI:29.50-46.31) for cousins of affected individuals. In sex-stratified analysis, relatives of affected females were found more likely to be affected than were relatives of affected males, with stronger aggregation observed for HCM. CONCLUSIONS: Familial risk of HCM and DCM is high and associated with genetic resemblance, with strongest aggregations observed in relatives of affected females with HCM, whereas this association was distinctly attenuated for DCM. The finding of a Carter effect, more pronounced in HCM, suggests a multifactorial threshold model of inheritance.

Ultrasound imaging of apoptosis in tumor response: novel preclinical monitoring of photodynamic therapy effects.

High-frequency ultrasound is a novel method to detect apoptotic cell death based on changes in cell morphology that cause alterations in the viscoelastic and, consequently, the acoustic properties of cell ensembles and tissues. In this study, we evaluated the first preclinical tumor-based use of high-frequency ultrasound spectroscopy to noninvasively monitor tumor treatment by following xenograft malignant melanoma tumor responses to photodynamic therapy (PDT) in vivo. We observed a time-dependant increase in ultrasound backscatter variables after treatment. The observed increases in spectroscopic variables correlated with morphologic findings, indicating increases in apoptotic cell death, which peaked at 24 hours after PDT. We analyzed the changes in spectral slope and backscatter in relation to apoptosis and histologic variations in cell nuclear size. Changes in spectral slope strongly correlated with the changes in mean nuclear size over time, associated with apoptosis, after PDT (P < 0.05). At 48 hours, a decrease in ultrasound backscatter was observed, which could be explained by an increase in cell nuclear degradation. In summary, we show that high-frequency ultrasound spectroscopic variables can be used noninvasively to monitor response after treatment in a preclinical tumor cancer model. These findings provide a foundation for future investigations regarding the use of ultrasound to monitor and aid the customization of treatments noninvasively based on responses to specific interventions.

GAP1(IP4BP)/RASA3 mediates Galphai-induced inhibition of mitogen-activated protein kinase.

The dopamine D2S receptor (short isoform) couples to inhibitory Galphai/o proteins to inhibit thyrotropin-releasing hormone (TRH)-stimulated p42/p44 mitogen-activated protein kinase (ERK1/2) phosphorylation in GH4ZR7 rat pituitary cells, consistent with its actions to inhibit prolactin gene transcription and cell proliferation. However, the underlying mechanism is unclear. To identify novel Galphai effectors, yeast two-hybrid screening of a GH4ZR7 cDNA library was done using constitutively active Galphai3-Q204L, and multiple clones of the RasGAP cDNA GAP1(IP4BP)/RASA3 were identified. In yeast mating assay, RASA3 preferentially interacted with activated forms of Galphai/o/z proteins, but not with Galphas. A direct interaction was indicated by in vitro pull-down assay, in which S-His-RASA3 preferentially bound guanosine 5'-O-(gamma-thio)triphosphate-activated Galphai3 and Galphai2 compared with guanosine 5'-O-(beta-thio)diphosphate-inactivated proteins. Similarly, in co-immunoprecipitation studies in HEK-293 cells, FLAG-tagged RASA3 preferentially interacted with activated mutants of Galphai3 and Galphai2 compared with wild type proteins. In GH4ZR7 cells, co-immunoprecipitation studies of endogenous proteins demonstrated a Galphai3-RASA3 complex that was induced upon TRH/D2S receptor co-activation. To address RASA3 function in dopamine D2S receptor-induced inhibition of ERK1/2 activity, endogenous RASA3 protein expression was suppressed (70% knockdown) in GH4ZR7 cells stably transfected with full-length antisense cDNA of RASA3. The selected antisense clones had similar levels of dopamine D2S receptor binding and D2S-induced inhibition of cAMP formation compared with parental GH4ZR7 cells. In these clones, D2S-mediated inhibition of TRH-induced phospho-ERK1/2 was reversed by 70-80% compared with parental GH4ZR7 cells. Our results provide a novel mechanism for dopamine D2S-induced inhibition of ERK1/2 and indicate that RASA3 links Galphai proteins to inhibit Gq-induced Ras/ERK1/2 activation.

Differential signaling of dopamine-D2S and -D2L receptors to inhibit ERK1/2 phosphorylation.

Although they have distinct functions, the signaling of dopamine-D(2) receptor short and long isoforms (D(2)S and D(2)L) is virtually identical. We compared inhibitory regulation of extracellular signal-regulated kinases (ERK1/2) in GH4 pituitary cells separately transfected with these isoforms. Activation of rat or human dopamine-D(2)S, muscarinic or somatostatin receptors inhibited thyrotropin-releasing hormone-induced ERK1/2 phosphorylation, while the D(2)L receptor failed to inhibit this response. In order to address the structural basis for the differential signaling of D(2)S and D(2)L receptors, we examined the D(2)L-SS mutant, in which a protein kinase C (PKC) pseudosubstrate site that is present in the D(2)L but not D(2)S receptor was converted to a consensus PKC site. In transfected GH4 cells, the D(2)L-SS mutant inhibited thyrotropin-releasing hormone-induced ERK1/2 phosphorylation almost as strongly as the D(2)S receptor. A D(2)S-triple mutant that eliminates PKC sites involved in D(2)S receptor desensitization also inhibited ERK1/2 activation. Similarly, in striatal cultures, the D(2)-selective agonist quinpirole inhibited potassium-stimulated ERK1/2 phosphorylation, indicating the presence of this pathway in neurons. In conclusion, the D(2)S and D(2)L receptors differ in inhibitory signaling to ERK1/2 due to specific residues in the D(2)L receptor alternatively spliced domain, which may account for differences in their function in vivo.

