Localization and functional consequences of a direct interaction between TRIOBP-1 and hERG/KCNH2 proteins in the heart

Reduced levels of hERG protein and the corresponding repolarizing current IKr can cause arrhythmia and sudden cardiac death, but the underlying cellular mechanisms controlling hERG surface expression are not well understood. We identified TRIOBP-1, an F-actin binding protein previously associated with actin polymerization, as a putative hERG-interacting protein in a yeast-two hybrid screen of a cardiac library. We corroborated this interaction using Forster resonance energy transfer (FRET) in HEK293 cells and co-immunoprecipitation in HEK293 cells and native cardiac tissue. TRIOBP-1 overexpression reduced hERG surface expression and current density, whereas reducing TRIOBP-1 expression via shRNA knockdown resulted in increased hERG protein levels. Immunolabeling in rat cardiomyocytes showed that native TRIOBP-1 overlapped predominantly with myosin binding protein C and secondarily with rat ERG. In human stem cell-derived cardiomyocytes, TRIOBP-1 overexpression caused intracellular co-sequestration of hERG signal, reduced native IKr, and disrupted action potential repolarization. Calcium currents were also reduced to a lesser degree and cell capacitance was increased. These findings establish that TRIOBP-1 interacts directly with hERG and can affect protein levels, IKr magnitude, and cardiac membrane excitability

hERG1a N-terminal eag domain–containing polypeptides regulate homomeric hERG1b and heteromeric hERG1a/hERG1b channels: A possible mechanism for long QT syndrome

Human ether-á-go-go–related gene (hERG) potassium channels are critical for cardiac action potential repolarization. Cardiac hERG channels comprise two primary isoforms: hERG1a, which has a regulatory N-terminal Per-Arnt-Sim (PAS) domain, and hERG1b, which does not. Isolated, PAS-containing hERG1a N-terminal regions (NTRs) directly regulate NTR-deleted hERG1a channels; however, it is unclear whether hERG1b isoforms contain sufficient machinery to support regulation by hERG1a NTRs. To test this, we constructed a series of PAS domain–containing hERG1a NTRs (encoding amino acids 1–181, 1–228, 1–319, and 1–365). The NTRs were also predicted to form from truncation mutations that were linked to type 2 long QT syndrome (LQTS), a cardiac arrhythmia disorder associated with mutations in the hERG gene. All of the hERG1a NTRs markedly regulated heteromeric hERG1a/hERG1b channels and homomeric hERG1b channels by decreasing the magnitude of the current–voltage relationship and slowing the kinetics of channel closing (deactivation). In contrast, NTRs did not measurably regulate hERG1a channels. A short NTR (encoding amino acids 1–135) composed primarily of the PAS domain was sufficient to regulate hERG1b. These results suggest that isolated hERG1a NTRs directly interact with hERG1b subunits. Our results demonstrate that deactivation is faster in hERG1a/hERG1b channels compared to hERG1a channels because of fewer PAS domains, not because of an inhibitory effect of the unique hERG1b NTR. A decrease in outward current density of hERG1a/hERG1b channels by hERG1a NTRs may be a mechanism for LQTS

Cholesterol selectively regulates IL-5 induced mitogen activated protein kinase signaling in human eosinophils.

Eosinophils function contributes to human allergic and autoimmune diseases, many of which currently lack curative treatment. Development of more effective treatments for eosinophil-related diseases requires expanded understanding of eosinophil signaling and biology. Cell signaling requires integration of extracellular signals with intracellular responses, and is organized in part by cholesterol rich membrane microdomains (CRMMs), commonly referred to as lipid rafts. Formation of these organizational membrane domains is in turn dependent upon the amount of available cholesterol, which can fluctuate widely with a variety of disease states. We tested the hypothesis that manipulating membrane cholesterol content in primary human peripheral blood eosinophils (PBEos) would selectively alter signaling pathways that depend upon membrane-anchored signaling proteins localized within CRMMs (e.g., mitogen activated protein kinase [MAPK] pathway), while not affecting pathways that signal through soluble proteins, like the Janus Kinase/Signal Transducer and Activator of Transcription [JAK/STAT] pathway. Cholesterol levels were increased or decreased utilizing cholesterol-chelating methyl-β-cyclodextrin (MβCD), which can either extract membrane cholesterol or add exogenous membrane cholesterol depending on whether MβCD is preloaded with cholesterol. Human PBEos were pretreated with MβCD (cholesterol removal) or MβCD+Cholesterol (MβCD+Chol; cholesterol delivery); subsequent IL-5-stimulated signaling and physiological endpoints were assessed. MβCD reduced membrane cholesterol in PBEos, and attenuated an IL-5-stimulated p38 and extracellular-regulated kinase 1/2 phosphorylation (p-p38, p-ERK1/2), and an IL-5-dependent increase in interleukin-1β (IL-1β) mRNA levels. In contrast, MβCD+Chol treatment elevated PBEos membrane cholesterol levels and basal p-p38, but did not alter IL-5-stimulated phosphorylation of ERK1/2, STAT5, or STAT3. Furthermore, MβCD+Chol pretreatment attenuated an IL-5-induced increase in cell survival at 48 hours, measured as total cellular metabolism. The reduction in cell survival following cholesterol addition despite unaltered STAT phosphorylation contradicts the current dogma in which JAK/STAT activation is sufficient to promote eosinophil survival, and suggests an additional, unidentified mechanism critically regulates IL-5-mediated human PBEos survival

exponential decay fits, y = A1*exp(-x/t1)+y0.

