Lactate up-regulates the expression of lactate oxidation complex-related genes in left ventricular cardiac tissue of rats

Abstract

Background: Besides its role as a fuel source in intermediary metabolism, lactate has been considered a signaling molecule modulating lactate-sensitive genes involved in the regulation of skeletal muscle metabolism. Even though the flux of lactate is significantly high in the heart, its role on regulation of cardiac genes regulating lactate oxidation has not been clarified yet. We tested the hypothesis that lactate would increase cardiac levels of reactive oxygen species and up-regulate the expression of genes related to lactate oxidation complex.

Methods/principal findings: Isolated hearts from male adult Wistar rats were perfused with control, lactate or acetate (20mM) added Krebs-Henseleit solution during 120 min in modified Langendorff apparatus. Reactive oxygen species (O2●-/H2O2) levels, and NADH and NADPH oxidase activities (in enriched microsomal or plasmatic membranes, respectively) were evaluated by fluorimetry while SOD and catalase activities were evaluated by spectrophotometry. mRNA levels of lactate oxidation complex and energetic enzymes MCT1, MCT4, HK, LDH, PDH, CS, PGC1α and COXIV were quantified by real time RT-PCR. Mitochondrial DNA levels were also evaluated. Hemodynamic parameters were acquired during the experiment. The key findings of this work were that lactate elevated cardiac NADH oxidase activity but not NADPH activity. This response was associated with increased cardiac O2●-/H2O2 levels and up-regulation of MCT1, MCT4, LDH and PGC1α with no changes in HK, PDH, CS, COXIV mRNA levels and mitochondrial DNA levels. Lactate increased NRF-2 nuclear expression and SOD activity probably as counter-regulatory responses to increased O2●-/H2O2.

Conclusions: Our results provide evidence for lactate-induced up-regulation of lactate oxidation complex associated with increased NADH oxidase activity and cardiac O2●-/H2O2 driving to an anti-oxidant response. These results unveil lactate as an important signaling molecule regulating components of the lactate oxidation complex in cardiac muscle.

Fig 1. NADPH and NADH oxidase activities and reactive oxygen species (O₂ ^●-/H₂O₂) levels in perfused and non-perfused hearts.

(A) NADPH oxidase, (B) NADH oxidase activities, (C) O₂ ^●-/H₂O₂ levels and (D) correlation between NADH oxidase activity and O₂ ^●-/H₂O₂ concentration in hearts perfused with KH or KH + lactate (20 mM) solutions during 120 min. Levels of O₂ ^●-/H₂O₂ in non-perfused hearts challenged with (E) lactate and (F) NADH. (G) Levels of O₂ ^●-/H₂O₂ concentration in perfused hearts challenged with acetate. Values are mean ± SE of 10 hearts; *indicates p<0.05 and **indicates p<0.01 vs. control group.

Fig 2. Activities of antioxidant enzymes and gene expression.

Activities of (A) catalase and (B) SOD. mRNA levels of (C) NRF-1, (D) NRF-2, (E) SOD1, (F) SOD2 and (G) SOD3. (H) Nuclear NRF-2 expression in hearts perfused with KH or KH + lactate (20 mM) solutions during 120 min. Values are mean ± SE of 6–9 hearts; *indicates p<0.05 vs. control group.

Fig 3. Lactate oxidation complex and energetic metabolic enzymes genes expression and mitochondrial DNA content.

(A) MCT1, (B) MCT4, (C) LDH, (D) PGC1α, (E) HK, (F) PDH, (G) CS, (H) COXIV mRNA levels and (I) mtDNA level in hearts perfused with KH or KH + lactate (20 mM) solutions during 120 min. Values are mean ± SE of 6–9 hearts. *indicates p<0.05 and **indicates p<0.01 vs. control group.

Fig 4. Activities of enzymes involved in lactate turnorver.

Activities of (A) HK, (B) LDH, (C) PDH, and (D) CS in hearts perfused with KH or KH + lactate (20 mM) solutions during 120 min. Values are mean ± SE of 6 hearts.

Fig 5. Hemodynamic parameters measured in perfused hearts.

(A) Left ventricular developed pressure, (B) Heart rate, (C) +dP/dt_max, (D)-dP/dt_max and (E) Perfusion pressure after 120 min of perfusion with KH or KH + lactate (20mM) solutions. Values are mean ± SE of 10–15 hearts; *indicates p<0.05 vs. control group.