myocardial tissues  (Elabscience Biotechnology)

Journal: Frontiers in Pharmacology

Article Title: β-Sitosterol attenuates obstructive sleep apnea-related myocardial injury via MALAT1-mediated HIF-1α regulation

doi: 10.3389/fphar.2025.1692758

Figure Lengend Snippet: β-Sitosterol alleviates CIH-induced myocardial injury in rats CIH rats were administrated with intermittent hypoxia, followed by low, middle and high dose of β-Sitosterol treatment. Rats exposed to normoxia (Normal) received olive oil as vehicle control. (A) Ratio of heart weight to body weight (HW/BW) in rats exposed to normoxia (Normal), CIH (Model), or CIH with low/middle/high-dose β-sitosterol treatment (10/15/20 mg/kg). (B) Quantitative analysis of fibrosis area in myocardial tissues. (C) Quantitative analysis of apoptosis rate (TUNEL staining). (D) Representative H&E staining showing myocardial pathology (black arrow: wavy arrangement; yellow arrow: necrotic cells; scale bar: 50 μm). (E) Representative Masson’s trichrome staining (blue: collagen fibrosis). (F) Representative TUNEL staining (green: apoptotic cells; red: nuclei; scale bar: 20 μm) and (F) quantitative analysis of apoptosis rate. Data are presented as mean ± SD; n = 8 per group. **P < 0.01 vs Normal; #P < 0.05, ##P < 0.01 vs Model; & P < 0.01 vs Middle-dose drug.

Article Snippet: Myocardial tissue sections were stained with Hematoxylin and Eosin Staining Kit (Beyotime, Shanghai, China), Masson Staining Kit (SenBeiJia, Nanjing, China) and Colorimetric TUNEL Apoptosis Assay Kit (Beyotime) following the manufacturer’s instructions.

Techniques: Control, TUNEL Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: β-Sitosterol attenuates obstructive sleep apnea-related myocardial injury via MALAT1-mediated HIF-1α regulation

doi: 10.3389/fphar.2025.1692758

Figure Lengend Snippet: β-Sitosterol downregulates MALAT1/HIF1A and apoptosis-related proteins in CIH rats CIH rats were administrated with intermittent hypoxia, followed by low, middle and high dose of β-Sitosterol treatment. (A,B) qRT-PCR analysis of MALAT1 and HIF1A mRNA levels in myocardial tissues. (C) Representative Western blot bands and (D–F) quantitative analysis of HIF-1α, Bax, and cleaved caspase-3 protein levels (β-actin: loading control). Data are presented as mean ± SD; n = 8 per group. ** P < 0.01 vs Normal; # P < 0.05, ## P < 0.01 vs Model; & P < 0.01 vs Middle-dose drug.

Article Snippet: Myocardial tissue sections were stained with Hematoxylin and Eosin Staining Kit (Beyotime, Shanghai, China), Masson Staining Kit (SenBeiJia, Nanjing, China) and Colorimetric TUNEL Apoptosis Assay Kit (Beyotime) following the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: β-Sitosterol attenuates obstructive sleep apnea-related myocardial injury via MALAT1-mediated HIF-1α regulation

doi: 10.3389/fphar.2025.1692758

Figure Lengend Snippet: β-Sitosterol alleviates myocardial injury via MALAT1 in vivo . CIH rats were induced by administration of intermittent hypoxia, followed by β-Sitosterol treatment or/and MALAT1 overexpression. (A) HW/BW ratio. (B) Representative H&E staining of myocardial pathology (scale bar: 50 μm). (C,D) Masson’s staining (fibrosis, blue) and (D) quantification. (E) TUNEL staining (apoptosis, green) and (F) quantification. (G,H) qRT-PCR of HIF1A mRNA. (I) Western blot bands and (J–L) quantification of HIF-1α, Bax, and cleaved caspase-3. Data are presented as mean ± SD; n = 8 per group. * P < 0.05 vs Model; # P < 0.05 vs β-Sitosterol + LV-NC; ns indicated P > 0.05.

