Hexarelin treatment preserves myocardial function and reduces cardiac fibrosis in a mouse model of acute myocardial infarction

Figures

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  Figure 1. Cardiac functional parameters measured by cMRI. Ejection fraction (%), LV end‐diastolic volume, LV end‐systolic volume measured post‐MI/sham procedure with or without HEX treatment. (A). EF 24 h postprocedure, (SHAM‐VEH (n = 5), SHAM‐HEX (5), MI‐VEH (7), MI‐HEX (7)); (B) EF 14 days postprocedure (n = 6/5/10/11); (C) LV EDV 14 days postprocedure (n = 6/5/10/11); (D) LV ESV 14 days postprocedure (n = 6/5/10/11). Data in all figures expressed as mean ± SEM; ***P < 0.001, ****P < 0.0001, versus SHAM‐VEH group; # P < 0.05, ## P < 0.01, versus MI‐VEH group; + P < 0.05, ++++ P< 0.0001, versus SHAM‐HEX group. cMRI, cardiac magnetic resonance imaging; EDV, end diastolic volume; ESV, end systolic volume.

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  Figure 2. Infarct size measured by cMRI. (A). Representative LGE images acquired using a T1‐weighted INTRAGATE gradient‐echo sequence 24 h post LAD ligation. The hyperintense region represents the infarcted tissue (indicated by white arrows); (a). HEX‐treated (b). VEH‐treated. (B) Myocardial infarct size assessed 24 h post‐MI (IV%), (n = 7/7). (C) Myocardial infarct size 14 days post‐MI, infarct size assessed via IV% approach, or (D) via midline‐length approach, (n = 10/10). ## P < 0.01 versus MI‐VEH group. IV%, Infarct volume %.

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  Figure 3. Assessment of LV mass and heart weight. LV myocardial mass (mg) assessed by cMRI after: (A) 24 h, (SHAM‐VEH/HEX (5), MI‐VEH/HEX (7)); (B) 14 days post‐MI/sham procedure with or without HEX treatment, (n = 6/5/10/11). (C) Heart weight compared 21 days postprocedure. Tibial length (TL) was used as an adjustment factor, (n = 6/5/5/6). *P < 0.05, **P < 0.01, ***P < 0.001, versus SHAM‐VEH group; ## P < 0.01, versus MI‐VEH group. TL, tibial length.

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  Figure 4. Histology and morphometric analysis. (A) Interstitial LV collagen content measured by picrosirus red staining, (n = 6/5/6/7); (B) LV collagen concentration, measured by hydroxyproline analysis, (n = 6/5/4/7); (C) TGF‐β1 (6/5/5/6), (D) α‐SMA (5/6/5/7), and (E) MMP‐13 immunohistochemistry staining (n = 6/5/5/6) measured 21 days post‐MI/sham procedure with or without HEX treatment. The right column displays representative sections from each treatment group corresponding to the selected markers and methods described above. *P < 0.05, ***P < 0.001, ****P < 0.0001, versus SHAM‐VEH group; ## P < 0.01, #### P < 0.0001, versus MI‐VEH group; + P < 0.05, ++ P < 0.05; versus SHAM‐HEX group.

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  Figure 5. Serum assessment of: (A) Troponin‐I (pg/mL) (n = 4/4/5/5); (B) TNF‐α (pg/mL) (n = 4/4/4/4); (C) IL‐1β (pg/mL) (n = 3/3/4/5); (D) IL‐6 (pg/mL) (n = 4/4/5/4). Serum levels assessed 24 h post‐MI/sham procedure with or without HEX treatment. *P < 0.05, **P < 0.01, versus SHAM‐VEH group; # P < 0.05, versus MI‐VEH group.

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  Figure 6. Heart rate variability analysis. Electrocardiographic data recorded 21 days post‐MI/sham procedure with or without HEX treatment. (A) Sympathetic nerve activity represented by low‐frequency power (LF/HFr), (n = 4/5/4/8). (B) Parasympathetic nerve activity represented by high‐frequency power (nHFr), (n = 4/5/4/8). *P < 0.05, versus SHAM‐VEH group; ## P < 0.01, versus MI‐VEH group. HF, high frequency; LF, low frequency; nHF, normalized high‐frequency power.

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  Figure 7. Hexarelin: proposed mechanisms of action (see text). MI induces dynamic alterations in fibroblast phenotype, initiates an inflammatory response and activates neurohormonal pathways that collectively result in cardiac remodeling, mechanical dysfunction, and electrical disturbances in the failing heart, increasing the risk of sudden cardiac death (Dixon et al. 2015). TFG‐β1 is markedly augmented following MI and is the most potent profibrotic cytokine known, causing excessive extracellular matrix production, inducing its own secretion and driving myofibroblast activation (Dixon et al. 2015). Inflammatory cytokines exert potent proinflammatory actions on cardiac fibroblasts and can negatively affect LV function. Similarly, the RAAS & SNA play a critical role in activation of myofibroblasts and influence LV remodeling & dysfunction (Sun and Weber 2000; Dixon et al. 2015). HEX may attenuate and actively reverse cardiac remodeling and myocardial fibrosis by targeting these pathways (indicated by “x”) and thus holds promise in returning the failing heart to a functional state. RAAS, renin angiotensin aldosterone system; SNA, sympathetic nervous activation.