# Patent

## Title
Method for modulating or controlling sodium channel current by reactive oxygen species (ROS) originating from mitochondria

## Abstract

Method for modulating or controlling sodium channel current of a cell includes inducing mitochondrial reactive oxygen species (ROS) production in the cell.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation-in-part (CIP) application of U.S. application Ser. No. 12/289,005, filed Oct. 17, 2008, which claims the priority benefit of U.S. Provisional Patent Application Ser. No. 60/960,883, filed Oct. 18, 2007, both of which are hereby incorporated herein in their entirety by reference. This application further claims the priority benefit of U.S. Provisional Patent Application Ser. No. 61/305,668, filed Feb. 18, 2010, which is also hereby incorporated herein in its entirety by reference.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
The invention was made with government supported under grants R01 HL085558, R01 HL073753, and P01 HL058000 awarded by the NIH. The government has certain rights in the invention.


FIELD AND BACKGROUND OF THE INVENTION
The present invention is generally directed to regulating sodium channels in a cell, and more particularly to a method for modulating sodium channel current in a cell by reactive oxygen species (ROS) originating from mitochondria.
Recently, we reported that mutations in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) protein, a gene associated with Brugada Syndrome and Sudden Infant Death Syndromes (References 1 and 2), cause reduced cardiac sodium channel (Nav1.5) function by modulating pyridine nucleotides (Reference 3). Elevated intracellular NADH results in a rapid decrease in cardiac Na+ current (INa) in cardiomyocytes that is large enough to be clinically significant (Reference 4) and of a magnitude seen in Brugada Syndrome (Reference 5). The effect is identical on heterologously expressed sodium channel in human embryonic kidney (HEK) cells. The immediacy of the NADH effect on reducing INa and the lack of change in mRNA abundance under various experimental conditions suggests that the effect of NADH is post-transcriptional.
NADH modulated Nav1.5 through PKC activation and increased oxidative stress (Reference 3). The finding that the balance of oxidized and reduced NAD(H) regulates INa suggests that the metabolic state of myocytes may influence Nav1.5. NADH is known to oscillate with myocardial ischemia, and mitochondrial injury is associated with increased NADH and ROS levels (References 6 and 7). These changes in NADH could contribute to reduced INa, conduction block, and arrhythmic risk known to exist with ischemia. Moreover, heart failure is associated with increased oxidative stress, reduced NAD+, and increased NADH (References 8-10). The increased NADH level may contribute to the increased oxidative stress and diminished INa in heart failure (References 11 and 12).
Several metabolic pathways are known to produce ROS, including uncoupled nitric oxide synthase (NOS), the NAD(P)H oxidase, xanthine oxidase, and the mitochondrial electron transport chain (ETC). Cardiac oxidation leads to NOS uncoupling and diastolic dysfunction (Reference 13). NAD(P)H oxidases are an important source of superoxide in human atherosclerosis (Reference 14). Xanthine oxidase plays an important role in various forms of ischemic injury and in chronic heart failure (Reference 15). In ischemia/reperfusion injury, the ETC serves as the source of ROS (Reference 16). In chronic heart failure, ROS levels increase (References 17 and 18) and myocardial antioxidant reserve decreases (References 19 and 20). In turn, ROS increases cell death by apoptosis, reduces cellular respiration, induces structural damage to proteins including ion channels, and impairs contractility (Reference 8).
Aspects of the Invention
The present disclosure is directed to various aspects of the present invention.
One aspect of the present invention includes discovery and/or demonstration that mitochondria are the main source of NADH-dependent ROS downregulating sodium channel current (INa) in cardiac cells.
Another aspect of the present invention includes discovery and/or demonstration that mitochondrial superoxide release is responsible for downregulation of INa.
Another aspect of the present invention includes discovery and/or demonstration that elevation in intracellular NADH results in activation of protein kinase C (PKE) and subsequent mitochondrial complex III release of reactive oxygen species (ROS) through the mitochondrial inner member anion channel (IMAC).
Another aspect of the present invention includes discovery and/or demonstration that inhibition of mitochondrial ROS overproduction by one or more strategies prevents or suppresses INa downregulation by NADH.
Another aspect of the present invention includes suggestions and/or development of possible therapeutic approaches or strategies to reduce or prevent arrhythmic risk generally associated with cardiomyopathy.
Another aspect of the present invention includes a method of modulating or controlling sodium channel current of a cell by activating or inducing mitochondrial reactive oxygen species (ROS) production in the cell.
Another aspect of the present invention includes a method of reducing arrhythmic risk by administering a mitochondrial targeted antioxidant to an individual.



