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. 2014 Sep 1;307(5):H710-21.
doi: 10.1152/ajpheart.00890.2013. Epub 2014 Jul 11.

NADPH oxidase-2 inhibition restores contractility and intracellular calcium handling and reduces arrhythmogenicity in dystrophic cardiomyopathy

Daniel R Gonzalez  1 , Adriana V Treuer  1 , Guillaume Lamirault  2 , Vera Mayo  2 , Yenong Cao  2 , Raul A Dulce  2 , Joshua M Hare  3
Affiliations

NADPH oxidase-2 inhibition restores contractility and intracellular calcium handling and reduces arrhythmogenicity in dystrophic cardiomyopathy

Daniel R Gonzalez et al. Am J Physiol Heart Circ Physiol. .

Abstract

Duchenne muscular dystrophy may affect cardiac muscle, producing a dystrophic cardiomyopathy in humans and the mdx mouse. We tested the hypothesis that oxidative stress participates in disrupting calcium handling and contractility in the mdx mouse with established cardiomyopathy. We found increased expression (fivefold) of the NADPH oxidase (NOX) 2 in the mdx hearts compared with wild type, along with increased superoxide production. Next, we tested the impact of NOX2 inhibition on contractility and calcium handling in isolated cardiomyocytes. Contractility was decreased in mdx myocytes compared with wild type, and this was restored toward normal by pretreating with apocynin. In addition, the amplitude of evoked intracellular Ca(2+) concentration transients that was diminished in mdx myocytes was also restored with NOX2 inhibition. Total sarcoplasmic reticulum (SR) Ca(2+) content was reduced in mdx hearts and normalized by apocynin treatment. Additionally, NOX2 inhibition decreased the production of spontaneous diastolic calcium release events and decreased the SR calcium leak in mdx myocytes. In addition, nitric oxide (NO) synthase 1 (NOS-1) expression was increased eightfold in mdx hearts compared with wild type. Nevertheless, cardiac NO production was reduced. To test whether this paradox implied NOS-1 uncoupling, we treated cardiac myocytes with exogenous tetrahydrobioterin, along with the NOX inhibitor VAS2870. These agents restored NO production and phospholamban phosphorylation in mdx toward normal. Together, these results demonstrate that, in mdx hearts, NOX2 inhibition improves the SR calcium handling and contractility, partially by recoupling NOS-1. These findings reveal a new layer of nitroso-redox imbalance in dystrophic cardiomyopathy.

Keywords: BH4; Duchenne; NADPH oxidase; NOS-1 uncoupling; mdx; phospholamban; ryanodine receptor; superoxide.

