Novel Splicing Factor RBM25 Modulates Bcl-x Pre-mRNA 5′ Splice Site Selection▿

RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-xS 5′ splice site (5′ ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-xL. Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-xS 5′ ss selection, leading to decreased Bcl-xS isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5′ ss; however, it was not required when a strong consensus 5′ ss was present. In support of a role for RBM25 in modulating the selection of a 5′ ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-xS 5′ ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A

Mitochondrial Ca2+ flux modulates spontaneous electrical activity in ventricular cardiomyocytes.

INTRODUCTION:Ca2+ release from sarcoplasmic reticulum (SR) is known to contribute to automaticity via the cytoplasmic Na+-Ca2+ exchanger (NCX). Mitochondria participate in Ca2+ cycling. We studied the role of mitochondrial Ca2+ flux in ventricular spontaneous electrical activity. METHODS:Spontaneously contracting mouse embryonic stem cells (ESC)-derived ventricular cardiomyocytes (CMs) were differentiated from wild type and ryanodine receptor type 2 (RYR2) knockout mouse ESCs and differentiated for 19-21 days. Automaticity was also observed in human induced pluripotent stem cell (hiPSC)-derived ventricular CMs differentiated for 30 days, and acute isolated adult mouse ventricular cells in ischemic simulated buffer. Action potentials (APs) were recorded by perforated whole cell current-clamp. Cytoplasmic and mitochondrial Ca2+ transients were determined by fluorescent imaging. RESULTS:In mouse ESC-derived ventricular CMs, spontaneous beating was dependent on the L-type Ca2+ channel, cytoplasmic NCX and mitochondrial NCX. Spontaneous beating was modulated by SR Ca2+ release from RYR2 or inositol trisphosphate receptors (IP3R), the pacemaker current (If) and mitochondrial Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU). In RYR2 knockout mouse ESC-derived ventricular CMs, mitochondrial Ca2+ flux influenced spontaneous beating independently of the SR Ca2+ release from RYR2, and the mitochondrial effect was dependent on IP3R SR Ca2+ release. Depolarization of mitochondria and preservation of ATP could terminate spontaneous beating. A contribution of mitochondrial Ca2+ flux to automaticity was confirmed in hiPSC-derived ventricular CMs and ischemic adult mouse ventricular CMs, confirming the findings across species and cell maturity levels. CONCLUSIONS:Mitochondrial and sarcolemma NCX fluxes are required for ventricular automaticity. Mitochondrial Ca2+ uptake plays a modulatory role. Mitochondrial Ca2+ uptake through MCU is influenced by IP3R-dependent SR Ca2+ release

The effect of mitochondrial Ca²⁺ flux on spontaneously beating in hiPSC-derived ventricular-like CMs.

<p><b>A</b>) Typical cytoplasmic Ca²⁺ transients in a hiPSC-derived CM. <b>B</b>) Inhibition effects of mitochondrial Ca²⁺ influx blocker, 1 μmol/L Ru360 (in pipette solution), on the beating rate of cytoplasmic Ca²⁺ transients. The spontaneous beating rate is reduced by 21.2 ± 4.3% (n = 10). <b>C</b>) The mitochondrial NCX (NCLX) blocker, 3 μmol/L CGP-37157, abolishes the automaticity of CMs (n = 11). <b>D</b>) Cytoplasmic Ca²⁺ transients are abolished by mitochondrial depolarization by 300 nmol/L FCCP and 1 μmol/L oligomycin (n = 9).</p

A cost-effective and enhanced mesenchymal stem cell expansion platform with internal plasma-activated biofunctional interfaces

Mesenchymal stem cells (MSCs) used for clinical applications require in vitro expansion to achieve therapeutically relevant numbers. However, conventional planar cell expansion approaches using tissue culture vessels are inefficient, costly, and can trigger MSC phenotypic and functional decline. Here we present a one-step dry plasma process to modify the internal surfaces of three-dimensional (3D) printed, high surface area to volume ratio (high-SA:V) porous scaffolds as platforms for stem cell expansion. To address the long-lasting challenge of uniform plasma treatment within the micrometre-sized pores of scaffolds, we developed a packed bed plasma immersion ion implantation (PBPI3) technology by which plasma is ignited inside porous materials for homogeneous surface activation. COMSOL Multiphysics simulations support our experimental data and provide insights into the role of electrical field and pressure distribution in plasma ignition. Spatial surface characterisation inside scaffolds demonstrates the homogeneity of PBPI3 activation. The PBPI3 treatment induces radical-containing chemical structures that enable the covalent attachment of biomolecules via a simple, non-toxic, single-step incubation process. We showed that PBPI3-treated scaffolds biofunctionalised with fibroblast growth factor 2 (FGF2) significantly promoted the expansion of MSCs, preserved cell phenotypic expression, and multipotency, while reducing the usage of costly growth factor supplements. This breakthrough PBPI3 technology can be applied to a wide range of 3D polymeric porous scaffolds, paving the way towards developing new biomimetic interfaces for tissue engineering and regenerative medicine

Regulation of abnormal automaticity by mitochondrial Ca²⁺ flux in ischemic mouse ventricular CMs.

