Preserving mitochondrial function prevents the proteasomal degradation of GTP cyclohydrolase I

Abstract

The development of pulmonary hypertension is a common accompaniment of congenital heart disease (CHD) with increased pulmonary blood flow. Our recent evidence suggests that asymmetric dimethylarginine (ADMA)-induced mitochondrial dysfunction causes endothelial nitric oxide synthase (eNOS) uncoupling secondary to a proteasome-dependent degradation of GTP cyclohydrolase I (GCH1) that results in a decrease in the NOS cofactor tetrahydrobiopterin (BH(4)). Decreases in NO signaling are thought to be an early hallmark of endothelial dysfunction. As l-carnitine plays an important role in maintaining mitochondrial function, in this study we examined the protective mechanisms and the therapeutic potential of l-carnitine on NO signaling in pulmonary arterial endothelial cells and in a lamb model of CHD and increased pulmonary blood flow (Shunt). Acetyl-l-carnitine attenuated the ADMA-mediated proteasomal degradation of GCH1. This preservation was associated with a decrease in the association of GCH1 with Hsp70 and the C-terminus of Hsp70-interacting protein (CHIP) and a decrease in its ubiquitination. This in turn prevented the decrease in BH(4) levels induced by ADMA and preserved NO signaling. Treatment of Shunt lambs with l-carnitine also reduced GCH1/CHIP interactions, attenuated the ubiquitination and degradation of GCH1, and increased BH(4) levels compared to vehicle-treated Shunt lambs. The increases in BH(4) were associated with decreased NOS uncoupling and enhanced NO generation. Thus, we conclude that L-carnitine may have a therapeutic potential in the treatment of pulmonary hypertension in children with CHD with increased pulmonary blood flow.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Figure 1. Acetyl L-carnitine prevents the ADMA-mediated mitochondrial dysfunction and decreases in GTP cyclohydrolase I in pulmonary arterial endothelial cells

PAEC were pretreated with acetyl-L-carnitine (ALC, 0–1mM) for 2h then exposed to ADMA (5μM, 2h). The effect on mitochondrial function was then determined by examining the effect on mitochondrial ROS levels, using MitoSOX red fluorescence. Representative images are shown. ADMA causes a significant increase in MitoSOX fluorescence (A) that is dose-dependently decreased in the presence of ALC (A) or when PAEC were preincubated with PEG-SOD (100U/ml, A). To confirm the MitoSOX data, mitochondrial fractions were prepared and superoxide levels were determined by EPR. ADMA increases mitochondrial superoxide levels in PAEC and ALC abolishes this increase (B). ADMA also caused a disruption of the mitochondrial membrane potential as estimated using the DePsipher compound and fluorescence microscopy (C). ALC pre-treatment preserved the mitochondrial membrane potential (C). Western blot analysis also revealed that ADMA decreased the levels of SOD2 (D) and increased the levels of UCP2 (E). Again ALC pre-treatment prevented these changes (D & E). Cumulatively, the mitochondrial dysfunction induced by ADMA led to a significant decrease in cellular ATP levels (F). The decrease in ATP levels was prevented by ALC (F). In addition protein extracts (G) and mRNA (H) were prepared from cells were treated with ALC (100μM) and ADMA and subjected to Western blot analysis and qRT-PCR respectively. A representative image is shown of the Western blot using an antibody specific for GCH1 and loading normalized by reprobing the membranes with an antibody specific to β-actin (G). ALC prevents the ADMA mediated decrease in GCH1 protein levels (G). ADMA either alone or in combination with ALC has no effect of GCH1 mRNA levels although ALC alone significantly increased GCH mRNA levels (H). Cells were incubated with ADMA for 2h then ALC was added (100μM, 0–2h post-ADMA exposure). Post-exposure with ALC preserves GCH1 protein levels (I). Further, increasing the redox scavenging potential of the cells by adding PEG-SOD (100U/ml) or PEG-catalase (100U/ml) prevents the ADMA-mediated decrease in GCH1 protein levels (J). Values are means ± SEM, n=8–10; *P

