A Novel Pak1 Activator Ameliorates ER Stress for HFpEF Therapy

Figures

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  FIGURE 1. Pak1 and UPR related genes were downregulated during HFpEF progression. (A) UMAP of cardiomyocytes isolated from mouse hearts subjected to HFD+L‐NAME treatment, illustrating clustering based on transcriptomic profiles. (B) Gene set enrichment analysis (GSEA) of single‐nucleus RNA sequencing reveals significant downregulation of the “HALLMARK UNFOLDED PROTEIN RESPONSE” pathway in cardiomyocytes from the HFpEF patients compared to controls. (C) Heatmap of endoplasmic reticulum (ER)‐related gene expression in mouse hearts after 5 and 15 weeks of HFD + L‐NAME treatment, n = 3 mice per group. (D) qPCR results of mice heart tissues, n = 3‐5 per group. (E,G) Representative western blots and quantification of Pak1 from human heart samples. n = 4 samples per group. (F,H) Representative western blots and quantification of Pak1 from the mouse heart tissues. n = 5 mice per group. (I) Representative western blots of proteins from primary mouse cardiomyocytes. Data are presented as means ± SEM. Statistical analysis was by one‐way ANOVA followed by Tukey's multiple comparisons test.

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  FIGURE 2. Knockdown of Pak1 in the heart impaired diastolic function at 6 weeks of HFpEF stress. (A) Representative image of echocardiography at 6 weeks. Top: M‐mode of short axis view, Middle: Doppler of E and A wave, Bottom: Tissue Doppler of E and e’ wave. (B) Preserved systolic function and reduced diastolic function in the left ventricle were detected by echocardiography at week 6. n = 5 to 7 mice per group. (C) Representative images of WGA staining. (D) The cross‐sectional areas of cardiomyocytes were assessed. (E) Representative images of Masson staining. (F) The percentage of fibrosis area was measured in transversal sections stained with Masson's trichrome. (G) The heart weight‐to‐tibia length ratio was calculated. Scale bars were set at 50 µm (WGA) and 100 µm (Masson). n = 5 to 8 samples per group. Data are presented as means ± SEM. Statistical analysis was by two‐way ANOVA followed by Tukey's multiple comparisons test.

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  FIGURE 3. Pak1 knockdown impaired ER function and promoted cardiomyocyte apoptosis. (A) Electron microscopy showed expanded ER lumens of Pak1 knockdown mouse hearts after 6 weeks of HFD+L‐NAME treatment. Arrowheads show normal ER (arrows) and fragmented ER with vacuolization. Scale bars = 1 µm. (B) Representative western blots. (C‐H) Quantification of ER stress‐related proteins. n = 6 mouse samples per group. (I) Representative western blots of t‐Pak1 and UPR pathway effectors and (J‐M) Quantification of panel (I) in AC16 cells. n = 3 samples per group. Data are presented as means ± SEM. Statistical analysis was by two‐way ANOVA followed by Tukey's multiple comparisons test.

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  FIGURE 4. Pak1 overexpression preserved cardiac diastolic function following HFD+L‐NAME treatment. (A) Representative image of echocardiography at 15 weeks of HFpEF stress. Top: M‐mode of parasternal short axis view, Middle: Doppler image of E and A wave, Bottom: Tissue Doppler of E and e’ wave. (B) Systolic function and diastolic function were analyzed. n = 5 to 8 mice per group. (C) Section morphology of hearts stained with WGA and (D) quantitative analysis. (E) Cardiac fibrosis stained with Masson and (F) quantitative analysis. (G) Proportion of heart weight to tibia length. Scale bars were set at 50 µm (WGA) and 100 µm (Masson). n = 5 to 8 samples per group. Data are presented as means ± SEM. Statistical analysis was by two‐way ANOVA followed by Tukey's multiple comparisons test.

