Heme Oxygenase-2 Localizes to Mitochondria and Regulates Hypoxic Responses in Hepatocytes

Hypoxia occurs as a part of multiple disease states, including hemorrhagic shock. Adaptive responses occur within the cell to limit the consequences of hypoxia. This includes changes in mitochondrial respiration, stress-induced cell signaling, and gene expression that is regulated by hypoxia inducible factor-1α (HIF-1α). Heme oxygenase-2 (HO-2) has been shown to be involved in oxygen sensing in several cell types. The purpose of these experiments was to test the hypothesis that HO-2 is a critical regulator of mitochondrial oxygen consumption and reactive oxygen species (ROS) production to influence hypoxia-adaptive responses such as HIF-1α protein levels and JNK signaling. Methods and Results. In vitro studies were performed in primary mouse hepatocytes. HO-2, but not HO-1, was expressed in mitochondria at baseline. Decreased oxygen consumption and increased mitochondrial ROS production in response to hypoxia were dependent upon HO-2 expression. HO-2 expression regulated HIF-1α and JNK signaling in a mitochondrial ROS-dependent manner. Furthermore, knockdown of HO-2 led to increased organ damage, systemic inflammation, tissue hypoxia, and shock in a murine model of hemorrhage and resuscitation. Conclusion. HO-2 signaling plays a role in hypoxic signaling and hemorrhagic shock. This pathway may be able to be harnessed for therapeutic effects

Hemorrhagic shock and resuscitation-induced skeletal muscle mitochondrial injury was limited by CO therapy.

<p>A. The change in respiratory control ratio (RCR; state 3:state4) from baseline to 2 hours after resuscitation in the porcine model demonstrated that HS/R led to mitochondrial injury. CO treatment resulted in an overall increase in the mean RCR, representing decreased mitochondrial injury (*P<0.05 compared to HS/R). B. Changes in RCR in murine thigh skeletal muscle from baseline to 2 hours after resuscitation demonstrated mitochondrial injury following HS/R, and inhaled CO protected against this injury (N = 8 mice per group; *P<0.05 compared to baseline; #P<0.05 compared to control treated-HS/R mice). ANOVA was utilized for above comparisons.</p

Arterial blood gas measurements in mice bled to and maintained at a MAP of 20 mm Hg for 30 minutes with and without inhaled CO (250 ppm).

<p>*P<0.05 compared to sham mice.</p><p>^§P<0.05 compared to shock mice.</p><p>Sham mice underwent anesthesia and surgical manipulation without hemorrhage. Mice were bled to a pressure of 20 mm Hg over 15 minutes. CO therapy was started once a pressure of 20 mm Hg was reached.</p

CO therapy protects against mortality in a model of murine hemorrhagic shock and resuscitation.

<p>A severe hemorrhagic shock and resuscitation model resulted in 80% mortality by 36 hours. CO therapy (250 ppm) limited mortality to 20% (P<0.05). Kaplan-Meier method was used to compare survival.</p

CO protects against organ injury and inflammation in a dose dependent fashion in murine model of hemorrhagic shock and resuscitation.

<p>Lung myeloperoxidase activity (MPO; <b>A.</b>) and serum ALT (<b>B.</b>) at 4 hours after resuscitation in mice demonstrates lung and liver injury, respectively. CO limits this injury in a dose-dependent fashion when treated for 30 minutes (25–500 ppm) starting 90 minutes into hypotension. C. Serum TNF-alpha and IL-6 levels were also increased by hemorrhagic shock and resuscitation at a 4 hour time point, and CO therapy limited these markers of inflammation in a dose dependent fashion. Results are mean±SEM for 8 mice per group. *P<0.05 compared to sham and #P<0.05 compared to shock. ANOVA was utilized for above comparisons.</p

CO decreases oxygen consumption and limits the development of cellular hypoxia in hepatocytes in vitro.

<p>A. Oxygen consumption rates of primary murine hepatocytes were demonstrated <i>in vitro</i> in normoxic cells or in hepatocytes immediately following 30 minutes of hypoxia. CO treatment (250ppm) occurred during this normoxic or hypoxic periods. Hypoxia decreased oxygen consumption rates (*P<0.01 compared to normoxic cells) and this was further decreased by CO therapy (#P<0.05 compared to hypoxia alone). Results of four independent experiments, with each condition performed in triplicate. <b>B, C.</b> Representative immunocytochemistry and quantitative mean fluorescence of hypoxyprobe staining in hepatocytes under normoxic, normoxic+CO, hypoxic, or hypoxic +CO conditions for 30 minutes. Increased green staining represents increased cellular hypoxia. ANOVA was utilized for above comparisons.</p

CO protects against hemorrhagic shock and resuscitation-induced platelet activation and mitochondrial injury.

<p><b>A</b>. HS/R results in decreased ATP linked respiration (*P<0.05 compared to sham), while CO treatment prevented these changes (#P<0.05 compared to HS/R). <b>B</b>. HS/R had a minimal effect on mitochondrial reserve capacity, while CO treated HS/R pigs demonstrated an increase in this parameter (*P<0.05 compared to sham and HS/R) <b>C</b>. HS/R increased platelet activation by 2.33±0.1 fold over sham pigs at a 2 hour time point as determined by staining for CD62p by FACS (*P<0.05 compared to sham). CO treatment limited this activation to only a 1.64±0.08 increase over sham (#P<0.05 compared to HS/R). n = 7–11 pigs per group in each experiment. ANOVA was utilized for above comparisons.</p

CO has minimal influence on gross cardiovascular parameters in porcine hemorrhagic shock and resuscitation.

<p>Hemodynamic data are shown at time points throughout the experiments [baseline, end of hemorrhage (H1), 30 minutes into hypotension (H30), 60 minutes into hypotension (H60), immediately prior to resuscitation (resusc), at the end of the initial hextend bolus (hextend), 2 hours into the resuscitation (Obs2h), and 4 hours into the resuscitation (Obs4h)]. Data is shown for mean arterial pressure (MAP, <b>A</b>.), heart rate (<b>B</b>.), central venous pressure (CVP, <b>C.</b>), mixed venous saturation (S_VO₂%, <b>D</b>.), and mean pulmonary arterial pressure (<b>E</b>.). Expected changes in hemodynamics are seen in shock and resuscitation, with no significant influences demonstrated in the CO treated pigs. ANOVA was utilized for above comparisons.</p