BRIEF DESCRIPTION OF THE DRAWINGS
One of the above and other aspects, novel features and advantages of the present invention will become apparent from the following detailed description of the non-limiting preferred embodiment(s) of invention, illustrated in the accompanying drawings, wherein:
FIG. 1 illustrates that the source of ROS induced by NADH is the mitochondria. (A) Representative traces of INa demonstrate the decrease in current in the presence of [NADH]i (100 μmol/L) was blocked by mitoTEMPO (5 μmol/L). (B) The downregulation of peak INa by [NADH]i at 100 μmol/L (**P<0.01 versus SCN5A group) is not reversed by L-NAME, apocynin, or allopurinol (P>0.05 versus NADH group), but is reversed by mitoTEMPO at 5 μmol/L (P>0.05 versus SCN5A group, P<0.01 versus NADH group). All these compounds have no effect on INa when applied alone (P>0.05 versus SCN5A group). Numbers in parentheses indicate the number of experiments.
FIG. 2 illustrates mitochondrial ROS production in response to [NADH]i monitored by MitoSOXTM Red with SCN5A cells and myocytes. The control groups were untreated, the PL groups were treated with 1 and 10 mmol/L pyruvate/lactate for 10 min, and the NAD-PL groups were incubated with 500 μmol/L NAD+ for  ̃6 hours and then treated with pyruvate/lactate buffer for 10 min. The color pictures in the upper panel are representative images of myocytes of three groups. The scale bar indicates 10 μm. The lower panel shows the relative MitoSOXTM Red fluorescent intensity, ***P<0.001 versus the untreated cells or NAD-PL groups. For each group, 9-16 samples were averaged.
FIG. 3 illustrates that PKC, the electron transport chain, and the IMAC are involved in downregulation of INa by [NADH]i. (A) Downregulation of INa by [NADH]i (**P<0.01 versus SCN5A) is reversed by rotenone (1 μmol/L), but not by 5-HD. Diazoxide does not affect INa (P>0.05 versus SCN5A). (B) Malonate (1 mmol/L) blocks the NADH effect on reducing INa, and antimycin A (20 μmol/L) reproduces the [NADH]i effect (**P<0.01 versus SCN5A group). The antimycin A-induced reduction in INa is prevented by [NAD+]o, forskolin, or rotenone. Azide failed to block the NADH effect. (C) Chelerythrine failed to block the antimycin A effect on reducing INa, confirming that PKC activation is required for ROS generation. (D) Downregulation of INa by [NADH]i is reversed by DIDS, PK11195 and 4′-CD, but not by CsA (**P<0.01 versus SCN5A groups). Numbers in parentheses indicate the number of experiments.
FIG. 4 illustrates neonatal ventricular myocytes analogous downregulation of INa by [NADH]i. Downregulation can be blocked by rotenone and 4′-CD, but not L-NAME. Antimycin A decreases INa similarly to that of [NADH]i (**P<0.01 and ***P<0.001 versus control myocytes). Numbers in parentheses indicate the number of experiments.
FIG. 5 illustrates downregulation of INa by A280V GPD1-L being reversed by mitoTEMPO, rotenone, and 4′-CD (**P<0.01 versus all other groups). Peak currents at −20 mV were normalized to cell capacitance and divided by the current obtained with SCN5A cells transfected with WT GPD1-L. Numbers in parentheses indicate the number of experiments.
FIG. 6 illustrates a proposed signaling pathway by which the mutant GPD1-L and NADH downregulate cardiac Na+ channel by causing PKC activation and ROS overproduction from the complex III of mitochondrial electron transport chain. Reactive oxygen species (ROS) are released from the mitochondria by the IMAC that is modulated by the mBzR. NAD+ upregulates the cardiac Na+ channel through PKA activation and inhibition of ROS overproduction.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S) OF THE INVENTION
The present invention is based, at least in part, on the discovery and/or demonstration that mitochondria are the main source of NADH-dependent ROS downregulating the cardiac Nav1.5.
Altered cardiac metabolism is associated with increased risk of arrhythmias and sudden death. In part, this occurs because of reduced electrical conduction in the cardiomyocytes, but the mechanisms for this are not clear. As noted above, we have shown previously that a mutation in GPD1-L protein, causing the sudden death condition Brugada syndrome, reduces INa by raising intracellular NADH levels and inducing ROS. Here, we investigated the source of ROS induced by elevated NADH. We found that elevated NADH induced ROS production from mitochondria and that ROS released from the mitochondria was mediated by the IMAC. NAD+, inhibition of mitochondrial electron transport, a mitochondrial targeted antioxidant, and an IMAC modulator could prevent the reduction in INa by reducing mitochondrial ROS production. These findings contribute to our understanding of the mechanisms of conduction block and arrhythmia when cardiac metabolism is disordered. In addition, they suggest possible therapeutic strategies to reduce arrhythmic risk associated with cardiomyopathy.
Methods
All chemicals were purchased from Sigma (St. Louis, Mo.) except: diazoxide and FGIN-1-27 (Enzo Life Sciences International, Inc., Plymouth Meeting, Pa.); chelerythrine and cyclosporin A (Alomone Labs, Jerusalem, Israel); and Hoechst 33342, MitoSOXTM Red, and tetramethylrhodamine methyl ester (TMRM) (Molecular Probes, Eugene, Oreg.). MitoTEMPO was a generous gift from Dr. Sergey Dikalov (Emory University, Atlanta, Ga.).
Cell Culture
We maintained a human embryonic kidney (HEK) cell line stably expressing the human cardiac Nav1.5 channel (SCN5A cells). Expression of Nav1.5 was linked to green fluorescent protein (GFP) expression by an internal ribosomal entry site (SCN5A-IRES-GFP). SCN5A cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 0.2 mg/mL geneticin (for antibiotic selection) and 1% penicillin/streptomycin in a 95% O2/5% CO2 incubator at 37° C. Rat neonatal ventricular myocytes (NVM) were isolated from neonatal rat hearts by collagenase treatment (Worthington Biochemical Corporation, Lakewook, N.J.).
Nearly undetectable levels of GPD1-L protein are expressed in HEK cells (Reference 1). Therefore, for whole-cell patch clamping experiments to study GPD1-L effects on Nav1.5, SCN5A cells were transiently transfected with WT or A280V GPD1-L (a generous gift from Dr. Barry London, University of Pittsburgh, Pa.) and an expression vector containing red fluorescent protein (RFP) as described previously (Reference 2). In these experiments, cells expressing both GFP and RFP were studied.
Electrophysiology
Na+ currents were measured using the whole-cell patch clamp technique in voltage-clamp mode at room temperature. Pipettes (1-2 MΩ) were filled with a pipette solution containing (in mmol/L): CsCl 80, cesium aspartate 80, EGTA 11, MgCl2 1, CaCl2 1, HEPES 10, and Na2ATP 5 (adjusted to pH 7.4 with CsOH). The bath solution consisted of (in mmol/L): NaCl 130, CsCl 5, CaCl2 2, MgCl2 1.2, HEPES 10 and glucose 5 (adjusted to pH 7.4 with CsOH). A stepped voltage protocol from −100 to +60 mV with a holding potential of −100 mV was applied to establish the presence of voltage-gated Nav1.5 channels. Peak currents obtained during steps to −20 or −30 mV were used for comparison in determining the relative reduction of INa. Steady state fast inactivation was assessed during voltage depolarization from a holding potential of −140 to −20 mV for 500 ms, and measuring current at −20 mV. In all recordings, 80% of the series resistance was compensated, yielding a maximum voltage error of  ̃1 mV. Data were sampled at 50 kHz and later low pass filtered at 10 kHz for analysis. Currents were recorded and analyzed with an Axopatch 200B amplifier, Axon Digidata 1320A A/D converter and pClamp software (Molecular Devices, Sunnyvale, Calif.). To minimize time-dependent drift in gating parameters, all protocols were initiated 2-5 min after whole-cell configuration was obtained. The currents were normalized with cell capacitance prior to deriving ratios.
Rat NVM action potentials were measured using the whole-cell patch clamp technique in current-clamp mode at room temperature. Pipettes (2-4 MΩ) were filled with a pipette solution containing (in mmol/L): NaCl 10, potassium glutamate 130, EGTA 1.0, MgCl2 0.5, KCl 9, HEPES 10, glucose 10, and MgATP 5 (adjusted to pH 7.4 with KOH). The bath solution consisted of (in mmol/L): NaCl 140, KCl 5, CaCl2 2, MgCl2 1.0, HEPES 10 and glucose 10 (adjusted to pH 7.4 with NaOH). Action potentials were evoked by brief (4 ms) current injections applied at 0.8-1 Hz. Eighty percent of the series resistance was compensated, yielding a maximum voltage error of  ̃1 mV. Data were sampled at 50 kHz and later low pass filtered at 10 kHz for analysis. Action potentials were recorded and analyzed with an Axopatch 200B amplifier, Axon Digidata 1320A A/D converter and pClamp software.
The following specific inhibitors or activators were applied directly in the pipette solution, alone or together: NADH (100-500 μmol/L), Nω-nitro-L-arginine methyl ester (L-NAME, 1-20 mmol/L), allopurinol (200 μmol/L), mitoTEMPO (5-20 μmol/L), rotenone (1-5 μmol/L), antimycin A (20-40 μmol/L), azide (10 mmol/L), 5-hydroxydecanoate (5-HD, 300 μmol/L), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 500 μmol/L), cycloporine A (CsA, 0.5 μmol/L), PK11195 (50 μmol/L), 4′-chlorodiazepam (4′-CD, 40-100 μmol/L), and FGIN-1-27 (500 μmol/L). Apocynin (100-300 μmol/L), forskolin (1-5 μmol/L), NAD (500 μmol/L) and malonate (1 mmol/L) were applied to bath solution, respectively. Concentrations were determined in our laboratory or by using the similar values used in literatures.
Intracellular NADH Level
Intracellular NADH levels ([NADH]i) were detected by using the EnzyChromTM NAD+/NADH Assay Kit (BioAssay Systems, Hayward, Calif.) in SCN5A cells with or without treatment of 1 mmol/L pyruvate and 10 mmol/L lactate for 10 min at room temperature. The intensity difference of the reduced product color, measured at 565 nm at time zero and 15 min later, was proportional to the change in [NADH]i.
Confocal Microscopy
To measure mitochondrial ROS, the fluorescent probe MitoSOXTM Red was used according to the manufacturer's protocol. Briefly, three groups of SCN5A cells or rat NVM were studied: untreated cells, the PL group (cells treated with 1 mmol/L pyruvate and 10 mmol/L lactate for 10 min at room temperature, under which condition intracellular NADH level was increased (References 21-23), see “Results” below), and the NAD-PL group (cells incubated with NAD+ for  ̃6 hours at 37° C. and then treated with 1 mmol/L pyruvate and 10 mmol/L lactate for 10 min at room temperature). The three groups of cells were then incubated with 2.5 μM MitoSOXTM Red in Hank's balanced salt solution (HBSS) for 10 min at 37° C., followed by three times wash with warm HBSS. Before treatment with MitoSOXTM Red, cells were first stained with Hoechst 33342 (0.4 μg/ml working concentration) for 20 min at 37° C. Images were taken on a Zeiss LSM510 META confocal microscope (Carl Zeiss GmbH, Oberkochen, Germany) using an argon laser excitation (514 nm) with emission collection at more than 560 nm (red). The cell area was calculated, and the whole cell fluorescence of MitoSOXTM Red was measured with ImageJ software. The number of pixels of cell fluorescence divided by the cell area was used to determine the mitochondrial ROS generation. For each of the groups, 9 to 16 cells were used. NADH in water has an emission peak at 460 nm and less than 20% of the maximum value above 560 nm (Reference 46). Therefore, NADH is unlikely to interfere with the fluorescence of MitoSOXTM Red in our experiments.
To measure the effect of elevated intracellular NADH level on the mitochondrial membrane potential (ΔΨm), the fluorescent membrane-permeant cationic probe TMRM, which is readily sequestered by mitochondria, was applied. SCN5A cells or rat NVM were loaded with TMRM (100 nmol/L) (Reference 24) for 30 min at 37° C. in the dark. Then, cells were washed gently twice and kept with the bath solution used in the patch experiments before being placed on the stage of a Zeiss LSM510 META confocal microscope (35° C.). TMRM was excited at 543 nm with a helium neon laser (3%), and the emission was collected through a 560 nm longpass filter. Images were collected at 30 s intervals for 10 min and then 2 min interval for 30 min. For the PL group, pyruvate and lactate were applied after the first image was taken. The mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 10 μmol/L) was incubated for 1 min at 35° C. with cells, which is sufficient to completely depolarize ΔΨm (Reference 25). Images were then collected every 2 min for 20 min. The resulting fluorescence images were processed using Zeiss LSM510 META software to obtain the time course of the TMRM fluorescence changes.
Statistical Evaluations
Data are shown as the mean±SEM. Determinations of statistical significance were performed with ANOVA with Bonferroni correction for comparisons of multiple means. A value of P<0.05 was considered statistically significant.







