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

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

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 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

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

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

Cardiomyocytes treated with palmitate have increased total and mitochondrial ROS.

<p>A. Representative experiment done with cardiomyocytes in triplicate, height is DCF fluorescence minus background, in live cells, expressed in arbitrary units, mean + SEM. Palmitate, but not oleate, increases ROS significantly. PA = palmitate 200 μM, OA = oleate 200 μM, B. Cardiomyocytes from the same experiment using mitosox red readout to indicate mitochondrial ROS. C. Inhibition of mitochondrial lipid uptake or beta-oxidation prevents the increase in ROS. ETO = etomoxir 200 μM, TMZ = trimetazidine 10 μM, MT = mito-TEMPO, 20 μM. D. Cardiomyocytes from the same experiment using mitosox red readout. For all panels, means are significantly different by ANOVA, * = sig different from control by post-hoc test. E. Confocal images of live isolated cardiomyocytes after control and palmitate treatment. Cells were stained with mitosox red and counter-stained with mitotracker green.</p

Palmitate depolarizes the mitochondrial inner membrane potential and causes mitochondrial calcium overload from increased sparks.

<p>A. Representative experiment done with cardiomyocytes in triplicate, height is TMRM fluorescence minus background, in live cells, expressed in arbitrary units, mean + SEM. PA = palmitate 200 μM. B. Dantrolene prevents the loss of inner membrane potential. Dan = dantrolene 1 μM. C. Similar experiment using H9c2 cells and TMRM readout. D. Cardiomyocytes with Rhod2AM readout indicating mitochondrial calcium. For all panels, means are significantly different by ANOVA, * = sig different from control by post-hoc test. E. Representative line-scan sparks experiments from cardiomyocytes; height is spark amplitude, over time. F. Graph of sparks, normalized to control, n = 10–11, * = p<0.05.</p