Distinct mechanisms of Rem inhibition of Ca_V1.2 differentially depend on Rem/β interaction.

<p>(A, B) Differential impact of CFP-Rem on surface density of wild-type (α_1C[BBS]-YFP+β_2a) and mutant (α_1C[BBS]-YFP+β_2aTM) Ca_V1.2 channels, respectively, using a surface channel quantum dot labeling method. Confocal images for corresponding imaging channels were obtained with identical instrument settings. Scale bar, 25 µm. (C) Rapid recruitment of CFP-Rem₂₆₅-C1_PKC to the plasma membrane induced by 1 µM PdBu. Scale bar, 8 µm. (D, E) PdBu-induced membrane translocation of CFP-Rem₂₆₅-C1_PKC concomitantly inhibits wild-type (α_1C+β_2a), but not mutant (α_1C+β_2aTM) Ca_V1.2 channels. (F, G) Rem inhibits gating currents and <i>Q</i>_max in both wild-type and mutant Ca_V1.2 channels. * <i>P</i><0.05 when compared to the corresponding without Rem data using Student's two-tailed unpaired <i>t</i> test.</p

Distinct RGKs differentially use β-binding-dependent and independent mechanisms to inhibit Ca_V1.2 channels.

<p>(A) Histogram showing impact of individual RGKs on wild-type (α_1C+β_2a) and mutant (α_1C+β_2aTM) Ca_V1.2 channels. *, #, $ <i>P</i><0.05 when compared to α_1C+β_2a, α_1C+β_2aTM, or α_1C+β_2a+RGK, respectively, using two-tailed unpaired Student's <i>t</i> test. (B) Cartoon showing dichotomy in the determinants used by distinct RGKs to inhibit Ca_V1.2 channels.</p

Rem interaction with α_1C N-terminus mediates β-binding-independent inhibition.

<p>(A) <i>Top</i>, topography of Ca_V2.2 α_1B subunit. <i>Bottom</i>, interaction of Ca_V2.2 α_1B intracellular domains with YFP-Rem probed using FRET. Dotted lines represent FRET data from YFP-Rem+CFP-α_1CNT and YFP-Rem+CFP, respectively. (B, C) Population <i>I</i>_peak-<i>V</i> relationships for wild type (α_1B+β_2a) and mutant (α_1B+β_2aTM) Ca_V2.2 channels, respectively, in the absence (▪, <i>n</i> = 5 for wild type channels, and <i>n</i> = 9 for mutant channels) or presence (red ▴, <i>n</i> = 5 for wild type channels, and <i>n</i> = 10 for mutant channels) of Rem. Data are means ± S.E.M. (D) Schematic showing rationale and predictions for α_1C N-terminus over-expression experiments. (E) Histogram showing impact of α_1C or α_1B N-terminus on wild-type (α_1C+β_2a) and mutant (α_1C+β_2aTM) Ca_V1.2 channels in the presence of Rem. * <i>P</i><0.05 when compared to α_1C+β_2a or α_1C+β_2aTM using two-tailed unpaired Student's <i>t</i> test. # <i>P</i><0.05 when compared to α_1C+β_2a+Rem or α_1C+β_2aTM+Rem using two-tailed unpaired Student's <i>t</i> test.</p

Rem binds α_1C N-terminus.

<p>(A) Schematic of α_1C showing four homologous transmembrane domains (I–IV), intracellular N/C termini and domain-connecting loops. (B) <i>Top</i>, interaction of individual CFP-tagged α_1C intracellular loops and termini with YFP-Rem probed using FRET. Dotted line represents YFP-Rem+CFP (<i>n</i> = 10). <i>Bottom</i>, confocal images. Scale bar, 8 µm. (C) CFP-tagged α_1CNT co-immunoprecipitates with YFP-Rem. All the co-ip lanes and the first input lane were from the same gel. The rest of the input lanes were from a second gel run simultaneously because there were insufficient lanes available in the first gel to accommodate all samples, including marker lanes. Hence, in the input gel image (<i>right</i>) the first lane (CFP-NT) was spliced onto the rest of the lanes (dotted line). The co-ip gels have been cropped to remove light chain IgG bands from the precipitating antibody. (D) Schematic of α_1CNT peptide fragments. (E) Co-immunoprecipitation of YFP-tagged α_1CNT peptide fragments with CFP-Rem. (F) Sequence comparison of last 22 N-terminus residues among distinct Ca_V1/Ca_V2 channel α₁ subunits.</p

Palmitate activates PKC, and palmitate-induced ROS production is inhibited by blocking PKC or NOX2.