TABLE 1







Parameters of voltage dependence of steady state activation and inactivation of all groups










Voltage dependence of activation
Voltage dependence of inactivation














V1/2, mV
k, mV
n
V1/2, mV
k, mV
n

















SCN5A
−44.7 ± 0.2
5.7 ± 0.1
14
−72.9 ± 0.3 
6.8 ± 0.2
15


+100 μM [NADH]i
−44.5 ± 0.3
5.5 ± 0.2
16
−75.6 ± 0.3#
7.0 ± 0.3
21


+100 μM [NADH]i + 10 mM [L-NAME]i
−45.9 ± 0.6
6.0 ± 0.5
8
−74.8 ± 0.6 
9.3 ± 0.5
7


+100 μM [NADH]i + 200 μM [apocynin]o
−43.2 ± 0.3
6.9 ± 0.2
14
−78.5 ± 0.6*
8.6 ± 0.5
14


+100 μM [NADH]i + 200 μM [allopurinol]i
−44.1 ± 1.1
6.6 ± 0.8
13
−77.5 ± 0.6*
6.9 ± 0.3
11


+100 μM [NADH]i + 5 μM [mitoTEMPO]i
−44.1 ± 0.2
5.9 ± 0.2
11
−77.4 ± 0.2 
6.1 ± 0.1
9


+100 μM [NADH]i + 1 μM [rotenone]i
−45.8 ± 0.4
5.2 ± 0.3
10
−78.1 ± 0.3*
7.6 ± 0.3
9


+20 μM [antimycin A]i
−46.1 ± 0.4
5.1 ± 0.3
13
−69.3 ± 0.8#
6.3 ± 0.3
7


+100 μM [NADH]i + 1 mM [malonate]o
−45.9 ± 0.4
5.9 ± 0.3
12
−75.9 ± 0.2 
6.6 ± 0.2
10


