Altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension

Abstract

Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have developed a model (shunt) of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Our previous studies have identified a progressive development of endothelial dysfunction in shunt lambs that is dependent, at least in part, on decreased nitric oxide (NO) signaling. The purpose of this study was to evaluate the possible role of a disruption in carnitine metabolism in shunt lambs and to determine the effect on NO signaling. Our data indicate that at 2 wk of age, shunt lambs have significantly reduced expression (P < 0.05) of the key enzymes in carnitine metabolism: carnitine palmitoyltransferases 1 and 2 as well as carnitine acetyltransferase (CrAT). In addition, we found that CrAT activity was inhibited due to increased nitration. Furthermore, free carnitine levels were significantly decreased whereas acylcarnitine levels were significantly higher in shunt lambs (P < 0.05). We also found that alterations in carnitine metabolism resulted in mitochondrial dysfunction, since shunt lambs had significantly decreased pyruvate, increased lactate, and a reduced pyruvate/lactate ratio. In pulmonary arterial endothelial cells cultured from juvenile lambs, we found that mild uncoupling of the mitochondria led to a decrease in cellular ATP levels and a reduction in both endothelial NO synthase-heat shock protein 90 (eNOS-HSP90) interactions and NO signaling. Similarly, in shunt lambs we found a loss of eNOS-HSP90 interactions that correlated with a progressive decrease in NO signaling. Our data suggest that mitochondrial dysfunction may play a role in the development of endothelial dysfunction and pulmonary hypertension and increased pulmonary blood flow.

Figures

Fig. 1.

Carnitine palmitoyltransferase (CPT) expression in peripheral lung tissue from control and shunt lambs at 2 wk of age. A: protein extracts (50 μg) prepared from peripheral lung of shunt and control lambs were analyzed by Western blot analysis using a specific antiserum raised against CPT1-B protein. CPT1-B expression was also normalized for loading using β-actin. A representative blot is shown. B: there was a significant decrease in normalized densitometric values for CPT1-B protein in peripheral lung tissue prepared from shunt compared with control lambs. Values are means ± SE; n = 6 control and 6 shunt lambs. *P < 0.05 vs. control. C: protein extracts (50 μg) prepared from peripheral lung of shunt or control lambs were analyzed by Western blot analysis using a specific antiserum raised against CPT2 protein. CPT2 expression was also normalized for loading using β-actin. A representative blot is shown. D: there was a significant decrease in normalized densitometric values for CPT2 protein in peripheral lung tissue prepared from shunt compared with control lambs. Values are means ± SE; n = 6 control and 6 shunt lambs. *P < 0.05 vs. control.

Fig. 2.

Carnitine acetyltransferase (CrAT) expression and activity in peripheral lung tissue from control and shunt lambs at 2 wk of age. A: protein extracts (50 μg) prepared from peripheral lung of shunt and control lambs were analyzed by Western blot analysis using a specific antiserum raised against CrAT protein. CrAT expression was also normalized for loading using β-actin. A representative blot is shown. B: there was a significant decrease in normalized densitometric values for CrAT protein in peripheral lung tissue prepared from shunt compared with control lambs. Values are means ± SE; n = 6 control and 6 shunt lambs. *P < 0.05 vs. control. C: CrAT activity was determined in protein extracts (40 μg) prepared from peripheral lung tissue from control and shunt lambs. There was a significant decrease in CrAT activity in peripheral lung tissue prepared from shunt compared with control lambs. Values are expressed as units of activity per μg of protein and are means ± SE; n = 4 control and 4 shunt lambs. *P < 0.05 vs. control.

Fig. 3.

Increased nitration of CrAT in peripheral lung tissue of shunt lambs at 2 wk of age. A: protein extracts (1,000 μg) prepared from peripheral lung of shunt and control lambs were subjected to immunoprecipitation (IP) using an antibody specific to 3-nitrotyrosine (3-NT) and then analyzed by Western blot analysis using a specific antiserum raised against CrAT protein. A representative immunoblot (IB) is shown with CrAT expression. Minimal binding was observed in the beads alone or in IgG preclear. B: there was a significant increase in nitrated CrAT protein in peripheral lung tissue prepared from shunt compared with control lambs. Values are means ± SE; n = 6 control and 6 shunt lambs. *P < 0.05 vs. control.

Fig. 4.

Peroxynitrite decreases CrAT activity. Purified pigeon breast muscle CrAT was exposed to authentic peroxynitrite or vehicle, and then the activity was determined. Peroxynitrite induced a significant decrease in CrAT activity. Values are means ± SD; n = 6. *P < 0.05 vs. vehicle.

Fig. 5.

Markers of mitochondrial dysfunction are increased in shunt lambs at 2 wk of age. A: protein extracts (25 μg) prepared from peripheral lung of shunt and control lambs were analyzed by Western blot analysis using a specific antiserum raised against SOD2 protein. SOD2 expression was also normalized for loading using β-actin. A representative blot is shown. B: there was a significant decrease in normalized densitometric values for SOD2 protein in peripheral lung tissue prepared from shunt compared with control lambs. Values are means ± SE; n = 6 control and 6 shunt lambs. *P < 0.05 vs. control. C: protein extracts (25 μg) prepared from peripheral lung of shunt and control lambs were analyzed by Western blot analysis using a specific antiserum raised against uncoupling protein-2 (UCP-2). UCP-2 expression was also normalized for loading using β-actin. A representative blot is shown. D: there was a significant increase in normalized densitometric values for UCP-2 protein in peripheral lung tissue prepared from shunt compared with control lambs. Values are means ± SE; n = 6 control and n = 6 shunt lambs. *P < 0.05 vs. control.

