Biventricular Increases in Mitochondrial Fission Mediator (MiD51) and Proglycolytic Pyruvate Kinase (PKM2) Isoform in Experimental Group 2 Pulmonary Hypertension-Novel Mitochondrial Abnormalities

Ping Yu Xiong, Lian Tian, Kimberly J Dunham-Snary, Kuang-Hueih Chen, Jeffrey D Mewburn, Monica Neuber-Hess, Ashley Martin, Asish Dasgupta, Francois Potus, Stephen L Archer, Ping Yu Xiong, Lian Tian, Kimberly J Dunham-Snary, Kuang-Hueih Chen, Jeffrey D Mewburn, Monica Neuber-Hess, Ashley Martin, Asish Dasgupta, Francois Potus, Stephen L Archer

Abstract

Introduction: Group 2 pulmonary hypertension (PH), defined as a mean pulmonary arterial pressure ≥25 mmHg with elevated pulmonary capillary wedge pressure >15 mmHg, has no approved therapy and patients often die from right ventricular failure (RVF). Alterations in mitochondrial metabolism, notably impaired glucose oxidation, and increased mitochondrial fission, contribute to right ventricle (RV) dysfunction in PH. We hypothesized that the impairment of RV and left ventricular (LV) function in group 2 PH results in part from a proglycolytic isoform switch from pyruvate kinase muscle (PKM) isoform 1 to 2 and from increased mitochondrial fission, due either to upregulation of expression of dynamin-related protein 1 (Drp1) or its binding partners, mitochondrial dynamics protein of 49 or 51 kDa (MiD49 or 51). Methods and Results: Group 2 PH was induced by supra-coronary aortic banding (SAB) in 5-week old male Sprague Dawley rats. Four weeks post SAB, echocardiography showed marked reduction of tricuspid annular plane systolic excursion (2.9 ± 0.1 vs. 4.0 ± 0.1 mm) and pulmonary artery acceleration time (24.3 ± 0.9 vs. 35.4 ± 1.8 ms) in SAB vs. sham rats. Nine weeks post SAB, left and right heart catheterization showed significant biventricular increases in end systolic and diastolic pressure in SAB vs. sham rats (LV: 226 ± 15 vs. 103 ± 5 mmHg, 34 ± 5 vs. 7 ± 1 mmHg; RV: 40 ± 4 vs. 22 ± 1 mmHg, and 4.7 ± 1.5 vs. 0.9 ± 0.5 mmHg, respectively). Picrosirius red staining showed marked biventricular fibrosis in SAB rats. There was increased muscularization of small pulmonary arteries in SAB rats. Confocal microscopy showed biventricular mitochondrial depolarization and fragmentation in SAB vs. sham cardiomyocytes. Transmission electron microscopy confirmed a marked biventricular reduction in mitochondria size in SAB hearts. Immunoblot showed marked biventricular increase in PKM2/PKM1 and MiD51 expression. Mitofusin 2 and mitochondrial pyruvate carrier 1 were increased in SAB LVs. Conclusions: SAB caused group 2 PH. Impaired RV function and RV fibrosis were associated with increases in mitochondrial fission and expression of MiD51 and PKM2. While these changes would be expected to promote increased production of reactive oxygen species and a glycolytic shift in metabolism, further study is required to determine the functional consequences of these newly described mitochondrial abnormalities.

Keywords: MiD51; PKM2 pyruvate kinase M2; aortic stenosis; group 2 PH; mitochondrial fission; mitochondrial pyruvate carrier; pulmonary hypertension; supra-coronary aortic banding (SAB).