Figure 1. Acetyl L-carnitine prevents the ADMA-mediated mitochondrial dysfunction and decreases in GTP cyclohydrolase I in pulmonary arterial endothelial cells

PAEC were pretreated with acetyl-L-carnitine (ALC, 0–1mM) for 2h then exposed to ADMA (5μM, 2h). The effect on mitochondrial function was then determined by examining the effect on mitochondrial ROS levels, using MitoSOX red fluorescence. Representative images are shown. ADMA causes a significant increase in MitoSOX fluorescence (A) that is dose-dependently decreased in the presence of ALC (A) or when PAEC were preincubated with PEG-SOD (100U/ml, A). To confirm the MitoSOX data, mitochondrial fractions were prepared and superoxide levels were determined by EPR. ADMA increases mitochondrial superoxide levels in PAEC and ALC abolishes this increase (B). ADMA also caused a disruption of the mitochondrial membrane potential as estimated using the DePsipher compound and fluorescence microscopy (C). ALC pre-treatment preserved the mitochondrial membrane potential (C). Western blot analysis also revealed that ADMA decreased the levels of SOD2 (D) and increased the levels of UCP2 (E). Again ALC pre-treatment prevented these changes (D & E). Cumulatively, the mitochondrial dysfunction induced by ADMA led to a significant decrease in cellular ATP levels (F). The decrease in ATP levels was prevented by ALC (F). In addition protein extracts (G) and mRNA (H) were prepared from cells were treated with ALC (100μM) and ADMA and subjected to Western blot analysis and qRT-PCR respectively. A representative image is shown of the Western blot using an antibody specific for GCH1 and loading normalized by reprobing the membranes with an antibody specific to β-actin (G). ALC prevents the ADMA mediated decrease in GCH1 protein levels (G). ADMA either alone or in combination with ALC has no effect of GCH1 mRNA levels although ALC alone significantly increased GCH mRNA levels (H). Cells were incubated with ADMA for 2h then ALC was added (100μM, 0–2h post-ADMA exposure). Post-exposure with ALC preserves GCH1 protein levels (I). Further, increasing the redox scavenging potential of the cells by adding PEG-SOD (100U/ml) or PEG-catalase (100U/ml) prevents the ADMA-mediated decrease in GCH1 protein levels (J). Values are means ± SEM, n=8–10; *P

Figure 1. Acetyl L-carnitine prevents the ADMA-mediated mitochondrial dysfunction and decreases in GTP cyclohydrolase I in pulmonary arterial endothelial cells

PAEC were pretreated with acetyl-L-carnitine (ALC, 0–1mM) for 2h then exposed to ADMA (5μM, 2h). The effect on mitochondrial function was then determined by examining the effect on mitochondrial ROS levels, using MitoSOX red fluorescence. Representative images are shown. ADMA causes a significant increase in MitoSOX fluorescence (A) that is dose-dependently decreased in the presence of ALC (A) or when PAEC were preincubated with PEG-SOD (100U/ml, A). To confirm the MitoSOX data, mitochondrial fractions were prepared and superoxide levels were determined by EPR. ADMA increases mitochondrial superoxide levels in PAEC and ALC abolishes this increase (B). ADMA also caused a disruption of the mitochondrial membrane potential as estimated using the DePsipher compound and fluorescence microscopy (C). ALC pre-treatment preserved the mitochondrial membrane potential (C). Western blot analysis also revealed that ADMA decreased the levels of SOD2 (D) and increased the levels of UCP2 (E). Again ALC pre-treatment prevented these changes (D & E). Cumulatively, the mitochondrial dysfunction induced by ADMA led to a significant decrease in cellular ATP levels (F). The decrease in ATP levels was prevented by ALC (F). In addition protein extracts (G) and mRNA (H) were prepared from cells were treated with ALC (100μM) and ADMA and subjected to Western blot analysis and qRT-PCR respectively. A representative image is shown of the Western blot using an antibody specific for GCH1 and loading normalized by reprobing the membranes with an antibody specific to β-actin (G). ALC prevents the ADMA mediated decrease in GCH1 protein levels (G). ADMA either alone or in combination with ALC has no effect of GCH1 mRNA levels although ALC alone significantly increased GCH mRNA levels (H). Cells were incubated with ADMA for 2h then ALC was added (100μM, 0–2h post-ADMA exposure). Post-exposure with ALC preserves GCH1 protein levels (I). Further, increasing the redox scavenging potential of the cells by adding PEG-SOD (100U/ml) or PEG-catalase (100U/ml) prevents the ADMA-mediated decrease in GCH1 protein levels (J). Values are means ± SEM, n=8–10; *P

