The c-fms gene encodes the receptor for macrophage colony-stimulating factor (CSF-1). The gene is expressed selectively in the macrophage and trophoblast cell lineages. Previous studies have indicated that sequences in intron 2 control transcript elongation in tissue-specific and regulated expression of c-fms. In humans, an alternative promoter was implicated in expression of the gene in trophoblasts. We show that in mice, c-fmstranscripts in trophoblasts initiate from multiple points within the 2-kilobase (kb) region flanking the first coding exon. A reporter gene construct containing 3.5 kb of 5′ flanking sequence and the downstream intron 2 directed expression of enhanced green fluorescent protein (EGFP) to both trophoblasts and macrophages. EGFP was detected in trophoblasts from the earliest stage of implantation examined at embryonic day 7.5. During embryonic development, EGFP highlighted the large numbers of c-fms–positive macrophages, including those that originate from the yolk sac. In adult mice, EGFP location was consistent with known F4/80-positive macrophage populations, including Langerhans cells of the skin, and permitted convenient sorting of isolated tissue macrophages from disaggregated tissue. Expression of EGFP in transgenic mice was dependent on intron 2 as no lines with detectable EGFP expression were obtained where either all of intron 2 or a conserved enhancer element FIRE (theFms intronic regulatory element) was removed. We have therefore defined the elements required to generate myeloid- and trophoblast-specific transgenes as well as a model system for the study of mononuclear phagocyte development and function.

Calcium and Ca(2+)-dependent signals play a crucial role in sperm motility and mammalian fertilization, but the molecules and mechanisms underlying these Ca(2+)-dependent pathways are incompletely understood. Here we show that homozygous male mice with a targeted gene deletion of isoform 4 of the plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA), which is highly enriched in the sperm tail, are infertile due to severely impaired sperm motility. Furthermore, the PMCA inhibitor 5-(and-6)-carboxyeosin diacetate succinimidyl ester reduced sperm motility in wild-type animals, thus mimicking the effects of PMCA4 deficiency on sperm motility and supporting the hypothesis of a pivotal role of the PMCA4 on the regulation of sperm function and intracellular Ca(2+) levels.

The main role of the plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA) is in the removal of Ca2+ from the cytosol. Recently, we and others have suggested a new function for PMCA as a modulator of signal transduction pathways. This paper shows the physical interaction between PMCA (isoforms 1 and 4) and alpha-1 syntrophin and proposes a ternary complex of interaction between endogenous PMCA, alpha-1 syntrophin, and NOS-1 in cardiac cells. We have identified that the linker region between the pleckstrin homology 2 (PH2) and the syntrophin unique (SU) domains, corresponding to amino acids 399-447 of alpha-1 syntrophin, is crucial for interaction with PMCA1 and -4. The PH2 and the SU domains alone failed to interact with PMCA. The functionality of the interaction was demonstrated by investigating the inhibition of neuronal nitric-oxide synthase-1 (NOS-1); PMCA is a negative regulator of NOS-1-dependent NO production, and overexpression of alpha-1 syntrophin and PMCA4 resulted in strongly increased inhibition of NO production. Analysis of the expression levels of alpha-1 syntrophin protein in the heart, skeletal muscle, brain, uterus, kidney, or liver of PMCA4-/- mice, did not reveal any differences when compared with those found in the same tissues of wild-type mice. These results suggest that PMCA4 is tethered to the syntrophin complex as a regulator of NOS-1, but its absence does not cause collapse of the complex, contrary to what has been reported for other proteins within the complex, such as dystrophin. In conclusion, the present data demonstrate for the first time the localization of PMCA1b and -4b to the syntrophin.dystrophin complex in the heart and provide a specific molecular mechanism of interaction as well as functionality.

