Sepsis Disrupts Mitochondrial Function and Diaphragm Morphology.

BACKGROUND: The diaphragm is the primary muscle of inspiration, and its dysfunction is frequent during sepsis. However, the mechanisms associated with sepsis and diaphragm dysfunction are not well understood. In this study, we evaluated the morphophysiological changes of the mitochondrial diaphragm 5 days after sepsis induction. METHODS: Male C57Bl/6 mice were divided into two groups, namely, cecal ligation and puncture (CLP, n = 26) and sham-operated (n = 19). Mice received antibiotic treatment 8 h after surgery and then every 24 h until 5 days after surgery when mice were euthanized and the diaphragms were collected. Also, diaphragm function was evaluated in vivo by ultrasound 120 h after CLP. The tissue fiber profile was evaluated by the expression of myosin heavy chain and SERCA gene by qPCR and myosin protein by using Western blot. The Myod1 and Myog expressions were evaluated by using qPCR. Diaphragm ultrastructure was assessed by electron microscopy, and mitochondrial physiology was investigated by high-resolution respirometry, Western blot, and qPCR. RESULTS: Cecal ligation and puncture mice developed moderated sepsis, with a 74% survivor rate at 120 h. The diaphragm mass did not change in CLP mice compared with control, but we observed sarcomeric disorganization and increased muscle thickness (38%) during inspiration and expiration (21%). Septic diaphragm showed a reduction in fiber myosin type I and IIb mRNA expression by 50% but an increase in MyHC I and IIb protein levels compared with the sham mice. Total and healthy mitochondria were reduced by 30% in septic mice, which may be associated with a 50% decrease in Ppargc1a (encoding PGC1a) and Opa1 (mitochondria fusion marker) expressions in the septic diaphragm. The small and non-functional OPA1 isoform also increased 70% in the septic diaphragm. These data suggest an imbalance in mitochondrial function. In fact, we observed downregulation of all respiratory chain complexes mRNA expression, decreased complex III and IV protein levels, and reduced oxygen consumption associated with ADP phosphorylation (36%) in CLP mice. Additionally, the septic diaphragm increased proton leak and downregulated Sod2 by 70%. CONCLUSION: The current model of sepsis induced diaphragm morphological changes, increased mitochondrial damage, and induced functional impairment. Thus, diaphragm damage during sepsis seems to be associated with mitochondrial dysfunction.

Effect of Sublethal Prenatal Endotoxaemia on Murine Placental Transport Systems and Lipid Homeostasis.

Infection alters the expression of transporters that mediate the placental exchange of xenobiotics, lipids and cytokines. We hypothesized that lipopolysaccharide (LPS) modifies the expression of placental transport systems and lipid homeostasis. LPS (150 µg/kg; i.p.) treatments were administered for 4 h or 24 h, animals were euthanized at gestational days (GD) 15.5 or 18.5, and maternal blood, fetuses and placentae were collected. Increased rates of fetal demise were observed at GD15.5 following LPS treatment, whereas at GD18.5, high rates of early labour occurred and were associated with distinct proinflammatory responses. Lipopolysaccharide did not alter ATP-binding cassette (ABC) transporter mRNA expression but decreased fatty acid binding protein associated with plasma membrane (Fabppm) at GD15.5 (LPS-4 h) and increased fatty acid translocase (Fat/Cd36) mRNA at GD18.5 (LPS-4 h). At the protein level, breast cancer-related protein (Bcrp) and ABC sub-family G member 1 (Abcg1) levels were decreased in the placental labyrinth zone (Lz) at GD15.5, whereas P-glycoprotein (P-gp) and Bcrp Lz-immunostaining was decreased at GD18.5. In the placental junctional zone (Jz), P-gp, Bcrp and Abcg1 levels were higher at GD18.5. Specific maternal plasma and placental changes in triacylglycerol, free fatty acid, cholesterol, cholesterol ester and monoacylglycerol levels were detected in a gestational age-dependent manner. In conclusion, LPS-increased risk of fetal death and early labour were associated with altered placental ABC and lipid transporter expression and deranged maternal plasma and placental lipid homeostasis. These changes may potentially modify fetal xenobiotic exposure and placental lipid exchange in cases of bacterial infection.

ZIKV Disrupts Placental Ultrastructure and Drug Transporter Expression in Mice.

Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome.

Sepsis Impairs Thyroid Hormone Signaling and Mitochondrial Function in the Mouse Diaphragm.

