these aldehydes include aliphatic and aromatic aldehydes from the environment , food , and those generated during cell metabolism . when aldh2 was overexpressed , the deleterious effects of acetaldehyde in the heart were diminished . a single point mutation in aldh2 greatly reduces aldh2 activity in east asians , which leads to accumulation of acetaldehyde and ultimately facial flushing   and other harmful effects . moreover , this mutation is reportedly associated with diabetic complications   and coronary diseases in east asians . it is suggested that the toxicity of reactive aldehydes plays main role in the pathogenesis . we focus on the secondary products of oxidative stress , that is , reactive aldehydes . toxic reactive aldehydes such as 4-hydroxy-2-nonenal ( 4hne ) are produced by lipid peroxidation during oxidative stress in the mitochondria . 4hne is especially toxic as it forms adducts with proteins and leads to cellular dysfunction [ 714 ] . in another recent report , we have shown that pharmacological inhibition of aldh2 per se induces mitochondrial dysfunction and cell death . to provide an extension of our previous results , we planned to investigate the effect of aldh2 inhibition using disulfiram ( dsf ) , an aldh2 inhibitor , on high glucose stress in h9c2 cardiomyocyte cell lines . h9c2 cell lines { atcc clr-1446 , routinely used as cardiomyocytes [ 1618 ] } , were cultured and grown in dmem medium . after 1  hr of starving , h9c2 cells were treated with dsf ( santa cruz chemicals , santa cruz , ca ) , an inhibitor of aldh2 or dmso for 2 hours , and then subjected to high glucose stress for 24 hours . the high glucose stress was induced by replacing the culture medium with 33  mm of d - glucose ( hg ) . we maintained the glucose concentration in the cell culture medium throughout the study period by measuring the glucose level using a glucometer . to nullify the osmotic stress caused by high concentration of solute in the culture medium is not the cause for the observed cellular stress , the culture medium of another set of cells was replaced with the same 33  mm d - mannitol ( ctrl ) . we chose 2 doses for dsf , that is , 10  m and 2  m after performing a dose - response curve using dsf in inhibiting aldh2 activity as shown in supplementary figure  1 ( see supplementary material available online at http://dx.doi.org/10.1155/2016/1390861 ) . we used 2  m for the mitochondrial studies as mitochondrial oxygen consumption rate ( ocr ) was decreased below detection level with 10  m dsf treatment . intracellular ros can oxidize the nonfluorescent 29,79-dichlorofluorescein diacetate ( dcfh - da ) to generate fluorescent 29,79-dichlorofluorescein ( dcf ) . fluorescence intensity of dcf can be measured using a fluorimeter . therefore to measure intracellular ros in h9c2 cells , we used the dcf kit and followed the manufacturer 's instructions ( abcam inc . briefly , cells were washed twice with pbs after the study protocol , and then the ros detection solution was added . the fluorescence intensity of dcf was measured using a fluorimeter at 504  nm excitation and 524  nm emission wavelengths . aldh2 activity was measured by following the procedure described elsewhere [ 19 , 20 ] . in brief , enzymatic activity of aldh2 from cell lysate was determined spectrophotometrically by using the reductive reaction of nad+ to nadh at 340  nm . all assays were carried out at 25c in 0.1  m sodium pyrophosphate buffer , ph = 9.5 with 2.4  mm nad+ as a cofactor and 10  mm acetaldehyde as the substrate . 4hne protein adducts were evaluated by western immunoblot as described earlier [ 21 , 22 ] . in brief , protein samples from cell lysates were separated using sds - page and the proteins were then transferred to immobilon - p membranes ( millipore , billerica , ma ) . anti-4hne - cys / his / lys rabbit antibody ( millipore ) and anti--actin mouse monoclonal antibody ( scbt , santa cruz , ca ) at a concentration of 1  :  1000 ( 4c overnight ) were used . the bound antibody was visualized with horseradish peroxidase- ( hrp- ) coupled secondary antibody and chemiluminescence detection reagents . the ratios of levels of 4hne protein adducts and -actin , the loading control , were calculated and plotted as graphs . cells grown at 6070% confluence were starved for 1  hr using dmem media without serum . the plates were then divided into the following treatment groups ( n = 6 plates each ) . at the end of the protocol , cells were washed with tbs buffer and trypsinized . trypan blue was added at a concentration of 1  :  1 and subsequently an automated cell counter ( bio - rad ) was used to determine % of live cells remaining . mitosox reagent was used to measure mitochondrial ros as explained in the manufacturer 's instructions . 1.02.0  ml of 5  m mitosox reagent working solution was used to cover h9c2 cells and incubated for 10 minutes at 37c , protected from light . after washing cells with warm buffer , we processed the cells for imaging using florescent microscope with absorption at 510  nm and emission 580  nm . we also quantified the fluorescence using synergy h1 multi - mode reader ( biotek inc . ) . 