Effect of FADD^−/− upon cardiac function as determined by echocardiography.

<p>(A) Representative echocardiographic recordings pre- and post-24 hours and 7 days of reperfusion. (B) and (C) Graphic summary of LV ejection fraction (LVEF) and LV fractional shortening (LVFS) in groups (n = 10–14 mice/group). *<i>P</i><0.05, **<i>P</i><0.01 FADD^−/− vs. NLC control (FADD∶GFP MHC-Cre⁻).</p

Azithromycin therapy reduces cardiac inflammation and mitigates adverse cardiac remodeling after myocardial infarction: Potential therapeutic targets in ischemic heart disease

<div><p>Introduction</p><p>Acute myocardial infarction (MI) is a primary cause of worldwide morbidity and mortality. Macrophages are fundamental components of post-MI inflammation. Pro-inflammatory macrophages can lead to adverse cardiac remodeling and heart failure while anti-inflammatory/reparative macrophages enhance tissue healing. Shifting the balance between pro-inflammatory and reparative macrophages post-MI is a novel therapeutic strategy. Azithromycin (AZM), a commonly used macrolide antibiotic, polarizes macrophages towards the anti-inflammatory phenotype, as shown in animal and human studies. We hypothesized that AZM modulates post-MI inflammation and improves cardiac recovery.</p><p>Methods and results</p><p>Male WT mice (C57BL/6, 6–8 weeks old) were treated with either oral AZM (160 mg/kg/day) or vehicle (control) starting 3 days prior to MI and continued to day 7 post-MI. We observed a significant reduction in mortality with AZM therapy. AZM-treated mice showed a significant decrease in pro-inflammatory (CD45⁺/Ly6G^-/F4-80⁺/CD86⁺) and increase in anti-inflammatory (CD45⁺/Ly6G^-/F4-80⁺/CD206⁺) macrophages, decreasing the pro-inflammatory/anti-inflammatory macrophage ratio in the heart and peripheral blood as assessed by flow cytometry and immunohistochemistry. Macrophage changes were associated with a significant decline in pro- and increase in anti-inflammatory cytokines. Mechanistic studies confirmed the ability of AZM to shift macrophage response towards an anti-inflammatory state under hypoxia/reperfusion stress. Additionally, AZM treatment was associated with a distinct decrease in neutrophil count due to apoptosis, a known signal for shifting macrophages towards the anti-inflammatory phenotype. Finally, AZM treatment improved cardiac recovery, scar size, and angiogenesis.</p><p>Conclusion</p><p>Azithromycin plays a cardioprotective role in the early phase post-MI through attenuating inflammation and enhancing cardiac recovery. Post-MI treatment and human translational studies are warranted to examine the therapeutic applications of AZM.</p></div

Effect of FADD^−/− on cardiac infarct size, cardiac remodeling and survival after 6 weeks MI.

<p>(A) representative TTC stained heart tissue section at 6 week post-MI in FADD^−/− and NLC control groups. (B) Graphic summary of infarct size expressed as the length of the scare/LV circumference, n = 8 animals in each group. (C) Graphic presentation of LV area in NLC and FADD^−/− groups, <i>P</i><0.05 FADD^−/− vs. NLC, n = 8 in each groups. (D) Graphic presentation of LVIDd measured by echocardiography. <i>P</i><0.05 FADD^−/− vs. NLC, n = 8 in each groups. (E) Graphic presentation the ratio of heart length (HL)/tibia length (TL) in sham or MI mice. <i>P</i><0.05 FADD^−/− vs. NLC, n = 8 in each groups. (F) Survival curve in 8 week post-sham (n = 8 in each group) or post-MI mice (n = 23 in each group). *<i>P</i><0.05, FADD^−/− vs. NLC control.</p

Effect of FADD^−/− upon cardiac function as determined by ventricular catheterization, spanning pre- to 7 days post-reperfusion.

<p>(A) Left ventricular systolic pressure (LVSP). (B) Left ventricular end diastolic pressure (LVEDP). (C) Rate of rise of left ventricular pressure (+dP/dt) and (D) Rate of reduction of left ventricular pressure (−dP/dt). n = 10–14 mice/group. *<i>P</i><0.05 FADD^−/− vs. NLC control.</p

Time course of FADD expression in each group post-myocardial ischemia/reperfusion (I/R).

