Lycium barbarum-Derived Extracellular Vesicles Ameliorate Myocardial Hypertrophy in Heart Failure with Preserved Ejection Fraction, Associated with Reduced Cardiomyocyte Apoptosis

Figures

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  Figure 1. Extraction, preparation and characterization of Gq-EVs. (A) Extraction process of Gq-EVs. (B) Transmission electron microscopy image of Gq-EVs stored at 4 °C. (C) Average particle size of Gq-EVs measured by nanoparticle tracking analysis (NTA) stored at 4 °C. (D) The particle size and (E) zeta potential measured using the Malvern Laser Particle Sizer stored at 4 °C. (F) Transmission electron microscopy image of Gq-EVs stored at −80 °C. (G) The particle size and (H) zeta potential measured using the Malvern Laser Particle Sizer stored at −80 °C. Data were summarized as means ± SD. NTA, nanoparticle tracking analysis.

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  Figure 2. Metabolomic analysis for the component identification of Gq-EVs. (A) Components of Gq-EVs. (B) Carbohydrates in Gq-EVs. (C) Lipids in Gq-EVs. (D) KEGG pathways.

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  Figure 3. Effects of nebulized inhalation of Gq-EVs on mice with HFpEF. (A) In vivo imaging and ex vivo imaging of the heart and lungs. The color progression from blue to yellow and then to red indicates an increase in fluorescence intensity, reflecting a higher degree of Gq-EVs enrichment. (B) Statistics of the average fluorescence intensity of ex vivo imaging of heart and lung tissues. (C) Schematic diagram of the intervention of nebulized inhalation of Gq-EVs. (D) Body weight. (E) Energy intake. (F) GTT assay. (G) Area under the curve of GTT. (H) Fasting blood glucose levels. The data were summarized as means ± SD. *, p < 0.05 vs. Ctrl; #, p < 0.05 vs. HFpEF + PBS. GTT, glucose tolerance test; HFD, high-fat diet; L-NAME, Nω-nitro-L-arginine methyl ester. Abbreviations are the same as in Figure 1.

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  Figure 4. Nebulized inhalation of Gq-EVs improved cardiac function in mice with HFpEF. (A) B-mode and M-mode echocardiography. The yellow lines are the M-mode cursor. Left ventricular structural indicators (B) IVSS, IVST, (C) LVEDd, LVEDs, (D) LVPWd, LVPWs were determined. (E) EF%. (F) LV Mass. (G) E/A ratio of mitral valve orifice blood flow pattern. (H) Pulsed-mode echocardiography. The green lines are heart rate of mice. (I) IVRT. (J) SBP and (K) DBP were measured. The data were summarized as means ± SD. *, p < 0.05 vs. Ctrl; #, p < 0.05 vs. HFpEF + PBS. DBP, diastolic blood pressure; EF%, ejection fraction percentage; LVEDd, left ventricular end-diastolic diameter; LVEDs, left ventricular end-systolic diameter; LVPWd, left ventricular posterior wall thickness at end-diastole; LVPWs, left ventricular posterior wall thickness at end-systole; IVRT, Isovolumic relaxation time; IVSs, interventricular septum thickness at end-systolic; IVSd, interventricular septum thickness at end-diastolic; SBP, systolic blood pressure. Abbreviations are the same as in Figure 3.

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  Figure 5. Nebulized inhalation of Gq-EVs alleviated cardiac pathological damage in mice with HFpEF. (A) Gross view of cardiac tissue. (B) H&E staining of cardiac tissue. (C) Masson’s trichrome staining was performed to assess (E) the deposition of mature fibers. (D) Cardiac organ index. (F) Statistics of (G) WGA staining in cardiac tissue. The data were summarized as means ± SD. *, p < 0.05 vs. Ctrl; #, p < 0.05 vs. HFpEF + PBS. Abbreviations are the same as in Figure 1.