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine iodide ( jc-1 ; sigma - aldrich ) was used to determine changes in mitochondrial transmembrane potential ( m ) . jc-1 ( 10  l ; 200  m ; final concentration 2  m ) was added to h9c2 cells on coverslips , incubated for 45  min in the dark , and washed twice with pbs . jc-1 fluorescence was measured from a single excitation wavelength ( 490  nm ) with dual emission ( shift from green at 530  nm to red at 590  nm ) using a fluorimeter . loss of mitochondrial m is presented as the relative ratio of green to red fluorescence . h9c2 cells were seeded at 30,000 cells / well onto bioscience v7 culture plates in growth medium containing 15% fbs ( fetal bovine serum ) on the first day . the next day , medium was replaced , and cells were grown in the culture growth medium . within 24  hrs of seeding , a confluent monolayer of h9c2 cells was formed and then cells were used as described below . xf24 analyzer ( seahorse biosciences ) was used to measure ocr to be monitored in real time function in h9c2 cardiomyocytes as mentioned in our earlier report . for all measurements , the culture medium was changed to serum - free media for 1  hr prior to assay . at the end of the treatment , cells were washed with buffer and cell mito stress test ( cmst ) media for cell bioenergetics measurements were added into each well . oligomycin , an atp synthase inhibitor ( to check the atp mediated respiration ) ; fccp , a known uncoupler and proton translocating ionophore ( to determine maximal respiration and the mitochondrial reserve capacity ) ; and antimycin a , an inhibitor of the mitochondrial electron transport chain ( to check ocr from nonmitochondrial respiration related ) prepared in cmst were injected sequentially through ports in the seahorse flux pak cartridges and then mitochondrial ocr was recorded . dsf pretreatment significantly increased ros levels in cultured h9c2 cardiomyocytes subjected to high glucose stress compared to dsf - untreated cells presented with equimolar concentrations of glucose or mannitol . surprisingly , dsf pretreatment significantly increased ros levels in the mannitol group compared to the dsf - untreated group . however , dsf pretreatment enhanced the ros increase significantly higher in h9c2 cells with high glucose stress compared to cells in the control ( mannitol ) group ( figure 1 ) . dsf pretreatment significantly reduced aldh2 activity in cultured h9c2 cardiomyocytes subjected to high glucose stress compared to dsf - untreated cells presented with equimolar concentrations of glucose or mannitol . furthermore , this dsf pretreatment significantly reduced aldh2 activity in h9c2 cells with high glucose stress compared to cells in the control ( mannitol ) group ( figure 2 ) . dsf pretreatment for 2 hours significantly augmented 4hne adducts formation in cells subjected to high glucose stress compared to dsf - untreated cells presented with equimolar concentrations of glucose or mannitol in their culture medium . moreover , among the dsf - pretreated cells , increase in 4hne adduct formation was found at a significantly higher amount in the hg group compared to the ctrl group ( figure 3 ) . pretreatment with dsf significantly increased cell death in the high glucose group compared to the untreated high glucose group . surprisingly , dsf pretreatment significantly increased cell death in the mannitol group compared to cells in the dsf - untreated group . among the dsf treated groups , cell death was significantly increased in the hg group compared to the mannitol group ( figure 4 ) . pretreatment with dsf reduced mitochondrial membrane potential in the hg group compared to the dsf - untreated hg and ctrl groups . furthermore , dsf enhanced the reduction in mitochondrial membrane potential in cultured h9c2 cardiomyocytes subjected to high glucose stress compared to cells presented with equimolar mannitol in their culture medium ( figure 5 ) . hg increased mitochondrial ros in cultured h9c2 cardiomyocytes which was further enhanced in both ctrl and hg groups with dsf pretreatment ( supplementary figure  2 ) . basal and fccp - induced maximal ocr were decreased in hg group compared to ctrl group . pretreatment with dsf enhanced maximal ocr reduction significantly in hg group but not in ctrl group ( figure 6 ) . in this study , we report that the pharmacological inhibition of aldh2 potentiates high glucose induced deleterious effects in cultured h9c2 cardiomyocyte cell lines . in a recent study , we reported that aldh2 inhibition by 75  m dsf per se attenuated mitochondrial respiration and induced cell death in cultured h9c2 cardiomyocyte cell lines without any stress . while dsf inhibited recombinant mitochondrial aldh2 enzyme with an ic50 value of 36.4  m , we found that dsf 75  m actually attenuated 50% of aldh2 activity in h9c2 cells without any stress . , we plan to pretreat cells with dsf in high glucose stress . in order to do this , we performed a pilot study to determine the dsf dose as explained in the method section . we first inhibited aldh2 activity in h9c2 cells using dsf and then subjected them to high glucose stress . to induce high glucose stress in vitro the cell culture medium was replaced with 33  mm d - glucose or 33  mm d - mannitol as per previous reports . as we explained in our previous report , h9c2 cardiomyocyte cell lines are suitable alternatives for primary cardiomyocytes to be used in hyperglycemic studies . in fact , it was shown that isolated cardiomyocytes and h9c2 cells respond similarly under high glucose stress . we and others reported that hyperglycemia decreases myocardial aldh2 activity in rodents [ 19 , 26 , 27 ] . the possible mechanism that we propose for this reduction is that