Article Snippet: Myocardial tissue sections were stained with Hematoxylin and Eosin Staining Kit (Beyotime, Shanghai, China), Masson Staining Kit (SenBeiJia, Nanjing, China) and Colorimetric TUNEL Apoptosis Assay Kit (Beyotime) following the manufacturer’s instructions.

Techniques: In Vivo, Over Expression, Staining, TUNEL Assay, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Disrupting the interaction between connexin 43 and calmodulin restores gap junction function and mitigates reperfusion arrhythmias

doi: 10.1038/s41598-025-18366-3

Figure Lengend Snippet: The establishment of isolated rat heart IR injury model. ( A ) Schematic representation of the experimental protocol of isolated rat heart IR injury model. ( B ) Representative TTC staining images showing myocardial infarct size after I/R injury. White areas indicate infarcted myocardium, while red areas represent viable (non-infarcted) myocardium. ( C ) Quantitative analysis of myocardial infarct size expressed as a percentage of the total myocardial area. ( D ) HE staining was performed to observe the I/R-induced histological changes. ( E ) Typical arrhythmias in the I/R group, the red arrows indicate premature electrical events, while the blue arrows indicate ST-segment elevation.

Article Snippet: Paraffin-embedded myocardial tissue Sect. (5 μm thick) were first deparaffinized in xylene (A530011, Sangon Biotech, China) and rehydrated through a series of graded ethanol solutions (A500737, Sangon Biotech, China).

Techniques: Isolation, Staining

Journal: Scientific Reports

Article Title: Disrupting the interaction between connexin 43 and calmodulin restores gap junction function and mitigates reperfusion arrhythmias

doi: 10.1038/s41598-025-18366-3

Figure Lengend Snippet: Interfering with the interaction between Cx43 and CaM improved myocardial electrophysiological parameters and reduced arrhythmia scores. (A) Recorded representative epicardial activation mapping of left ventricular. (B-D) Quantification of conduction velocity, absolute inhomogeneity, and inhomogeneity index of left ventricular. (E) Quantification of arrhythmia score in different groups. Data are expressed as the mean ± SD ( n = 6). Statistical differences were determined using one-way ANOVA, followed by post hoc Tukey’s test. *** P < 0.001 I/R group vs. control group; # P < 0.05 and ## P < 0.01 IR + sh-CaM#1 group vs. I/R group; & P < 0.05, && P < 0.01 and &&& P < 0.001 IR + SP15 group vs. I/R group.

Article Snippet: Paraffin-embedded myocardial tissue Sect. (5 μm thick) were first deparaffinized in xylene (A530011, Sangon Biotech, China) and rehydrated through a series of graded ethanol solutions (A500737, Sangon Biotech, China).

Techniques: Activation Assay, Control

Journal: Physiological Reports

Article Title: Left ventricular pressure‐loading improves pressure‐induced right ventricular remodeling by redistributing mechanical load and reducing mechanosignaling

doi: 10.14814/phy2.70546

Figure Lengend Snippet: Comparisons between control, PAB, and DB simulations and representative experimental PV loops. Control simulations are depicted in the red column, PAB simulations in the blue column, and DB simulations in the purple column. Rows represent simulated parameters, from top to bottom: PV‐loops of the LV (red) and RV (dark blue), myofiber strain, flow, and myofiber stress. (Middle Column: PAB) Pulmonary banding dilates the RV, reduces RV pump contractility (Ees), induces mechanical discoordination during ventricular relaxation (i.e., increased LVEDSI), increases isovolumic relaxation time and RVfw work density, but decreases LV and septal work density. (Right Column: DB) Double banding reduces RV volume, increases RV Ees, while RVfw myocardial workload (i.e., work density) is decreased. In contrast, the LV dilates and LV myocardial work density increases with aortic banding. The interventricular mechanical imbalance, both in terms of myocardial work density and mechanical relaxation dyssynchrony (LVEDSI) is reduced in the double banding setup, when compared to the pulmonary banding simulation. (Figure . Continued) representative RV (top row) and LV (bottom row) PV loops measured from rats that underwent control, PAB, and DB surgeries. AV, aortic valve; DB, double banding; Ees, end‐systolic elastance; LV, left ventricle; LVEDSI, left ventricular end‐diastolic shape index; LVfw, left ventricular free wall; MV, mitral valve; PAB, pulmonary artery banding; PV, pressure‐volume; PV, pulmonary valve; RV, right ventricle; RVfw, right ventricular free wall; Sept, septum; TV, tricuspid valve.