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Figures

Fig. 1.
Fig. 1.
NADPH oxidase (NOX) isoforms and associated subunits expression. A: Western blot analysis of the isoforms of NOX and NOX2-associated subunits in wild-type (WT) and dystrophic (mdx) hearts. GAPDH is used as load control. The bar graph depicts the normalized expression of NOX and NOX2-associated subunits; n = 6 hearts for each strain. B: real-time PCR analysis for NOX1, NOX2, and NOX4 (n = 4 for WT and 5 for mdx). *P < 0.05 vs. WT. **P < 0.001 vs. WT.
Fig. 2.
Fig. 2.
NOX-derived reactive oxygen species (ROS) production. A: fresh sections of hearts stained with dihydroethidium (DHE) to detect the production of ROS. The bar graph depicts the quantification of the fluorescence intensity. WT under control conditions (n = 16 sections from 6 hearts), mdx in control conditions (n = 10 sections from 6 hearts), WT treated with 100 μM apocynin (APO) (n = 17 sections from 4 hearts), and mdx treated with APO (n = 12 sections from 3 hearts) are shown. *P < 0.001 vs. all of the other groups. Scale bar indicates 100 μm. B: NADPH-dependent ROS production assessed by lucigenin chemiluminescence in WT and mdx cardiac homogenates (n = 5 each strain). RLU, relative light units. *P = 0.038. C: NOX-derived ROS production in myocytes. Freshly isolated cardiac myocytes from WT and mdx myocytes were loaded with 2′,7′-dichlorofluorescein diacetate (DCF) and treated with vehicle (DMSO) or the NOX inhibitors APO (100 μM) or VAS2870 (20 μM) for 20 min and then fixed and visualized under confocal microscopy. For each group, n = 60 cells from 4 WT hearts and 3 mdx hearts. Scale bar indicates 10 μm. *P < 0.05, **P < 0.001, and ***P < 0.0001 vs. WT control. ††P < 0.001 and †††P < 0.0001 vs. mdx control.
Fig. 3.
Fig. 3.
Sarcomere shortening-frequency relationship. A: contractility was evaluated in cardiac myocytes isolated from controls (WT) and mdx mice. A, left: representative traces of twitches from cardiomyocytes electrically paced between 0.5 and 4 Hz. Right: the response (sarcomere shortening) to the train of stimulation (0.5–4 Hz) from WT (n = 31, from 7 hearts), WT plus APO (n = 23 from 6 hearts), mdx (n = 25 from 6 hearts), and mdx myocytes with APO (n = 15, from 4 hearts). ΔL/L0, change in length/optimal length. *P < 0.0001 vs. WT, 2-way ANOVA. B: intracellular Ca2+. Left: representative traces of the amplitude of evoked intracellular Ca2+ concentration ([Ca2+]i) transients in isolated cardiomyocytes. Right: the response of WT (n = 35 from 7 hearts), mdx (n = 21, from 6 hearts), WT treated with APO (n = 12 from 3 hearts), and mdx treated with APO myocytes (n = 10 from 3 hearts) to the train of stimulation (0.5–4 Hz). *P < 0.05 vs. all of the other groups (two-way ANOVA).
Fig. 4.
Fig. 4.
Ca2+ re-uptake kinetics. A: the graph depicts the values of time constant (τ) of [Ca2+]i decay, a measurement of Ca2+ re-uptake, as function of frequency of stimulation. Mdx myocytes display higher values of τ at all frequencies studies. *P < 0.05 and **P < 0.01, mdx vs. WT. B: phospholamban (PLB) phosphorylation. Western blot analysis of heart homogenates from WT and mdx mice, under control conditions or treated with 100 μM APO during 45 min is shown. Left, top: Western blots for phosphorylated PLB in serine 16 (Ser16). Bottom: total levels of PLB (Total PLB) for each heart. Right: the bar graphs depict the quantification of the densitometry for each phosphorylation (n = 3 hearts for each group). *P < 0.05 vs. WT control (ANOVA).
Fig. 5.
Fig. 5.