hyperglycemia increases ros generation which attenuates aldh2 activity and thereby increases 4hne adduct formation , which ultimately leads to cellular dysfunction and then death . increased ros in type 1 and type 2 diabetes is implicated in the development of diabetic cardiomyopathy [ 28 , 29 ] . this result suggests that aldh2 inhibition by dsf itself can induce ros generation independent of high glucose stress . dsf treatment further enhanced the increase in ros levels in high glucose stress compared to control . it appears dsf pretreatment made the cells more susceptible to high glucose mediated aldh2 impairment . next , we found a significant increase in 4hne protein adducts only in dsf - pretreated cells with high glucose stress . this increase was not present in the control group or the dsf - untreated hg group . though we observed increased ros levels and reduced aldh2 activity in the control group with dsf treatment , we did not observe an increase in the level of 4hne protein adducts . however we suppose the 20% aldh2 activity remaining may be sufficient to metabolize 4hne in the control group . perhaps , this could have caused the observed reduction in the level of 4hne protein adducts . the mitochondrial membrane potential was reduced in the dsf treated hg group compared to the dsf - untreated hg group and dsf treated control group . it unequivocally suggests that dsf increases the susceptibility in cells subjected to high glucose stress to a drop in mitochondrial membrane potential despite the difference in glucose levels . progressive decrease in mitochondrial transmembrane potential is associated with apoptotic cell death . in our study , there was a decrease in the mitochondrial potential under the hyperglycemic condition , suggesting that hyperglycemia could act as an apoptosis - inducing stimulus . next , we found mitochondrial ocr at basal and fccp - induced maximal response were reduced in hg stress . aldh2 inhibition per se attenuated ocr and further this reduction in ocr is linked with cell death . but dsf pretreatment increased the cell death to more than 90% in the high glucose group . this data again implicates the role of aldh2 inhibition in maintaining cell viability and potentiating high glucose mediated cell death . this was also seen in our earlier study as aldh2 inhibition by dsf and aldh2 sirna contributed to cell death by decreasing mitochondrial function in h9c2 cells during 24 hours . decrease in aldh2 activity by dsf without causing 4hne adducts formation itself was noted to cause significant reduction in mitochondrial reserve capacity and subsequently cell death in h9c2 cells . similarly we observed an increase in cell death in control cells perhaps due to the reduction in mitochondrial respiratory defects demonstrated previously . strikingly , the aldh2 inhibition by dsf led to a significant increase in 4hne adducts as well as a decrease in mitochondrial membrane potential in cells under high glucose stress . earlier reports have shown that hyperglycemia reduced the activity and levels of myocardial aldh2 [ 27 , 32 ] . overexpression of aldh2 conferred cardioprotection by attenuating 4hne toxicity in stz - injected hyperglycemic mice . taken altogether , we suggest aldh2 activity plays an important role in the process of hyperglycemia - induced cardiac damage . similarly we speculate that the hyperglycemic stress in aldh22 carriers with diabetes or noncarriers with other comorbidities with low aldh2 activity may be vulnerable to cardiac tissue damage . our previous observation points out that long - term diabetes in patients reduces aldh2 activity and increases 4hne adduct formation in diabetic hearts . it also implies that chronic hyperglycemia can lead to an inevitable decrease in aldh2 activity and cause an exacerbation of hyperglycemia - induced cardiac damage . we conclude this report by stating that the inhibition of aldh2 activity potentiates high glucose stress - induced deleterious effects in cardiac cells . our study implicates individuals with compromised aldh2 activity may have higher propensity to develop diabetic cardiac complications or increase the severity of the damage .
aldehyde dehydrogenase ( aldh ) gene superfamily consists of 19 isozymes . 
 they are present in various organs and involved in metabolizing aldehydes that are biologically generated . for instance , aldh2 , a cardiac mitochondrial aldh isozyme , is known to detoxify 4-hydroxy-2-nonenal , a reactive aldehyde produced upon lipid peroxidation in diabetic conditions . 
 we hypothesized that inhibition of aldh leads to the accumulation of unmetabolized 4hne and consequently exacerbates injury in cells subjected to high glucose stress . 
 h9c2 
 cardiomyocyte cell lines were pretreated with 10  m disulfiram ( dsf ) , an inhibitor of aldh2 or vehicle ( dmso ) for 2 hours , and then subjected to high glucose stress { 33  mm d - glucose ( hg ) or 33  mm d - mannitol as an osmotic control ( ctrl ) } for 24  hrs . 
 the decrease in aldh2 activity with dsf pretreatment was higher in hg group when compared to ctrl group . increased 4hne adduct formation with dsf pretreatment was higher in hg group compared to ctrl group . 
 pretreatment with dsf leads to potentiated hg - induced cell death in cultured h9c2 cardiomyocytes by lowering mitochondrial membrane potential . 
 our results indicate that aldh2 activity is important in preventing high glucose induced cellular dysfunction .