Fig 2: Cardiomyocyte‐specific PHLDA3 overexpression blunts the aortic banding (AB)‐induced hypertrophic response. A, Schematic illustration of the construction of cardiomyocyte‐specific PHLDA3 transgenic mouse lines. B, Representative Western blots for cardiomyocyte‐specific PHLDA3 expression in transgenic mice and their non‐transgenic mice controls (n=3 independent experiments). C through E, Statistical results of the ratios of heart weight/body weight (C), lung weight/body weight (D), and heart weight/TL (E) in non‐transgenic mice and transgenic mice at 4 weeks after sham or AB surgery (n=12–13 mice per group). F, Representative hematoxylin and eosin staining images of the gross morphology and left ventricular muscle of hearts from the indicated groups (n=6 mice per group; scale bar, 50 μm). G, Statistical results of the cardiomyocyte cross‐sectional area from the indicated groups (n>100 cells per group). H through J, Echocardiographic assessment of the left ventricle end‐diastolic diameter (H), left ventricle end‐systolic diameter (I), and fractional shortening (J) in the indicated groups at 4 weeks after sham or AB surgery (n=11–13 mice per group). K, Representative images of picrosirius red staining of perivascular and myocardial interstitial regions of the hearts from the indicated groups (n=6 mice per group; scale bar, 100 μm). L, Statistical results of fibrotic areas from the indicated groups (n>30 fields per group). M and N, Real‐time PCR showing the mRNA levels of fetal genes (M) and fibrotic markers (N) in the heart tissues from the indicated groups at 4 weeks after sham or AB surgery (n=4 per group). **P<0.01 vs non‐transgenic mice sham; ## P<0.01 vs non‐transgenic mice AB in (C through E, G through J, L through N). By Tamhane T2 analysis (C, E, G, I, L through N) or Bonferroni post hoc analysis (D, H, and J). AB indicates aortic banding; Anp, atrial natriuretic peptide; Bnp, B‐type natriuretic peptide; BW, body weight; Ctgf, connective tissue growth factor; FS, fractional shortening; H&E, hematoxylin and eosin; HW, heart weight; LV, left ventricle; LVEDd, left ventricle end‐diastolic diameter; LVESd, left ventricle end‐systolic diameter; LW, lung weight; Myh7, myosin heavy chain 7; NTG, non‐transgenic mice; PHLDA3, pleckstrin homology‐like domain family A, member 3; TG, conditional PHLDA3 transgenic mice; TL, tibia length.

Fig 3: PHLDA3 attenuates Ang II‐induced cardiomyocyte hypertrophy in vitro. A, Representative Western blots verify PHLDA3 expression in cultured neonatal rat cardiomyocytes (NRCMs) infected with AdshRNA, AdshPHLDA3, AdGFP and AdPHLDA3. B, Representative immunofluorescence images of NRCMs infected with AdshRNA, AdshPHLDA3, AdGFP and AdPHLDA3 after treatment with PBS or Ang II for 48 hours (green: α‐actinin; blue: nuclear; scale bar, 40 μm). C, Quantitative results of the cell surface area of NRCMs infected with AdshRNA or AdshPHLDA3 after treatment with PBS or Ang II for 48 hours. D, Quantitative results of the cell surface area of NRCMs infected with AdGFP or AdPHLDA3 after treatment with PBS or Ang II for 48 hours. E, Transcript levels of the hypertrophic biomarkers Anp and Myh7 in NRCMs infected with AdshRNA or AdshPHLDA3. F, Transcript levels of the hypertrophic biomarkers Anp and Myh7 in NRCMs infected with AdGFP or AdPHLDA3. **P<0.01 vs AdshRNA+PBS, ## P<0.01 vs AdshRNA+Ang II in (C and E); **P<0.01 vs AdGFP+PBS, # P<0.05 or ## P<0.01 vs AdGFP+Ang II in (D and F). n=3 independent experiments in (A through F). n>50 cells per group in (C and D). By Tamhane T2 analysis (C, E [Myh7] and F) or Bonferroni post hoc analysis (D and E [Anp]). AdGFP indicates adenoviral green fluorescent protein; AdPHLDA3, adenoviral vectors containing the PHLDA3 cDNA; AdshPHLDA3, adenoviral vectors harboring the PHLDA3 short hairpin RNA; AdshRNA, adenoviral short hairpin RNA; Ang II, angiotensin II; Anp, atrial natriuretic peptide; Myh7, myosin heavy chain 7; PHLDA3, pleckstrin homology‐like domain family A, member 3.

