Rivaroxaban Attenuates Right Ventricular Remodeling in Rats with Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive condition that frequently results in right ventricular (RV) remodeling. The objectives of this study are to investigate effects of rivaroxaban on RV remodeling in a rat model of PAH, created with Sugen5416 and chronic hypoxia, and the in vitro effects of rivaroxaban on human cardiac microvascular endothelial cells (HCMECs). To create the PAH model, male Sprague-Dawley rats were subcutaneously injected with Sugen5416 (20 mg/kg) and exposed to 2 weeks of hypoxia (10% O2), followed by 2 weeks of exposure to normoxia. The animals were then divided into 2 groups with or without administration of rivaroxaban (12 mg/kg/d) for a further 4 weeks. HCMECs were cultured under hypoxic conditions (37 °C, 1% O2, 5% CO2) with Sugen5416 and with or without rivaroxaban. In the model rats, RV systolic pressure and Fulton index increased by hypoxia with Sugen5416 were significantly decreased when treated with rivaroxaban. In HCMECs, hypoxia with Sugen5416 increased the expression of protease-activated receptor-2 (PAR-2) and the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor-kappa B (NF-κB), while treatment with rivaroxaban significantly suppressed the expression of these proteins. Rivaroxaban attenuated RV remodeling in a rat model of PAH by reducing ERK, JNK and NF-κB activation. Rivaroxaban has the possibility of providing additive effects on RV remodeling in patients with PAH.

GABAB receptor regulates proliferation in the high-grade chondrosarcoma cell line OUMS-27 via apoptotic pathways.

BACKGROUND: High-grade chondrosarcoma, which has a high incidence of local recurrence and pulmonary metastasis despite surgical resection, is associated with poor prognosis. Therefore, new and effective adjuvant therapies are urgently required for this disease. Gamma-aminobutyric acid (GABA), which acts as a neurotrophic factor during nervous system development, is related to the proliferation and migration of certain cancer cells. The GABAergic system, which is composed of GABA, the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), and GABA receptors, has an important function in nerve growth and development of neural crest. Therefore, the GABAergic system may play important functional roles in the proliferation of chondrosarcoma cells, which are derived from neural crest cells. We examined the anti-tumor effects of the GABAergic system on a chondrosarcoma cell line. METHODS: We evaluated the underlying mechanisms of the anti-tumor effects of the GABAergic system, such as the involvement of different signaling pathways, apoptosis, and cell cycle arrest, in the high-grade chondrosarcoma cell line OUMS-27. In addition, we performed whole-cell patch-clamp recordings for Ca2+ currents and evaluated the changes in intracellular Ca2+ concentration via Ca2+ channels, which are related to the GABAB receptor in high-grade chondrosarcoma cells. RESULTS: The GABAB receptor antagonist CGP had anti-tumor effects on high-grade chondrosarcoma cells in a dose-dependent manner. The activities of caspase 3 and caspase 9 were significantly elevated in CGP-treated cells compared to in untreated cells. The activity of caspase 8 did not differ significantly between untreated cells and CGP-treated cells. However, caspase 8 tended to be up-regulated in CGP-treated cells. The GABAB receptor antagonist exhibited anti-tumor effects at the G1/S cell cycle checkpoint and induced apoptosis via dual inhibition of the PI3/Akt/mTOR and MAPK signaling pathways. Furthermore, the changes in intracellular Ca2+ via GABAB receptor-related Ca2+ channels inhibited the proliferation of high-grade chondrosarcoma cells by inducing and modulating apoptotic pathways. CONCLUSIONS: The GABAB receptor antagonist may improve the prognosis of high-grade chondrosarcoma by exerting anti-tumor effects via different signaling pathways, apoptosis, cell cycle arrest, and Ca2+ channels in high-grade chondrosarcoma cells.

Presenilin 2 regulates the systolic function of heart by modulating Ca2+ signaling.