<p>exponential decay fits, y = A1*exp(-x/t1)+y0.</p

Doxorubicin induced DNA damage in stroma, theca, and granulosa cells by 4 hours post-injection.

<p><i>A.</i> Summary graph of DXR-induced DNA damage time line in stroma/theca cells. DXR-induced DNA damage reached detectable levels at 2 hours post-injection as a 30% increase in comet moment (p<0.05, ANOVA, Bonferroni, pooled data from n = 4 exps., 2 mice/point, >100 cells/mouse/point). By 24 hours, DNA damage (comet moment) in stroma/theca cells climbed to over a 2-fold increase compared to control. <i>B.</i> Summary graph of DXR-induced DNA damage in granulosa cells plotted vs. time. DXR-induced DNA damage reached detectable levels at 4 hours post-injection as a roughly 2-fold increase in comet moment over control (p<0.05, ANOVA, n = 4 exps., >100 cells/point). Damage was maintained at this level until 24 hours, when cells exhibit a ≥4-fold increase in comet moment reflecting an increase in apoptotic comets. <i>C.</i> Summary graph of oocyte comet moments from treated mice showed a gradual increase in DNA damage over time, reaching a 50% increase 10–12 hours post-DXR injection (p<0.05, ANOVA, Bonferroni, n = 4 exps., >30 cells/point). Representative images of comets are shown in lower panel; oocytes are boxed in white to distinguish from granulosa cells.</p

Acute Doxorubicin Insult in the Mouse Ovary Is Cell- and Follicle-Type Dependent

<div><p>Primary ovarian insufficiency (POI) is one of the many unintended consequences of chemotherapy faced by the growing number of female cancer survivors. While ovarian repercussions of chemotherapy have long been recognized, the acute insult phase and primary sites of damage are not well-studied, hampering efforts to design effective intervention therapies to protect the ovary. Utilizing doxorubicin (DXR) as a model chemotherapy agent, we defined the acute timeline for drug accumulation, induced DNA damage, and subsequent cellular and follicular demise in the mouse ovary. DXR accumulated first in the core ovarian stroma cells, then redistributed outwards into the cortex and follicles in a time-dependent manner, without further increase in total ovarian drug levels after four hours post-injection. Consistent with early drug accumulation and intimate interactions with the blood supply, stroma cell-enriched populations exhibited an earlier DNA damage response (measurable at 2 hours) than granulosa cells (measurable at 4 hours), as quantified by the comet assay. Granulosa cell-enriched populations were more sensitive however, responding with greater levels of DNA damage. The oocyte DNA damage response was delayed, and not measurable above background until 10–12 hours post-DXR injection. By 8 hours post-DXR injection and prior to the oocyte DNA damage response, the number of primary, secondary, and antral follicles exhibiting TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive granulosa cells plateaued, indicating late-stage apoptosis and suggesting damage to the oocytes is subsequent to somatic cell failure. Primordial follicles accumulate significant DXR by 4 hours post-injection, but do not exhibit TUNEL-positive granulosa cells until 48 hours post-injection, indicating delayed demise. Taken together, the data suggest effective intervention therapies designed to protect the ovary from chemotherapy accumulation and induced insult in the ovary must act almost immediately to prevent acute insult as significant damage was seen in stroma cells within the first two hours.</p></div

DXR accumulation in the ovary and follicle was time-dependent.

<p><i>A.</i> Graph of total DXR fluorescence intensity in the ovary as a function of time reveals DXR accumulation in the ovary follows an exponential fit, with signal rapidly rising through 4 hours post-injection, followed by plateau (n = 4). <i>B.</i> The ratio of DXR in follicles vs. stroma was calculated as a function normalized to area. The graph demonstrates the ratio of DXR fluorescence in follicles vs. stroma increases steadily over time (n = 4).</p

Time-dependent apoptosis following DXR insult is follicle type-dependent.

<p><i>A.</i> Graph plots mean apoptotic index for secondary, early antral, and late antral follicles over a 48-hour time period post-DXR injection. <i>B</i>. Graph plots mean apoptotic index for primordial and primary follicles over a 48-hour time period post-DXR injection.</p