Figure 1. Acetyl L-carnitine prevents the ADMA-mediated mitochondrial dysfunction and decreases in GTP cyclohydrolase I in pulmonary arterial endothelial cells

PAEC were pretreated with acetyl-L-carnitine (ALC, 0–1mM) for 2h then exposed to ADMA (5μM, 2h). The effect on mitochondrial function was then determined by examining the effect on mitochondrial ROS levels, using MitoSOX red fluorescence. Representative images are shown. ADMA causes a significant increase in MitoSOX fluorescence (A) that is dose-dependently decreased in the presence of ALC (A) or when PAEC were preincubated with PEG-SOD (100U/ml, A). To confirm the MitoSOX data, mitochondrial fractions were prepared and superoxide levels were determined by EPR. ADMA increases mitochondrial superoxide levels in PAEC and ALC abolishes this increase (B). ADMA also caused a disruption of the mitochondrial membrane potential as estimated using the DePsipher compound and fluorescence microscopy (C). ALC pre-treatment preserved the mitochondrial membrane potential (C). Western blot analysis also revealed that ADMA decreased the levels of SOD2 (D) and increased the levels of UCP2 (E). Again ALC pre-treatment prevented these changes (D & E). Cumulatively, the mitochondrial dysfunction induced by ADMA led to a significant decrease in cellular ATP levels (F). The decrease in ATP levels was prevented by ALC (F). In addition protein extracts (G) and mRNA (H) were prepared from cells were treated with ALC (100μM) and ADMA and subjected to Western blot analysis and qRT-PCR respectively. A representative image is shown of the Western blot using an antibody specific for GCH1 and loading normalized by reprobing the membranes with an antibody specific to β-actin (G). ALC prevents the ADMA mediated decrease in GCH1 protein levels (G). ADMA either alone or in combination with ALC has no effect of GCH1 mRNA levels although ALC alone significantly increased GCH mRNA levels (H). Cells were incubated with ADMA for 2h then ALC was added (100μM, 0–2h post-ADMA exposure). Post-exposure with ALC preserves GCH1 protein levels (I). Further, increasing the redox scavenging potential of the cells by adding PEG-SOD (100U/ml) or PEG-catalase (100U/ml) prevents the ADMA-mediated decrease in GCH1 protein levels (J). Values are means ± SEM, n=8–10; *P

Figure 1. Acetyl L-carnitine prevents the ADMA-mediated mitochondrial dysfunction and decreases in GTP cyclohydrolase I in pulmonary arterial endothelial cells