Article Snippet: In the computer simulation, we assumed that the LV myocardial tissue was healthy (normal contractility and passive stiffness), whereas previous studies showed that pulmonary hypertension is associated with impaired LV function (Menzel et al., ).

Techniques: Control

Journal: Circulation Research

Article Title: Targeting Cardiomyocyte PCNA and POLD1 Prevents Pathologic Myocardial Hypertrophy

doi: 10.1161/CIRCRESAHA.124.325647

Figure Lengend Snippet: Cardiomyocyte p21 (cyclin-dependent kinase inhibitor 1) is increased in Mybpc3 (myosin-binding protein C3) −/− mice during the early stages of hypertrophic cardiomyocyte growth. A , Representative immunoblot of p21 (Santa Cruz Biotechnology [SCBT], sc-6246) from control (Ctl, wild-type C57BL/6) and Mybpc3 −/− left ventricular (LV) tissue at postnatal day (P) 2, P7, P25, P60, and P180. Total protein stain (TPS) was used as the loading Ctl. B , Immunoblot of p21 (SCBT, sc-6246) and β-actin in Ctl and Mybpc3 −/− LV tissue at P2, P7, P25, P60, and P180. C , Quantification of p21 protein expression normalized to β-actin in Ctl (n=4) and Mybpc3 −/− (n=4) LV tissue at P2, P7, P25, P60, and P180. p21 protein expressions are shown relative to Ctl of each time point. D , Measurement of p21 gene expression (Cdkn1a [cyclin-dependent kinase inhibitor 1a]) from Ctl and Mybpc3 −/− LV tissue at P7 (n=3), P25 (n=4), and P180 (n=3). Cdkn1a expression was normalized to Rpl32 (ribosomal protein L32) expression. Fold changes are shown relative to the Ctl gene expression at each time point. E and F , Immunoblot and quantification of p21 (eBioscience, 14-6715-81) from cardiomyocyte (CM) and noncardiomyocyte (non-CM) populations from Ctl and Mybpc3 −/− myocardial tissue at P25 (n=4). Immunoblot of Sar. α-actinin (sarcomeric α-actinin) was used to check the CM purity. TPS was used as the loading Ctl. Fold changes are shown relative to Ctl CMs. G and H , Immunoblot and quantification of p21 (eBioscience, 14-6715-81) from cytosolic and nuclear protein fractions from Ctl and Mybpc3 −/− myocardial tissue at P25 (n=5). Immunoblot of GAPDH and lamin B1 were used as loading Ctl. Fold changes are shown relative to Ctl for each fraction. All results are shown as mean±SEM. The Mann-Whitney U test is used for C , D , F , and H .

Article Snippet: In addition, Ki67 (proliferation marker protein Ki-67) antibody staining was performed on both myocardial tissue (Ki67 [CST (Cell Signaling Technology), 9449]) or hiPSC-CM (Ki67 [CST, 9129]).

