Restoring autophagy-apoptosis balance in diabetic cardiomyopathy via a biomimetic, ROS-responsive nanocarrier associated with Mst1 pathway modulation

Fig. 1. Construction and characterization of the cardiomyocyte-specific APT-modified hybrid biomimetic nanoplatform NP-APT. (A) Schematic illustration of APT-modified biomimetic nanoplatform preparation; (B) Representative TEM image (scale bar = 200 nm); (C) Dynamic light scattering (DLS) size distribution; (D) Particle size stability at 4 °C and 37 °C; (E) in vitro drug-release curves under different pH conditions. Data are presented as mean ± SD (n = 3); (F) PDI measured by DLS for 7 consecutive days in water, PBS, and DMEM containing 10% FBS; (G) ζ potential measured for 7 consecutive days in water, PBS, and DMEM containing 10% FBS; (H) TEM images of NP-APT from day 0 to day 7 (scale bar = 100 nm); (I) Hydrodynamic size stability of the nanoplatform in DMEM with or without 10% FBS; (J) surface-characterization spectrum of NP-APT; (K) CLSM images of NP and NP-APT internalized by RAW264.7 cells (scale bar = 15 μm); (L) FACS results showing cellular uptake of NP and NP-APT in RAW264.7 cells; (M) Quantification of cellular uptake of NP and NP-APT in RAW264.7 cells (n = 3, mean ± SD); (N) in vitro drug release of NP-APT with or without 1 mM H₂O₂. Statistical significance was determined by…

Fig. 2. NP-APT in a high-glucose-induced cardiomyocyte injury model. (A) Fluorescence images showing uptake of DiI-labeled nanoparticles by cardiomyocytes; (B-C) Flow cytometric analysis and quantification of DiI-positive cardiomyocytes after incubation with DiI-labeled nanoparticles; HG + DMF was not included in the DiI uptake assay; (D) JC-1 staining (scale bar = 25 μm), with red fluorescence indicating high membrane potential and green fluorescence indicating low membrane potential; (E) ROS and MitoSOX staining images (scale bar = 25 μm), with enhanced red fluorescence indicating increased mitochondrial oxidative stress; (F-H) Quantitative analysis of JC-1 red/green ratio, ROS and MitoSOX fluorescence intensity, and ATP content. Data are presented as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3. NP-APT exerts cardioprotective effects associated with regulation of the Mst1-autophagy-apoptosis axis. (A) qPCR analysis of Mst1 mRNA expression. (B-C) Western blot analysis and quantification of Mst1/GAPDH and p-Mst1/Mst1 ratio; the p-Mst1/Mst1 ratio is interpreted cautiously because total Mst1 and p-Mst1 change synchronously. (D-E) Western blot analysis and quantification of autophagy-related proteins (ULK1, Beclin-1, p62, LC3-I/II). (F-G) Representative TEM images and quantitative analysis of autophagosome numbers. (H-I) Western blot analysis and quantification of apoptosis-related proteins, including Bax, Bcl-2, and cleaved caspase-3/total caspase-3 ratio. (J-K) TUNEL staining and quantitative analysis of TUNEL-positive cells. Data are presented as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Groups are presented in the following order: Control, HG, HG + empty NP, HG + DMF, HG + NP, HG + NP-APT

Fig. 4. In vitro pathway intervention reveals that NP-APT exerts protective effects by regulating the Mst1-autophagy-apoptosis axis. (A-C) Representative images and quantitative analysis of JC-1, total ROS, and MitoSOX staining; JC-1 green fluorescence indicates low membrane potential and red fluorescence indicates high membrane potential; MitoSOX red fluorescence indicates mitochondrial ROS; ROS green fluorescence indicates total ROS; (D) ATP content assay; (E-F) Western blot analysis and quantification of Mst1, p-Mst1, autophagy-related proteins (Beclin-1, LC3-I/II, and p62), and apoptosis-related proteins (Bax and Bcl-2); (G-H) Representative TUNEL staining images and quantitative analysis; (I) RT-qPCR analysis and quantification of p62 mRNA expression; (J) Mitochondrial respiration OCR measured during a Seahorse mitochondrial stress assay; (K) ECAR trace recorded during the indicated glucose-FCCP-Rot/AA metabolic perturbation sequence. Data are presented as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5. Establishment of the diabetic phenotype and in vivo validation of NP-APT cardiac targeting. (A) Schematic illustration of the HFHSD/STZ model-establishment protocol; (B) fasting blood glucose levels; (C) body weight; (D) whole-body fluorescence retention profiles expressed as radiant efficiency over 0.5–48 h; (E) representative in vivo fluorescence images at 4, 12, and 24 h; (F) quantitative analysis of cardiac-region fluorescence intensity; (G-H) ex vivo fluorescence imaging of major organs and corresponding quantitative analysis; (I) heart-to-liver fluorescence ratio; and (J) representative fluorescence images showing NP-APT signals in cTnI-positive cardiomyocyte, Vimentin-positive fibroblast, and CD31-positive endothelial regions in ex vivo hearts from DCM mice. Data are presented as mean ± SD (n = 8). Statistical significance was determined by one-way ANOVA; ****p < 0.0001

