Plasma Proteomics Identifies B2M as a Regulator of Pulmonary Hypertension in Heart Failure With Preserved Ejection Fraction

Abstract

Background: Pulmonary hypertension (PH) represents an important phenotype in heart failure with preserved ejection fraction (HFpEF). However, management of PH-HFpEF is challenging because mechanisms involved in the regulation of PH-HFpEF remain unclear.

Methods: We used a mass spectrometry-based comparative plasma proteomics approach as a sensitive and comprehensive hypothesis-generating discovery technique to profile proteins in patients with PH-HFpEF and control subjects. We then validated and investigated the role of one of the identified proteins using in vitro cell cultures, in vivo animal models, and independent cohort of human samples.

Results: Plasma proteomics identified high protein abundance levels of B2M (β2-microglobulin) in patients with PH-HFpEF. Interestingly, both circulating and skeletal muscle levels of B2M were increased in mice with skeletal muscle SIRT3 (sirtuin-3) deficiency or high-fat diet-induced PH-HFpEF. Plasma and muscle biopsies from a validation cohort of PH-HFpEF patients were found to have increased B2M levels, which positively correlated with disease severity, especially pulmonary capillary wedge pressure and right atrial pressure at rest. Not only did the administration of exogenous B2M promote migration/proliferation in pulmonary arterial vascular endothelial cells but it also increased PCNA (proliferating cell nuclear antigen) expression and cell proliferation in pulmonary arterial vascular smooth muscle cells. Finally, B2m deletion improved glucose intolerance, reduced pulmonary vascular remodeling, lowered PH, and attenuated RV hypertrophy in mice with high-fat diet-induced PH-HFpEF.

Conclusions: Patients with PH-HFpEF display higher circulating and skeletal muscle expression levels of B2M, the magnitude of which correlates with disease severity. Our findings also reveal a previously unknown pathogenic role of B2M in the regulation of pulmonary vascular proliferative remodeling and PH-HFpEF. These data suggest that circulating and skeletal muscle B2M can be promising targets for the management of PH-HFpEF.

Keywords: beta 2-microglobulin; heart failure; hypertension, pulmonary; proteomics; sirtuin 3.

Figure 1.

Changes in plasma protein abundance profiles in patients with pulmonary hypertension (PH)–heart failure with preserved ejection fraction (HFpEF). A, Schematic overview of mass spectrometry (MS)–based plasma proteomic analysis. B, Volcano plot showing the fold change and statistical significance (P value was determined using the Student t test) for protein abundance levels. The solid curves represent the significance threshold at P<0.05 and an S₀ of 0.868. C, Changing protein abundances in PH-HFpEF (age, 69.8±7.9 years; male sex, 8; body mass index, 39.1±10.4; mean pulmonary artery pressure [mPAP], 39.4±8.1 mm Hg; pulmonary capillary wedge pressure [PCWP], 20.2±4.5 mm Hg; World Health Organization function class II: 1 [6%], III: 14 [88%], and IV: 1 [6%]). D, Relative protein abundance of B2M (β2-microglobulin). Data are mean±SEM. P value was determined by Mann-Whitney U test. CA3 indicates carbonic anhydrase 3; CHGA, chromogranin-A; CRP, C-reactive protein; SAA1, serum amyloid A1; TMT, tandem mass tag; and WWC3, WW and C2 domain containing protein 3.

Figure 2.

Circulating levels of B2M (β2-microglobulin) are elevated in patients with pulmonary hypertension (PH)–heart failure with preserved ejection fraction (HFpEF). A, Circulating levels of B2M were measured by ELISA in plasma of the validation cohort of control (Ctrl) subjects, HFpEF patients without PH, and PH-HFpEF patients (diagnosis is confirmed by right heart catheterization [RHC] when the resting mean pulmonary artery pressure [mPAP] was ≥25 mm Hg, pulmonary capillary wedge pressure [PCWP] was ≥15 mm Hg, and transpulmonary pressure gradient [TPG] was ≥12 mm Hg or during exercise mPAP was >30 mm Hg, PCWP ≥20 mm Hg, and total pulmonary resistance [TPR] ≥3). Data are mean±SEM. P value was determined using 1-way ANOVA followed by Tukey post hoc test. B through F, Correlation between circulating levels of B2M and resting mPAP (B), PCWP (C), TPG (D), pulmonary vascular resistance (PVR; E), or right atrial pressure (RAP; F) in patients with PH-HFpEF whose plasma samples were collected within a month of RHC. Spearman r is shown. The upper and lower dotted lines represent the 95% CIs of the regression.