PAEC were pretreated with acetyl-L-carnitine (ALC, 0–1mM) for 2h then exposed to ADMA (5μM, 2h). The effect on mitochondrial function was then determined by examining the effect on mitochondrial ROS levels, using MitoSOX red fluorescence. Representative images are shown. ADMA causes a significant increase in MitoSOX fluorescence (A) that is dose-dependently decreased in the presence of ALC (A) or when PAEC were preincubated with PEG-SOD (100U/ml, A). To confirm the MitoSOX data, mitochondrial fractions were prepared and superoxide levels were determined by EPR. ADMA increases mitochondrial superoxide levels in PAEC and ALC abolishes this increase (B). ADMA also caused a disruption of the mitochondrial membrane potential as estimated using the DePsipher compound and fluorescence microscopy (C). ALC pre-treatment preserved the mitochondrial membrane potential (C). Western blot analysis also revealed that ADMA decreased the levels of SOD2 (D) and increased the levels of UCP2 (E). Again ALC pre-treatment prevented these changes (D & E). Cumulatively, the mitochondrial dysfunction induced by ADMA led to a significant decrease in cellular ATP levels (F). The decrease in ATP levels was prevented by ALC (F). In addition protein extracts (G) and mRNA (H) were prepared from cells were treated with ALC (100μM) and ADMA and subjected to Western blot analysis and qRT-PCR respectively. A representative image is shown of the Western blot using an antibody specific for GCH1 and loading normalized by reprobing the membranes with an antibody specific to β-actin (G). ALC prevents the ADMA mediated decrease in GCH1 protein levels (G). ADMA either alone or in combination with ALC has no effect of GCH1 mRNA levels although ALC alone significantly increased GCH mRNA levels (H). Cells were incubated with ADMA for 2h then ALC was added (100μM, 0–2h post-ADMA exposure). Post-exposure with ALC preserves GCH1 protein levels (I). Further, increasing the redox scavenging potential of the cells by adding PEG-SOD (100U/ml) or PEG-catalase (100U/ml) prevents the ADMA-mediated decrease in GCH1 protein levels (J). Values are means ± SEM, n=8–10; *P

Figure 1. Acetyl L-carnitine prevents the ADMA-mediated mitochondrial dysfunction and decreases in GTP cyclohydrolase I in pulmonary arterial endothelial cells

PAEC were pretreated with acetyl-L-carnitine (ALC, 0–1mM) for 2h then exposed to ADMA (5μM, 2h). The effect on mitochondrial function was then determined by examining the effect on mitochondrial ROS levels, using MitoSOX red fluorescence. Representative images are shown. ADMA causes a significant increase in MitoSOX fluorescence (A) that is dose-dependently decreased in the presence of ALC (A) or when PAEC were preincubated with PEG-SOD (100U/ml, A). To confirm the MitoSOX data, mitochondrial fractions were prepared and superoxide levels were determined by EPR. ADMA increases mitochondrial superoxide levels in PAEC and ALC abolishes this increase (B). ADMA also caused a disruption of the mitochondrial membrane potential as estimated using the DePsipher compound and fluorescence microscopy (C). ALC pre-treatment preserved the mitochondrial membrane potential (C). Western blot analysis also revealed that ADMA decreased the levels of SOD2 (D) and increased the levels of UCP2 (E). Again ALC pre-treatment prevented these changes (D & E). Cumulatively, the mitochondrial dysfunction induced by ADMA led to a significant decrease in cellular ATP levels (F). The decrease in ATP levels was prevented by ALC (F). In addition protein extracts (G) and mRNA (H) were prepared from cells were treated with ALC (100μM) and ADMA and subjected to Western blot analysis and qRT-PCR respectively. A representative image is shown of the Western blot using an antibody specific for GCH1 and loading normalized by reprobing the membranes with an antibody specific to β-actin (G). ALC prevents the ADMA mediated decrease in GCH1 protein levels (G). ADMA either alone or in combination with ALC has no effect of GCH1 mRNA levels although ALC alone significantly increased GCH mRNA levels (H). Cells were incubated with ADMA for 2h then ALC was added (100μM, 0–2h post-ADMA exposure). Post-exposure with ALC preserves GCH1 protein levels (I). Further, increasing the redox scavenging potential of the cells by adding PEG-SOD (100U/ml) or PEG-catalase (100U/ml) prevents the ADMA-mediated decrease in GCH1 protein levels (J). Values are means ± SEM, n=8–10; *P

Figure 2. Acetyl L-carnitine prevents the ADMA-induced decrease in GCH1-Hsp90 interactions and GCH1 ubiquitination in pulmonary arterial endothelial cells

