Activation of Toll‐like receptor 7 provides cardioprotection in septic cardiomyopathy‐induced systolic dysfunction

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  FIGURE 1. TLR7 expression was upregulated in response to LPS in vivo and in vitro. C57BL/6J mice (n = 24) were subjected to LPS (10 mg/kg) or saline for 24 hours. A, Representative images of western blot and statistical analysis of TLR7 expression in myocardial tissue (n = 6). P = .0008, analyzed by t‐test. B, qPCR analysis of TLR7 mRNA level in myocardial tissue (n = 4). Normalized to GAPDH. P < .0001, analyzed by t‐test. C, Representative images of western blot and statistical analysis of TLR7 expression in isolated AMCMs (n = 6). P = .0357, analyzed by t‐test. D, qPCR analysis of TLR7 mRNA level in isolated AMCMs (n = 4). Normalized to β‐Actin. P = .0237, analyzed by t‐test. E, Immunofluorescence double‐labeling with TLR7 and α‐actin after NRVMs were stimulated with LPS (10 µg/mL) or PBS for 24 hours (n = 16‐25cells). Scale bar: 20 µm. F, Representative images of western blot and statistical analysis of TLR7 expression in NRVMs in response to LPS or PBS (n = 6). P = .0007, analyzed by t‐test. G, qPCR analysis of TLR7 mRNA level in NRVMs in response to LPS or PBS (n = 4). P = .0083, analyzed by t‐test. The data are shown as the mean ± SEM, with each data point representing a sample of…

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  FIGURE 2. TLR7 deficiency aggravated cardiac dysfunction in septic cardiomyopathy. C57BL/6J WT and TLR7−/− mice were subjected to intraperitoneal injection of LPS (10 mg/kg) or saline. A, Protocol. B, Representative M‐Mode images of the studied groups after LPS administrated for 24 hours (n = 8‐12). White line indicated LVIDs. C, Arrangement of cardiomyocytes was assessed by hematoxylin‐eosin (HE) staining when LPS stimulated for 24 hours (n = 3‐4). Scale bar: 10 µm. D‐G, Echocardiographic examination was used to evaluate cardiac function when LPS administrated for 6, 12, and 24 hours (n = 8‐10). D, Left ventricular ejection fraction (LVEF), P = .0072 at 12 hours and P = .0248 at 24 hours (vs WT‐LPS group), analyzed by t‐test. E, Left ventricle fractional shortening (LVFS), P = .0006 at 6 hours, P = .0316 at 12 hours and P = .0248 at 24 hours (vs WT‐LPS group), analyzed by t‐test. F, Left ventricular internal diameter at systole (LVIDs), P = .0069 at 12 hours and P = .0231 at 24 hours (vs WT‐LPS group), analyzed by t‐test, and G, interventricular septal systolic thickness (IVSs) were calculated from echocardiography. P = .0329 at 12 hours and P = .0274 at 24 hours (vs WT‐LPS group),…

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  FIGURE 3. Myocyte size, cardiac inflammation, and apoptosis were unchanged in TLR7 deficiency mice. AMCMs were isolated from WT and TLR7−/− mice subjected to disposable intraperitoneal injection of LPS (10 mg/kg) for 24 hours. A, Representative morphology of AMCMs, and cell length and width were detected (n = 20‐30 cells). Scale bar: 10 µm. P = .8274 in length and P = .9274 in width, analyzed by one‐way ANOVA with the Bonferroni post‐hoc test. B, Representative images of immunohistochemistry and quantification of CD68 (n = 27‐35 from 3 mice). Scale bar: 120 µm. P = .7189, analyzed by one‐way ANOVA with the Bonferroni post‐hoc test. C, Representative images of TUNEL (TdT mediated dUTP Nick End Labeling) and statistical analysis, and the black arrow indicated positive cell (n = 24‐31 from three mice). Scale bar: 50 µm. P = .8817, analyzed by one‐way ANOVA with the Bonferroni post‐hoc test. The data are shown as the mean ± SEM, with each data point representing a cell or a mouse Abbreviation: ns, no significance.

