Nicorandil, a Nitric Oxide Donor and ATP-Sensitive Potassium Channel Opener, Protects Against Dystrophin-Deficient Cardiomyopathy

Abstract

Background: Dystrophin-deficient cardiomyopathy is a growing clinical problem without targeted treatments. We investigated whether nicorandil promotes cardioprotection in human dystrophin-deficient induced pluripotent stem cell (iPSC)-derived cardiomyocytes and the muscular dystrophy mdx mouse heart.

Methods and results: Dystrophin-deficient iPSC-derived cardiomyocytes had decreased levels of endothelial nitric oxide synthase and neuronal nitric oxide synthase. The dystrophin-deficient cardiomyocytes had increased cell injury and death after 2 hours of stress and recovery. This was associated with increased levels of reactive oxygen species and dissipation of the mitochondrial membrane potential. Nicorandil pretreatment was able to abolish these stress-induced changes through a mechanism that involved the nitric oxide-cyclic guanosine monophosphate pathway and mitochondrial adenosine triphosphate-sensitive potassium channels. The increased reactive oxygen species levels in the dystrophin-deficient cardiomyocytes were associated with diminished expression of select antioxidant genes and increased activity of xanthine oxidase. Furthermore, nicorandil was found to improve the restoration of cardiac function after ischemia and reperfusion in the isolated mdx mouse heart.

Conclusion: Nicorandil protects against stress-induced cell death in dystrophin-deficient cardiomyocytes and preserves cardiac function in the mdx mouse heart subjected to ischemia and reperfusion injury. This suggests a potential therapeutic role for nicorandil in dystrophin-deficient cardiomyopathy.

Keywords: cardiomyopathy; induced pluripotent cells; muscular dystrophy; nicorandil.

Figure 1

Assessment of dystrophin exon deletions and dystrophin expression in induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Nonaffected (N) iPSCs and 2 unrelated iPSC lines generated from subjects with Duchenne muscular dystrophy (Dys1-iPSC, containing an exon 3-6 deletion and Dys2-iPSC, containing an exon 45-53 deletion) were differentiated into cardiomyocytes (iCMs) and assessed for specific dystrophin exon deletions by reverse transcription polymerase chain reaction (RT-PCR) and dystrophin protein expression by immunostaining. A, Polymerase chain reaction (PCR) amplification confirms exons 3 to 5 and 47 to 48 in N-iCMs. In Dys1-iCMs, exons 3 to 5 are not amplified, but exons 47 to 48 are present. In the Dys2-iCMs, exons 3 to 5 are amplified but not exons 47 to 48, thus confirming the expected mutations in the Dys-iCMs. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was a positive control. B, Representative immunostaining of iCMs using antibodies against dystrophin (upper panel) and costaining of dystrophin and cardiac troponin T (cTNT; middle panel). Dystrophin is detected in troponin-positive N-iCMs but not in Dys1-iCMs. The Dys2-iCMs display weaker dystrophin staining in troponin-positive cells. The expression of the cardiac-specific eGFP to label cardiomyocytes is shown in the lower panel. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI).