Fig 4: PHLDA3 inhibits the activation of AKT signaling induced by prohypertrophic stimuli. A and B, Representative Western blots and quantitative analysis of phosphorylated and total MEK1/2, ERK1/2, JNK1/2 and P38 levels in PHLDA3‐Flox and PHLDA3‐CKO mice (A) or transgenic and non‐transgenic mice (B) at 4 weeks after sham or aortic banding surgery (n=4 mice per group; n.s. means no significant difference vs PHLDA3‐Flox aortic banding or non‐transgenic mice aortic banding). C and D, Representative Western blots and quantitative analysis of phosphorylated and total AKT, mTOR, GSK3β, and P70S6K levels in PHLDA3‐Flox and PHLDA3‐CKO mice (C) or transgenic and non‐transgenic mice (D) at 4 weeks after sham or aortic banding surgery (n=4 mice per group; **P<0.01 vs PHLDA3‐Flox aortic banding or non‐transgenic mice aortic banding). E and F, Representative Western blots and quantitative analysis of phosphorylated and total AKT, mTOR, GSK3β, and P70S6K levels in neonatal rat cardiomyocytes infected with AdshRNA and AdshPHLDA3 (E) or AdGFP and AdPHLDA3 (F) and treated with PBS or Ang II (n=3 independent experiments; **P<0.01 vs AdshRNA+Ang II or AdGFP+Ang II). By 2‐tailed Student t‐test (A through C [p‐AKT], C [p‐GSK3β], C [p‐P70S6K], D [p‐mTOR], F [p‐mTOR]) or Welch test (C [p‐mTOR], D [p‐AKT], D [p‐GSK3β], D [p‐P70S6K], E and F [p‐AKT], F [p‐GSK3β], F [p‐P70S6K]). AB indicates aortic banding; AdGFP, adenoviral green fluorescent protein; AdPHLDA3, adenoviral vectors containing the PHLDA3 cDNA; AdshPHLDA3, adenoviral vectors harboring the PHLDA3 short hairpin RNA; AdshRNA, adenoviral short hairpin RNA; Ang II, angiotensin II; CKO, cardiomyocyte‐specific conditional PHLDA3 knockout mice; ERK, extracellular regulated protein kinase; Flox, PHLDA3loxP/loxP (PHLDA3‐Flox) mice; GSK3β, glycogen synthase kinase 3β; JNK, c‐Jun N‐terminal kinase; MEK, MAPK/ERK kinase; mTOR, mammalian target of rapamycin; NTG, non‐transgenic mice; p38, protein 38; P70S6K, ribosomal protein S6 kinase beta‐1; PHLDA3, pleckstrin homology‐like domain family A, member 3; TG, conditional PHLDA3 transgenic mice.

Fig 5: PHLDA3 expression is reduced in the development of pathological cardiac hypertrophy and heart failure. A, Representative Western blots and quantitative analysis of PHLDA3 and ANP protein levels in heart tissues from C57BL/6J mice subjected to sham or AB surgery at the indicated time points (n=6 per group). B, Representative Western blots and quantitative analysis of PHLDA3 and ANP protein levels in cultured neonatal rat cardiomyocytes incubated with PBS or Ang II at the indicated time points (n=3 independent experiments). *P<0.05 or **P<0.01 vs sham or PBS; # P<0.05 or ## P<0.01 vs AB 4w or Ang II 24 hours. By Tamhane's T2 analysis (A and B). AB indicates aortic banding; Ang II, angiotensin II; ANP, atrial natriuretic peptide; PHLDA3, pleckstrin homology‐like domain family A, member 3.