Metformin ameliorates gender-and age-dependent hemodynamic instability and myocardial injury in murine hemorrhagic shock

Abstract

Severity of multiple organ failure is significantly impacted by age and gender in patients with hemorrhagic shock. However, the molecular mechanisms underlying the enhanced organ injury are not fully understood. AMP-activated protein kinase (AMPK) is a pivotal orchestrator of metabolic responses during stress. We investigated whether hemorrhage-induced myocardial injury is age and gender dependent and whether treatment with metformin, an AMPK activator, affords cardioprotective effects. C57/BL6 young (3-5months) and mature (9-12months) male and female mice were subjected to hemorrhagic shock by blood withdrawing followed by resuscitation with blood and Lactated Ringer's solution. Vehicle-treated young and mature mice of both genders had a similar elevation of plasma inflammatory cytokines at 3h after resuscitation. However, vehicle-treated male mature mice experienced hemodynamic instability and higher myocardial damage than young male mice, as evaluated by echocardiography, histology and cardiovascular injury biomarkers. There was also a gender-dependent difference in cardiovascular injury in the mature group as vehicle-treated male mice exhibited more severe organ injury than female mice. At molecular analysis, vehicle-treated mature mice of both genders exhibited a marked downregulation of AMPKα activation and nuclear translocation of peroxisome proliferator-activated receptor γ co-activator α when compared with young mice. Treatment with metformin improved cardiovascular function and survival in mature animals of both genders. However, specific cardioprotective effects of metformin were gender-dependent. Metformin did not affect hemodynamic or inflammatory responses in young animals. Thus, our data suggest that targeting metabolic recovery with metformin may be a potential treatment approach in severe hemorrhage in adult population.

Keywords: AMP-activated protein kinase (AMPK); Age; Gender; Hemorrhagic shock; Metformin; Peroxisome proliferator-activated receptor-γ co-activator α (PGC-1α).

Fig. 1

Effect of in vivo treatment with metformin on mean arterial blood pressure (A and B) and heart rate (C and D) in young and mature mice of both genders subjected to hemorrhage and resuscitation. Data represents the mean ± SD of 10 mice in each group. Vehicle (normal saline) or metformin (100 mg/kg) was administered intra-arterially at the time of resuscitation. Arrows indicate time of induction of hemorrhage and initiation of resuscitation and metformin or vehicle administration. *P<0.05 vs baseline values at time 0 of the same group; ‡P<0.05 vs young group of the same gender; #P<0.05 vs vehicle-treated group of the same age.

Fig. 2

Effect of in vivo treatment with metformin on blood levels of glucose (A) and lactate (B) before hemorrhage (control baseline), at the end of the hemorrhagic hypovolemic period (End Hem), and at 3 hours after reperfusion (hemorrhagic shock). Data represents the mean ± SEM of 7 mice for each group. *P<0.05 vs baseline values of age- and gender-matched control mice.

Fig. 3

Representative histology photomicrographs of heart sections. Normal myocardium architecture in control young male (A) and female (D) and mature male (G) and female mice (J). Myocardial damage in vehicle-treated young male (B) and female (E) and mature male (H) and females (K) after hemorrhagic shock presented with perivascular and endocardial edema (arrows). Significant amelioration of heart architecture in metformin-treated young (C and F) and mature mice (I and L). Magnification x400.

Fig. 4

Effect of in vivo treatment with metformin on plasma levels of follistatin (A), endocan-1 (B) and CXCL16 (C). Data represents the mean ± SEM of 5 control mice, 10 vehicle- or metformin-treated mice for each gender and age group. *P<0.05 vs baseline values of age- and gender-matched control mice; ‡P<0.05 vs young group of the same gender; #P<0.05 vs vehicle-treated group of the same age; ΩP<0.05 vs age-matched male mice.

Fig. 5

Effect of in vivo treatment with metformin on plasma levels of IL-6 (A), KC (B), TNFα (C), IFNγ (D), IL-10 (E) and IL-1β (F). Data represents the mean ± SEM of 5 control mice and 10 vehicle- or metformin-treated mice for each gender and age group. *P<0.05 vs baseline values of age- and gender-matched control mice; ‡P<0.05 vs young group of the same gender; #P<0.05 vs vehicle-treated group of the same age; ΩP<0.05 vs age-matched male mice.

Fig. 6

Myeloperoxidase activity in lungs and liver. Data represents the mean ± SEM of 5 control mice and 10 vehicle- or metformin-treated mice for each gender and age group. *P<0.05 vs baseline values of age- and gender-matched control mice; ‡P<0.05 vs young group of the same gender; #P<0.05 vs vehicle-treated group of the same age.

Fig. 7

Image analyses of expression of cytosolic pAMPKα (A), nuclear pAMPKα (B), nuclear PGC-1α (C) and nuclear SIRT1 (D). Data represents the mean ± SEM of 4 control mice and 6 vehicle- or metformin-treated mice for each gender and age group. *P<0.05 vs baseline values of age- and gender-matched control mice; #P<0.05 vs vehicle-treated group of the same age.

Fig 8

Activity of the p65 subunit of NF-κB (A) and PPARγ (B) in cardiac nuclear extracts at 3 hours after resuscitation. Data represents the mean ± SEM of 3 control mice and 6 vehicle- or metformin-treated mice for each gender and age group. (C) Cardiac content of FAD. Data represents the mean ± SEM of 4 control mice and 7 vehicle- or metformin-treated mice for each gender and age group. *P<0.05 vs baseline values of age- and gender-matched control mice; ‡P<0.05 vs young group of the same gender; #P<0.05 vs vehicle-treated group of the same age; Ω P<0.05 vs age-matched male mice.

Fig. 9

Effect of metformin on survival rates of young and mature male and female mice subjected to hemorrhagic shock. Vehicle or metformin (100 mg/kg intra-arterially) was administered as single bolus at the time of resuscitation. ‡P<0.05 vs young group of the same gender; #P<0.05 vs vehicle-treated group of the same age.