PGC1α-mediated mitofusin-2 deficiency in female rats and humans with pulmonary arterial hypertension

Abstract

Rationale: Pulmonary arterial hypertension (PAH) is a lethal, female-predominant, vascular disease. Pathologic changes in PA smooth muscle cells (PASMC) include excessive proliferation, apoptosis-resistance, and mitochondrial fragmentation. Activation of dynamin-related protein increases mitotic fission and promotes this proliferation-apoptosis imbalance. The contribution of decreased fusion and reduced mitofusin-2 (MFN2) expression to PAH is unknown.

Objectives: We hypothesize that decreased MFN2 expression promotes mitochondrial fragmentation, increases proliferation, and impairs apoptosis. The role of MFN2's transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α), was assessed. MFN2 therapy was tested in PAH PASMC and in models of PAH.

Methods: Fusion and fission mediators were measured in lungs and PASMC from patients with PAH and female rats with monocrotaline or chronic hypoxia+Sugen-5416 (CH+SU) PAH. The effects of adenoviral mitofusin-2 (Ad-MFN2) overexpression were measured in vitro and in vivo.

Measurements and main results: In normal PASMC, siMFN2 reduced expression of MFN2 and PGC1α; conversely, siPGC1α reduced PGC1α and MFN2 expression. Both interventions caused mitochondrial fragmentation. siMFN2 increased proliferation. In rodent and human PAH PASMC, MFN2 and PGC1α were decreased and mitochondria were fragmented. Ad-MFN2 increased fusion, reduced proliferation, and increased apoptosis in human PAH and CH+SU. In CH+SU, Ad-MFN2 improved walking distance (381 ± 35 vs. 245 ± 39 m; P < 0.05); decreased pulmonary vascular resistance (0.18 ± 0.02 vs. 0.38 ± 0.14 mm Hg/ml/min; P < 0.05); and decreased PA medial thickness (14.5 ± 0.8 vs. 19 ± 1.7%; P < 0.05). Lung vascularity was increased by MFN2.

Conclusions: Decreased expression of MFN2 and PGC1α contribute to mitochondrial fragmentation and a proliferation-apoptosis imbalance in human and experimental PAH. Augmenting MFN2 has therapeutic benefit in human and experimental PAH.

Figure 1.

Mitofusin-2 (MFN2) levels are decreased in human pulmonary arterial hypertension (PAH). (A) Representative image of increased mitochondrial fragmentation observed in human pulmonary arterial smooth muscle cells (PASMC) from patients with PAH compared with PASMC from control patients. Photoactivated mitochondria (pseudocolored to white/red) highlights the mitochondrial network (blue) connectivity in control and PAH hPASMCs. (B) Quantitative reverse-transcriptase polymerase chain reaction revealed decreased expression of the profusion protein MFN2 in human PAH PASMCs and increased expression of the profission proteins dynamin-related protein 1 (DRP1) and FIS1 (n = 5 independent control cell lines and 5 independent PAH cell lines). (C) Immunoblotting confirmed down-regulation of MFN2 (n = 4 independent control cell lines and 4 independent PAH cell lines). No significant difference in MFN1 expression was observed. Expression was normalized to translocase of the outer mitochondrial membrane (TOM20). (D) Immunohistochemistry for MFN2 in human lungs showing decreased MFN2 in vascular media in PAH versus control subjects. Lower panel is a higher magnification subsegment of the upper panel highlighting the decreased intensity in the vascular media. Staining intensity was quantified throughout the vasculature in arbitrary units (AU). ***P < 0.001, *P < 0.05. FIS1 = fission 1; MIEF = mitochondrial elongation factor; OPA1 = optic atrophy 1.

Figure 2.