PAEC were pretreated with ALC (100μM, 2h) prior to ADMA exposure (5μM, 2h). Whole cell lysates (1mg) were then subjected to immunoprecipitation (IP) using antibodies specific to Hsp90 (A), Hsp70 (B), or CHIP (C) then analyzed by Western blot (IB) analysis using a specific antiserum raised against GCH1. A representative image is shown for each Western blot. Blots were stripped and reprobed for Hsp90, Hsp70, or CHIP to normalize for the immunoprecipitation efficiency. Densitometric values were then obtained for each. ADMA significantly reduces the interaction of GCH1 with Hsp90 and significantly enhances its interaction with Hsp70 and CHIP. ALC attenuates the ADMA-induced decrease in Hsp90-GCH1 and the increase in Hsp70-GCH1 and CHIP-GCH1. In addition, cell lysates (1mg) were subjected to ubiquitinated protein enrichment (AP) followed by Western blot using specific antiserum raised against GCH1 (IB). A representative image is shown. ADMA significantly increases the ubiquitination of GCH1 and this is attenuated in the presence of ALC (D). PAEC were also incubated with ADMA for 2h in the presence or absence of the proteasome inhibitor, lactacystin (20 μM). GCH1 protein levels were determined by Western blotting with loading normalized by reprobing the membranes with an antibody specific to β-actin (E). Proteasomal inhibition prevents the decrease in GCH1 induced by ADMA (E). ALC and lactacystin also attenuate the ADMA-mediated decrease in GCH1 activity (F). ALC, ADMA, PEG-SOD, or PEG-catalase either alone or in combination had no affect on PAEC 20s proteasome activity (G). Values are mean ± SEM, n=3–6. *P

Figure 2. Acetyl L-carnitine prevents the ADMA-induced decrease in GCH1-Hsp90 interactions and GCH1 ubiquitination in pulmonary arterial endothelial cells

PAEC were pretreated with ALC (100μM, 2h) prior to ADMA exposure (5μM, 2h). Whole cell lysates (1mg) were then subjected to immunoprecipitation (IP) using antibodies specific to Hsp90 (A), Hsp70 (B), or CHIP (C) then analyzed by Western blot (IB) analysis using a specific antiserum raised against GCH1. A representative image is shown for each Western blot. Blots were stripped and reprobed for Hsp90, Hsp70, or CHIP to normalize for the immunoprecipitation efficiency. Densitometric values were then obtained for each. ADMA significantly reduces the interaction of GCH1 with Hsp90 and significantly enhances its interaction with Hsp70 and CHIP. ALC attenuates the ADMA-induced decrease in Hsp90-GCH1 and the increase in Hsp70-GCH1 and CHIP-GCH1. In addition, cell lysates (1mg) were subjected to ubiquitinated protein enrichment (AP) followed by Western blot using specific antiserum raised against GCH1 (IB). A representative image is shown. ADMA significantly increases the ubiquitination of GCH1 and this is attenuated in the presence of ALC (D). PAEC were also incubated with ADMA for 2h in the presence or absence of the proteasome inhibitor, lactacystin (20 μM). GCH1 protein levels were determined by Western blotting with loading normalized by reprobing the membranes with an antibody specific to β-actin (E). Proteasomal inhibition prevents the decrease in GCH1 induced by ADMA (E). ALC and lactacystin also attenuate the ADMA-mediated decrease in GCH1 activity (F). ALC, ADMA, PEG-SOD, or PEG-catalase either alone or in combination had no affect on PAEC 20s proteasome activity (G). Values are mean ± SEM, n=3–6. *P

Figure 2. Acetyl L-carnitine prevents the ADMA-induced decrease in GCH1-Hsp90 interactions and GCH1 ubiquitination in pulmonary arterial endothelial cells