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  FIGURE 5. Pak1 overexpression potentiated protective UPR through ATF4 and ATF6 signaling. (A) Representative TEM images from n = 3 samples per group. Yellow arrows indicate ER. Scale bar = 1 µm. (B) Representative western blot of ER stress‐related proteins. (C–F) Quantification of ER stress‐related proteins. n = 6 samples per group. (G) Representative western blots of t‐Pak1 and UPR pathway effectors, and (H–K) Quantification of panel (G). n = 3 samples per group. Data are presented as means ± SEM. Statistical analysis was by two‐way ANOVA followed by Tukey's multiple comparisons test.

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  FIGURE 6. Pak1 overexpression restores protein synthesis via ERK‐MNK1‐eIF4E signaling in PA‐treated AC16 cells. (A) Representative puromycin incorporation assay and corresponding quantification of total protein synthesis in AC16 cells. n = 3 samples per group. (B) Immunofluorescence images showing puromycin for nascent peptide (red) and Calnexin for ER (green). Nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. (C,D) Western blots and quantification of the total and phosphorylation of eIF4E, and eIF4EBP1 expression. n = 6 samples per group. (E,F) Western blots and quantification of MNK1 and ERK1/2. n = 6 samples per group. (G–J) Western blots and quantification of phosphorylated eIF4E, phosphorylated PERK, and ATF4 expression in adGFP‐ and adPak1‐transduced cells treated with PA in the presence or absence of the MNK1/2 inhibitor tomivosertib. n = 3 samples per group. Data are presented as means ± SEM. Statistical analysis was by two‐way ANOVA followed by Tukey's multiple comparisons test.

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  FIGURE 7. Therapeutic targeting of Pak1 with compound JB2019 improved cardiac function and alleviated ER stress in HFpEF. (A) Experimental Overview. After 10‐weeks of HFpEF treatment, mice were subjected to compound treatment until 15‐weeks. Functional analysis was performed every 5 weeks. (B) Representative left ventricular M‐mode (top), pulse wave doppler (middle), and tissue Doppler (bottom) electrocardiographic films. (C–E) Tissue doppler analysis of E/e’ wave, E/A wave, and IVRT at 15 weeks. n = 5–10 mice per group. (F) Representative images of WGA staining (Scale bar = 50 µm), Masson's trichrome staining (Scale bar = 100 µm), and DHE staining (Scale bar = 100 µm). (G) Quantification of WGA staining to assess cardiomyocyte cross sectional area. (H) Quantification of Masson's trichrome staining to assess interstitial fibrosis. (I) Quantification of DHE staining to assess superoxide generation. (J) Heart weight/tibial length ratio. n = 5‐10 samples per group. (K–L) Relative mRNA expression of hypertrophic (NppA and NppB) and fibrotic (Col1a2) gene markers. n = 5 samples per group (M) Representative immunoblots of key markers of the ER stress response pathway and (N‐Q) Quantification of…

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  FIGURE 8. JB2019 alleviated PA‐induced ER stress in cardiac organoids. (A) Representative brightfield images of cardiac organoids beating activity, I indicates intensity. Scale bar = 1000 µm. (B) Representative calcium images and the synchronized contraction was shown by calcium flux frequency. Results were expressed as F/F0 (F0 refers to baseline intensity). Scale bar = 1000 µm. (C) Immunofluorescence images of hESC‐CMs with PA and JB2019 treatment showing colocalization of ROS (CellROX, green) and ER marker (ER Tracker, red); nuclear marker DAPI (blue). Scale bar = 200 µm. (D) Cardiac organoids stained with ER‐Tracker Red and DAPI blue visualized by confocal microscopy. Scale bar = 50 µm. (E) Cells from (D) were quantified for the percentage of ER whorls (n = 5 independent samples per group; >100 cells analyzed per sample). (F) Quantification of ER whorl number in cells from (D). n = 5 independent samples per group; >100 cells analyzed per sample. (G) Representative western blots of UPR pathway effectors and (H‐K) Quantification of panel (G). n = 5 independent samples per group; Data are presented as means ± SEM. Statistical analysis was by two‐way ANOVA followed by Tukey's multiple…