Mitofusin-2 (MFN2) is decreased in the chronic hypoxia plus Sugen-5416 (CH+SU) and monocrotaline rat. (A) Representative image of increased mitochondrial fragmentation observed in CH+SU pulmonary arterial smooth muscle cells (PASMC) compared with PASMC from control rats. Mitochondrial fragmentation count (MFC) quantified the significantly increased fragmentation in CH+SU PASMC (n = 5 cell lines in each group). Mitochondria labeled in green with BacMam virus. (B) Immunoblotting confirmed MFN2 is decreased in CH+SU versus control cell lines (n = 4 each). Expression was normalized to smooth muscle actin for each sample. (C) Quantitative reverse-transcriptase polymerase chain reaction revealed decreased expression of MFN2 in CH+SU versus control PASMCs (n = 5 cell lines each). (D) Representative images show increased expression of the activated form of DRP1 (characterized by phosphorylation at Ser616) in the vascular media in CH+SU versus control lungs. DRP1 p-Ser616 was indexed to medial area and converted to arbitrary units (AU). Total of 25 vessels studied in each group. Blue = DAPI, green = DRP1 p-Ser616. **P < 0.01, *P < 0.05. (E) Increased mitochondrial fragmentation count in monocrotaline rat PASMC compared with control. Representative images with BacMam virus. (F) Immunoblotting confirmed MFN2 is decreased in monocrotaline versus control rats (n = 4 each). Expression was normalized to actin for each sample. DRP1 = dynamin-related protein 1; FIS1 = fission 1; MIEF = mitochondrial elongation factor; OPA1 = optic atrophy 1; SD = Sprague-Dawley rats; TOM20 = translocase of the outer mitochondrial membrane.

Figure 3.

Mitofusin-2 (MFN2) overexpression reduces mitochondrial fragmentation and proliferation in pulmonary arterial smooth muscle cells (PASMC). (A) Increased mitochondrial fragmentation is observed in pulmonary arterial hypertension (PAH) PASMC compared with control in cells loaded with the mitochondrial targeted dye tetramethylrhodamine methyl ester. Mitochondria are labeled in red. (B) MFN2 overexpression reduces the mitochondrial fragmentation count. Total of 20–25 cells studied in each group. (C) MFN2 overexpression reduced proliferation in control PASMC and PAH PASMC (multiplicity of infection = 50). (D) TUNEL staining of PASMC after adenovirus carrying MFN2 (Ad-MFN2) showing increased apoptosis in control and PAH PASMC compared with Ad-empty. TUNEL-positive stains in bright green. Nuclei stained blue with DAPI. (E) Ad-MFN2 increased the amount of TUNEL-positive cells in control and PAH PASMC. ***P < 0.001, *P < 0.05.

Figure 4.

Fragmentation of mitochondria by hypoxia-inducible factor-1α is reduced by overexpression of mitofusin-2 (MFN2). (A) Increased mitochondrial fragmentation observed in pulmonary arterial smooth muscle cells (PASMC) of chronic hypoxia plus Sugen-5416 (CH+SU) and monocrotaline rats is decreased with overexpression of MFN2. Mitochondria labeled with BacMam virus and colored in green. (B) Graphic quantification of mitochondrial fragmentation count in animal models of pulmonary hypertension treated with adenovirus carrying MFN2. (C) We cotransfected PASMC with mitochondrial matrix-targeted DsRed and photo-activatable green fluorescent protein (GFP). PASMC were then studied in normoxia, hypoxia (5% O₂), and after exposure to cobalt chloride (CoCl₂). Hypoxia and cobalt cause mitochondrial fragmentation. MFN2 at multiplicity of infection 50 restored the mitochondrial network. Mitochondria network labeled in red with photo-activated GFP shown in green. (D) The rate of decline in the mito-PA-GFP signal (measured in relative fluorescence intensity units) is directly proportional to the extent of mitochondrial fusion. PASMC from control rats with MFN2 overexpression had the fastest decrease in relative intensity of mito-PA-GFP, followed by control rats PASMC. Control rat PASMC exposed to either CoCl₂ or hypoxia had the highest relative intensity of mito-PA-GFP consistent with the fragmented nature of these mitochondria. SDR = Sprague-Dawley rats. *P < 0.05. MCT = monocrotaline.