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  FIGURE 4. TLR7 deficiency exacerbated sepsis‐induced Ca2+ handling disorder in cardiomyocytes. Cardiomyocytes isolated from WT and TLR7−/− mice administrated with LPS or saline were incubated with Fluo‐4 AM to detected Ca2+ handling. A, Protocol. B, Representative Ca2+ transient curve and (lower panel) Fluo‐4 fluorescence of Ca2+ transient peak (n = 10‐14). Scale bar: 20 µm. C, Ca2+‐transient amplitudes quantification of AMCMs in indicated groups (n = 10‐14). P = .0007 (vs NS‐WT) and P = .0052 (vs LPS‐WT), analyzed by one‐way ANOVA with the Bonferroni post‐hoc test. F = 30.72. D, Decay of Ca2+ transient was quantified (n = 15‐20). E‐G, the level of Ca2+ in the sarcoplasmic reticulum was detected by pretreated with caffeine (10 mM). P = .0004 (vs NS‐WT) and P = .0009 (vs LPS‐WT), analyzed by one‐way ANOVA with the Bonferroni post‐hoc test. F = 79.91. E, Fluo‐4 fluorescence of caffeine‐induced Ca2+ transient peak, Scale bar: 20 µm. F, Typical curve, and G, quantification of the caffeine‐induced Ca2+ transient peak in indicated groups (n = 10‐15). P = .0002, analyzed by t‐test. The data are shown as the mean ± SEM, with each data point representing an adult mice cardiomyocyte *P < .05. **P <…

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  FIGURE 5. TLR7 knockout aggravated sepsis‐induced downregulated RyR2 and Serca, and reduced Serca activity by phospholamban dephosphorylation. After LPS stimulated for 24 hours, cardiac tissue and AMCMs were harvested from WT and TLR7−/− mice. A, Representative images of western blot and statistical analysis of RyR2 expression in cardiac tissue (n = 6), P = .0225 (vs NS‐WT) and P = .0005 (vs LPS‐WT), analyzed by two‐way ANOVA with the Bonferroni post‐hoc test. F = 12.38, and B, qPCR analysis of RyR2 mRNA level in cardiac tissue (n = 4), P = .0411 (vs NS‐WT) and P = .0072 (vs LPS‐WT), analyzed by two‐way ANOVA with the Bonferroni post‐hoc test. F = 8.597. Normalized to GAPDH. C, Representative tracing of sarcomere shortening in AMCMs isolated from indicated mice, and D, accumulated data of sarcomere shortening (n = 10‐15 cells), P = .0095 (vs NS‐WT) and P = .0325 (vs LPS‐WT), analyzed by one‐way ANOVA with the Bonferroni post‐hoc test. F = 22.24. E, Ca2+ uptake activity of Serca in sarcoplasmic reticulum was analyzed by incubating with Fura 2 (n = 8‐12), P = .0052 (vs NS‐WT) and P = .0077 (vs LPS‐WT), analyzed by one‐way ANOVA with the Bonferroni post‐hoc test. F = 17.87, and F, ATPase…

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  FIGURE 6. Effect of TLR7 on Ca2+ handling in cultured neonatal rat ventricular myocytes. Neonatal rat ventricular myocytes (NRVMs) transfected with Ad‐TLR7 or Ad‐LacZ were stimulated with LPS (10 µg/mL) or PBS for 24 hours. A, Protocol showing the method of neonatal rat ventricular myocytes (NRVMs) culture. B, Representative images of western blot and statistical analysis of RyR2 expression (n = 6), P = .0276 (vs PBS‐Ad‐LacZ) and P = .0269 (vs LPS‐Ad‐LacZ), analyzed by two‐way ANOVA with the Bonferroni post‐hoc test. F = 7.385. C, Representative images of western blot and statistical analysis of Serca expression (n = 6), P = .0382 (vs PBS‐Ad‐LacZ) and P = .0274 (vs LPS‐Ad‐LacZ), analyzed by two‐way ANOVA with the Bonferroni post‐hoc test. F = 8.798. D, qPCR analysis of mRNA level for the gene coding for Serca (ATP2A2) and RyR2 (n = 3‐4). Normalized to β‐Actin. For RyR2 mRNA, P = .0064 (vs PBS‐Ad‐LacZ) and P = .0092 (vs LPS‐Ad‐LacZ), analyzed by one‐way ANOVA with the Bonferroni post‐hoc test. F = 8.527. For SERCA mRNA, P = .0037 (vs PBS‐Ad‐LacZ) and P = .031 (vs LPS‐Ad‐LacZ), analyzed by one‐way ANOVA with the Bonferroni post‐hoc test. F = 6.725. E, Typical Ca2+ transient curve and (lower…

