Expression and function of voltage gated proton channels (Hv1) in MDA-MB-231 cells.

Expression of the voltage gated proton channel (Hv1) as identified by immunocytochemistry has been reported previously in breast cancer tissue. Increased expression of HV1 was correlated with poor prognosis and decreased overall and disease-free survival but the mechanism of its involvement in the disease is unknown. Here we present electrophysiological recordings of HV1 channel activity, confirming its presence and function in the plasma membrane of a breast cancer cell line, MDA-MB-231. With western blotting we identify significant levels of HV1 expression in 3 out of 8 "triple negative" breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of HV1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of HV1 using shRNA (knock-down; KD) and by eliminating HV1 using CRISPR/Cas9 gene editing (knock-out; KO). Surprisingly, these two approaches produced incongruous effects. Knock-down of HV1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H2O2 release. In contrast, HV1 Knock-out cells did not show reduced migration or H2O2 release. HV1 KO but not KD cells showed an increased glycolytic rate accompanied by an increase in p-AKT (phospho-AKT, Ser473) activity. The expression of CD171/LCAM-1, an adhesion molecule and prognostic indicator for breast cancer, was reduced in HV1 KO cells. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from HV1 KO cells grew less than WT in mass, with lower staining for the Ki-67 marker for cell proliferation rate. Therefore, deletion of HV1 expression in MDA-MB-231 cells limits tumor growth rate. The limited growth thus appears to be independent of oxidant production by NADPH oxidase molecules and to be mediated by cell adhesion molecules. Although HV1 KO and KD affect certain cellular mechanisms differently, both implicate HV1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.

ß-Adrenergic induced SR Ca2+ leak is mediated by an Epac-NOS pathway.

Cardiac ß-adrenergic receptors (ß-AR) and Ca2+-Calmodulin dependent protein kinase (CaMKII) regulate both physiological and pathophysiological Ca2+ signaling. Elevated diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) contributes to contractile dysfunction in heart failure and to arrhythmogenesis. ß-AR activation is known to increase SR Ca2+ leak via CaMKII-dependent phosphorylation of the ryanodine receptor. Two independent and reportedly parallel pathways have been implicated in this ß-AR-CaMKII cascade, one involving exchange protein directly activated by cAMP (Epac2) and another involving nitric oxide synthase 1 (NOS1). Here we tested whether Epac and NOS function in a single series pathway to increase ß-AR induced and CaMKII-dependent SR Ca2+ leak. Leak was measured as both Ca2+ spark frequency and tetracaine-induced shifts in SR Ca2+, in mouse and rabbit ventricular myocytes. Direct Epac activation by 8-CPT (8-(4-chlorophenylthio)-2'-O-methyl-cAMP) mimicked ß-AR-induced SR Ca2+ leak, and both were blocked by NOS inhibition. The same was true for myocyte CaMKII activation (assessed via a FRET-based reporter) and ryanodine receptor phosphorylation. Inhibitor and phosphorylation studies also implicated phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) downstream of Epac and above NOS activation in this pathway. We conclude that these two independently characterized parallel pathways function mainly via a single series arrangement (ß-AR-cAMP-Epac-PI3K-Akt-NOS1-CaMKII) to mediate increased SR Ca2+ leak. Thus, for ß-AR activation the cAMP-PKA branch effects inotropy and lusitropy (by effects on Ca2+ current and SR Ca2+-ATPase), this cAMP-Epac-NOS pathway increases pathological diastolic SR Ca2+leak. This pathway distinction may allow novel SR Ca2+ leak therapeutic targeting in treatment of arrhythmias in heart failure that spare the inotropic and lusitropic effects of the PKA branch.

Ischemia/Reperfusion injury protection by mesenchymal stem cell derived antioxidant capacity.

Mesenchymal stem cell (MSC) transplantation after ischemia/reperfusion (I/R) injury reduces infarct size and improves cardiac function. We used mouse ventricular myocytes (VMs) in an in vitro model of I/R to determine the mechanism by which MSCs prevent reperfusion injury by paracrine signaling. Exposure of mouse VMs to an ischemic challenge depolarized their mitochondrial membrane potential (Ψmito), increased their diastolic Ca(2+), and significantly attenuated cell shortening. Reperfusion of VMs with Ctrl tyrode or MSC-conditioned tyrode (ConT) resulted in a transient increase of the Ca(2+) transient amplitudes in all cells. ConT-reperfused cells exhibited a decreased number early after depolarization (EADs) (ConT: 6.3% vs. Ctrl: 28.4%) and prolonged survival (ConT: 58% vs. Ctrl: 33%). Ψmito rapidly recovered in Ctrl as well as ConT-treated VMs on reperfusion; however, in Ctrl solution, an exaggerated hyperpolarization of Ψmito was determined that preceded the collapse of Ψmito. The ability of ConT to attenuate the hyperpolarization of Ψmito was suppressed on inhibition of the PI3K/Akt signaling pathway or IK,ATP. However, protection of Ψmito was best mimicked by the reactive oxygen species (ROS) scavenger mitoTEMPO. Analysis of ConT revealed a significant antioxidant capacity that was linked to the presence of extracellular superoxide dismutase (SOD3) in ConT. In conclusion, MSC ConT protects VMs from simulated I/R injury by its SOD3-mediated antioxidant capacity and by delaying the recovery of Ψmito through Akt-mediated opening of IK,ATP. These changes attenuate reperfusion-induced ROS production and prevent the opening of the permeability transition pore and arrhythmic Ca(2+) release.