Figure 2

Nitric oxide synthase protein localization and gene expression levels in nonaffected and dystrophin-deficient induced pluripotent stem cell (iPSC)-derived myocytes. Nonaffected (N)-iPSCs and 2 dystrophin-deficient (Dys1 and Dys2) iPSC lines were differentiated into cardiomyocytes (iCMs) and assessed for dystrophin and either neuronal nitric oxide synthase (nNOS) or endothelial NOS (eNOS) cellular location by confocal microscopy. A, Dystrophin (upper panels) is distributed to the sarcolemma in N-iCMs and to a smaller extent in Dys2-iCMs, whereas nNOS (middle panels) is detected in the cytosol in all groups. The merged images (lower panels) show that dystrophin and nNOS staining do not overlap at the sarcolemma in the iCMs. Scale bar = 50 µmol/L. B, Dystrophin (upper panels) is localized to the sarcolemma along with eNOS (middle panels) in N-iCMs. The close proximity of dystrophin and eNOS is confirmed in the merged lower panel. The proximity of eNOS with dystrophin cannot be assessed in the Dys1-iCM line, which does not express dystrophin, however, in the Dys2-iCMs that express an internally truncated form of dystrophin, eNOS and dystrophin are found in close proximity at the sarcolemma. Scale bar = 50 µmol/L. C and D, In N-iPSC-derived skeletal myocytes, dystrophin is colocalized with nNOS at the sarcolemma (scale bar = 25 µmol/L; C) but dystrophin does not colocalized with eNOS (scale bar = 25 mmol/L; D). E and F, Quantitative real-time PCR (qPCR) reveals that nNOS (E) and eNOS (F) messenger RNA (mRNA) levels are decreased in Dys-iCMs when compared to N-iCMs. Data are mean ± standard error of the mean (SEM) of values from 3 to 6 pooled independent cardiac differentiations, run in triplicate. *P ≤ .05 versus N-iCMs.

Figure 3

Concentration-dependent effects of nicorandil to protect against stress and recovery (S/R)-induced cell injury and reactive oxygen species (ROS) production in nonaffected induced pluripotent stem cell (iPSC)-derived cardiomyocytes. The N-iCMs were subjected to 2 hours of oxidative stress followed by recovery. Protective effects of different concentrations of nicorandil (0.1, 1, 10, and 100 mmol/L) on cell injury and ROS production were assessed after 4 hours of recovery by measuring lactate dehydrogenase (LDH) release into the supernatant and evaluating dihydroethidium (DHE) fluorescent intensity, respectively. A, Stress induces an increase in LDH release in N-iCMs, which is mitigated by 10 and 100 µmol/L nicorandil but not 0.1 and 1 µmol/L. B, Stress induced an increase in ROS levels as indicated by DHE fluorescence, which is abolished by 10 and 100 µmol/L nicorandil but not 0.1 and 1 µmol/L. Data are mean ± standard error of the mean (SEM) of values from 3 independent experiments (n = 3/group). *P ≤ .05 versus control.

Figure 4

Nicorandil prevents stress-induced cell injury and death in dystrophin-deficient induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Cells were treated for 24 hours with nicorandil with or without 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of nitric oxide (NO)-sensitive guanylyl cyclase, and 5-hydroxydecanoate (5-HD), an inhibitor of mitoK_ATP channel opening prior to 2 hours of oxidative stress. Cell injury was assessed after 4 hours of recovery by measuring LDH release into the supernatant, and cell death was measured after 24 hours by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive staining. A, Stress induces an increase in LDH release in all groups but is significantly elevated in Dys1-iCMs and Dys-2 iCMs. Nicorandil is able to prevent the stress-induced injury. The ODQ and 5-HD partially reverse nicorandil’s protective effects. B, Cell death was assessed by TUNEL staining after 24 hours of recovery. The Dys1-iCMs have a higher rate of death under control conditions. Stress increases the rate of cell death in iCMs and even more so in Dys-iCMs. Nicorandil is able to prevent the increase in stress-induced death. The ODQ and 5-HD partially abolish the protective effects of nicorandil. Data are mean ± standard error of the mean (SEM) of values from 3 independent experiments (n = 3/group). *P ≤ .05 versus control, ^#P ≤ .05 versus N-iCMs, ^&P ≤ .05 versus stress, ^γP ≤ .05 versus stress + nicorandil (nico).