Figure 5.

Mitofusin-2 (MFN2) overexpression in pulmonary arterial smooth muscle cells (PASMC) increases profusion proteins. (A) Adenovirus carrying MFN2 (Ad-MFN2) infection of rat PASMC in culture increased MFN2 and other fusion genes including MFN1 and optic atrophy 1 (OPA1). Ad-MFN2 also increased and peroxisome proliferator-activated receptor γ coactivator-1 α (PGC1α). Note logarithmic scale. (B) Representative immunoblot and graph confirming Ad-MFN2 increased MFN2 and PGC1α in human control PASMC (n = 4 cell lines each). (C) Immunoblot showing decreased PGC1α in PASMC from patients with pulmonary arterial hypertension (PAH) compared with control (n = 4 cell lines each). (D) Immunoblot showing decreased PGC1α in PASMC from chronic hypoxia plus Sugen-5416 (CH+SU) rats compared with control (n = 4 cell lines each). (E) Immunoblot showing decreased PGC1α in PASMC from monocrotaline rats compared with control (n = 4 each). (F) Decreased mRNA expression of PGC1α in PASMC from patients with PAH and CH+SU rats compared with their respective controls (n = 5 cell lines in each group). ***P < 0.001, *P < 0.05. DRP1 = dynamin-related protein 1; FIS1 = fission 1; TFAM = transcription factor A, mitochondrial.

Figure 6.

Peroxisome proliferator-activated receptor γ coactivator-1 α (PGC1α) plays a regulatory role in determining mitofusin-2 (MFN2) expression and mitochondrial fragmentation in human pulmonary arterial smooth muscle cells (PASMC). (A) siMFN2 decreased in MFN2 and PGC1α expression in control human PASMC. Conversely, administration of siPGC1α decreases PGC1α and MFN2 expression. (B) siPGC1α increased mitochondrial fragmentation in human control PASMC. (C) siMFN2 increased mitochondrial fragmentation in human control PASMC. (D) Immunoblot showing decreased MFN2 expression in setting of siMFN2. (E) Representative images of mitochondrial fragmentation in the setting of siPGC1α and siMFN2. (F) siMFN2 caused a significant increase in proliferation in human control PASMC, indicating that MFN2 acts as a brake on PASMC proliferation. ***P < 0.001, **P < 0.01, *P < 0.05. GAPDH = glyceraldehyde phosphate dehydrogenase.

Figure 7.

Therapeutic benefit of mitofusin-2 (MFN2) in the chronic hypoxia plus Sugen-5416 (CH+SU) model. (A) CH+SU led to a significant decrease in treadmill exercise time compared with control subjects. Exercise capacity was improved in rats treated with adenovirus carrying MFN2 (Ad-MFN2). Pulmonary artery acceleration time (PAAT) and tricuspid annular plane systolic excursion (TAPSE), which were decreased in CH+SU rats, were improved by Ad-MFN2, reflective of an improvement of pulmonary artery pressures and right ventricular function, respectively. (B) Ad-MFN2 significantly increased cardiac output (CO) and reduced pulmonary vascular resistance (PVR) in CH+SU rats (PVR = mean pulmonary artery pressure [mPAP] – left ventricular end diastolic pressure divided by the cardiac output). (C) Ad-MFN2 significantly decreased PA muscularization in CH+SU and had an antiproliferative effect on the PA vasculature, as evidenced by the decreased percentage of proliferating cell nuclear antigen (PCNA) positive smooth muscle cells (SMC) in Ad-MFN2–treated animals. (D) MFN2 and peroxisome proliferator-activated receptor γ coactivator-1 α (PGC1α) mRNA expression were reduced in CH+SU lungs and their expression was normalized in the Ad-MFN2–treated group. (E) Representative computed tomography (CT) angiogram. Note that compared with control, CH+SU rats have decreased percentage of small vessels (< 0.1 μm) and treatment with Ad-MFN2 significantly increased the percentage of small vessels. ***P < 0.001, **P < 0.01, *P < 0.05. GFP = green fluorescent protein.