Mesenchymal stem cells improve cardiac conduction by upregulation of connexin 43 through paracrine signaling.

Mesenchymal stem cells (MSCs) were shown to improve cell survival and alleviate cardiac arrhythmias when transplanted into cardiac tissue; however, little is known about the mechanism by which MSCs modify the electrophysiological properties of cardiac tissue. We aimed to distinguish the influence of cell-cell coupling between myocytes and MSCs from that of MSC-derived paracrine factors on the spontaneous activity and conduction velocity (θ) of multicellular cardiomyocyte preparations. HL-1 cells were plated on microelectrode arrays and their spontaneous activity and θ was determined from field potential recordings. In heterocellular cultures of MSCs and HL-1 cells the beating frequency was attenuated (t(0h): 2.26 ± 0.18 Hz; t(4h): 1.98 ± 0.26 Hz; P < 0.01) concomitant to the intercellular coupling between MSCs and cardiomyocytes. In HL-1 monolayers supplemented with MSC conditioned media (ConM) or tyrode (ConT) θ significantly increased in a time-dependent manner (ConT: t(0h): 2.4 cm/s ± 0.2; t(4h): 3.1 ± 0.4 cm/s), whereas the beating frequency remained constant. Connexin (Cx)43 mRNA and protein expression levels also increased after ConM or ConT treatment over the same time period. Enhanced low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation after ConT treatment implicates the Wnt signaling pathway. Suppression of Wnt secretion from MSCs (IWP-2; 5 µmol/l) reduced the efficacy of ConT to induce phospho-LRP6 and to increase θ. Inhibition of ß-catenin (cardamonin; 10 µmol/l) or GSK3-α/ß (LiCl; 5 mmol/l) also suppressed changes in θ, further supporting the hypothesis that MSC-mediated Cx43 upregulation occurs in part through secreted Wnt ligands and activation of the canonical Wnt signaling pathway.

IP3-dependent nuclear Ca2+ signalling in the mammalian heart.

In cardiac myocytes the type-2 inositol 1,4,5-trisphosphate receptor (IP(3)R2) is the predominant isoform expressed. The IP(3)R2 channel is localized to the SR and to the nuclear envelope. We studied IP(3)-dependent nuclear Ca(2+) signals ([Ca(2+)](Nuc)) in permeabilized atrial myocytes and in isolated cardiac nuclei. In permeabilized myocytes IP(3) (20 microm) and the more potent IP(3)R agonist adenophostin (5 microm) caused an elevation of [Ca(2+)](Nuc). An IP(3)-dependent increase of [Ca(2+)](Nuc) was still observed after pretreatment with tetracaine to block Ca(2+) release from ryanodine receptors (RyRs), and the effect of IP(3) was partially reversed or prevented by the IP(3)R blockers heparin and 2-APB. Isolated nuclei were superfused with an internal solution containing the Ca(2+) indicator fluo-4 dextran. Exposure to IP(3) (10 microm) and adenophostin (0.5 microm) increased [Ca(2+)](Nuc) by 25 and 27%, respectively. [Ca(2+)](Nuc) increased to higher levels than [Ca(2+)](Cyt) immediately adjacent to the outer membrane of the nuclear envelope, suggesting that a significant portion of nuclear IP(3) receptors are facing the nucleoplasm. When nuclei were pretreated with heparin or 2-APB, IP(3) failed to increase [Ca(2+)](Nuc). Isolated nuclei were also loaded with the membrane-permeant low-affinity Ca(2+) probe fluo-5N AM which compartmentalized into the nuclear envelope. Exposure to IP(3) and adenophostin resulted in a decrease of the fluo-5N signal that could be prevented by heparin. Stimulation of IP(3)R caused depletion of the nuclear Ca(2+) stores by approximately 60% relative to the maximum depletion produced by the ionophores ionomycin and A23187. The fluo-5N fluorescence decrease was particularly pronounced in the nuclear periphery, suggesting that the nuclear envelope may represent the predominant nuclear Ca(2+) store. The data indicate that IP(3) can elicit Ca(2+) release from cardiac nuclei resulting in localized nuclear Ca(2+) signals.

Biosensors to measure inositol 1,4,5-trisphosphate concentration in living cells with spatiotemporal resolution.

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an approximately 11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent K(d) = 31.3 +/- 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to approximately 30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.