PAEC were pretreated with ALC (100μM, 2h) prior to ADMA exposure (5μM, 2h). Whole cell lysates (1mg) were then subjected to immunoprecipitation (IP) using antibodies specific to Hsp90 (A), Hsp70 (B), or CHIP (C) then analyzed by Western blot (IB) analysis using a specific antiserum raised against GCH1. A representative image is shown for each Western blot. Blots were stripped and reprobed for Hsp90, Hsp70, or CHIP to normalize for the immunoprecipitation efficiency. Densitometric values were then obtained for each. ADMA significantly reduces the interaction of GCH1 with Hsp90 and significantly enhances its interaction with Hsp70 and CHIP. ALC attenuates the ADMA-induced decrease in Hsp90-GCH1 and the increase in Hsp70-GCH1 and CHIP-GCH1. In addition, cell lysates (1mg) were subjected to ubiquitinated protein enrichment (AP) followed by Western blot using specific antiserum raised against GCH1 (IB). A representative image is shown. ADMA significantly increases the ubiquitination of GCH1 and this is attenuated in the presence of ALC (D). PAEC were also incubated with ADMA for 2h in the presence or absence of the proteasome inhibitor, lactacystin (20 μM). GCH1 protein levels were determined by Western blotting with loading normalized by reprobing the membranes with an antibody specific to β-actin (E). Proteasomal inhibition prevents the decrease in GCH1 induced by ADMA (E). ALC and lactacystin also attenuate the ADMA-mediated decrease in GCH1 activity (F). ALC, ADMA, PEG-SOD, or PEG-catalase either alone or in combination had no affect on PAEC 20s proteasome activity (G). Values are mean ± SEM, n=3–6. *P

Figure 3. Over-expression of a dominant negative Hs90 in pulmonary arterial endothelial cells

PAEC were transiently transfected with a DN Hsp90 or pcDNA3 (as a control). Forty-eight hours after transfection, whole cell lysates (20μg) were prepared and subjected to Western blot analysis using specific antisera raised against Hsp90 (A), Hsp70 (B), and CHIP (C) with loading normalized by reprobing the membranes with an antibody specific to β-actin. A representative image is shown for each. Transfection of DN Hsp90 increases Hsp90 protein levels by ~2-fold (A). Similarly, Hsp70 levels are also significantly increased (B). However, CHIP protein levels are unaffected (C). Values are means ± SEM, n=4–6. *P

Figure 4. Inhibition of Hsp90 alone is sufficient to stimulate GCH1 ubiquitination and degradation in pulmonary arterial endothelial cells

PAEC were transiently transfected with a DN Hsp90 or pcDNA3 (as a control). Forty-eight hours after transfection, whole cell lysates (1mg) were subjected to immunoprecipitation (IP) using antibodies specific to Hsp90 (A), Hsp70 (B) or CHIP (C) then analyzed by Western blot (IB) analysis using an anti-GCH1 antibody. Blots were stripped and reprobed with the appropriate IP antibody to normalize for immunoprecipitation efficiency. Representative images are shown for each. The over-expression of DN Hsp90 decreases GCH1-Hsp90 interactions (A) but increases both GCH1-Hsp70 (B) and GCH1-CHIP (C) interactions. Ubiquitinated protein enrichment (AP) followed by Western blot with an anti-GCH1 antibody was also used to measure GCH1 ubquitination (IB). A representative image is shown (D). There is a significant increase in ubquitinated GCH1 (D). Whole cell lysates (20μg) were also subjected to Western blot analysis using a specific antiserum raised against GCH1 with loading normalized by reprobing the membranes with an antibody specific to β-actin. A representative image is shown along with the densitometric analysis indicating that DN Hsp90 over-expression significantly decreased GCH1 protein levels (E). Values are means ± SEM, n=4–6. *P

Figure 5. Depletion of Hsp70 in pulmonary arterial endothelial cells

PAEC were transiently transfected with a siRNA for Hsp70 or a control siRNA. Forty-eight hours after transfection, whole cell lysates (20μg) were prepared and subjected to Western blot analysis using specific antisera raised against Hsp70 (A), Hsp90 (B), and CHIP (C) with loading normalized by reprobing the membranes with an antibody specific to β-actin. A representative image is shown for each. Transfection with the Hsp70 siRNA reduces Hsp70 protein levels by ~50% (A) without altering Hsp90 (B) or CHIP (C) protein levels. Values are means ± SEM, n=6. *P