<p>MCU blocker, 1 μmol/L Ru360 (in the pipette solution), reduces the ischemia-induced spontaneous beating rate of cytoplasmic Ca²⁺ transients in mouse ventricular CMs (*<i>p</i><0.05, compared with control groups. n = 11).</p

Modulation of spontaneous beating by blocking ion channels and receptors in Wt ventricular-like ESC-derived CMs.

<p><b>A</b>) Typical APs of Wt ventricular-like ESC-derived CM recorded by perforated whole-cell current-clamp technique (n = 15). <b>B</b>) Effects of sarcolemmal ion channel or transporter blockers. Beating rate is reduced by the I_f specific blocker, 10 μmol/L ivabradine (n = 6). Automaticity is eliminated by the L-type Ca²⁺ channel blocker, 10 μmol/L nifedipine (n = 6), and the NCX blocker, 10 μmol/L KB-R7943 (n = 5). <b>C</b>) Effects of sarcoplasmic reticulum ion channel blockers. Spontaneous beating is modulated by the SR Ca²⁺ release blocker, 10 μmol/L Ryanodine (n = 5), and the IP₃R blocker, 2 μmol/L 2-APB (n = 4). <b>D</b>) Effects of altering mitochondrial Ca²⁺ handling. Spontaneous beating is modulated by mitochondrial Ca²⁺ influx blocker, 1 μmol/L Ru360 (in pipette solution, n = 5), and mitochondrial NCX (NCLX) blocker, 3 μmol/L CGP-37157 (n = 6). <b>E</b>) Summary of effects on spontaneous beating. *<i>p</i><0.05, compared with control group.</p

The parameters of cytoplasmic Ca²⁺ transients recorded in Wt and RYR2^-/- ventricular-like CM.

<p>The parameters of cytoplasmic Ca²⁺ transients recorded in Wt and RYR2^-/- ventricular-like CM.</p

Mitochondrial Ca2+ Flux Modulates Spontaneous Electrical Activity in Ventricular Cardiomyocytes

Ca²⁺ release from sarcoplasmic reticulum (SR) is known to contribute to automaticity via the cytoplasmic Na⁺-Ca²⁺ exchanger (NCX). Mitochondria participate in Ca²⁺ cycling. We studied the role of mitochondrial Ca²⁺ flux in spontaneous electrical activity

Proposed model of ion channels and transporters modulating spontaneous beating in ventricular-CMs.

<p>Sarcolemmal and mitochondrial NCXs are necessary for spontaneous beating. The mitochondrial membrane potential, L-type Ca²⁺ channels, and Na⁺ channels are also required. NCLX: mitochondrial NCX. NCX1: NCX type 1. Solid lines represent mitochondrial Ca²⁺ flux pathways for automaticity. Dashed lines show Ca²⁺ clock and dash dot line is M-clock (Voltage clock).</p

Mitochondrial Ca²⁺ flux modulates spontaneous electrical activity in ventricular cardiomyocytes

<div><p>Introduction</p><p>Ca²⁺ release from sarcoplasmic reticulum (SR) is known to contribute to automaticity via the cytoplasmic Na⁺-Ca²⁺ exchanger (NCX). Mitochondria participate in Ca²⁺ cycling. We studied the role of mitochondrial Ca²⁺ flux in ventricular spontaneous electrical activity.</p><p>Methods</p><p>Spontaneously contracting mouse embryonic stem cells (ESC)-derived ventricular cardiomyocytes (CMs) were differentiated from wild type and ryanodine receptor type 2 (RYR2) knockout mouse ESCs and differentiated for 19–21 days. Automaticity was also observed in human induced pluripotent stem cell (hiPSC)-derived ventricular CMs differentiated for 30 days, and acute isolated adult mouse ventricular cells in ischemic simulated buffer. Action potentials (APs) were recorded by perforated whole cell current-clamp. Cytoplasmic and mitochondrial Ca²⁺ transients were determined by fluorescent imaging.</p><p>Results</p><p>In mouse ESC-derived ventricular CMs, spontaneous beating was dependent on the L-type Ca²⁺ channel, cytoplasmic NCX and mitochondrial NCX. Spontaneous beating was modulated by SR Ca²⁺ release from RYR2 or inositol trisphosphate receptors (IP₃R), the pacemaker current (I_f) and mitochondrial Ca²⁺ uptake by the mitochondrial Ca²⁺ uniporter (MCU). In RYR2 knockout mouse ESC-derived ventricular CMs, mitochondrial Ca²⁺ flux influenced spontaneous beating independently of the SR Ca²⁺ release from RYR2, and the mitochondrial effect was dependent on IP₃R SR Ca²⁺ release. Depolarization of mitochondria and preservation of ATP could terminate spontaneous beating. A contribution of mitochondrial Ca²⁺ flux to automaticity was confirmed in hiPSC-derived ventricular CMs and ischemic adult mouse ventricular CMs, confirming the findings across species and cell maturity levels.</p><p>Conclusions</p><p>Mitochondrial and sarcolemma NCX fluxes are required for ventricular automaticity. Mitochondrial Ca²⁺ uptake plays a modulatory role. Mitochondrial Ca²⁺ uptake through MCU is influenced by IP₃R-dependent SR Ca²⁺ release.</p></div