Genetic studies of families with familial Alzheimer's disease have implicated presenilin 2 (PS2) in the pathogenesis of this disease. PS2 is ubiquitously expressed in various tissues including hearts. In this study, we examined cardiac phenotypes of PS2 knockout (PS2KO) mice to elucidate a role of PS2 in hearts. PS2KO mice developed normally with no evidence of cardiac hypertrophy and fibrosis. Invasive hemodynamic analysis revealed that cardiac contractility in PS2KO mice increased compared with that in their littermate controls. A study of isolated papillary muscle showed that peak amplitudes of Ca2+ transients and peak tension were significantly higher in PS2KO mice than those in their littermate controls. PS2KO mouse hearts exhibited no change in expression of calcium regulatory proteins. Since it has been demonstrated that PS2 in brain interacts with sorcin, which serves as a modulator of cardiac ryanodine receptor (RyR2), we tested whether PS2 also interacts with RyR2. Immmunoprecipitation analysis showed that PS2, sorcin, and RyR2 interact with each other in HEK-293 cells overexpressing these proteins or in mouse hearts. Immunohistochemistry of heart muscle indicated that PS2 colocalizes with RyR2 and sorcin at the Z-lines. Elevated Ca2+ attenuated the association of RyR2 with PS2, whereas the association of sorcin with PS2 was enhanced. The enhanced Ca2+ transients and contractility in PS2KO mice were observed at low extracellular [Ca2+] but not at high levels of [Ca2+]. Taken together, our results suggest that PS2 plays an important role in cardiac excitation-contraction coupling by interacting with RyR2.

Sarcolipin regulates sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by binding to transmembrane helices alone or in association with phospholamban.

Phospholamban (PLN), a regulator of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs), interacts with both the cytosolic N domain and transmembrane helices M2, M4, M6, and M9 of SERCA. Amino acids in the transmembrane domain of PLN that are predicted to interact with SERCA1a are conserved in sarcolipin (SLN), a functional PLN homologue. Accordingly, the effects of critical mutations in SERCA1a, PLN, and NF-SLN (SLN tagged N-terminally with a FLAG epitope) on NF-SLN/SERCA1a and PLN/NF-SLN/SERCA1a interactions were compared. Critical mutations in SERCA1a and NF-SLN diminished functional interactions between SERCA1a and NF-SLN, indicating that NF-SLN and PLN interact with some of the same amino acids in SERCA1a. Mutations in PLN or NF-SLN affected the amount of SERCA1a that was coimmunoprecipitated in each complex with antibodies against either PLN or SLN, but not the pattern of coimmunoprecipitation. PLN mutations had more dramatic effects on SERCA1a coimmunoprecipitation than SLN mutations, suggesting that PLN dominates in the primary interaction with SERCA1a. Coimmunoprecipitation also confirmed that PLN and NF-SLN form a heterodimer that interacts with SERCA1a in a regulatory fashion to form a very stable PLN/NF-SLN/SERCA1a complex. Modeling showed that the SLN/SERCA1a complex closely resembles the PLN/SERCA1a complex, but with the luminal end of SLN extending to the loop connecting M1 and M2, where Tyr-29 and Tyr-31 interact with aromatic residues in SERCA1a. Modeling of the PLN/SLN/SERCA1a complex predicts that the regulator binding cavity in the E(2) conformation of SERCA1a can accommodate both SLN and PLN helices, but not two PLN helices.

Sarcolipin inhibits polymerization of phospholamban to induce superinhibition of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs).

Sarcolipin (SLN), a regulator of the sarco(endo)plasmic reticulum Ca(2+)-ATPase of fast-twitch skeletal muscle (SERCA1a), is also expressed in cardiac and slow-twitch skeletal muscles where phospholamban (PLN) and SERCA2a are expressed. Co-expression in HEK-293 cells of SLN tagged N-terminally with a FLAG epitope (NF-SLN), PLN, and SERCAs followed by measurement of the Ca(2+) dependence of Ca(2+) transport activity in isolated microsomal fractions showed that NF-SLN can reduce the apparent Ca(2+) affinity of both SERCA1a (DeltaK(Ca) = -0.22 +/- 0.01 pCa units) and SERCA2a (DeltaK(Ca) = -0.37 +/- 0.04 pCa units). When SERCA1a or SERCA2a were co-expressed with both NF-SLN and PLN, inhibition was synergistic, reducing DeltaK(Ca) by about -1.0 pCa units. Co-immunoprecipitation showed that NF-SLN increased the binding of PLN to SERCA, whereas PLN did not increase the binding of NF-SLN to SERCA. Elevated Ca(2+) dissociates both PLN and NF-SLN from their complexes with both SERCA1a and SERCA2a, but NF-SLN induced resistance to Ca(2+) dissociation of the PLN.SERCA complex. Co-immunoprecipitation of PLN and NF-SLN without SERCA showed that NF-SLN binds directly to PLN and that NF-SLN inhibits the formation of PLN pentamers. Thus the ability of NF-SLN to elevate the content of PLN monomers can account, at least in part, for the superinhibitory effects of NF-SLN in the presence of PLN.