Techniques: Binding Assay, Western Blot, Control, Staining, Expressing, Gene Expression, MANN-WHITNEY

Journal: Circulation Research

Article Title: Targeting Cardiomyocyte PCNA and POLD1 Prevents Pathologic Myocardial Hypertrophy

doi: 10.1161/CIRCRESAHA.124.325647

Figure Lengend Snippet: p21 (cyclin-dependent kinase inhibitor 1) deficiency increases hypertrophic remodeling in Mybpc3 (myosin-binding protein C3) −/− mice. A , Schematic of Cdkn1a (cyclin-dependent kinase inhibitor 1a) −/− Mybpc3 −/− murine model generated by crossing a Cdkn1a −/− mouse with a Mybpc3 −/− mouse. B , Western blot of p21 (Santa Cruz Biotechnology [SCBT], sc-6246) from control (Ctl, wild-type C57BL/6), Mybpc3 −/− , and Cdkn1a −/− Mybpc3 −/− left ventricular (LV) tissue at postnatal day (P) 25. Immunoblot of β-actin used as a loading Ctl. C , Immunofluorescence staining of p21 (eBioscience, 14-6715-81; red) in Ctl, Mybpc3 −/− , and Cdkn1a −/− Mybpc3 −/− myocardial tissue at P25. Cardiomyocytes were identified with PCM1 (pericentriolar material 1; green) and nuclei labeled by 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 20 µm. D , Hematoxylin and eosin–stained cross-sections of Ctl, Mybpc3 −/− , and Cdkn1a −/− Mybpc3 −/− myocardial tissue at P25. Scale bars, 1 mm. E , Heart weight (HW) and ( F ) heart weight to tibia length ratio (HW/TL) of Ctl (n=10), Cdkn1a −/− (n=5), Mybpc3 −/− (n=10), and Cdkn1a −/− Mybpc3 −/− (n=11) mice at P25. Echocardiography assessment of ( G ) interventricular septal thickness at end-diastole (IVSd), ( H ) left ventricular posterior wall thickness at end-diastole (LVPWd), ( I ) left ventricular internal diameter at end-diastole (LVIDd), and ( J ) left ventricular fractional shortening (FS) in Ctl, Cdkn1a −/− , Mybpc3 −/− , and Cdkn1a −/− Mybpc3 −/− hearts at P25 and P180 (n=5–7). K , Representative wheat germ agglutinin (WGA) staining (green) with nuclei labeled by DAPI (blue) from Ctl, Cdkn1a −/− , Mybpc3 −/− , and Cdkn1a −/− Mybpc3 −/− myocardial tissue at P25 and P180. Scale bars, 50 µm. L , Cardiomyocyte cross-sectional area at P25 and P180 of Ctl, Cdkn1a −/− , Mybpc3 −/− , and Cdkn1a −/− Mybpc3 −/− mice as assessed by WGA staining of left ventricular cross-sections (n=5). Minimum 100 cardiomyocytes (CMs)/sample. All results are shown as mean±SEM. Two-way ANOVA with the Tukey multiple comparison test is used for E through J and L . ANOVA factor: Cdkn1a −/− and Mybpc3 −/− . Normality was assumed based on the central limit theorem for L .

Article Snippet: In addition, Ki67 (proliferation marker protein Ki-67) antibody staining was performed on both myocardial tissue (Ki67 [CST (Cell Signaling Technology), 9449]) or hiPSC-CM (Ki67 [CST, 9129]).

Techniques: Binding Assay, Generated, Western Blot, Control, Immunofluorescence, Staining, Labeling, Comparison

Journal: Circulation Research

Article Title: Targeting Cardiomyocyte PCNA and POLD1 Prevents Pathologic Myocardial Hypertrophy

