Inhibiting pyruvate kinase muscle isoform 2 regresses group 2 pulmonary hypertension induced by supra-coronary aortic banding

Abstract

Introduction: Group 2 pulmonary hypertension (PH) has no approved PH-targeted therapy. Metabolic remodelling, specifically a biventricular increase in pyruvate kinase muscle (PKM) isozyme 2 to 1 ratio, occurs in rats with group 2 PH induced by supra-coronary aortic banding (SAB). We hypothesize that increased PKM2/PKM1 is maladaptive and inhibiting PKM2 would improve right ventricular (RV) function.

Methods: Male, Sprague-Dawley SAB rats were confirmed to have PH by echocardiography and then randomized to treatment with a PKM2 inhibitor (intraperitoneal shikonin, 2 mg/kg/day) versus 5% DMSO (n = 5/group) or small interfering RNA-targeting PKM2 (siPKM2) versus siRNA controls (n = 7/group) by airway nebulization.

Results: Shikonin-treated SAB rats had milder PH (PAAT 32.1 ± 1.3 vs 22.1 ± 1.2 ms, P = .0009) and lower RV systolic pressure (RVSP) (31.5 ± 0.9 vs 55.7 ± 1.9 mm Hg, P < .0001) versus DMSO-SAB rats. siPKM2 nebulization reduced PKM2 expression in the RV, increased PAAT (31.7 ± 0.7 vs 28.0 ± 1.3 ms, P = .025), lowered RVSP (30.6 ± 2.6 vs 42.0 ± 4.0 mm Hg, P = .032) and reduced diastolic RVFW thickness (0.69 ± 0.04 vs 0.85 ± 0.06 mm, P = .046). Both shikonin and siPKM2 regressed PH-induced medial hypertrophy of small pulmonary arteries.

Conclusion: Increases in PKM2/PKM1 in the RV contribute to RV dysfunction in group 2 PH. Chemical or molecular inhibition of PKM2 restores the normal PKM2/PKM1 ratio, reduces PH, RVSP and RVH and regresses adverse PA remodelling. PKM2 merits consideration as a therapeutic cardiac target for group 2 PH.

Keywords: heart failure with preserved ejection fraction; left ventricular hypertrophy; pyruvate kinase muscle isoform 2; right ventricular hypertrophy aortic stenosis; shikonin; uncoupled glycolysis.

Figure 1.. Experimental design for the supra-coronary aortic banding (SAB) study of Group 2 PH A) shikonin therapy protocol and B) silencing PKM2 by nebulization of small inhibitory RNA targeting PKM2 (siPKM2) protocol.

SD, Sprague-Dawley; IP, intraperitoneal; DMSO, dimethyl sulfoxide, Neb, nebulization.

Figure 2.. Echocardiography results before and after 2-weeks of therapy with shikonin (2mg/kg IP daily) or silencing pyruvate kinase muscle isoform 2 (siPKM2) nebulization treatment (1nM nebulized every 3 days x 3).

Tricuspid annular plane systolic excursion (TAPSE) A) before and B) after 2-week of shikonin and siPKM2 therapy. Shikonin did not affect TAPSE, but siPKM2 significantly improved TAPSE. Pulmonary arterial acceleration time (PAAT) C) before and D) after shikonin and siPKM2 therapy. Both shikonin and siPKM2 treatment markedly improved PAAT. Systolic left ventricular free wall thickness (LVFWT) E) before and F) after shikonin and siPKM2 therapy. Diastolic LVFWT G) before and H) after shikonin and siPKM2 therapy. Shikonin and siPKM2 treatment did not affect both systolic and diastolic LVFWT. Systolic right ventricular free wall thickness (RVFWT) I) before and J) after shikonin and siPKM2 therapy. Diastolic RVFWT K) before and L) after shikonin and siPKM2 therapy. Shikonin and siPKM2 treatment significantly reduced both systolic and diastolic RVFWT.