Sarcoplasmic reticulum (SR) Ca2+ stores and arrhythmogenic Ca2+ release in mdx myocytes: impact of NOX inhibition. A: assessment of intra-SR Ca2+ stores. Representative traces of calcium signal (fura 2) from myocytes paced at 4 Hz and challenged with caffeine (20 mM) are shown. The bar graph on the right depicts total SR Ca2+ content in WT myocytes (n = 12 cells from 4 hearts), mdx (n = 12, from 4 hearts), WT treated with APO (n = 6 from 3 hearts), and mdx with APO (n = 6, from 3 hearts). *P < 0.05 vs. all the other groups. B, left: the protocol to assess the sensitivity of the ryanodine receptor (RyR). The myocytes are paced at 4 Hz for 10 s, then stimulation is stopped, and the bathing Tyrode solution is rapidly changed for one free of Na+ and Ca2+, to rule out influences of sarcolemmal channels (Na+ current, Ca2+ current) and the sodium/calcium exchanger. Under this condition, the Ca2+ release evoked is the consequence of activated RyR2 channels. The bar graph on the right indicates the number of events in 40 s in each group: WT control (n = 18 cells from 3 hearts), mdx control (n = 11 cells from 3 hearts), WT treated with APO (n = 9 cells from 3 hearts), and mdx with APO (n = 5 from 3 hearts). *P < 0.01 vs. all other groups and †P < 0.05 vs. WT in control conditions.
Fig. 6.
Fig. 6.
SR Ca2+ leak: impact of NOX inhibition. A: typical trace of the protocol used to evaluate SR calcium leak using tetracaine. The tetracaine-induced drop in diastolic fura 2 fluorescence (F) ratio is used as an estimate of SR Ca2+ leak, which is insensitive to changes in SR Ca2+ uptake. B: load-leak function. N = 7–8 cells in each group, from 5 WT and 4 mdx hearts. ***P = 0.0002. C: comparison of the amount of SR calcium leak at similar (matched) SR calcium loads. *P = 0.0243, mdx vs. WT and mdx treated with APO.
Fig. 7.
Fig. 7.
Nitric oxide synthase (NOS)-1 uncoupling. A: Western blot analysis for NOS1 in WT and mdx cardiac homogenates. *P < 0.05. B: nitrate levels in cardiac tissue from WT and mdx cardiac homogenates. *P < 0.05. C: S-nitrosothiol levels in cardiac myocytes. Cardiomyocytes from WT (n = 85) and mdx myocytes (n = 43) were assessed for S-nitrosothiol levels by immunostaining for S-nitrocysteine residues. Tetrahydrobioterin (BH4) supplementation (300 μM, 30 min) increased the S-nitrosylation levels in mdx myocytes (n = 55). As control, WT myocytes were treated with HgCl2 (n = 7) a redox agent that abolishes S-nitrosylation. In addition, cells were treated with an irrelevant IgG as control for the antibody specificity. **P < 0.005, mdx vs. WT and mdx + BH4.
Fig. 8.
Fig. 8.
Real-time nitric oxide (NO) production and PLB phosphorylation. A: isolated cardiomyocytes were loaded with the NO-sensitive probe 4,5-diaminofluorescein (DAF)-diacetate (DA) and stimulated at 1 Hz (baseline). Then the frequency stimulation is switched to 4 Hz, and the signal for DAF-NO is registered for 5 min. The graph depicts the average signal ± SE for WT (n = 4), mdx (n = 5), and mdx myocytes treated either with BH4 (300 μM, 15 min, n = 6) or the NOX inhibitor VAS2870 (20 μM, 20 min, n = 5). †P < 0.05 vs. mdx control. B: levels of PLB phosphorylation at Ser16 in isolated hearts from control mice, mdx mice, and mdx treated with BH4 (100 μM, 15 min), or mdx treated with VAS2870 (20 μM, 20 min); n = 5 hearts each group. The graph (left) depicts average ± SE. *P < 0.05, mdx vs. all other groups. Right: representative Western blot for phosphorylated PLB (top) and total PLB (bottom).
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References

    1. Alkaitis MS, Crabtree MJ. Recoupling the cardiac nitric oxide synthases: tetrahydrobiopterin synthesis and recycling. Curr Heart Fail Rep 9: 200–210, 2012 - PMC - PubMed
    1. Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87: 245–313, 2007 - PubMed
    1. Beigi F, Gonzalez DR, Minhas KM, Sun QA, Foster MW, Khan SA, Treuer AV, Dulce RA, Harrison RW, Saraiva RM, Premer C, Schulman IH, Stamler JS, Hare JM. Dynamic denitrosylation via S-nitrosoglutathione reductase regulates cardiovascular function. Proc Natl Acad Sci U S A 109: 4314–4319, 2012 - PMC - PubMed
    1. Beynon RP, Ray SG. Cardiac involvement in muscular dystrophies. QJM 101: 337–344, 2008 - PubMed
    1. Cai H, Griendling KK, Harrison DG. The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases. Trends Pharmacol Sci 24: 471–478, 2003 - PubMed

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