Figure 5

Nicorandil normalizes reactive oxygen species (ROS) levels and maintains the mitochondrial after stress in dystrophin-deficient induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Cells were subjected to 2 hours of oxidative stress followed by 4 hours of recovery with or without 24-hour pretreatment with nicorandil and with or without 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of nitric oxide (NO)-sensitive guanylyl cyclase, and 5-hydroxydecanoate (5-HD), an inhibitor of mitoK_ATP channel opening. Cells were stained with tetramethylrhodamine ethyl ester (TMRE) to assess the mitochondrial membrane potential or with dihydroethidium (DHE) to detect the production of ROS. A, Quantitative analysis of DHE fluorescence. Stress induced an increase in ROS levels, which was mitigated by pretreatment of nicorandil. The ODQ inhibits nicorandil in N-iCMs and Dys2-iCMs but not Dys1-iCMs, and 5-HD has no effect on nicorandil. B, Quantitative analysis of TMRE fluorescence. Stress decreased mitochondrial membrane potential in Dys-iCMs, and nicorandil abrogates the effects of stress. The ODQ and 5-HD abolish the protective effects of nicorandil. Data are mean ± standard error of the mean (SEM) of values from 3 independent experiments (n = 3/group). *P ≤ .05 versus control, ^#P ≤ .05 versus N-iCMs, ^&P ≤ .05 versus stress, ^γP ≤ .05 versus stress + nico.

Figure 6

Nicorandil increases superoxide dismutase 2 (SOD2) expression and decreases mitochondrial reactive oxygen species (ROS) levels after stress. A, Quantitative polymerase chain reaction (PCR) analysis of SOD2 gene transcripts revealed that SOD2 expression levels are decreased in the Dys1-iCMs and Dys2-iCMs at baseline when normalized to N-iCMs. B, N-iCMs, Dys1-iCMs, and Dys2-iCMs were subjected to 2 hours of oxidative stress followed by 4 hours of recovery with or without nicorandil. The SOD2 expression levels were normalized to control conditions within each group. Stress increases SOD2 expression in N-iCMs but not Dys-iCMs. Nicorandil restores SOD2 levels after stress in the Dys-iCMs. C, The iCMs were transduced with a fluorescent probe that detects hydrogen peroxide in the mitochondria (HyPer-Mito) and subjected to control or stress and recovery (S/R) conditions. The S/R induced an increase in mitochondrial hydrogen peroxide, which was mitigated by the pretreatment with nicorandil. Data are mean ± standard error of the mean (SEM) of values from 3 independent experiments (n = 3/group). *P ≤ .05 versus control, ^#P ≤ .05 versus N-iCMs, ^&P ≤ .05 versus stress.

Figure 7

Xanthine oxidase inhibitors decrease stress-induced reactive oxygen species (ROS) production in induced pluripotent stem cell (iPSC)-derived cardiomyocytes, and nicorandil decreases cytosolic ROS level. A, Cells were subjected to either control (no stress) or to stress and recovery (S/R) conditions (2 hours of stress followed by 4 hours of recovery) with or without xanthine oxidase inhibitors allopurinol and febuxostat. Cells were stained with dihydroethidium (DHE) to detect ROS, and a defined area in each cardiomyocyte was quantitated for the intensity of DHE fluorescence. Allopurinol and febuxostat prevented the increase of ROS after stress. B, Cells were transduced with a fluorescent probe that detects hydrogen peroxide in the cytosol (HyPer-Cyto) and subjected to control or S/R conditions. There is increased fluorescence in the Dys1-iCMs and Dys2-iCMs after S/R compared to the N-iCMs. Nicorandil decreased cytosolic ROS levels. Data are mean + standard error of the mean (SEM) of values from 3 independent experiments (n = 3/group). *P ≤ .05 versus control, ^#P ≤ .05 versus N-iCMs, ^&P ≤ .05 versus stress.

Figure 8

Nicorandil increases recovery of left ventricular developed pressure (LVDP) after ischemia and reperfusion in the isolated mdx heart. Isolated hearts from mdx and C57 control mice were subjected to 30 minutes of global ischemia followed by 2 hours of reperfusion with/without pretreatment with nicorandil (Nico). Nicorandil improves recovery of LVDP in both mdx and control hearts. Data are mean + standard error of the mean (SEM), n = 8 hearts/group. *P ≤ .05 versus C57 + vehicle, #P ≤ .05 versus mdx-vehicle.