Figure 3.. Hemodynamic studies.

Left and right heart catheterization results show significant reduction of right ventricular systolic pressure (RVSP) in the shikonin and silencing pyruvate kinase muscle isoform 2 (siPKM2) treated supra-coronary aortic banding (SAB) rats vs. DMSO and silencing control (siControl) treated SAB rats. After 2-weeks of shikonin (2mg/kg IP daily) or siPKM2 (1 nmol nebulized every 3 days) treatment, A) there was no significant difference in left ventricular systolic pressure (LVSP); B) there was a trend toward reduction in left ventricular end diastolic pressure (LVEDP); C) there was a reduction right ventricular systolic pressure (RVSP); and D) there was no significant difference in right ventricular end diastolic pressure (RVEDP) in both shikonin and siPKM2 treated SAB rats vs. their respective control SAB rats.

Figure 4.. Inhibiting pyruvate kinase muscle isoform 2 (PKM2) with either shikonin or siPKM2 reduces right ventricle (RV) myocyte cross-sectional size.

Hematoxylin and eosin stain of lung cross-section showing supra-coronary aortic banding (SAB) caused marked B) increase in RV myocyte cross-sectional area compared to A) sham rats treated with 5% dimethyl sulfoxide (DMSO). There was marked reduction of RV myocyte cross-sectional area in D) shikonin and F) siPKM2 treated supra-coronary aortic banding (SAB) rats compared to their respective controls B) and E). C) shikonin treatment does not affect RV myocyte size in sham rats. G) illustrated the summary statistics.

Figure 5.. Inhibiting pyruvate kinase muscle isoform 2 (PKM2) with either shikonin or siPKM2 reduces distal pulmonary arterial muscularization.

Hematoxylin and eosin stain of lung cross-section showing supra-coronary aortic banding (SAB) caused marked B) increase in pulmonary vascular wall thickness (%) compared to A) sham rats treated with 5% dimethyl sulfoxide (DMSO). There was marked reduction of pulmonary vascular wall thickness (%) in D) shikonin and F) siPKM2 treated supra-coronary aortic banding (SAB) rats compared to their respective controls B) and E). C) shikonin treatment does not affect pulmonary arterial muscularization in sham rats. G) illustrated the summary statistics.

Figure 6.. Immunoblot showing reduction of PKM2/PKM1 ratio in shikonin or siPKM2 treated SAB rats compared to SAB rats treated with DMSO and siControl.

A) Representative Western blot showing upregulation of PKM2 and down-regulation of PKM1 in the SAB-DMSO rats, and treatment with shikonin (2 mg IP daily for 2-week) in SAB rat reduced the expression of PKM2/PKM1 ratio. B) Representative Western blot showing nebulization of siPKM2 (1nM) every 3 days for 2-week in SAB rats reduced the expression of PKM2/PKM1 ratio.

Figure 7.. Immunofluorescence (IF) of lung stained with PKM1 antibody showing no significant changes in PKM1 expression in the lungs or small PAs of siPKM2 treated animals vs. siControl.

Representative IF images stained with PKM1 antibody of A-D) lung cross-section or F-I) small pulmonary arteries. There is a trend towards PKM1 expression reduction in the E) lung cross-section of siPKM2 treated supra-coronary aortic banding rats (SAB) rats but not in the siControl treated SAB rats. There is no significant difference in PKM1 expression in the J) small pulmonary arteries.

Figure 8.. Immunofluorescence (IF) of lung stained with PKM2 antibody showing significant reduction in PKM2 expression in lungs (but not small PAs) of siPKM2-treated animals vs. siControl.

Representative IF images stained with PKM2 antibody of A-D) lung section or F-I) small pulmonary arteries. There is significant reduction in PKM2 expression in the E) lung cross-section of siPKM2 treated supra-coronary aortic banding rats (SAB) rats but not in siControl treated SAB rats. There is no significant difference in PKM2 expression in the J) small pulmonary arteries.