Background: Sepsis can cause the nonthyroidal illness syndrome (NTIS), resulting in perturbed thyroid hormone (TH) signaling and reduced thyroxine (T4) levels. TH is a major regulator of muscle function, via its influence on mitochondria. This study aimed at evaluating the relationship between TH signaling, mitochondrial function, and the antioxidant defense system in the diaphragms of septic mice. Methods: Male C57Bl/6 mice were divided into two groups: cecal ligation and puncture (CLP) and sham. Twenty-four hours after surgery, plasma, diaphragms, and livers were collected. TH metabolism and responses were analyzed by measuring messenger RNA (mRNA) expression of Dio1 in the liver, and Thra, Thrb, Dio2, Slc16a10, and Slc16a2 (encodes MCT 10 and 8), in the diaphragm. T4 plasma levels were measured by radioimmunoassay. Damage to diaphragm mitochondria was assessed by electron microscopy and real-time polymerase chain reaction (qPCR), and function with oxygraphy. The diaphragm antioxidative defense system was examined by qPCR, analyzing superoxide dismutase (SOD) 1 (Sod1), mitochondrial superoxide dismutase (SOD 2; Sod2), extracellular superoxide dismutase (SOD 3; Sod3), glutathione peroxidase 1 (Gpx1), and catalase (Cat) expression. The effect of TH replacement was tested by treating the mice with T4 and triiodothyronine (T3) (CLP+TH) after surgery. Results: CLP mice presented reduced total plasma T4 concentrations, downregulated Dio1, and upregulated Il1b mRNA expression in the liver. CLP mice also displayed downregulated Thra, Thrb, Slc16a10, and Slc16a2 expression in the diaphragm, suggesting that TH signaling was compromised. The expression of Ppargc1a (encoding PGC1a) was downregulated, which correlated with the decrease in the number of total mitochondria, increase in the percentage of injured mitochondria, downregulation of respiratory chain complex 2 and 3 mRNA expression, and reduced maximal respiration. In addition, septic animals presented a three-fold increase in Ucp3 and G6pdh expression; downregulated Sod3, Gpx1, and Cat expression; and upregulated Sod2 expression, potentially due to elevated reactive oxygen species levels. The mitochondrial number and the percentage of injured mitochondrial were similar between sham and CLP+TH mice. Conclusions: Sepsis induced responses consistent with NTIS, resulted in mitochondrial damage and functional impairment, and modulated the expression of key antioxidant enzymes in the diaphragm. Thus, impaired diaphragm function during sepsis seems to involve altered local TH signaling, mitochondrial dysfunction, and oxidative stress defense.

Reduced mitochondrial respiration and increased calcium deposits in the EDL muscle, but not in soleus, from 12-week-old dystrophic mdx mice.

Mitochondria play an important role in providing ATP for muscle contraction. Muscle physiology is compromised in Duchenne muscular dystrophy (DMD) and several studies have shown the involvement of bioenergetics. In this work we investigated the mitochondrial physiology in fibers from fast-twitch muscle (EDL) and slow-twitch muscle (soleus) in the mdx mouse model for DMD and in control C57BL/10J mice. In our study, multiple mitochondrial respiratory parameters were investigated in permeabilized muscle fibers from 12-week-old animals, a critical age where muscle regeneration is observed in the mdx mouse. Using substrates of complex I and complex II from the electron transport chain, ADP and mitochondrial inhibitors, we found in the mdx EDL, but not in the mdx soleus, a reduction in coupled respiration suggesting that ATP synthesis is affected. In addition, the oxygen consumption after addition of complex II substrate is reduced in mdx EDL; the maximal consumption rate (measured in the presence of uncoupler) also seems to be reduced. Mitochondria are involved in calcium regulation and we observed, using alizarin stain, calcium deposits in mdx muscles but not in control muscles. Interestingly, more calcium deposits were found in mdx EDL than in mdx soleus. These data provide evidence that in 12-week-old mdx mice, calcium is accumulated and mitochondrial function is disturbed in the fast-twitch muscle EDL, but not in the slow-twitch muscle soleus.

High levels of circulating triiodothyronine induce plasma cell differentiation.

The effects of hyperthyroidism on B-cell physiology are still poorly known. In this study, we evaluated the influence of high-circulating levels of 3,5,3'-triiodothyronine (T3) on bone marrow, blood, and spleen B-cell subsets, more specifically on B-cell differentiation into plasma cells, in C57BL/6 mice receiving daily injections of T3 for 14 days. As analyzed by flow cytometry, T3-treated mice exhibited increased frequencies of pre-B and immature B-cells and decreased percentages of mature B-cells in the bone marrow, accompanied by an increased frequency of blood B-cells, splenic newly formed B-cells, and total CD19(+)B-cells. T3 administration also promoted an increase in the size and cellularity of the spleen as well as in the white pulp areas of the organ, as evidenced by histological analyses. In addition, a decreased frequency of splenic B220(+) cells correlating with an increased percentage of CD138(+) plasma cells was observed in the spleen and bone marrow of T3-treated mice. Using enzyme-linked immunospot assay, an increased number of splenic immunoglobulin-secreting B-cells from T3-treated mice was detected ex vivo. Similar results were observed in mice immunized with hen egg lysozyme and aluminum adjuvant alone or together with treatment with T3. In conclusion, we provide evidence that high-circulating levels of T3 stimulate plasma cytogenesis favoring an increase in plasma cells in the bone marrow, a long-lived plasma cell survival niche. These findings indicate that a stimulatory effect on plasma cell differentiation could occur in untreated patients with Graves' disease.