doi: 10.1161/CIRCRESAHA.124.325647

Figure Lengend Snippet: Overexpression of cardiomyocyte p21 (cyclin-dependent kinase inhibitor 1) reduces cardiomyocyte hypertrophy in Mybpc3 (myosin-binding protein C3) −/− mice. A , Schematic of the Cdkn1a (cyclin-dependent kinase inhibitor 1a) SUPER Mybpc3 −/− Myh6Cre (Tg(Myh6-cre)1Jmk) murine model generated by crossing a Cdkn1a SUPER Myh6Cre mouse with a Mybpc3 −/− mouse. B , Immunoblot for p21 (Santa Cruz Biotechnology [SCBT], sc-6246) identified tdTomato-tagged p21 (p21-tdT) and endogenous p21 (p21-endo) from control (Ctl, wild-type C57BL/6), Mybpc3 −/− Myh6Cre, and Cdkn1a SUPER Mybpc3 −/− Myh6Cre left ventricular (LV) tissue at postnatal day (P) 25. Western blot of β-actin used as a loading Ctl. C , Immunofluorescence staining for tdTomato protein (tdTomato; red) in Ctl, Mybpc3 −/− Myh6Cre, and Cdkn1a SUPER Mybpc3 −/− Myh6Cre myocardial tissue at P25. Cardiomyocytes were identified with sarcomeric α-actinin (green) and nuclei labeled by 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 5 µm. D , Hematoxylin and eosin–stained cross-sections of Ctl, Mybpc3 −/− Myh6Cre, and Cdkn1a SUPER Mybpc3 −/− Myh6Cre myocardial tissue at P25. Scale bars, 1 mm. E , Heart weight (HW) and ( F ) heart weight to tibia length ratio (HW/TL) of Ctl (n=7), Cdkn1a SUPER Myh6Cre (n=6), Mybpc3 −/− Myh6Cre (n=7), and Cdkn1a SUPER Mybpc3 −/− Myh6Cre (n=9) at P25. Echocardiography assessment of ( G ) interventricular septal thickness at end-diastole (IVSd), ( H ) left ventricular posterior wall thickness at end-diastole (LVPWd), ( I ) left ventricular internal diameter at end-diastole (LVIDd), and ( J ) left ventricular fractional shortening (FS) in Ctl, Cdkn1a SUPER Myh6Cre, Mybpc3 −/− Myh6Cre, and Cdkn1a SUPER Mybpc3 −/− Myh6Cre hearts at P25 and P180 (n=5–9). K , Representative wheat germ agglutinin (WGA) staining (green) with nuclei labeled by DAPI (blue) from Ctl, Mybpc3 −/− Myh6Cre, and Cdkn1a SUPER Mybpc3 −/− Myh6Cre myocardial tissue at P25. Scale bars, 50 µm. L , Cardiomyocyte cross-sectional area at P25 of Ctl, Mybpc3 −/− Myh6Cre, and Cdkn1a SUPER Mybpc3 −/− Myh6Cre myocardial tissue as assessed by WGA staining of left ventricle cross-sections (n=5). Minimum 100 cardiomyocytes (CMs)/sample. M , Quantification of total CMs per left ventricular (LV) wall section in Ctl, Cdkn1a −/− , Mybpc3 −/− , Cdkn1a −/− Mybpc3 −/− , Cdkn1a SUPER Myh6Cre, and Cdkn1a SUPER Mybpc3 −/− Myh6Cre hearts (n=4–5). All results are shown as mean±SEM. Two-way ANOVA with the Tukey multiple comparison test is used for E through J and M . ANOVA factor: Cdkn1a −/− , Cdkn1a SUPER Myh6Cre, and Mybpc3 −/− Myh6Cre. One-way ANOVA with the Tukey multiple comparison test is used for L . Normality was assumed based on the central theorem for L and M .

Article Snippet: In addition, Ki67 (proliferation marker protein Ki-67) antibody staining was performed on both myocardial tissue (Ki67 [CST (Cell Signaling Technology), 9449]) or hiPSC-CM (Ki67 [CST, 9129]).