+100 μM [NADH]i + 10 mM [azide]i
−46.6 ± 0.6
5.5 ± 0.5
16
−79.3 ± 0.2*
5.0 ± 0.2
9


+100 μM [NADH]i + 300 μM [5-HD]i
 −39.9 ± 0.2*
6.7 ± 0.1
13
−78.8 ± 0.2*
5.9 ± 0.2
7


+200 μM [diazoxide]i
−45.1 ± 0.5
7.1 ± 0.5
11
−71.0 ± 0.3 
7.5 ± 0.3
5


+100 μM [NADH]i + 500 μM [DIDS]i
−45.7 ± 0.3
5.1 ± 0.2
13
−79.2 ± 0.2*
6.2 ± 0.2
9


+100 μM [NADH]i + 0.5 μM [CsA]i
 −41.2 ± 1.0*
5.3 ± 0.8
13
−75.5 ± 0.5 
7.0 ± 0.4
9


+100 μM [NADH]i + 50 μM [PK11195]i
 −4 1.3 ± 0.7*
5.9 ± 0.5
15
−79.0 ± 0.2*
6.0 ± 0.2
7


+100 μM [NADH]i + 40 μM [4′-CD]i
−45.6 ± 0.5
6.0 ± 0.4
14
−78.6 ± 0.4*
7.5 ± 0.4
10


+500 μM [FGIN-1-27]i
−46.7 ± 0.4
5.4 ± 0.3
9
−78.9 ± 0.5#
8.4 ± 0.5
7


+100 μM [NADH]i + 500 μM [FGIN-1-27]i
−46.7 ± 0.4
5.4 ± 0.3
9
−83.3 ± 0.3*
7.0 ± 0.2
5





Data are shown as mean ± SEM, n is sample number. For V1/2,


# P < 0.05 vs. SCN5A, and


*P < 0.05 vs. +100 μM [NADH]i.






