Hepatic mitochondrial bioenergetics in aged C57BL/6 mice exhibit delayed recovery from severe burn injury

Abstract

Severe burn injuries initiate a cascade of downstream events, culminating in multiple organ dysfunction, sepsis, and even death. The elderly are in particular vulnerable to such outcomes, due primarily to a scarcity of knowledge on trauma progression at the biomolecular level in this population. Mitochondria, the cellular powerhouses, have been increasingly scrutinized recently for their contribution to trauma outcomes. We hypothesized that elderly have a worse outcome compared to adult patients due to failed recovery of hepatic mitochondria. Using a murine model of burn injury, Seahorse respirometry and functional proteomic assays, we demonstrate the impact of thermal trauma on hepatic mitochondrial respiration in adult and aged mice. While the mitochondria in adults rebound from the initial insult within 7days of the injury, the older animals display delayed recovery of mitochondrial bioenergetics accompanied by uncoupling and an oxidative environment. This is associated with a state of increased protein oxidation and nitrosylation, along with increases in circulating mtDNA, a known damage-associated molecular pattern. These findings suggest that hepatic mitochondria fail to normalize after trauma in aged mice and we suggest that this cellular failure is associated with organ damage and subsequently increased morbidity and mortality in elderly burn patients.

Keywords: Elderly; Electron transport chain; Oxidative phosphorylation; Oxidative stress; Trauma.

Figure 1

An analysis of mitochondrial dynamics in adult and aged mice at 24 h post-burn. (A) Respiration profiles of adult sham (■) and burn (30% TBSA; ■) hepatic mitochondria from a Seahorse XF96 analyzer. (B) Representative images from in-gel enzyme activity assays for complexes I–V of the ETC in adult mice. (C) Respiration profiles of aged sham (■) and burn (■) hepatic mitochondria from a Seahorse XF96 analyzer (D) Representative images from in-gel enzyme activity assays and densitometric measurements of complexes I–V of the ETC in aged mice. (E) Survival curve of adult and aged mice following a 30% TBSA burn. n=3 and SEM for sham mice; n=5 and SEM for burnt mice. *p ≤ 0.05; **p≤ 0.01.

Figure 2

ROS production and oxidative defense in adult and aged mice at 24 h post-burn. DCFDA oxidation of adult (A) and aged (B) hepatic mitochondria at basal levels (DCFDA; no substrate), 5 min with substrate (5 mM pyruvate, 3 mM malate) and 15 min with substrate. Densitometric measurements and representative images of mitochondrial glutathione peroxidase (mGPx) from adult (C) and aged (D) mice. Representative Western blots and densitometry for MnSOD from adult (E) and aged (F) mice. n=3 and SEM for sham mice; n=5 and SEM for burnt mice. *p ≤ 0.05 (□, Sham; ■, Burn).

Figure 3

Activity of cytoplasmic enzymes mediating defense against ROS in adult and aged mice at 24 h post-burn. Representative in-gel activity assays and densitometric measurements for catalase from adult (A) and aged (B) mice post-burn. Densitometric measurements and representative activity gel images for cytoplasmic glutathione peroxidase (cGPx) from adult (C) and aged (D) mice post-burn. n=3 and SEM for sham mice; n=5 and SEM for burnt mice. *p ≤ 0.05 (□, Sham; ■, Burn).

Figure 4

An analysis of mitochondrial dynamics in adult mice at 7 days post-burn. (A) Respiration profiles of adult sham (■) and burn (■) hepatic mitochondria from a Seahorse XF96 analyzer. (B) Coupling percentage of sham and burn mitochondria as determined by the Seahorse XF Stress Test Report Generator. (C) DCFDA oxidation of adult hepatic mitochondria at basal levels (DCFDA; no substrate), 5 min with substrate (5 mM pyruvate, 3 mM malate) and 15 min with substrate. (D) Representative images from in-gel activity assays and densitometric measurements for complexes I–V of the ETC in adult mice. n=3 and SEM for sham mice; n=5 and SEM for burnt mice. *p ≤ 0.05; **p≤ 0.01.

Figure 5

ROS markers and enzymes mediating defense against ROS in adult mice at 7 days post-burn. (A) Protein carbonyl concentrations in hepatic mitochondria. (B) Representative Western blot image and densitometric measurements of nitro-tyrosine levels normalized to GAPDH. (C) Representative image and densitometric measurements of mGPx following in-gel enzyme activity assays. (D) Densitometric measurements and representative Western blot image of MnSOD (E) Densitometric measurements and representative image of catalase following in-gel enzyme activity assays. (F) Representative image from in-gel enzyme activity assays for cGPx with associated densitometric measurements. n=3 and SEM for sham mice; n=5 and SEM for burnt mice. *p ≤ 0.05 (□, Sham; ■, Burn).

Figure 6

An analysis of mitochondrial dynamics in aged mice at 7 days post-burn. (A) Respiration profiles of aged sham (■) and burn (■) hepatic mitochondria from a Seahorse XF96 analyzer. (B) Coupling percentage of sham and burn mitochondria as determined by the Seahorse XF Stress Test Report Generator. (C) DCFDA oxidation of aged hepatic mitochondria at basal levels (DCFDA; no substrate), 5 min with substrate (5 mM pyruvate, 3 mM malate) and 15 min with substrate. (D) Representative images and densitometric measurements from in-gel enzyme activity assays for complexes I–V of the ETC in aged mice. n=3 and SEM for sham mice; n=5 and SEM for burnt mice. *p ≤ 0.05; **p≤ 0.01

Figure 7

ROS markers, mitochondrial damage and enzymes mediating defense against ROS in aged mice at 7 days post-burn. (A) Protein carbonyl concentrations in hepatic mitochondria. (B) Representative Western blot image and densitometric measurements of nitro-tyrosine levels normalized to GAPDH. (C) A comparison of plasma mtDNA levels in sham and burned animals from adult and aged cohorts. (D) A comparison of liver mtDNA levels in sham and burned animals from adult and aged cohorts. (E) Densitometric measurements of GPx with a representative image following in-gel enzyme activity assays. (F) Densitometric measurements of catalase activity with a representative image following in-gel enzyme activity assays. (F) Representative Western blot of MnSOD with associated densitometry. n=3 and SEM for sham mice; n=5 and SEM for burnt mice. *p ≤ 0.05 (□, Sham; ■, Burn).