Techniques: Over Expression, Binding Assay, Generated, Western Blot, Control, Immunofluorescence, Staining, Labeling, Comparison

Journal: Circulation Research

Article Title: Targeting Cardiomyocyte PCNA and POLD1 Prevents Pathologic Myocardial Hypertrophy

doi: 10.1161/CIRCRESAHA.124.325647

Figure Lengend Snippet: p21 (cyclin-dependent kinase inhibitor 1) negatively regulates cardiomyocyte polyploidy in Mybpc3 (myosin-binding protein C3) −/− mice. A , Schematic of 5-ethynyl-2′-deoxyuridine (EdU) injection in mice from postnatal day (P) 21 to P25. B , Representative click-EdU (green) and PCM1 (pericentriolar material 1; red) staining with nuclei labeled by 4′,6-diamidino-2-phenylindole (DAPI; blue) and ( C ) quantification of EdU-positive cardiomyocytes (CMs) from control (Ctl, wild-type C57BL/6; n=5), Cdkn1a (cyclin-dependent kinase inhibitor 1a) −/− (n=5), Mybpc3 −/− (n=6), Cdkn1a −/− Mybpc3 −/− (n=6), and Cdkn1a SUPER Mybpc3 −/− Myh6Cre (Tg(Myh6-cre)1Jmk) (n=5) myocardial tissue. Minimum 100 nuclei/sample. Arrow indicates cardiomyocyte nuclei, and arrowhead indicates noncardiomyocyte nuclei. Scale bars, 25 µm. D , Schematic representation of left ventricular tissue nuclei isolation followed by PCM1 labeling and Hoechst staining followed by flow cytometry (FC) analysis. E , Cardiomyocyte nuclear ploidy from Ctl, Cdkn1a −/− , Mybpc3 −/− , Cdkn1a −/− Mybpc3 −/− , and Cdkn1a SUPER Mybpc3 −/− Myh6Cre nuclei by FC analysis. F , Quantitative representation of nuclear ploidy through FC analysis shown as percentage of diploid (2n) and greater than diploid (>2n) from Ctl, Cdkn1a −/− , Mybpc3 −/− , Cdkn1a −/− Mybpc3 −/− , and Cdkn1a SUPER Mybpc3 −/− Myh6Cre tissue samples (n=5). Minimum 30000 nuclei/sample. ANOVA factor: genotype and ploidy. G , Linear regression analysis of the relationship between ploidy measured by FC and DNA content measured by immunohistochemistry (IHC) in Ctl, Mybpc3 −/− , and Cdkn1a −/− Mybpc3 −/− left ventricular (LV) tissue samples. H , Determination of cardiomyocyte ploidy (DNA content) using immunofluorescence staining by measuring DAPI intensity from Ctl, Cdkn1a −/− , Mybpc3 −/− , Cdkn1a −/− Mybpc3 −/− , and Cdkn1a SUPER Mybpc3 −/− Myh6Cre cardiomyocyte nuclei. Minimum 100 nuclei/sample (n=5). All results are shown as mean±SEM. Two-way ANOVA with the Tukey multiple comparison test is used for C , F , and H . ANOVA factor: Cdkn1a −/− , Mybpc3 −/− , and Cdkn1a SUPER Myh6Cre. The simple linear regression analysis is used for G . Normality was assumed based on the central limit theorem for C , F , and H .

Article Snippet: In addition, Ki67 (proliferation marker protein Ki-67) antibody staining was performed on both myocardial tissue (Ki67 [CST (Cell Signaling Technology), 9449]) or hiPSC-CM (Ki67 [CST, 9129]).

Techniques: Binding Assay, Injection, Staining, Labeling, Control, Isolation, Flow Cytometry, Immunohistochemistry, Immunofluorescence, Comparison

Journal: Circulation Research

Article Title: Targeting Cardiomyocyte PCNA and POLD1 Prevents Pathologic Myocardial Hypertrophy