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  FIGURE 7. Cardiac‐specific overexpression of TLR7 alleviated sepsis‐induced cardiac dysfunction. A, Protocol and schematics of TLR7 transgenic construct. B, Representative M‐Mode images of the studied groups after LPS administrated for 24h (n = 10‐12). C, Representative HE‐stained heart sections from littermate WT (LWT) and cardiac‐specific overexpression TLR7 gene (cTG‐TLR7) mice pretreated with or without LPS stimulated for 24 hours (n = 3‐4). Scale bar: 50 µm. D‐F, Echocardiographic examination was used to evaluate cardiac function when LPS administrated for 24 hours (n = 10‐12). D, Left ventricular ejection fraction, P < .0001 (vs NS‐LWT) and P = .0041 (vs LPS‐LWT), analyzed by two‐way ANOVA with the Bonferroni post‐hoc test. F = 11.35, E, left ventricle fractional shortening (LVFS), P < .0001 (vs NS‐LWT) and P = .0067 (vs LPS‐LWT), analyzed by two‐way ANOVA with the Bonferroni post‐hoc test. F = 12.02, and F, left ventricular end systolic diameter (LVIDs) were calculated from echocardiography, P < .0001 (vs NS‐LWT) and P = .0074 (vs LPS‐LWT), analyzed by two‐way ANOVA with the Bonferroni post‐hoc test. F = 17.76. G‐I, Pressure‐volume (PV) loop measurements were recorded when LPS…

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  FIGURE 8. Inhibition of PKA hindered TLR7's cardioprotective effects in response to sepsis. A, Protocol. B, Typical Ca2+ transient curve and (lower panel) Fluo‐4 fluorescence of Ca2+ transient peak (n = 10‐14). Scale bar: 20 µm. C, Ca2+‐transient amplitudes quantification in AMCMs from LWT and cTG‐TLR7 mice in indicated groups (n = 10‐15), P = .0007 (vs NS‐LWT‐DMSO), P = .0328 (vs LPS‐LWT‐DMSO) and P = .0078 (vs LPS‐cTG‐DMSO) analyzed by one‐way ANOVA with the Bonferroni post‐hoc test. F = 21.03. D, Decay of Ca2+ transient was quantified (n = 10‐15), P = .0004 (vs NS‐LWT‐DMSO), P = .0084 (vs LPS‐LWT‐DMSO) and P = .0388 (vs LPS‐cTG‐DMSO) analyzed by one‐way ANOVA with the Bonferroni post‐hoc test. F = 36.73. E, Representative tracing of sarcomere shortening from in AMCMs isolated from indicated mice, and F, accumulated data of sarcomere shortening (n = 9‐12), P = .0065 (vs NS‐LWT‐DMSO), P = .0157 (vs LPS‐LWT‐DMSO) and P = .0205 (vs LPS‐cTG‐DMSO) analyzed by one‐way ANOVA with the Bonferroni post‐hoc test. F = 33.84. G‐I, The level of Ca2+ in the sarcoplasmic reticulum was detected by pretreated with caffeine (10 mM). G, Fluo‐4 fluorescence of caffeine‐induced Ca2+ transient peak, H, typical…