Results
Sources of ROS Induced by NADH
Since SOD is able to block the effect of NADH (Reference 3), ROS are implicated in the signaling cascade whereby NADH reduces INa. Sources of ROS within a cell include uncoupled NOS, the NAD(P)H oxidases, xanthine oxidase, and mitochondria. By using specific inhibitors, we tested which of these was the source of ROS modulating INa in response to increased cytosolic NADH.
FIG. 1 shows that apocynin, Nω-nitro-L-arginine methyl ester (L-NAME), and allopurinol did not affect INa, when they were applied alone in SCN5A cells. When applied with 100 μmol/L NADH, none of these blockers were able to inhibit the NADH effect on reducing cardiac INa. Steady state activation (SSA) was minimally affected by these compounds, and there were physiologically nonsignificant trends for hyperpolarizing shifts in steady state inactivation (SSI) with apocynin and allopurinol in the presence of NADH (Table 1). These experiments indicate that the NAD(P)H oxidases, uncoupled NOS, and xanthine oxidases are not the source of ROS induced by NADH.
MitoTEMPO is a highly positively charged TEMPO derivative that is concentrated in the mitochondria matrix and acts there as a superoxide scavenger (References 26 and 27). MitoTEMPO at 5 μM blocked the NADH effect on reducing INa but had no effect on INa when applied alone (FIG. 1). The SSA and SSI were not affected by mitoTEMPO with or without the presence of NADH (Table 1). This implied that the mitochondria were a likely source of ROS induced by increased NADH.
Mitochondrial ROS Generation Induced by Elevated NADH
Mitochondrial ROS generation was monitored with MitoSOXTM Red in SCN5A cells and rat NVM, respectively. MitoSOXTM Red is a membrane permeant, fluorogenic dye for selective detection of superoxide in the mitochondria. Once in the mitochondria, the dye is oxidized by superoxide and exhibits red fluorescence. Application of MitoSOXTM Red in untreated cells revealed a low level of red fluorescence, indicating low levels of mitochondrial ROS (FIG. 2). SCN5A cells and rat NVM were treated with 1 and 10 mmol/L PL buffer (PL group in FIG. 2). This PL buffer increased intracellular NADH level by 1.7±0.1-fold and decreased INa to 0.54±0.04 of control (P<0.01) (Reference 3). Treatments showed 2.06±0.09-fold and 2.18±0.15-fold increases in mitochondrial ROS levels for SCN5A cells and rat NVM as compared to untreated cells, respectively. This increase in ROS was blocked by NAD+ pre-incubation (NAD-PL group in FIG. 2, 0.96±0.06 and 1.11±0.18-fold of untreated cells, respectively). These observations are in agreement with the electrophysiological studies and confirm that mitochondria are the source of ROS overproduction induced by elevated NADH.
The ETC as a Source of NADH-Induced ROS
Our previous work has shown that PKC activation is required for ROS production in response to NADH (Reference 3). The ETC and mitochondrial ATP-sensitive K+ channel (mitoKATP) are targets of PKC activation (Reference 28), and both have been shown to be involved in ROS generation and release from mitochondria (References 6, 16, 29 and 30). An inhibitor and an opener of the mitoKATP channel, 5-hydroxydecanoate (5-HD) (Reference 31) and diazoxide (Reference 32) respectively, were applied to study whether they would have any effect on INa. As shown in FIG. 3A, 5-HD neither blocked the NADH effect on reducing INa nor showed any effect on INa when applied alone. Diazoxide did not affect INa, either. For 5-HD, there were minor shifts of V1/2 values of the SSA and SSI relationships that were not enough to affect the evaluation of the peak currents (Table 1). These experiments indicate that the mitoKATP channel is not involved in NADH modulation of Nav1.5.
Complexes I and III are the main sources of ROS production of ETC (References 6, 16 and 33). Rotenone, which decreases ROS generation by inhibiting complex I (i.e. NADH dehydrogenase) (References 6 and 33), blocked entirely the NADH effect on INa (FIG. 3A), indicating that the ETC was the source of ROS overproduction induced by NADH. Malonate, an inhibitor complex II, (Reference 33) also blocked the NADH effect and reversed the decrease in INa (FIG. 3B). FIG. 3B also shows that azide, which inhibits complex IV, (Reference 33) failed to block NADH effect on reducing INa. Antimycin A blocks the electron transfer from the Qi to Qo sites of complex III and increases ROS generation in the intermembrane space of mitochondria (References 6 and 33). We found that antimycin A gave rise to an equivalent decrease of INa as did NADH. Comparably to NADH, the antimycin A effect was blocked by NAD+, forskolin, or rotenone as shown in FIG. 3B (Reference 3). A PKC inhibitor, chelerythrine, failed to block the antimycin A effect to reduce INa, as shown in FIG. 3C. This confirmed that PKC activation was necessary for ROS generation from complex III (Reference 3). Shifts of V1/2 values of SSI were observed with rotenone and azide in the presence of NADH, and with antimycin A alone. These were minor and unlikely to be sufficient to affect Na+ channel availability significantly at the holding potential used (Table 1).
NADH-Induced ROS Release from Mitochondria was through the Mitochondrial Inner Membrane Anion Channel (IMAC)
Mitochondrial respiration is ordinarily accompanied by low-level ROS generation. In the event of significant cellular ROS, mitochondria respond by increasing their own ROS production, a phenomenon termed ROS-induced ROS release (RIRR) (References 34 and 35). Two modes of RIRR have been reported: the mitochondrial inner membrane anion channel (IMAC)-dependent and the mitochondrial permeability transition pore (MPTP)-dependent mechanisms. These two anions channels along with the voltage-dependent anion channel (VDAC) are thought to be the predominant paths for cytosolic release of superoxide generated by the ETC. Cycloporine A (CsA) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) are inhibitors of MPTP and IMAC/VDAC, respectively. FIG. 3D shows that DIDS blocked the NADH effect on reducing INa, but CsA did not. Measurements of the mitochondrial ΔΨm with TMRM showed that elevated NADH levels did not affect the ΔΨm (data not shown). This indicated that the IMAC or VDAC but not MPTP are involved in ROS release in response to NADH.
IMAC is regulated by the mitochondrial benzodiazepine receptor (mBzR). It has been reported that ROS generation and oscillations are prevented by inhibiting IMAC with mBzR ligands such as 4′-chlorodiazepam (4′-CD) and PK11195 (Reference 6). Inhibition of mitochondria ROS release by 4′-CD is thought to prevent reperfusion arrhythmias (Reference 24). As shown in FIG. 3D, both 4′-CD and PK11195 were capable of blocking the NADH effect on INa. Since the mBzR modifies ROS release through the IMAC, these data strengthen the idea that IMAC is involved in mitochondrial ROS release in response to NADH. FGIN-1-27 (500 μmol/L), an activator of mBzR (References 6 and 24), showed that simply opening the mBzR was not enough to decrease INa (1.01±0.14 of SCN5A group, P>0.05). When FGIN-1-27 and NADH were applied together, FGIN-1-27 showed no influence on the reduction in INa mediated by NADH. NADH (100 μmol/L) alone reduced INa to 0.54±0.04 of SCN5A group (P<0.01) (Reference 3), while in the presence of FGIN-1-27 (500 μmol/L), the reduction of INa by NADH was 0.51±0.04 (P<0.01). This implies that the mBzR is fully activated in the presence of NADH.
Neonatal Ventricular Myocytes Show Similar Results
Analogous experiments were repeated using rat NVM to confirm the effects of rotenone, antimycin A, 4′-CD, and L-NAME on NADH regulation of Nav1.5. As shown in FIG. 4, rotenone and 4′-CD blocked the NADH effect on INa, while L-NAME did not. Antimycin A reduced INa to 55±7% in myocytes. These results were in agreement with the findings obtained with SCN5A cells, confirming the mitochondrial role on NADH regulation of Nav1.5 in myocytes.