Diacylglycerol and ceramide formation induced by dopamine D2S receptors via Gbeta gamma -subunits in Balb/c-3T3 cells.

Diacylglycerol (DAG) and ceramide are important second messengers affecting cell growth, differentiation, and apoptosis. Balb/c-3T3 fibroblast cells expressing dopamine-D2S (short) receptors (Balb-D2S cells) provide a model of G protein-mediated cell growth and transformation. In Balb-D2S cells, apomorphine (EC(50) = 10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at 1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicating G(i)/G(o) proteins. To address which G proteins are involved, Balb-D2S clones expressing individual PTX-insensitive Galpha(i) proteins were treated with PTX and tested for apomorphine-induced responses. Neither PTX-insensitive Galpha(i2) nor Galpha(i3) rescued D2S-induced DAG or ceramide formation. Both D2S-induced DAG and ceramide signals required Gbetagamma-subunits and were blocked by inhibitors of phospholipase C [1-(6-[([17beta]-3-methoxyestra-1,2,3[10]-trien- 17yl)amino]hexyl)-1H-pyrrole-2,5-dione (U-73122) and partially by D609]. The similar G protein specificity of D2S-induced calcium mobilization, DAG, and ceramide formation indicates a common Gbetagamma-dependent phospholipase C-mediated pathway. Both D2 agonists and ceramide specifically induced mitogen-activated protein kinase (ERK1/2), suggesting that ceramide mediates a novel pathway of D2S-induced ERK1/2 activation, leading to cell growth.

Growth hormone-induced diacylglycerol and ceramide formation via Galpha i3 and Gbeta gamma in GH4 pituitary cells. Potentiation by dopamine-D2 receptor activation.

Growth hormone (GH) secretion is regulated by indirect negative feedback mechanisms. To address whether GH has direct actions on pituitary cells, lipid signaling in GH(4)ZR(7) somatomammotroph cells was examined. GH (EC(50) = 5 nm) stimulated diacylglycerol (DAG) and ceramide formation in parallel by over 10-fold within 15 min and persisting for >3 h. GH-induced DAG/ceramide formation was blocked by pertussis toxin (PTX) implicating G(i)/G(o) proteins and was potentiated 1.5-fold by activation of G(i)/G(o)-coupled dopamine-D2S receptors, which had no effect alone. Following PTX pretreatment, only PTX-resistant Galpha(i)3, not Galpha(o) or Galpha(i)2, rescued GH-induced DAG/ceramide signaling. GH-induced DAG/ceramide formation was also blocked in cells expressing Gbetagamma blocker GRK-ct. In GH(4)ZR(7) cells, GH induced phosphorylation of JAK2 and STAT5, which was blocked by PTX and mimicked by ceramide analogue C2-ceramide or sphingomyelinase treatment to increase endogenous ceramide. We conclude that in GH(4) pituitary cells, GH induces formation of DAG/ceramide via a novel Galpha(i)3/Gbetagamma-dependent pathway. This novel pathway suggests a mechanism for autocrine feedback regulation by GH of pituitary function.

Dopamine-D2S receptor inhibition of calcium influx, adenylyl cyclase, and mitogen-activated protein kinase in pituitary cells: distinct Galpha and Gbetagamma requirements.

The G protein specificity of multiple signaling pathways of the dopamine-D2S (short form) receptor was investigated in GH4ZR7 lactotroph cells. Activation of the dopamine-D2S receptor inhibited forskolin-induced cAMP production, reduced BayK8644- activated calcium influx, and blocked TRH-mediated p42/p44 MAPK phosphorylation. These actions were blocked by pretreatment with pertussis toxin (PTX), indicating mediation by G(i/o) proteins. D2S stimulation also decreased TRH-induced MAPK/ERK kinase phosphorylation. TRH induced c-Raf but not B-Raf activation, and the D2S receptor inhibited both TRH-induced c-Raf and basal B-Raf kinase activity. After PTX treatment, D2S receptor signaling was rescued in cells stably transfected with individual PTX-insensitive Galpha mutants. Inhibition of adenylyl cyclase was partly rescued by Galpha(i)2 or Galpha(i)3, but Galpha(o) alone completely reconstituted D2S-mediated inhibition of BayK8644-induced L-type calcium channel activation. Galpha(o) and Galpha(i)3 were the main components involved in D2S-mediated p42/44 MAPK inhibition. In cells transfected with the carboxyl-terminal domain of G protein receptor kinase to inhibit Gbetagamma signaling, only D2S-mediated inhibition of calcium influx was blocked, but not inhibition of adenylyl cyclase or MAPK. These results indicate that the dopamine-D2S receptor couples to distinct G(i/o) proteins, depending on the pathway addressed, and suggest a novel Galpha(i)3/Galpha(o)-dependent inhibition of MAPK mediated by c-Raf and B-Raf-dependent inhibition of MAPK/ERK kinase.