Figure 8.

Combination of fusion (adenovirus carrying mitofusin-2 [Ad-MFN2]) and antifission (mitochondrial division inhibitor-1 [Mdivi1]) therapy has additive effect in regressing established pulmonary hypertension in chronic hypoxia plus Sugen-5416 (CH+SU) rats. (A) Mdivi1 alone failed to improve treadmill distance in CH+SU animals, which was most markedly improved in Ad-MFN2–treated group (note this group has the highest cardiac output [CO]). *Treadmill distance of CH+SU + dimethyl sulfate (DMSO) was significantly less than control rats (P < 0.05). ^†Treadmill distance of CH+SU+Mdivi1 and treadmill distance of CH+SU+ Mdivi1+Ad-MFN2 was significantly longer than CH+SU+DMSO (P < 0.05). (B) Prolongation of pulmonary artery acceleration time (PAAT) (indicating a decreased mean pulmonary artery pressure [mPAP]) was achieved with Mdivi1 monotherapy and with the combination therapy of Mdivi1 plus Ad-MFN2. ****PAAT was significantly longer in control rats compared with all other groups (P < 0.0001). ^†PAAT is significantly longer in CH+SU+Mdivi1+Ad-MFN2 compared with CH+SU+DMSO (P < 0.05). (C) mPAP was significantly decreased by Ad-MFN2+Mdivi1, indicating the benefits of inhibiting fission and promoting fusion. ****mPAP was significantly lower in control rats compared with all other groups (P < 0.0001). ^†mPAP was significantly lower in CH+SU+Ad-MFN2 compared with CH+SU+DMSO (P < 0.05). ^$mPAP was significantly lower in CH+SU+Mdivi1+Ad-MFN2 compared with CH+SU+DMSO (P < 0.01). (D) The highest CO was observed in Ad-MFN2 treated group. ****CO was significantly reduced in CH+SU + DMSO rats compared with control (P < 0.0001). *CO was significantly lower in CH+SU+DMSO compared with all other treatment groups (P < 0.05). ^†CO is higher in CH+SU+Ad-MFN2 compared with the other treatment groups (P < 0.01). (E) Pulmonary vascular resistance (PVR) was significantly decreased in Ad-MFN2–treated CH+SU rats compared with untreated CH+SU rats or CH+SU rats treated with Mdivi1 alone (*P < 0.05). There was a trend to significance in difference in PVR between untreated CH+SU rats and Mdivi1+Ad-MFN2 treated CH+SU rats (P = 0.08). (F) Right ventricular free wall (RVFW) thickness was improved most successfully with combined Ad-MFN2+Mdivi1 with no observed benefit observed with Mdivi1 alone. **RVFW was thinner in control rats compared with CH+SU+DMSO and CH+SU+Mdivi1 (P < 0.01). ^†RVFW was significantly reduced in CH+SU+Ad-MFN2 compared with CH+SU+Mdivi1 (P < 0.01). ^$RVFW in CH+SU+Mdivi1+Ad-MFN2 was significantly reduced compared with CH+SU+Ad-MFN2 (P < 0.01). (G) Reduction in % medial thickness of small PAs (Ad-MFN2+Mdivi1 > Ad-MFN2 > Mdivi1 > DMSO. ***% medial thickness was less in control rats compared with CH+SU+DMSO and CH+SU+Mdivi1 (P < 0.001). ^†% medial thickness was less in CH+SU+Ad-MFN2 and CH+SU+Mdivi1+Ad-MFN2 compared with CH+SU+Mdivi1 (P < 0.01). (H) Representative images of smooth muscle actin staining showed decreased medial thickness in CH+SU animals treated with Mdivi1, Ad-MFN2, or Mdivi1 plus Ad-MFN2. Blue = DAPI; red = smooth muscle actin; green = proliferating cell nuclear antigen. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.