Nitrite treatment rescues cardiac dysfunction in aged mice treated with conjugated linoleic acid

Abstract

Conjugated linoleic acid (cLA) is a commercially available weight-loss supplement that is not currently regulated by the U.S. FDA. Numerous studies suggest that cLA mediates protection against diseases including cancer, diabetes, atherosclerosis, immune function, and obesity. Based upon these reports, it was hypothesized that supplementation with cLA would improve heart function in aged wild-type (WT) mice. At 10 months of age, mice were treated with cLA, nitrite, or the combination of the two. Echocardiograms revealed that cardiac function was decreased in aged compared to young WT mice, as determined by percentage of fractional shortening. Also, contrary to the hypothesis, mice that received cLA (6-week treatment) had significantly worse cardiac function compared to controls. This effect was attenuated when mice were cotreated with cLA and nitrite. Taken together, these results suggest that cLA-mediated cardiac injury can be circumvented by nitrite supplementation in a murine model of aging.

Keywords: Conjugated linoleic acid; Free radicals; Heart function; Nitrite; eNOS.

Fig. 1

Aged mice have significant cardiac dysfunction compared to young mice. Echocardiograms in M mode show cardiac dysfunction in aged mice (A). 10 month-old mice have a significantly lower %FS than 3 month old mice (68±3 vs. 35±6, *p<0.05) (B).

Fig. 2

cLA significantly decreases cardiac function in aged mice. Echocardiograms in M mode demonstrate significant dysfunction in cLA treated mice compared to controls (A). 10 month-old mice treated with cLA have significantly lower %FS compared to controls (35±6 vs. 19±9, *p<0.05) (B). Heart rate remained constant during heart function analysis (C).

Fig. 3

cLA decreases intracellular nitric oxide (NO) levels in human umbilical vein endothelial cells (HUVECs). Nitrite (A) and nitrate (B) levels are significantly decreased in HUVECs treated with increasing concentrations of cLA, (*p<0.05), while NO levels in media remain unchanged (C). Consequently, eNOS protein expression was decreased following treatment with cLA (D) and significantly reduced at 2.5μM (*p<0.05) (E).

Fig. 3

cLA decreases intracellular nitric oxide (NO) levels in human umbilical vein endothelial cells (HUVECs). Nitrite (A) and nitrate (B) levels are significantly decreased in HUVECs treated with increasing concentrations of cLA, (*p<0.05), while NO levels in media remain unchanged (C). Consequently, eNOS protein expression was decreased following treatment with cLA (D) and significantly reduced at 2.5μM (*p<0.05) (E).

Fig. 4

cLA decreases intracellular nitric oxide (NO) levels in bovine aortic endothelial cells (BAECs). Nitrite (A) levels are reduced with a 10μM cLA treatment (*p<0.05), and nitrate (B) levels are reduced in samples treated with both 5μM and 10μM cLA in BAECs, while NO levels in media remain unchanged (C). eNOS protein expression is unaffected following cLA treatment in BAECs (D and E). Treatment with increasing concentrations of cLA induces Nox2 protein expression in BAECs (D). Nox2 expression was significantly increased from controls, although no differences were seen between concentrations of cLA (*p<0.05) (F). Subsequently, LT PAGE analysis demonstrated that cLA treatment increased eNOS monomerization in BAECs compared to control treated samples (G), indicating uncoupling of eNOS.

Fig. 4

cLA decreases intracellular nitric oxide (NO) levels in bovine aortic endothelial cells (BAECs). Nitrite (A) levels are reduced with a 10μM cLA treatment (*p<0.05), and nitrate (B) levels are reduced in samples treated with both 5μM and 10μM cLA in BAECs, while NO levels in media remain unchanged (C). eNOS protein expression is unaffected following cLA treatment in BAECs (D and E). Treatment with increasing concentrations of cLA induces Nox2 protein expression in BAECs (D). Nox2 expression was significantly increased from controls, although no differences were seen between concentrations of cLA (*p<0.05) (F). Subsequently, LT PAGE analysis demonstrated that cLA treatment increased eNOS monomerization in BAECs compared to control treated samples (G), indicating uncoupling of eNOS.

Fig. 5

Co-administration of cLA + nitrite increases plasma nitrite in WT mice. Mice treated with both nitrite and the combination of cLA and nitrite, demonstrate a significant increase in plasma nitrite levels compared to control or cLA treated animals (*p<0.05) (A). Nitrite and combination cLA and nitrite treated mice also demonstrated a significant increase in plasma nitrate levels (*p<0.05) (B). 10 month-old mice have significantly more plasma NO compared to 3 month old mice (*p<0.05) (C). Following cLA treatment, 10 month-old mice have significantly reduced NO levels from age matched controls (#p<0.05) (C).

Fig. 6

cLA decreases eNOS protein expression in murine cardiac tissue. 10 month-old mice treated with cLA demonstrate a decrease in eNOS protein compared to controls (A). Quantitation of eNOS expression, normalized to GAPDH, indicates that cLA treatment causes a significant decrease in eNOS protein (*p<0.05, compared to control) and co-administration of cLA and nitrite resulted in a significant increase of eNOS expression (#p<0.05, compared to cLA) (B). cLA induces Nox2 expression in murine cardiac tissue. 10 month-old mice treated with cLA demonstrate a modest increase in Nox2 expression (A). Moreover, co-administration of cLA and nitrite results in a significant decrease when compared to cLA-treated mice (*p<0.05) (A, C). Animals treated with cLA indicate a loss of eNOS coupling. Mice treated with cLA show a marked decrease in eNOS dimerization, compared to controls. This is moderately corrected with the combination of cLA and nitrite. Importantly, incubation with BH₄ shifts all samples towards the dimer form, indicating coupling of eNOS.

Fig. 6

cLA decreases eNOS protein expression in murine cardiac tissue. 10 month-old mice treated with cLA demonstrate a decrease in eNOS protein compared to controls (A). Quantitation of eNOS expression, normalized to GAPDH, indicates that cLA treatment causes a significant decrease in eNOS protein (*p<0.05, compared to control) and co-administration of cLA and nitrite resulted in a significant increase of eNOS expression (#p<0.05, compared to cLA) (B). cLA induces Nox2 expression in murine cardiac tissue. 10 month-old mice treated with cLA demonstrate a modest increase in Nox2 expression (A). Moreover, co-administration of cLA and nitrite results in a significant decrease when compared to cLA-treated mice (*p<0.05) (A, C). Animals treated with cLA indicate a loss of eNOS coupling. Mice treated with cLA show a marked decrease in eNOS dimerization, compared to controls. This is moderately corrected with the combination of cLA and nitrite. Importantly, incubation with BH₄ shifts all samples towards the dimer form, indicating coupling of eNOS.

Fig. 7

Nitrite supplementation rescues cLA-exacerbated cardiac dysfunction in aged mice. Echocardiograms in M mode demonstrate that cLA-mediated cardiac dysfunction is attenuated with co-administration of cLA and nitrite (A). %FS is rescued in cLA + nitrite treated animals (B).