<p>(A) Top: Representative photomicrographs of FADD expression in cardiac tissue by western blot in WT C57/BL6 (lanes 1–2) and FADD^+/−-FADD∶GFP-MHC-Cre⁻ (lanes 3–4) control mice, FADD^−/−-FADD∶GFP-MHC-Cre⁻ (NLC, line 5–6) and FADD^−/−-FADD∶GFP-MHC-Cre⁺ (FADD^−/−, line 7–8) mice. Bottom: Ratio of FADD expression (FADD or FADD+FADD∶GFP to GAPDH). (B) Top: Representative photomicrographs of FADD∶GFP expression in cardiac tissue pre- and post-MI/R by western blot in FADD^−/−-FADD∶GFP-MHC-Cre⁻ (NLC) and FADD^−/−-FADD∶GFP-MHC-Cre⁺ (FADD^−/−) groups. Bottom: Ratio of FADD expression (FADD∶GFP to GAPDH). n = 4. **<i>P</i><0.01 vs. FADD^−/−-FADD∶GFP-MHC-Cre⁻ (NLC). ^#<i>P</i><0.05 vs pre-I/R condition in NLC and FADD^−/− groups respectively, n = 4.</p

FADD^−/− ameliorates post-I/R myocardial infarct size.

<p>(A) Representative photograph of TTC stained heart tissue section obtained 24 hours I/R in FADD^−/− and NLC groups. (B) Graphic summery of infarct size expressed as percentage of area-at-risk (AAR) and the size of AAR. n = 8–10 mice/group. **<i>P</i><0.01, FADD^−/− vs. NLC control.</p

AZM treatment exerts immunomodulatory effects on cytokines expression following MI.

<p>mRNA expression of pro-inflammatory cytokines in HT (Panel A) and PB (Panel B), demonstrate significant reduction in gene expression of these cytokines in the early inflammatory phase following injury with AZM therapy compared to vehicle controls (red line demarcates the level of gene expression in sham operated mice). The mRNA expression of anti-inflammatory cytokines is augmented with AZM therapy compared to vehicle control (n = 4 mice/group/time point, *P<0.05, **P<0.01, ***P<0.001 and **** P<0.0001 compared to vehicle controls). Data presented as mean ± SEM. AZM, azithromycin; HT, heart; IL-1β, interleukin 1 beta; IL-6, interleukin 6; IL-4, interleukin 4; iNOS, inducible nitric oxide synthase; MCP-1, monocyte chemoattractant protein-1; PB, peripheral blood; PPARγ, peroxisome proliferator-activated receptor gamma; TGF-1β, tissue growth factor 1 beta; TNF-α, tumor necrosis factor-alpha; YM1 (Chil3), chitinase-like 3.</p

Time course of post-MI (A) TNFα and (B) LTα production.

<p>N = 14–16/group. *P<0.05, **P<0.01 vs. control.</p

Restoring diabetes-induced autophagic flux arrest in ischemic/reperfused heart by ADIPOR (adiponectin receptor) activation involves both AMPK-dependent and AMPK-independent signaling

<p>Macroautophagy/autophagy is increasingly recognized as an important regulator of myocardial ischemia-reperfusion (MI-R) injury. However, whether and how diabetes may alter autophagy in response to MI-R remains unknown. Deficiency of ADIPOQ, a cardioprotective molecule, markedly increases MI-R injury. However, the role of diabetic hypoadiponectinemia in cardiac autophagy alteration after MI-R is unclear. Utilizing normal control (NC), high-fat-diet-induced diabetes, and <i>Adipoq</i> knockout (<i>adipoq</i>^−/−) mice, we demonstrated that autophagosome formation was modestly inhibited and autophagosome clearance was markedly impaired in the diabetic heart subjected to MI-R. <i>adipoq</i>^−/− largely reproduced the phenotypic alterations observed in the ischemic-reperfused diabetic heart. Treatment of diabetic and <i>adipoq</i>^−/− mice with AdipoRon, a novel ADIPOR (adiponectin receptor) agonist, stimulated autophagosome formation, markedly increased autophagosome clearance, reduced infarct size, and improved cardiac function (P < 0.01 vs vehicle). Mechanistically, AdipoRon caused significant phosphorylation of AMPK-BECN1 (Ser93/Thr119)-class III PtdIns3K (Ser164) and enhanced lysosome protein LAMP2 expression both in vivo and in isolated adult cardiomyocytes. Pharmacological AMPK inhibition or genetic <i>Prkaa2</i> mutation abolished AdipoRon-induced BECN1 (Ser93/Thr119)-PtdIns3K (Ser164) phosphorylation and AdipoRon-stimulated autophagosome formation. However, AdipoRon-induced LAMP2 expression, AdipoRon-stimulated autophagosome clearance, and AdipoRon-suppressed superoxide generation were not affected by AMPK inhibition. Treatment with MnTMPyP (a superoxide scavenger) increased LAMP2 expression and stimulated autophagosome clearance in simulated ischemic-reperfused cardiomyocytes. However, no additive effect between AdipoRon and MnTMPyP was observed. Collectively, these results demonstrate that hypoadiponectinemia impairs autophagic flux, contributing to enhanced MI-R injury in the diabetic state. ADIPOR activation restores AMPK-mediated autophagosome formation and antioxidant-mediated autophagosome clearance, representing a novel intervention effective against MI-R injury in diabetic conditions.</p