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  Figure 6. The TP53-related signaling pathway may potentially or partially participate in the therapeutic effects of Gq-EVs on HFpEF. (A) Venn diagram of targets between Gq-EVs and HFpEF. (B) Interaction network diagram of the top 25 targets. (C) Tp53 mRNA levels. (D) Bcl2 mRNA levels. (E) Bax mRNA levels. (F) β-Mhc mRNA levels. (G) Bnp mRNA levels. (H) Statistics of BCL2 protein levels. (I) Statistics of BAX protein levels. (J) Statistics of P53 protein levels. (K) Expression of BCL2, BAX, and P53 protein. (L) Co-staining of cardiomyocytes in cardiac tissue with TUNEL. The data were summarized as means ± SD. *, p < 0.05 vs. Ctrl; #, p < 0.05 vs. HFpEF + PBS. Bnp, brain natriuretic peptide; β-Mhc, β-myosin heavy chain. Abbreviations are the same as in Figure 1.

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  Figure 7. Gq-EVs improved cardiomyocyte apoptosis and regulated the TP53 pathway. (A) Schematic diagram of the intervention of Gq-EVs and PFT-α in mice. (B) WGA staining and (C) TUNEL staining of cardiac tissue. (D) Trans-pulmonary cell experiment of Gq-EVs. (E) Average particle size of Gq-EVs measured by NTA in the upper chamber of the transwell chamber. (F) Average particle size of Gq-EVs measured by NTA in the lower chamber. (G) Co-staining of Gq-EVs and cTnT in cardiomyocytes. (H) CCK-8 assay of cells treated with Gq-EVs. (I) TP53 mRNA levels. (J) β-MHC mRNA levels. (K) BNP mRNA levels. (L) BCL2 mRNA levels. (M) BAX mRNA levels. The data were summarized as means ± SD. *, p < 0.05 vs. Ctrl; #, p < 0.05 vs. PA. BAX, Bcl-2-associated X protein; BCL2, B-cell lymphoma 2; BNP, brain natriuretic peptide; β-MHC, β-myosin heavy chain; cTnT, cardiac troponin T; PFT-α, Pifithrin-α. Abbreviations are the same as in Figure 1.

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  Figure 8. Gq-EVs alleviated the PA-induced cardiomyocyte apoptosis and were potentially or partially associated with alterations in the TP53 mitochondrial pathway. (A) KEGG pathway map related to the enrichment of Gq-EVs and HFpEF. (B) TP53 mRNA levels. (C) Phalloidin staining. (D) TUNEL staining. (E) JC-1 staining of cardiomyocytes. (F) Expression of BAX, BCL2, and P53 proteins. (G) Statistics of BAX protein levels. (H) Statistics of BCL2 protein levels. (I) Statistics of P53 protein levels. (J) β-MHC mRNA levels. (K) BNP mRNA levels. (L) BCL2 mRNA levels. (M) BAX mRNA levels. (N) MFN2 mRNA levels. (O) OPA1 mRNA levels. (P) DRP1 mRNA levels. (Q) PGC-1α mRNA levels. The data were summarized as means ± SD. *, p < 0.05 vs. Ctrl; #, p < 0.05 vs. PA. DRP1, Dynamin-related protein 1; OPA1, Optic atrophy 1; PFT-μ, Pifithrin-μ; PGC-1α, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; MFN2, Mitofusin 2. Abbreviations are the same as in Figure 1.

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  Figure 9. Schematic diagram illustrating the proposed actions of Gq-EVs in HFpEF. (A) H&E staining of lung, spleen, liver, and kidney tissues. (B) Serum ALT level. (C) Serum AST level. (D) Mechanism diagram for the actions of Gq-EVs on HFpEF. Nebulized inhalation of Gq-EVs allows their delivery to cardiac tissue. In HFpEF, cardiomyocyte apoptosis and myocardial hypertrophy are associated with increased TP53 expression, activation of mitochondrial apoptotic signaling (e.g., BAX), and upregulation of heart failure markers (β-MHC and BNP), accompanied by reduced anti-apoptotic signaling (BCL2). Gq-EVs treatment is associated with attenuation of cardiomyocyte apoptosis and improvement of cardiac hypertrophy, potentially involving modulation of TP53-associated mitochondrial pathways and mitochondrial dynamics–related proteins. The data were summarized as means ± SD. *, p < 0.05 vs. Ctrl; #, p < 0.05 vs. ALT, alanine aminotransferase; AST, Aspartate Aminotransferase. Abbreviations are the same as in Figure 1.