Figure 6. Depletion of Hsp70 attenuates the ADMA-mediated degradation of GCH1 in pulmonary arterial endothelial cells

PAEC were transiently transfected with a siRNA for Hsp70 or a control siRNA. Forty-eight hours after transfection, whole cell lysates (1mg) were subjected to immunoprecipitation (IP) or ubiquitinated protein enrichment (AP) using an antibody specific to CHIP. IP samples were run on duplicate Western blot (IB), one blot was analyzed using an antibody specific for GCH1, the other blot was probed with an antibody for CHIP to normalize for the immunoprecipitation efficiency. AP samples were analyzed by Western blot (IB) analysis using an anti-GCH1 antibody. A representative image is shown for each. Depleting Hsp70 protein levels attenuated the ADMA-induced GCH1 interaction with CHIP (A) and prevented the increase in GCH-1 ubiquitination (B). Whole cell lysates (20μg) were also subjected to Western blot analysis using a specific antiserum raised against GCH1, loading was normalized by reprobing the membranes with an antibody specific to β-actin. A representative image is shown along with the densitometric analysis indicating that that reducing hsp70 protein levels preserves GCH1 protein levels in PAEC challenged with ADMA (C). Values are means ± SEM, n=6. *P

Figure 7. Depletion of CHIP in pulmonary arterial endothelial cells

PAEC were transiently transfected with a siRNA for CHIP or a control siRNA. Forty-eight hours after transfection, whole cell lysates (20μg) were prepared and subjected to Western blot analysis using specific antisera raised against CHIP (A), Hsp90 (B), and Hsp70 (C) with loading normalized by reprobing the membranes with an antibody specific to β-actin. A representative image is shown for each. Transfection with the CHIP siRNA reduces Hsp70 protein levels by ~50% (A) without altering Hsp90 (B) or Hsp70 (C) protein levels. Values are means ± SEM, n=6. *P

Figure 8. Depletion of CHIP attenuates ADMA-induced GCH1 degradation but not its interaction with Hsp70 in pulmonary arterial endothelial cells

PAEC were transiently transfected with a siRNA for CHIP or a control siRNA. Forty-eight hours after transfection, whole cell lysates (1mg) were subjected to immunoprecipitation (IP) or ubiquitinated protein enrichment (AP) using an antibody specific to Hsp70 then analyzed by Western blot (IB) analysis using an anti-GCH1 antibody. The IP blots were stripped and reprobed for Hsp70 to normalize for the immunoprecipitation efficiency. A representative image is shown for each Western blot. Depleting CHIP protein levels did not prevent the ADMA-induced interaction of GCH1 with Hsp70 (A). However, the ADMA-mediated increase in GCH1 ubiquitination was blocked (B). Whole cell lysates (20μg) were also subjected to Western blot analysis using a specific antiserum raised against GCH1, loading was normalized by reprobing the membranes with an antibody specific to β-actin. A representative image is shown along with the densitometric analysis indicating that that reducing CHIP protein levels preserves GCH1 protein levels in PAEC challenged with ADMA (C). Values are means ± SEM, n=6. *P

Figure 9. Acetyl L-carnitine attenuates the ADMA mediated decrease in tetrahydrobiopterin levels and increased eNOS uncoupling in pulmonary arterial endothelial cells

PAEC were pretreated with ALC (100μM, 2h) prior to ADMA exposure (5μM) and the effect on cellular BH4 levels determined. The presence of ALC attenuated the ADMA-mediated and BH4 levels (A). In addition, ALC prevented the decrease in NO generation in response to shear stress (20 dyn/cm2, 15 min) mediated by ADMA (B) and decreased the ADMA-mediated increases in eNOS-derived superoxide levels (C). Values are mean ± SEM, n=6. *P < 0.05 vs. untreated cells; † P<0.05 vs. ADMA alone.