doi: 10.1161/CIRCRESAHA.124.325647

Figure Lengend Snippet: p21 (cyclin-dependent kinase inhibitor 1) and PCNA (proliferating cell nuclear antigen) protein interactions are increased in cardiomyocyte hypertrophy. A , Schematic of p21 coimmunoprecipitation from control (Ctl, wild-type C57BL/6) and Mybpc3 (myosin-binding protein C3) −/− left ventricular (LV) lysates followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. List of p21-bound cell cycle regulatory proteins detected in Mybpc3 −/− LV tissue samples after proteomic analysis. Representative images from in situ proximity ligation assay (PLA) for ( B ) p21 and PCNA protein complexes, ( C ) p21 and CDK (cyclin-dependent kinase) 1 protein complexes, and ( D ) p21 and CDK2 protein complexes in Ctl and Mybpc3 −/− left ventricular tissue. Protein complexes are red with nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 10 µm. E , Quantification of protein complexes per 100 nuclei in Ctl and Mybpc3 −/− myocardial tissue (n=4). Minimum of 100 nuclei/sample (p21 antibody Millipore, MABE1816). F , Coimmunoprecipitation of p21 from left ventricular tissue lysate from Ctl and Mybpc3 −/− mice and immunoblotting for PCNA and p21 (Santa Cruz Biotechnology [SCBT], sc-6246; upper ). Whole myocardial tissue lysate immunoblotting for PCNA, p21, and β-actin as input ( lower ). G , Representative PLA images and ( H ) quantification of p21 and PCNA protein complexes in Ctl, Mybpc3 −/− Myh6Cre (Tg(Myh6-cre)1Jmk), Cdkn1a (cyclin-dependent kinase inhibitor 1a) −/− Mybpc3 −/− , and Cdkn1a SUPER Mybpc3 −/− Myh6Cre myocardial tissue (n=5). Positive p21 and PCNA complexes are red, with nuclei counterstained with DAPI (blue). Scale bars, 10 µm. Minimum of 100 nuclei/sample. I , Human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) were exposed to either scrambled Ctl small interfering RNA (siRNA) or PCNA targeting siRNA. Quantification of PCNA protein was assessed by immunoblot, and β-actin was used as a loading (Ctl siRNA n=5; PCNA siRNA n=6). J , Representative immunofluorescence staining and ( K ) quantification of Ki67 (Marker Of Proliferation Ki-67) (red) from Ctl and PCNA siRNA knockdown hiPSC-CMs stimulated with or without serum (n=4). Cardiomyocytes (CMs) were identified with sarcomeric α-actinin (green), and nuclei were labeled by DAPI (blue). Scale bars, 50 µm. Minimum 100 nuclei/sample. ANOVA factor: siRNA and serum. L , Representative staining of wheat germ agglutinin (WGA; red) with nuclei labeled by DAPI (blue) from Ctl and PCNA siRNA knockdown hiPSC-CMs stimulated with or without serum. Scale bars, 70 µm. M , Relative quantification of cardiomyocyte DNA content (relative to Ctl siRNA without serum) and ( N ) quantification of cardiomyocyte area in Ctl and PCNA siRNA knockdown hiPSC-CMs stimulated with or without serum (n=4). Minimum 100 CMs/sample. ANOVA factor: siRNA and serum. All results are shown as mean±SEM. The Welch t test is used for E . The Mann-Whitney U test is used for H and I . Two-way ANOVA with the Tukey multiple comparison test is used for K , M , and N . Normality was assumed based on the central limit theorem for E , K , M , and N .

Article Snippet: In addition, Ki67 (proliferation marker protein Ki-67) antibody staining was performed on both myocardial tissue (Ki67 [CST (Cell Signaling Technology), 9449]) or hiPSC-CM (Ki67 [CST, 9129]).

Techniques: Control, Binding Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, In Situ, Proximity Ligation Assay, Staining, Western Blot, Derivative Assay, Small Interfering RNA, Immunofluorescence, Marker, Knockdown, Labeling, Quantitative Proteomics, MANN-WHITNEY, Comparison