Figure 3.

Skeletal muscle levels of B2M (β2-microglobulin) are increased in mice with skeletal muscle SIRT3 (sirtuin-3) deficiency or high-fat diet (HFD)–induced pulmonary hypertension (PH)–heart failure with preserved ejection fraction (HFpEF). A and B, Skeletal muscle (A) and plasma (B) levels of B2M in WT (wild-type) and skeletal muscle–specific SIRT3 knockout (Sirt3^skm−/−) mice. C and D, Skeletal muscle (C) and plasma (D) levels of B2M in mice fed a regular diet (RD; 10% lipids/kcal) or HFD (60% lipids/kcal) for 16 weeks. Data are mean±SEM. P value was analyzed by Mann-Whitney U test.

Figure 4.

Muscle expression levels of B2M (β2-microglobulin) are increased in patients with pulmonary hypertension (PH)–heart failure with preserved ejection fraction (HFpEF). A, B2M expression levels were measured in muscle biopsies of patients with PH-HFpEF and control subjects by Western blots. Data are mean±SEM. P value was analyzed by Mann-Whitney U test. B and C, Correlation between skeletal muscle B2M expression with resting pulmonary capillary wedge pressure (PCWP; B) or right atrial pressure (RAP; C). Spearman r is shown. The upper and lower dotted lines represent the 95% CIs of the regression.

Figure 5.

Treatment with B2M (β2-microglobulin) induces pulmonary arterial vascular endothelial cell (PAVEC) migration/proliferation and promotes pulmonary arterial vascular smooth muscle cell (PAVSMC) proliferation. A and B, Human PAVECs were administered with exogenous B2M (10 μg/mL) for 4 days. Representative images of cell migration and related quantitative data (A). Cell proliferation assessed by cell counts (B). C and D, Human PAVSMCs were exposed to B2M (10 μg/mL) for 5 days. Representative images of cell numbers and cell proliferation assessed by cell counts (C). Representative Western blots for PCNA (proliferating cell nuclear antigen) protein expression levels (D). Data are mean±SEM. P value was determined using unpaired Student t test after testing for normality with Shapiro-Wilk test and equal variance.

Figure 6.

B2M (β2-microglobulin) whole-body KO (knockout) mice protect against metabolic syndrome–associated pulmonary hypertension (PH)–heart failure with preserved ejection fraction (HFpEF). A, Eight-week-old WT (wild-type) and whole-body B2M KO mice (B2m^−/−) were fed an regular diet (RD) or high-fat diet (HFD) for 16 weeks. B through G, At week 16, body weights (B), glucose tolerant abilities (C), right ventricular systolic pressures (RVSP; D), and left ventricular end-diastolic pressure (LVEDP; G) were measured. E and F, Weights of right ventricle (RV; E) and left ventricle (LV)+septum (S; F) normalized to tibial length were used as an index of ventricular hypertrophy. H, Representative images of lung sections stained with α-SMA (α-smooth muscle actin; 40×) and quantification of wall thickness from the mean of 5 to 6 vessels per lung section from 3 mice per group. Scale bar, 30 µm. I, PCNA (proliferating cell nuclear antigen) levels were analyzed by Western blot in pulmonary arterial vascular smooth muscle cells (PAVSMCs) of B2m^−/− and WT mice. Data are mean±SEM. P value was determined using 2-way ANOVA followed by Tukey post hoc test. For Figure 6D and 6G, P value was determined using the Kruskal-Wallis test followed by Dunn post hoc test. For glucose tolerance test, 2-way repeated measures ANOVA followed by Bonferroni post hoc test was performed. ****P<0.0001 WT HFD vs WT RD, and ^##P<0.01 B2m^−/− HFD vs WT HFD.