<p>A. Western blot from membrane preparation, arbitrary units, H9c2 cells. KDEL is a loading control for sarcoplasmic reticulum. B. Graph of membrane protein band quantification from panel A, expressed in arbitrary units. C. Western blot from cytosolic fraction, arbitrary units, H9c2 cells. D. Graph of membrane protein band quantification from panel C. No significant difference by ANOVA. E. Representative experiment done with cardiomyocytes in triplicate, height is DCF fluorescence minus background, in live cells, mean + SEM. F. Cardiomyocytes from the same experiment using mitosox red readout. G. Cardiomyocytes from the same experiment using TMRM signal. H. Cardiomyocytes from the same experiment using Rhod2 signal. I. Time course of NOX2 activation with oleate or palmitate, using H9c2 cells, expressed in arbitrary units. J. Etomoxir and PKC inhibitors prevent NOX2 activation, representative experiment using H9c2 cells. For all panels except D, means are significantly different by ANOVA, * = sig different from control by Dunnett post-hoc test. PA = palmitate 200 μM, OA = oleate 200 μM, ETO = etomoxir 200 gp = gp91ds peptide 50 μM, Ln = L-NAME 10 μM, Go = Go6983 5 μM, LY = LY333531 50 nM.</p

Diagram of proposed pathway.

<p>Saturated fats are internalized by cardiomyocytes and transported to the mitochondria to be used as a fuel source for beta-oxidation, causing a low level of mitochondrial ROS. This activates PKC, which in turn activates NOX2. ROS from NOX2 and mitochondrial ROS amplify each other in a feed-forward cycle that promotes greater ROS production and sarcoplasmic reticulum (SR) calcium leak.</p

NOX2 KO cardiomyocytes do not have an increase in ROS in response to palmitate or PKC activation.

<p>A. Representative experiment done with cardiomyocytes in triplicate, height is DCF fluorescence minus background, in live cells, expressed in arbitrary units, mean + SEM. B. Cardiomyocytes from the same experiment using mitosox red readout. C. Cardiomyocytes from the same experiment using TMRM readout. For all panels, means are significantly different by ANOVA, * = sig different from control by Dunnett post-hoc test. PA = palmitate 200 μM, gp = gp91ds peptide 50 μM, Go = Go6983 5 μM, LY = LY333531 50 nM, Cre = cresol 3 μM, PMA 100 nM.</p

Palmitate causes a decrease in mitochondrial oxygen consumption in cardiomyocytes.

<p>A. Oxygen consumption rate is decreased by palmitate. B. Extracellular acidification rate from the same experiment. Data from WT cardiomyocytes. Measurements from three time-points were obtained under each condition, using triplicate wells. b = baseline, o = oligomycin, F = FCCP, AA = antimycin-A and rotenone. C. Respiratory control ratio is reduced significantly by palmitate. D. Electron transport chain complex III activity is significantly reduced by palmitate. Units are nanomoles substrate utilized/min/mg protein. The means of all graphs are significantly different by ANOVA, * = sig different from control by post-hoc test.</p

Blocking electron transport chain with antimycin-A causes ROS and mitochondrial depolarization.

<p>A. Representative experiment done with cardiomyocytes in triplicate, height is DCF fluorescence minus background, in live cells, mean + SEM expressed in arbitrary units. B. Cardiomyocytes from the same experiment using mitosox red readout. C. Cardiomyocytes from the same experiment using TMRM signal. For all panels, means are significantly different by ANOVA, * = sig different from control by post-hoc test. gp = gp91ds peptide 50 μM, AA = antimycin-A at 10, 20 or 40 μM.</p

NOX2 inhibition reduces PMA-induced and cresol-induced ROS.

<p>A. Representative PMA experiment done with cardiomyocytes in triplicate, height is DCF fluorescence minus background, in live cells, expressed in arbitrary units, mean + SEM. B. PMA experiment using mitosox red signal. C. PMA experiment using TMRM signal. The NOX2 inhibitor and PKC inhibitors reduce depolarization. D. PMA experiment using Rhod2 signal. The NOX2 inhibitor and PKC inhibitors reduce calcium overload. E,F. Cresol increases total ROS and mitochondrial ROS. G. Cresol depolarized the mitochondrial inner membrane. H. Cresol increases mitochondrial calcium. For all panels, means are significantly different by ANOVA, * = sig different from control by post-hoc test, # = sig different from PMA by post-hoc test. PMA 100 nM, gp = gp91ds peptide 50 μM, Go = Go6983 5 μM, LY = LY333531 50 nM, Cre = cresol 1 or 3 μM.</p