Mitochondrial integrity in a neonatal bovine model of right ventricular dysfunction

Abstract

Right ventricular (RV) function is a key determinant of survival in patients with both RV and left ventricular (LV) failure, yet the mechanisms of RV failure are poorly understood. Recent studies suggest cardiac metabolism is altered in RV failure in pulmonary hypertension (PH). Accordingly, we assessed mitochondrial content, dynamics, and function in hearts from neonatal calves exposed to hypobaric hypoxia (HH). This model develops severe PH with concomitant RV hypertrophy, dilation, and dysfunction. After 2 wk of HH, pieces of RV and LV were obtained along with samples from age-matched controls. Comparison with control assesses the effect of hypoxia, whereas comparison between the LV and RV in HH assesses the additional impact of RV overload. Mitochondrial DNA was unchanged in HH, as was mitochondrial content as assessed by electron microscopy. Immunoblotting for electron transport chain subunits revealed a small increase in mitochondrial content in HH in both ventricles. Mitochondrial dynamics were largely unchanged. Activity of individual respiratory chain complexes was reduced (complex I) or unchanged (complex V) in HH. Key enzymes in the glycolysis pathway were upregulated in both HH ventricles, alongside upregulation of hypoxia-inducible factor-1α protein. Importantly, none of the changes in expression or activity were different between ventricles, suggesting the changes are in response to HH and not RV overload. Upregulation of glycolytic modulators without chamber-specific mitochondrial dysfunction suggests that mitochondrial capacity and activity are maintained at the onset of PH, and the early RV dysfunction in this model results from mechanisms independent of the mitochondria.

Keywords: cardiac; mitochondria; pulmonary hypertension; right ventricle.

Fig. 1.

Two weeks of exposure to hypoxia do not significantly alter mitochondrial shape and content in the right ventricle (RV) and left ventricle (LV). A–D: representative electron micrographs of control (CO) LV (A), CO RV (B), hypobaric hypoxia (HH) LV (C), and HH RV (D). E: expression of mitochondrial electron transport chain complexes was assessed by immunoblotting, using an antibody against one subunit of each complex; n = 10 experiments in each condition. F: mitochondrial DNA copy number was assessed by real-time PCR and expressed relative to a nuclear DNA housekeeping gene; n = 6 in each condition.

Fig. 2.

Mitochondrial content is diminished in both the LV and the RV in response to pulmonary hypertension (PH), with minimal changes in mitochondrial phenotype. A: representative images of midventricle transverse sections stained for total oxidative phosphorylation (OXPHOS), troponin I (TnI), and DAPI for myonuclear identification. B: quantification of mitochondrial content; n = 1/condition, 5 fields/image.

Fig. 3.

Exposure to hypobaric hypoxia suppresses complex I activity in both the LV and the RV (A), with no effect on complex V activity (B). Complex I and V activity was assessed spectrophotometrically using commercially available kits and are expressed as a change in absorbance over time; n = 10 in each condition.

Fig. 4.

Mitochondrial dynamics are generally unchanged in the hypoxic cow heart. Mitofusin 1 (Mfn1, A), mitofusin 2 (Mfn2, B), fission 1 (Fis1, C), and dynamin-1-like protein (Drp1, D) were assessed by immunoblotting; n = 10 in each condition.

Fig. 5.

Two weeks of exposure to hypobaric hypoxia significantly increases hypoxia-inducible factor-1α (HIF-1α) protein expression in both the right and left ventricles (A) without altering vascular endothelial growth factor (VEGF) expression (B). HIF-1α and VEGF were assessed by immunoblotting; n = 10 in each condition.

Fig. 6.

PH alters the metabolic phenotype in both the RV and the LV. Protein expression of pyruvate kinase (PKM) 1 (A) and PKM2 (B) was unchanged by exposure to hypobaric hypoxia; however, the ratio between 1 and 2 isoforms (C) was significantly diminished as a result of HH. PKM1 and -2 were assessed by immunoblotting and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH); n = 10 in each condition. Real-time PCR was used to assess transcriptional changes in PKM1 and PKM2 isoforms as a result of hypobaric hypoxia. PKM1 (D) and PKM2 (E) were unchanged by HH; however, the ratio between 1 and 2 was attenuated in HH (F), with a significant interaction between the right and left ventricles; n = 6 in each condition. PFK1 (G) and glucose transporter 4 (GLUT4, H) protein expression was upregulated in both ventricles, or unchanged, respectively, as a result of HH; n = 10 in each condition.