Cardiac Overexpression of S100A6 Attenuates Cardiomyocyte Apoptosis and Reduces Infarct Size After Myocardial Ischemia‐Reperfusion

Figures

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  Figure 1. Localization and confirmation of in vitro transduction. Immunocytochemical staining of (A) exogenous GFP‐tagged human S100A6 (green) in adenoviral‐transduced neonatal cardiomyocytes showing perinuclear and cytoplasmic localization (magnification ×60) and (B) endogenous rat S100A6 (green) in neonatal cardiomyocytes showing cytoplasmic and nuclear localization (magnification ×60). C, Staining of endogenous S100A6 (top panel—green), S100A6‐His fusion protein (middle panel—red) after cytoplasmic uptake of exogenously added S100A6 in the presence of 2 mmol/L Ca2+ after 1 hour, and colocalization (both cytoplasmic and nuclear) with endogenous S100A6 (bottom panel) (magnification ×20). D, Expression of rat‐S100A6, rat‐S100B, rat‐S100A1 and exogenous human/rat‐S100A6 mRNA in S100A6‐overexpressing and S100A6‐knockdown neonatal cardiomyocytes by qRT‐PCR. Data expressed as mean±SEM, *P<0.05 vs nontransduced controls, N=7 for each. Overexpression and downregulation of S100A6 did not affect the expression of S100A1 or S100B in vitro. E, Western blot in S100A6‐overexpressing and S100A6‐knockdown cardiomyocytes compared to null‐plasmid– and nontransduced controls. DAPI,…

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  Figure 2. Calcium transients and calcineurin (CaN) activity in vitro. A, Representative calcium transient imaging for nontransduced control, S100A6‐knockdown (KD) and S100A6‐overexpressing (OE) cardiomyocytes. B, The calcium release amplitude, the rate of calcium release and uptake in S100A6‐overexpressing and S100A6‐knockdown neonatal cardiomyocytes compared to nontransduced controls during spontaneous action potentials (mean±SEM, *P<0.05 vs nontransduced controls, N=7 for each). C, CaN phosphatase activity during normoxia and after H/R (24/6 hours) in S100A6‐overexpressing and S100A6‐knockdown neonatal cardiomyocytes compared to null‐transduced and nontransduced controls and relative expression of CaN A and CaN B in S100A6‐overexpressing, S100A6‐knockdown, null‐transduced, and nontransduced cardiomyocytes during normoxia and hypoxia/reoxygenation (mean±SEM, *P<0.05 vs null‐transduced and nontransduced controls, N=7 for each).

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  Figure 3. Effect of S100A6 on apoptosis after hypoxia/reoxygenation in vitro. S100A6 overexpression prevented hypoxia/reoxygenation (24/6 hours)‐induced apoptosis in vitro, whereas S100A6‐downregulated cells showed greater apoptosis under normal conditions and after hypoxia/reoxygenation (24/6 hours). A, TUNEL staining in (i) nontransduced controls, (ii) S100A6‐overexpressing, and (iii) S100A6‐knockdown neonatal cardiomyocytes. Nuclei are stained by DAPI (blue) and apoptotic cells are rhodamine positive (red), ×60. B, Rhodamine‐positive apoptotic neonatal cardiomyocytes detected by TUNEL staining in S100A6‐overexpressing and S100A6‐knockdown neonatal cardiomyocytes compared to null‐transduced and nontransduced cells (mean±SEM, *P<0.05 vs null‐transduced controls, N=10 for each). C, Caspase 3/7 activity in S100A6‐overexpressing and S100A6‐knockdown neonatal cardiomyocytes compared to null‐transduced and nontransduced control cells after hypoxia/reoxygenation (24/6 hours) (mean±SEM, *P<0.05 vs null‐transduced controls, N=10 for each). DAPI, 4ʹ,6‐diamidino‐2‐phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