NADH treatment did not affect the maximum diastolic membrane potential. The value for untreated NVM was −66.9±1.4 mV and was −64.3±1.8 mV for myocytes treated with 500 μmol/L NADH (p=NS). On the other hand, treatment with NADH decreased the maximum upstroke velocity of the action potential to 0.68±0.12 of untreated NVM (P<0.05).
A280V GPD1-L and NADH Affect INa Correspondingly
Previously, we have found that the mutant A280V GPD1-L reduces INa by increasing intracellular NADH (Reference 3). Similarly to the NADH-mediated INa reduction, mitoTEMPO, rotenone, and 4′-CD all reversed the INa decrease caused by A280V GPD1-L (FIG. 5). When these compounds were applied to cells expressing WT GPD1-L, the INa was unvaried (data not shown). These results imply that increased NADH mediates the effect of A280V GPD1-L to downregulate Nav1.5 and that mitochondrial ETC and IMAC are involved in the pro-arrhythmic effect of this mutation.
Discussion
Many signaling pathways involved in cardiomyopathy and cardioprotection converged on the mitochondria. Mitochondria comprise  ̃30-40% of the myocyte volume and generate >90% of the ATP (References 36 and 37). Also, they are a major site of physiological ROS production in the cardiomyocyte, with 1-3% of the electrons flowing through the ETC leaking to produce ROS (References 38 and 39). ROS generation within the mitochondrial matrix depends critically on the proton motive force, the NADH/NAD+ ratio, the CoQH2/CoQ ratio, and the local O2 concentration. Under conditions of a high NADH/NAD+ ratio, complex I and perhaps other enzymes linked to the NADH pool may contribute to ROS production (Reference 40).
In the present study, we discovered that the oxidative stress induced by NADH is derived from mitochondria. Experiments with different inhibitors for the uncoupled NOS, NAD(P)H oxidases, xanthine oxidases, mitoKATP, and the ETC revealed that the mitochondrial ETC plays a critical role in NADH regulation of Nav1.5. Blockade of the NADH effect to reduce INa was observed with rotenone and malonate, complex I and II blockers, respectively. Because malonate inhibited the NADH-induced ROS, but cannot prevent ROS release from complex I, it seemed likely that complex III was the source of ROS in our study. Another possibility is reverse electron transfer from complex II to complex I can also lead to ROS production (Reference 41). This is also blocked by malonate and rotenone. Antimycin A inhibits complex III at the Qi center and increases superoxide generation from the Qo center (Reference 42). In the present study, antimycin A caused a significantly reduced INa, supporting the idea that complex III is the source of ROS induced by NADH. At the same time, the antimycin A effect could be blocked by NAD+, forskolin, and rotenone. These results are comparable to the inhibition of the NADH effect on INa reported in this and previous work (Reference 3). Taken together, the data suggest that complex III is the main source of NADH-induced ROS generation and that blockade of electron flow upstream of complex III minimizes ROS production induced by NADH.
ROS produced by leakage of electrons from the ETC can trigger the opening of the mitochondrial IMAC and subsequent release of O2.− to the cytoplasm (References 6 and 34). IMAC-dependent ROS release is regulated by the mBzR. Localized mitochondrial ROS release can propagate throughout cardiac cells in the form of oscillations or waves (References 6 and 34). Mitochondrial depolarization associated with increase ROS and activation of the MPTP has been correlated with opening of the mitoKATP channel and conduction block, referred to as a metabolic sink (Reference 43). We show a second possible mechanism for conduction impairment involving mitochondrial ROS, ROS induced decreased INa, which is dependent on the mBzR and IMAC but not the MPTP. CsA failed to block the NADH effect on reducing INa while PK11195 and 4′-CD inhibited the NADH effect. This suggests that Na+ channel-mediated changes in conduction may precede those of mitoKATP, since the mitoKATP effect requires mitochondrial MPTP activation and mitochondrial depolarization whereas the NADH effect requires less extreme mitochondrial ROS production.
Studies of metabolic stress in isolated cardiac cells reveal that energy-sensitive K+ channels in the sarcolemmal membrane can be activated spontaneously in an oscillatory manner (Reference 44). These K+ current oscillations are closely associated with whole cell metabolic oscillations. Modulation of the cellular action potential by these metabolic oscillations could result in arrhythmias in the heart after ischemia-reperfusion. Mitochondria have been identified as the source of the oscillations. K+ channel opening compounds like diazoxide and nicorandil have been found to protect heart cells from ischemic or oxidative stress through a mechanism that involves the opening of mitoKATP channel (Reference 32). In our work, the blocker for mitoKATP, 5-HD, was unable to protect against the NADH-mediated reduction in INa, and an opener of mitoKATP, diazoxide, did not affect INa, either. These results indicate that the NADH effect is unique and independent of mitoKATP.
In summary, elevated intracellular NADH leads to mitochondrial ROS overproduction that results in downregulation of the cardiac Na+ channel. Mitochondrial ROS overproduction is mainly derived from complex III of the electron transport chain, and ROS is probably released into the cytoplasm through the IMAC, which is regulated by the mBzR (FIG. 6). A similar mechanism likely explains the arrhythmia syndromes induced by mutant GPD1-L protein (References 1 and 3), since the mutant GPD1-L A280V leads to an increase of intracellular NADH level and mitoTEMPO, rotenone, and 4′-CD block the A280V GPD1-L effect to reduce INa. Valdivia et al. (Reference 45) presented a somewhat different possible signaling pathway to explain the reduction in INa with mutations of GPD1-L. Nevertheless, the two proposals share many elements, including elevated NADH and PKC activation being involved in the signaling cascade. In experiments not shown, the lack of effect on INa of raising intracellular dihydroxyacetone phosphate, which should increase glycerol-3-phosphate production by glycerol-3-phosphate dehydrogenase catalysis without raising NADH levels, suggests that NADH and not glycerol-3-phosphate is mediating the reduction in current.
Our experiments do not unequivocally establish a mechanism by which mitochondrial ROS reduce INa. ROS could be having a direct effect on the channel, cause the channel to be excluded from the membrane, or alter channel post-translational modifications known to decrease INa. Preliminary experiments suggest that the disulfide reducing agent, dithiothreitol, does not prevent the NADH effect. Moreover, preliminary total internal reflection fluoroscopy experiments with labeled sodium channels do not show any channel internalization in response to NADH. It seems reasonable that PKC acts directly on the channel, as proposed by Valdivia et al. (Reference 45). Changes in the SSA and SSI relationships support this assertion. It is interesting to note, however, that the effect of only one of two GPD1-L mutations known to cause sudden death is fully reversed by eliminating a Na+ channel PKC phosphorylation site, suggesting the possibility of multiple mechanisms or sites being involved in the current reduction. Our results represent a heretofore unknown regulation of the cardiac Na+ channel by NADH through mitochondria ROS production that may help explain the link between altered metabolism and arrhythmic risk.
The following is a list of abbreviation and acronyms used herein:

    4′-CD, 4′-chlorodiazepam
    5-HD, 5-hydroxydecanoate
    AP, action potential
    CCCP, carbonyl cyanide 3-chlorophenylhydrazone
    CsA, Cycloporine A
    DIDS, 4′-diisothiocyanatostilbene-2,2′-disulfonic acid
    ETC, electron transport chain
    FGIN-1-27, [N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide
    GFP, green fluorescent protein
    GPD1-L, glycerol-3-phosphate dehydrogenase 1-like
    HEK, human embryonic kidney
    IMAC, the mitochondrial inner membrane anion channel
    L-NAME, Nω-nitro-L-arginine methyl ester
    mBzR, the mitochondrial benzodiazepine receptor
    mitoKATP, mitochondrial ATP-sensitive K+ channel
    MPTP, the mitochondrial permeability transition pore
    Nav1.5, cardiac sodium channel
    NOS, nitric oxide synthase
    NVM, neonatal ventricular myocyte
    PK, protein kinase
    PL, pyruvate/lactate
    RFP, red fluorescent protein
    RIRR, ROS-induced ROS release
    SCN5A, cardiac sodium channel
    SOD, superoxide dismutase
    SSA, steady state activation
    SSI, steady state inactivation
    TMRM, tetramethylrhodamine methyl ester
    VDAC, the voltage-dependent anion channel