Fig. 6. NP-APT improves body-weight trajectory, glycemic status, and cardiac function in DCM mice. (A) Body weight changes over weeks 1–8. (B) Fasting blood glucose levels. (C-D) Cardiac remodeling indices, including heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL). (E) Representative left ventricular M-mode echocardiographic images. (F-H) Quantitative analysis of left ventricular ejection fraction (EF%), fractional shortening (FS%), and E/A ratio. (I) Heart rate (bpm), LVIDd, LVIDs, and LVPWd as additional echocardiographic remodeling parameters. Data are presented as mean ± SD (n = 8). Statistical significance for the body-weight time course was determined by repeated-measures two-way ANOVA; endpoint bar-graph data were analyzed by one-way ANOVA. ***p < 0.001, ****p < 0.0001. Groups are presented in the following order: Control, DCM, DCM + empty NP, DCM + DMF, DCM + NP, DCM + NP-APT

Fig. 7. NP-APT alleviates myocardial structural injury, fibrosis, and cardiomyocyte hypertrophy in DCM mice. (A) Representative images of H&E, Masson’s trichrome, and WGA staining of myocardial tissues from each group (scale bar = 50 μm); (B) Quantification of inflammatory cell infiltration (cells/HPF); (C) Semi-quantitative scoring of myocardial fiber disorganization; (D) Collagen volume fraction (CVF); (E) Quantitative analysis of cardiomyocyte cross-sectional area (CSA). Data are presented as mean ± SD (n = 8). Statistical significance was determined by one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 8. NP-APT improves abnormal activation of the Mst1-autophagy-apoptosis signaling pathway in DCM mice. (A-F) Western blot analysis and quantitative assessment of total Mst1, the p-Mst1/Mst1 ratio, Beclin-1, p62, Bax/Bcl-2, and LC3-II/LC3-I protein expression in myocardial tissues from each group; (G) Representative TEM images showing myocardial autophagic structures (scale bar = 1 μm); (H) Quantitative analysis of autophagosome number; (I) Representative TUNEL staining images showing cardiomyocyte apoptosis (scale bar = 50 μm); (J) Quantification of TUNEL-positive cells. Data are presented as mean ± SD (n = 8). Statistical significance was determined by one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 9. Mst1 knockout and NP-APT improve glycemic status and cardiac function in DCM mice. (A) Fasting blood glucose levels; (B-C) Cardiac remodeling indices, including HW/BW and HW/TL; (D) Representative left ventricular M-mode echocardiographic images; (E-G) Quantitative analysis of EF%, FS%, and the E/A ratio. Direct comparisons between Mst1-KO and Mst1-KO + NP-APT were shown where applicable to indicate whether NP-APT provided additional benefit after Mst1 knockout. Data are presented as mean ± SD (n = 8). Statistical significance was determined by one-way ANOVA; ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 10. Combined Mst1-KO and NP-APT treatment markedly alleviates autophagy-apoptosis imbalance in DCM mice. (A-E) Western blot analysis and quantitative assessment of autophagy- and apoptosis-related proteins, including Beclin-1, p62, LC3-II/LC3-I, and Bax/Bcl-2, in myocardial tissues from each group. LC3-II/LC3-I was reported as an observed autophagy-related endpoint and interpreted together with p62, Beclin-1, TEM, and CQ-based flux assays rather than as a standalone flux readout; (F-G) Representative TEM images showing autophagosome formation in cardiomyocytes (scale bar = 1 μm) and corresponding quantitative analysis; (H-I) Representative TUNEL staining images (scale bar = 50 μm) and quantitative analysis of apoptotic cardiomyocytes. For Western blot quantification in panels B-E, data are presented as mean ± SD from three independent biological samples. For TEM-based autophagosome quantification and TUNEL quantification in panels G and I, data are presented as mean ± SD (n = 8). Statistical significance was determined by one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 11. scRNA-seq profiling reveals remodeling of the cardiac microenvironment following NP-APT intervention in DCM. (A) t-SNE visualization showing major cardiac cell populations in heart tissues from DCM and DCM + NP-APT groups; (B) Feature plot showing Mst1 expression across cardiac cell populations; (C) Violin plots of Mst1 expression in cardiomyocytes; (D) Violin plots of Mst1 activation-related gene set scores; (E) Violin plots of autophagy-related gene set scores; (F) Violin plots of apoptosis-related gene set scores. Single-cell RNA sequencing was performed with n = 1 biological sample per group and used as exploratory, hypothesis-generating mechanistic evidence; cell-level comparisons were not used for biological-replicate statistical inference