Figure 10. L-carnitine supplementation preserved GCH1-Hsp90 interactions in a lamb model of increased pulmonary blood flow

Protein extracts prepared from peripheral lung of L-carnitine (carnitine) or vehicle treated Shunt lambs were subjected to immunoprecipitation (IP) using antibodies specific to Hsp90, Hsp70, or CHIP then analyzed by Western blot (IB) analysis using a specific antiserum raised against GCH1. In addition, tissue lysates were also subjected to ubiquitinated protein enrichment (AP) followed by Western blot with an anti-GCH1 antibody (IB). A representative image is shown for each. The levels of GCH1 associated with Hsp90 (A) is significantly increased in L-carnitine treated Shunt lambs while the interaction of GCH1 with Hsp70 (B) and CHIP (C) are significantly decreased. There is also a significant decrease in ubquitinated GCH1 in carnitine treated Shunt lambs (D). Densitomeric values are mean ± SEM, n=5. *P

Figure 10. L-carnitine supplementation preserved GCH1-Hsp90 interactions in a lamb model of increased pulmonary blood flow

Protein extracts prepared from peripheral lung of L-carnitine (carnitine) or vehicle treated Shunt lambs were subjected to immunoprecipitation (IP) using antibodies specific to Hsp90, Hsp70, or CHIP then analyzed by Western blot (IB) analysis using a specific antiserum raised against GCH1. In addition, tissue lysates were also subjected to ubiquitinated protein enrichment (AP) followed by Western blot with an anti-GCH1 antibody (IB). A representative image is shown for each. The levels of GCH1 associated with Hsp90 (A) is significantly increased in L-carnitine treated Shunt lambs while the interaction of GCH1 with Hsp70 (B) and CHIP (C) are significantly decreased. There is also a significant decrease in ubquitinated GCH1 in carnitine treated Shunt lambs (D). Densitomeric values are mean ± SEM, n=5. *P

Figure 10. L-carnitine supplementation preserved GCH1-Hsp90 interactions in a lamb model of increased pulmonary blood flow

Protein extracts prepared from peripheral lung of L-carnitine (carnitine) or vehicle treated Shunt lambs were subjected to immunoprecipitation (IP) using antibodies specific to Hsp90, Hsp70, or CHIP then analyzed by Western blot (IB) analysis using a specific antiserum raised against GCH1. In addition, tissue lysates were also subjected to ubiquitinated protein enrichment (AP) followed by Western blot with an anti-GCH1 antibody (IB). A representative image is shown for each. The levels of GCH1 associated with Hsp90 (A) is significantly increased in L-carnitine treated Shunt lambs while the interaction of GCH1 with Hsp70 (B) and CHIP (C) are significantly decreased. There is also a significant decrease in ubquitinated GCH1 in carnitine treated Shunt lambs (D). Densitomeric values are mean ± SEM, n=5. *P

Figure 11. L-Carnitine supplementation protects tetrahydrobiopterin levels and enhances NOS coupling in lambs with increased pulmonary blood flow

BH4 levels in the peripheral lung of L-carnitine or vehicle treated Shunt lambs were measured by HPLC. BH4 levels are significantly increased after carnitine treatment (A). In addition, L-carnitine enhances tissue NOx levels (B) and decreases NOS-derived superoxide (C) indicative of enhanced eNOS coupling. Values expressed are mean ± SEM, n=5; *P<0.05 vs. Vehicle treated Shunt lambs.

Figure 12. A possible mechanism by which ADMA disrupts mitochondrial function and attenuates NO signaling in lambs with increased pulmonary blood flow

ADMA-induces mitochondrial dysfunction reducing cellular ATP levels. As the activity of Hsp90 is ATP-dependent, GCH1/Hsp90 interactions are disrupted. This leads to increased binding of Hsp70 to GCH1 leading to the recruitment of CHIP. CHIP then ubiquitinates GCH1 targeting it for proteasomal degradation. The resulting BH4 deficiency induces eNOS uncoupling. L-carnitine supplementation short-circuits this degradation pathway by maintaining mitochondrial function.