Journal: Circulation Research

Article Title: Targeting Cardiomyocyte PCNA and POLD1 Prevents Pathologic Myocardial Hypertrophy

doi: 10.1161/CIRCRESAHA.124.325647

Figure Lengend Snippet: Targeting cardiomyocyte endoreplication pathways can prevent the development of myocardial hypertrophy and diastolic dysfunction in Myh6 (myosin heavy chain 6) R404Q/WT mice. A , Representative click 5-ethynyl-2′-deoxyuridine (EdU; green) and PCM1 (pericentriolar material 1; red) staining with nuclei labeled by 4′,6-diamidino-2-phenylindole (DAPI; blue) from control (Ctl, wild-type C57BL/6) and Myh6 R404Q/WT at postnatal day (P) 10. Mice were injected with EdU for 3 consecutive days (P7–P9) and euthanized at P10. Arrow indicates cardiomyocyte nuclei, and arrowhead indicates noncardiomyocyte nuclei. Scale bars, 25 µm. B , Quantification of EdU-positive cardiomyocytes (CMs) from Ctl (n=6) and Myh6 R404Q/WT (n=5) myocardial tissue. Minimum of 100 nuclei/sample. C , Determination of cardiomyocyte DNA content (relative to Ctl) by measuring DAPI intensity from Ctl and Myh6 R404Q/WT (n=5) mice at P25. Minimum of 100 nuclei/sample. D , Echocardiography assessment of left ventricular posterior wall thickness at end-diastole (LVPWd) from Ctl and Myh6 R404Q/WT mice at P25 (n=5). E , Immunoblot and ( F ) relative quantification of p21 protein expression (relative to Ctl; Santa Cruz Biotechnology [SCBT], sc-6246) from Ctl (n=4) and Myh6 R404Q/WT (n=5) myocardial tissue at P7. β-Actin was used as loading Ctl. G , Representative proximity ligation assay (PLA) images and ( H ) quantification of PCNA and POLD1 (DNA polymerase delta 1) complexes from Ctl (n=4), Myh6 R404Q/WT (n=5), and Cdkn1a (cyclin-dependent kinase inhibitor 1a) −/− Myh6 R404Q/WT (n=5) myocardial tissue at P7. Cardiomyocytes were identified with PCM1 (green). Scale bars, 10 µm. Minimum of 50 nuclei/sample. I , Schematic of cardiomyocyte p21 overexpression strategy using cardiomyocytes-selective promoter (cTnT [cardiac troponin T]) and adeno-associated virus (AAV) 9 in Myh6 R404Q/WT animals. Echocardiography assessment of ( J ) interventricular septal thickness at end-diastole (IVSd), ( K ) left ventricular posterior wall thickness at end-diastole (LVPWd), ( L ) left ventricular internal diameter at end-diastole (LVIDd), ( M ) fractional shortening (FS), ( N ) isovolumic relaxation time (IVRT), and ( O ) early transmitral valve flow velocity to early mitral annulus tissue velocity ratio (E/e’) in Ctl, Myh6 R404Q/WT , Cdkn1a −/− Myh6 R404Q/WT , AAV9-cTnT-GFP (green flourescent protein)–injected Myh6 R404Q/WT , and AAV9-cTnT-Cdkn1a-injected Myh6 R404Q/WT animals at 3 and 8 months (n=4–7). P , Heart weight to tibia length ratio (HW/TL) at 8 months from the groups outlined above (n=5–7). Q , Graphic abstract , created in BioRender. All results are shown as mean±SEM. The unpaired Student t test is used for B through D . The Mann-Whitney U test is used for F . One-way ANOVA with the Tukey multiple comparison test is used for H . Two-way ANOVA with the Tukey multiple comparison test is used for J through P . ANOVA factor: AAV and genotype. Normality was assumed based on the central limit theorem for B , C , and H .

Article Snippet: In addition, Ki67 (proliferation marker protein Ki-67) antibody staining was performed on both myocardial tissue (Ki67 [CST (Cell Signaling Technology), 9449]) or hiPSC-CM (Ki67 [CST, 9129]).

Techniques: Staining, Labeling, Control, Injection, Western Blot, Quantitative Proteomics, Expressing, Proximity Ligation Assay, Over Expression, Virus, MANN-WHITNEY, Comparison