The brunt of cardiovascular disease (CVD) burden globally now resides within low- and middle-income countries, including Indonesia. However, little is known regarding cardiovascular health in Indonesia. This study aimed to estimate the prevalence of elevated CVD risk in a specific region of Indonesia. We conducted full household screening for cardiovascular risk factors among adults aged 40 years and older in 8 villages in Malang District, East Java Province, Indonesia, in 2016–2017. 10-year cardiovascular risk scores were calculated based on the World Health Organization/International Society of Hypertension’s region-specific charts that use age, sex, blood pressure, diabetes status and smoking behaviour. Among 22,093 participants, 6,455 (29.2%) had high cardiovascular risk, defined as the presence of coronary heart disease, stroke or other atherosclerotic disease; estimated 10-year CVD risk of ≥ 30%; or estimated 10-year CVD risk between 10% to 29% combined with a systolic blood pressure of > 140 mmHg. The prevalence of high CVD risk was greater in urban (31.6%, CI 30.7–32.5%) than in semi-urban (28.7%, CI 27.3–30.1%) and rural areas (26.2%, CI 25.2–27.2%). Only 11% and 1% of all the respondents with high CVD risk were on blood pressure lowering and statins treatment, respectively. High cardiovascular risk is common among Indonesian adults aged ≥40 years, and rates of preventive treatment are low. Population-based and clinical approaches to preventing CVD should be a priority in both urban and rural areas.

Background— Neuronal nitric oxide synthase (nNOS) has recently been shown to be a major regulator of cardiac contractility. In a cellular system, we have previously shown that nNOS is regulated by the isoform 4b of plasma membrane calcium/calmodulin-dependent ATPase (PMCA4b) through direct interaction mediated by a PDZ domain (PSD 95, Drosophilia Discs large protein and Zona occludens-1) on nNOS and a cognate ligand on PMCA4b. It remains unknown, however, whether this interaction has physiological relevance in the heart in vivo. Methods and Results— We generated 2 strains of transgenic mice overexpressing either human PMCA4b or PMCA ct120 in the heart. PMCA ct120 is a highly active mutant form of the pump that does not interact with or modulate nNOS function. Calcium was extruded normally from PMCA4b-overexpressing cardiomyocytes, but in vivo, overexpression of PMCA4b reduced the β-adrenergic contractile response. This attenuated response was not observed in ct120 transgenic mice. Treatment with a specific nNOS inhibitor ( N ω-propyl- l -arginine) reduced the β-adrenergic response in wild-type and ct120 transgenic mice to levels comparable to those of PMCA4b transgenic animals. No differences in lusitropic response were observed in either transgenic strain compared with wild-type littermates. Conclusions— These data demonstrate the physiological relevance of the interaction between PMCA4b and nNOS and suggests its signaling role in the heart.

Background and Purpose: The Hippo pathway has emerged as a potential therapeutic target to control pathological cardiac remodelling. The core components of the Hippo pathway, mammalian Ste‐20 like kinase 1 (Mst1) and mammalian Ste‐20 like kinase 2 (Mst2), modulate cardiac hypertrophy, apoptosis, and fibrosis. Here, we study the effects of pharmacological inhibition of Mst1/2 using a novel inhibitor XMU‐MP‐1 in controlling the adverse effects of pressure overload‐induced hypertrophy.Experimental Approach: We used cultured neonatal rat cardiomyocytes (NRCM) and C57Bl/6 mice with transverse aortic constriction (TAC) as in vitro and in vivo models, respectively, to test the effects of XMU‐MP‐1 treatment. We used luciferase reporter assays, western blots and immunofluorescence assays in vitro, with echocardiography, qRT‐PCR and immunohistochemical methods in vivo.Key Results: XMU‐MP‐1 treatment significantly increased activity of the Hippo pathway effector yes‐associated protein and inhibited phenylephrine‐induced hypertrophy in NRCM. XMU‐MP‐1 improved cardiomyocyte survival and reduced apoptosis following oxidative stress. In vivo, mice 3 weeks after TAC, were treated with XMU‐MP‐1 (1 mg·kg⁻¹) every alternate day for 10 further days. XMU‐MP‐1‐treated mice showed better cardiac contractility than vehicle‐treated mice. Cardiomyocyte cross‐sectional size and expression of the hypertrophic marker, brain natriuretic peptide, were reduced in XMU‐MP‐1‐treated mice. Improved heart function in XMU‐MP‐1‐treated mice with TAC, was accompanied by fewer TUNEL positive cardiomyocytes and lower levels of fibrosis, suggesting inhibition of cardiomyocyte apoptosis and decreased fibrosis.Conclusions and Implications: The Hippo pathway inhibitor, XMU‐MP‐1, reduced cellular hypertrophy and improved survival in cultured cardiomyocytes and, in vivo, preserved cardiac function following pressure overload.