Figures

Figure 1
Figure 1
Echocardiographic measurements of sham vs. supra-coronary aortic banding (SAB) rats at 4 weeks post banding. (A) Representative echocardiographic images showing development of group 2 PH in SAB rats. (1–2) Left ventricular free wall thickness (LVFW), (3–4) right ventricular free wall thickness (RVFW), and (5–6) pulmonary artery acceleration time (PAAT), and (7–8) tricuspid annular plane systolic excursion (TAPSE) of sham vs. supra-coronary aortic banding (SAB) rats. (B) Summary statistics showing significant increase of biventricular thickness and decrease of PAAT and TAPSE in SAB vs. sham. (1) LVFW thickness, (2) diastolic LVFW thickness, (3) RVFW thickness, (4) diastolic RVFW thickness, (5) PAAT, (6) TAPSE of sham vs. supra-coronary aortic banding rats.
Figure 2
Figure 2
Hemodynamic studies of sham vs. SAB rats. Left carotid catheterization showing (A) hemodynamic measurement of (B) systolic, and (C) diastolic blood pressure distal to the band (dSBP and dDBP) and (D) systolic and (E) diastolic blood pressure proximal to the band (pSBP and pDBP). Left heart catheterization showing (F) hemodynamic measurement of (G) left ventricular end systolic pressure (LVESP) and (H) left ventricular end diastolic pressure (LVEDP). Right heart catheterization showing (I) hemodynamic measurement of (J) right ventricular end systolic pressure (RVESP) and (K) right ventricular end diastolic pressure (RVEDP). (L) Schematic drawing showing the anatomical definition of distal and proximal to aortic band.
Figure 3
Figure 3
Histology and confocal microscopy images of sham vs. supra-coronary aortic banding (SAB) rats. Picrosirius red (PSR) staining of left ventricle (LV), and right ventricle (RV) of (A,D) sham vs. (B,E) SAB rats showing (C,F) increased myocardial fibrosis in SAB rat. Hematoxylin and eosin (H,E) stain of lung cross section of (G) sham vs. (H) SAB rats showing (I) increased number of muscular pulmonary arteries (PAs) per 50x field. Each dot represents tissue from a unique rat.
Figure 4
Figure 4
Representative confocal microscopy image showing left ventricular (LV) and right ventricular (RV) mitochondrial morphology and membrane potential of (A,D) sham rats vs. (B,E) supra-coronary aortic banding rats. The mitochondria (red) are labeled with tetramethylrhodamin, methyl ester (TMRM) and the nuclei (blue) are labeled with DAPI. (C) LV and (F) RV TMRM intensity. Each dot represents a heart from a unique rat.
Figure 5
Figure 5
Representative transmission electron microscopy (TEM) images showing reduced average mitochondria area in the left ventricle (LV) and right ventricle (RV) at 2 different magnifications. (A) Low magnification of sham LV, (B) supra-coronary aortic banding (SAB) LV, (C) sham RV, and (D) SAB LV. High magnification showing mitochondrial ultra-sturcture in (E) sham LV, (F) SAB LV, (G) sham RV, and (H) SAB RV. (I) Average LV mitochondrial area, and (J) average RV mitochondrial area. Each dot represents a single mitochondrion.
Figure 6
Figure 6
Expression levels of mitochondrial fission/fusion mediators in sham vs. supra-coronary aortic banding (SAB) rats. (A) Western blot showing no change in the expression level of total dynamin-related protein 1 (Drp1) in the (1) left ventricle (LV) and (2) right ventricle (RV) of supra-coronary aortic banding (SAB) rats vs. sham rats. (B) Western blot showing significant upregulation of mitochondrial dynamics of 51 kDa protein (MiD51) in the (1) LV and (2) RV of supra-coronary aortic banding (SAB) rats vs. sham rats. (C) Western blot showing significant downregulation of mitofusin-2 (Mfn2) in the (1) LV and (2) RV of supra-coronary aortic banding (SAB) rats vs. sham rats. Each band represents a unique animal. Two experimental cohorts were done; hence two groups are shown, labeled as 1 and 2. The same vinculin loading control is used for proteins probed on the same membrane.
Figure 7
Figure 7
Pyruvate kinase muscle (PKM) isoform 2 to 1 ratio is increased in supra-coronary aortic banding (SAB) vs. sham rats. Western blot showing upregulation of PKM2 and downregulation of PKM1, thus resulting in significant increase of PKM2/PKM1 ratio in the (A) left ventricle (LV) and (B) right ventricle (RV) of supra-coronary aortic banding (SAB) rats vs. sham rats. Each band represents a unique animal. Two experimental cohorts were done; hence two groups are shown, labeled as 1 and 2. The same vinculin loading control is used for proteins probed on the same membrane.
Figure 8
Figure 8
Mitochondrial pyruvate carrier 1 (MPC1) is upregulated in supra-coronary aortic banding (SAB) vs. sham rats. (A) Western blot showing significant upregulation of MPC1, but no change in MPC2 level in the left ventricle (LV) of SAB vs. sham rats. (B) Western blot showing no significant change of MPC 1&2 expression in the right ventricle (RV) of SAB vs. sham rats. Each band represents a unique animal. Three experimental cohorts are done; hence three groups are shown, labeled as 1–3. The same vinculin loading control is used for proteins probed on the same membrane.

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