Irisin Preserves Cardiac Performance and Insulin Sensitivity in Response to Hemorrhage

Abstract

Irisin, a cleaved product of the fibronectin type III domain containing protein-5, is produced in the muscle tissue, which plays an important role in modulating insulin resistance. However, it remains unknown if irisin provides a protective effect against the detrimental outcomes of hemorrhage. Hemorrhages were simulated in male CD-1 mice to achieve a mean arterial blood pressure of 35-45 mmHg, followed by resuscitation. Irisin (50 ng/kg) and the vehicle (saline) were administrated at the start of resuscitation. Cardiac function was assessed by echocardiography, and hemodynamics were measured through femoral artery catheterization. A glucose tolerance test was used to evaluate insulin sensitivity. An enzyme-linked immunosorbent assay was performed to detect inflammatory factors in the muscles and blood serum. Western blot was carried out to assess the irisin production in skeletal muscles. Histological analyses were used to determine tissue damage and active-caspase 3 apoptotic signals. The hemorrhage suppressed cardiac performance, as indicated by a reduced ejection fraction and fractional shortening, which was accompanied by enhanced insulin resistance and hyperinsulinemia. Furthermore, the hemorrhage resulted in a marked decrease in irisin and an increase in the production of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1). Additionally, the hemorrhage caused marked edema, inflammatory cell infiltration and active-caspase 3 positive signals in skeletal muscles and cardiac muscles. Irisin treatment led to a significant improvement in the cardiac function of animals exposed to a hemorrhage. In addition, irisin treatment improved insulin sensitivity, which is consistent with the suppressed inflammatory cytokine secretion elicited by hemorrhages. Furthermore, hemorrhage-induced tissue edema, inflammatory cell infiltration, and active-caspase 3 positive signaling were attenuated by irisin treatment. The results suggest that irisin protects against damage from a hemorrhage through the modulation of insulin sensitivity.

Keywords: hemorrhage; inflammation; insulin resistance; irisin; myocardial function.

Figure 1

Time course and experimental protocol for the hemorrhagic shock model. (A) BP of mice were maintained at 35-45 mmHg of mean arterial blood pressure (MAP) for one hour before fluid resuscitation with lactated ringer’s solution (LRS) in hemorrhagic shock groups. Irisin treatment was conducted simultaneously with resuscitation. The sham groups underwent operation without blood withdrawal; (B) MAP of animals while in hemorrhagic shock (Data are shown as Mean ± SEM, n = 4/each group); (C) The time course of MAP during the hemorrhage.

Figure 2

Echocardiographic measurements of cardiac function in hemorrhagic mice; Left ventricular (LV) function was assessed by two-dimensional M-mode echocardiography in sham and hemorrhage groups 2 h after sham operation and resuscitation. Bar graphs for (A) Ejection Fraction (EF); (B) Fractional Shortening (FS); (C) LV Internal Dimension in diastole (LVIDd); (D) LV Internal Dimension in systole LVIDs; (E) Left Ventricular Posterior Wall in diastole (LVPWd); (F) LV Posterior Wall in systole (LVPWs); and (G) Heart Rate (HR); (H) A representative image for the two dimensional and M-mode (Data are shown as Mean ± SEM, n = 4/each group).

Figure 3

Glucose tolerance test and insulin measurement. Mice fasted 6 h before glucose tolerance test. (A) Time dependent glycemic levels post glucose injection (1.5 g/kg body weight of glucose was administered intraperitoneally. Data are shown as Mean ± SEM, n = 5/each group, *** p < 0.001); (B) A bar graph for blood glucose area under the curve analysis from GTT (Data are shown as Mean ± SEM, n = 5/each group, *** p < 0.001); (C) The measurement of insulin level in serum among the groups (Data are shown as Mean ± SEM, n = 5/each group, *** p < 0.001).

Figure 4

ELISA analysis of IL-1 and TNF-α in serum and skeletal muscle. Bar graphs for (A) Levels of IL-1 in skeletal muscle; (B) Serum levels of IL-1; (C) Levels of TNF-α in skeletal muscle; (D) Serum levels of TNF-α. (data are shown as Mean ± SEM, n = 4/each group); (E) Immunostaining showing that irisin signals were attenuated by hemorrhage, (Hemo: hemorrhage, Scale bar: 50µm); (F) Immunoblot of irisin and β actin in skeletal muscle of mice exposed to hemorrhage; (G) Densitometric analysis of irisin in hemorrhage and sham groups. Results were expressed as the percentage of sham group (Data are shown as Mean ± SEM, n = 5/each group).

Figure 5

Histological analysis of injury in skeletal muscle, myocardium, and lung. (A) Skeletal muscle histology (H&E staining) of sham operated and hemorrhagic groups (200×, scale bars indicate 100 μm); (B) A bar graph for data of inflammatory cell infiltration in skeletal muscle (Data are shown as Mean ± SEM, n = 4/each group); (C) Cardiomyocyte histology (H&E staining) of sham operated and hemorrhagic groups (200×, scale bars indicate 100 μm); (D) A bar graph for data of neutrophilic infiltration in cardiac muscle (Data are shown as Mean ± SEM, n = 4/each group); (E) Histopathological images by H&E staining showed pathologic tissue damage in lungs from sham and hemorrhagic groups (H&E staining, original magnification, 400×, scale bars indicate 50 μm). Alveolar septal thickness was used to assess the extent of lung injury; (F) A bar graph for data of alveolar septal thickness of the lung (Data are shown as Mean ± SEM, n = 4/each group). Hemo: Hemorrhage.

Figure 6

Immunostaining of active-caspase 3 signals in the skeletal muscle and cardiac muscle. (A) Immunohistochemical staining of caspase-3 in skeletal muscle (original magnification, 400×); (B) A bar graph for statistical data of positive staining of capase-3 from A (Data are shown as Mean ± SEM, n = 4/each group); (C) Immunohistochemical staining of caspase-3 in cardiac muscle (original magnification, 400×); (D) A bar graph for statistical data of positive staining of capase-3 from C; (E) Immunohistochemical staining of caspase-3 in lungs (original magnification, 400×); (F) A bar graph for statistical data of positive staining of capase-3 from (E) (Data are shown as Mean ± SEM, n = 4/each group). Hemo: Hemorrhage, Scale bar = 50 µm.

Figure 7

(A) Immunohistochemical staining of superoxide dismutase (SOD) in skeletal muscles; (B) A bar graph for statistical data of SOD intensity from A (data are shown as Mean ± SEM, n = 4/each group). Hemo: Hemorrhage, Scale bar: 50 µm.