While this invention has been described as having preferred sequences, ranges, steps, materials, structures, components, features, and/or designs, it is understood that it is capable of further modifications, uses, and/or adaptations of the invention following in general the principle of the invention, and including such departures from the present disclosure as those come within the known or customary practice in the art to which the invention pertains, and as may be applied to the central features hereinbeforesetforth, and fall within the scope of the invention and of the limits of the appended claims.
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## Claims

1. A method of reducing arrhythmic risk in an individual with elevated intracellular NADH level, comprising the steps of:
a) providing an individual with elevated intracellular NADH level;
b) lowering the intracellular NADH level to a predetermined normal level by administering to the individual a therapeutic amount of a mitochondrial targeted antioxidant; and
c) wherein achieving the normal intracellular NADH level reduces arrhythmic risk in the individual.

2. The method of claim 1, wherein the amount of antioxidant is effective to reduce arrhythmic risk.

3. The method of claim 1, wherein the antioxidant prevents reduction in sodium channel current (INa) by reducing or suppressing mitochondrial ROS production.

4. The method of claim 1, wherein the antioxidant is administered orally or intravenously.

5. The method of claim 1, wherein the individual is suffering from arrhythmia.

6. The method of claim 1, wherein the antioxidant comprises at least one member selected from the group consisting of a powder, a tablet, a capsule, a solution, a suspension, and an injectable formulation.

7. The method of claim 1, wherein the antioxidant prevents a change in sodium channel current (INa) by reducing or suppressing mitochondrial ROS production.