Fig. 6.

2,4-Dinitrophenol (2,4-DNP) decreases ATP levels and increases mitochondrial oxidative stress in pulmonary arterial endothelial cells (PAEC). A: PAEC were treated with 2,4-DNP (25 μM, 0–8 h), and the cellular ATP levels were then determined. 2,4-DNP significantly decreased cellular ATP levels. Values are means ± SE; n = 6. T, time. *P < 0.05 vs. control. B: PAEC were treated with 2,4-DNP (25 μM, 4 h). Cells were then exposed to MitoSox (10 μM, 15 min) to measure mitochondrial superoxide levels (as a marker for mitochondrial dysfunction). 2,4-DNP induced a significant increase in MitoSox fluorescence (representative images are shown as an inset). Values are means ± SE; n = 4. *P < 0.05 vs. untreated.

Fig. 7.

2, 4-DNP decreases NO signaling in PAEC. A: PAEC were treated with 2,4-DNP (25 μM, 4 h) and washed with PBS, and lysates were prepared with modified RIPA buffer. IP was performed using an antibody to endothelial nitric oxide synthase (eNOS) and then Western blot analysis was done using a specific antiserum raised against heat shock protein 90 (HSP90). Blots were also stripped and reprobed for eNOS to normalize the IP. A representative blot is shown. B: 2,4-DNP treatment caused a significant reduction in eNOS-HSP90 interaction. Values are means ± SE; n = 3. *P < 0.05 vs. untreated. C: cells were treated with 2,4-DNP (25 μM, 4 h) and exposed to laminar shear stress (20 dyn/cm2, 15 min), and then the medium was assayed for nitrate/nitrite (NOx) as an indirect determination of NO production. 2,4-DNP significantly decreased the shear-mediated increase in NOx. Values are means ± SE; n = 6. *P < 0.05 vs. no shear. †P < 0.05 vs. control. D: cells were treated with 2,4-DNP (25 μM, 4 h) in the presence and absence of the NOS inhibitor 3-ethylisothiourea (ETU; 100 μM) and then exposed to laminar shear stress (20 dyn/cm2, 15 min). Superoxide levels were then determined using the spin trap 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine·HCl. 2,4-DNP significantly increased NOS-dependent superoxide generation in response to shear. Values are means ± SE; n = 3. *P < 0.05 vs. no shear. †P < 0.05 vs. shear + 2,4-DNP.

Fig. 8.

Progressive decreases in the interaction of eNOS with HSP90 in shunt compared with control lambs. A: the interaction of eNOS with HSP90 was determined by IP using specific antiserum raised against eNOS in tissue extracts prepared from peripheral lung of shunt and control lambs at 2 and 4 wk of age. IP extracts were analyzed using antisera against either eNOS or HSP90. A representative image is shown. No specific protein bands were observed in the beads alone or in IgG preclear. B: the levels of eNOS protein associated with HSP90 relative to total eNOS protein were calculated. The data obtained indicate that there was a progressive decrease in the association of eNOS with HSP90 in shunt compared with control lambs between 2 (bars at left) and 4 wk of age (bars at right). Values are means ± SE; n = 5 shunt and 5 control lambs at each age. *P < 0.05 compared with age-matched control. †P < 0.05 vs. 2-wk shunt. C: relative eNOS activity was estimated in shunt and age-matched control lambs at 2 and 4 wk of age by dividing peripheral lung tissue NOx levels by total lung eNOS activity (determined by calcium-dependent [3H]arginine to [3H]citrulline conversion). Relative eNOS activity was significantly lower in the shunt lambs at both 2 and 4 wk of age. Values are means ± SE; n = 4 shunt and 4 control lambs at each age. *P < 0.05 compared with age-matched control. †P < 0.05 vs. 2-wk shunt. D: superoxide anion levels determined by electron paramagnetic resonance (EPR) in snap-frozen lung tissue from shunt and age-matched control lambs at 4 wk of age in the presence and absence of the NOS inhibitor ETU (100 μM). A bar graph representing the cumulative data is shown. ETU-inhibitable superoxide levels were significantly higher in the shunt lambs. Values are means ± SD; n = 6 shunt and 6 control lambs at each age. *P < 0.05 compared with age-matched control. †P < 0.05 vs. 2-wk shunt. E: superoxide was generated in vitro by cross-reacting xanthine oxidase (XO; 1 U/ml) with xanthine (X; 1 mM) in the presence or absence of ETU (100 μM). Two control reactions, one lacking X and the other lacking XO, were included to ensure the absence of nonspecific reactions of either reagent with the EPR spin trap. The significant increase in superoxide generated by the X/XO reaction was not significantly quenched by the presence of ETU. Values are means ± SE; n = 3 for each condition. *P < 0.05 compared with X alone. †P < 0.05 vs. XO alone.