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  Figure 4. Effect of UTMD of S100A6 on myocardial ischemia‐reperfusion. Exogenous human S100A6 gene expression by q RT‐PCR in various regions of myocardium (anterior LV, posterior LV, and RV) at (A) day 1, (B) day 3, and (C) day 28 post–ischemia/reperfusion (I/R) normalized to normal nonischemic control myocardium (mean±SEM, *P<0.05 vs all other groups, N=7 per group). D, Mortality rate during/after myocardial I/R in UTMD human‐S100A6 plasmid‐pretreated and UTMD empty‐plasmid‐pretreated rats compared to nontreated controls, N=29 per group. E, Representative M‐mode echo image for (i) nontreated control, (ii) empty‐plasmid‐treated control, and (iii) UTMD S100A6‐plasmid‐treated. LV systolic function parameters ([F] ejection fraction and [G] fractional area change) following I/R in human‐S100A6 plasmid‐treated and empty plasmid‐treated rats compared to nontreated controls (mean±SEM, *P<0.05 vs nontreated and empty plasmid‐treated controls, N=14‐20 per group). H, Circumferential extent of LV akinesis following I/R in human‐S100A6 plasmid‐treated and empty plasmid‐treated rats compared to nontreated control rats (mean±SEM, *P<0.05 vs non‐treated and empty plasmid‐treated controls, N=14‐20 per…

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  Figure 5. Cardiac hypertrophy data post–myocardial ischemia‐reperfusion. A, Ratio of heart weight to tibia length at day 28 post‐I/R (mean±SEM, *P<0.05 vs nontreated and empty plasmid–treated controls, N=14‐20 in each group). B, Ratio of heart weight to body weight at day 28 post‐I/R (mean±SEM, *P<0.05 vs nontreated and empty plasmid–treated controls, N=14‐20 in each group). C, Representative H&E‐stained images of noninfarct sections of LV myocardium at day 28 post‐I/R in (i) control, (ii) empty plasmid, and (iii) S100A6 plasmid delivered rats. D, Cardiomyocyte size at day 28 post‐I/R (magnification ×100) (mean±SEM, *P<0.05 vs nontreated and empty plasmid–treated controls, N=50‐60 cells in each group). E, Relative expression of βMHC in the infarct and noninfarct regions of myocardium 28 days post‐I/R (mean±SEM, *P<0.05 vs nontreated and empty plasmid–treated controls, N=7 for all groups). F, Relative expression of ANF in the infarct and noninfarct regions of myocardium 28 days post‐I/R (mean±SEM, *P<0.05 vs nontreated and empty plasmid–treated controls, N=7 for all groups). ANF, atrial natriuretic factor; βMHC, β‐myosin heavy chain; H&E, hematoxylin and eosin; I/R, ischemia‐reperfusion.

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  Figure 6. Infarct size and apoptosis data post–myocardial ischemia‐reperfusion. A, Representative Evans blue–stained myocardial sections during ligation of the left anterior descending coronary artery, outlining the area at risk in nontreated controls, empty plasmid–treated controls, and human‐S100A6 plasmid–treated rats. B, Representative TTC‐stained myocardial cryosections 1 day post‐I/R in nontreated controls, empty plasmid–treated controls, and human‐S100A6 plasmid–treated rats. The nonviable/infarcted myocardium is white, and viable myocardium is red/pink. C, Representative MTC‐stained myocardial cryosections 28 days post‐I/R in nontreated controls, empty plasmid–treated controls, and human‐S100A6 plasmid–treated rats. The collagen debris in the central compact necrotic region of the infarcted area is shown in blue, myocardial fibers in pink, and nuclei in purple. D, Risk area by Evans blue as percentage of the LV (mean±SEM, N=6 per group). E, Nonviable myocardium/infarct area by TTC staining as percentage of the LV (mean±SEM, *P<0.05 vs nontreated and empty plasmid–treated controls, N=9 per group). F, Infarct area/scar size by MTC staining as a percentage of the LV (mean±SEM, *P<0.05…