The von Hippel-Lindau Chuvash mutation in mice alters cardiac substrate and high-energy phosphate metabolism

Abstract

Hypoxia-inducible factor (HIF) appears to function as a global master regulator of cellular and systemic responses to hypoxia. HIF pathway manipulation is of therapeutic interest; however, global systemic upregulation of HIF may have as yet unknown effects on multiple processes. We used a mouse model of Chuvash polycythemia (CP), a rare genetic disorder that modestly increases expression of HIF target genes in normoxia, to understand what these effects might be within the heart. An integrated in and ex vivo approach was employed. Compared with wild-type controls, CP mice had evidence (using in vivo magnetic resonance imaging) of pulmonary hypertension, right ventricular hypertrophy, and increased left ventricular ejection fraction. Glycolytic flux (measured using [(3)H]glucose) in the isolated contracting perfused CP heart was 1.8-fold higher. Net lactate efflux was 1.5-fold higher. Furthermore, in vivo (13)C-magnetic resonance spectroscopy (MRS) of hyperpolarized [(13)C1]pyruvate revealed a twofold increase in real-time flux through lactate dehydrogenase in the CP hearts and a 1.6-fold increase through pyruvate dehydrogenase. (31)P-MRS of perfused CP hearts under increased workload (isoproterenol infusion) demonstrated increased depletion of phosphocreatine relative to ATP. Intriguingly, no changes in cardiac gene expression were detected. In summary, a modest systemic dysregulation of the HIF pathway resulted in clear alterations in cardiac metabolism and energetics. However, in contrast to studies generating high HIF levels within the heart, the CP mice showed neither the predicted changes in gene expression nor any degree of LV impairment. We conclude that the effects of manipulating HIF on the heart are dose dependent.

Keywords: hyperpolarized pyruvate; hypoxia-inducible factor; magnetic resonance imaging.

Fig. 1.

Cardiac and lung gene expression. Quantitative real-time PCR was used to measure expression of key metabolic genes in hearts from wild-type (WT, n = 9) and Chuvash polycythemia (CP, n = 7) mice, aged 6–7 mo. To confirm the methodology, expression of endothelin-1 (Edn 1; dark gray) was also determined in lungs from 13 WT and 11 CP mice. Values are means ± 95% confidence interval. VEGF, vascular endothelial growth factor; GLUT, glucose transporter; PFKM, phosphofructokinase; PDK, pyruvate dehydrogenase kinase; LDHA, lactate dehydrogenase A; PKM, pyruvate kinase muscle; PPARa, peroxisome proliferator-activated receptor α. ****P < 0.001.

Fig. 2.

In vivo cardiac function. In vivo cine magnetic resonance imaging was used to measure cardiac mass and function in aging WT and CP mice (n = 3–8 mice/group). Open bars, WT; closed bars, CP. A: left ventricle (LV) mass; B: right ventricle (RV) wall thickness; C: LV cardiac output, corrected to body mass; D: LV ejection fraction. WT mice had normal cardiac morphology (E, representative image of mouse aged 3–6 mo). CP mice demonstrated marked interventricular septal bowing, particularly in early diastole (F; arrowheads, representative image of mouse aged 3–6 mo). *P < 0.05, **P < 0.02, and ***P < 0.01.

Fig. 3.

Perfused-heart energetics. ³¹P-magnetic resonance spectroscopy was performed on perfused hearts from WT and CP mice (n = 5 in each group), aged 6–7 mo. Body mass 30.1 ± 2.1 g for WT vs. 28.7 ± 1.2 for CP (not significant). Open bars, WT; closed bars, CP. Hearts were perfused under normal Langendorff conditions using palmitate buffer, and spectra were acquired, both without and with an infusion of isoproterenol to increase the rate pressure product (RPP). As expected, isoproterenol significantly increased the RPP (P < 0.001) and decreased the PCR-to-ATP ratio (P < 0.001). No significant difference between WT and CP was detected without isoproterenol, but following the increase in RPP the PCr/ATP ratio was significantly lower in the CP hearts compared with WT controls. A: RPP; B: PCr/ATP ratio. **P < 0.02.

Fig. 4.

Perfused heart metabolism. Glycolytic flux (A) and net lactate efflux (B) were determined in WT (n = 4) and CP (n = 7) mice, aged 15–17 mo. Body mass 33.8 ± 1.8 g for WT vs. 29.4 ± 1.6 for CP (not significant). Glycolytic and lactate rates were highly correlated (Pearson r = 0.935, P < 0.001). Palmitate oxidation rates (C) were determined in separate mice (n = 5 in each group), aged 11–17 mo. Body mass 27.4 ± 1.5 g for WT vs. 26.2 ± 2.0 for CP (P = 0.034). ***P < 0.01.

Fig. 5.

In vivo real-time cardiac metabolism. In vivo magnetic resonance spectroscopy of hyperpolarized [¹³C₁]pyruvate was performed in WT (n = 5) and CP (n = 4) mice, aged 9–15 mo. Body mass 32.0 ± 3.0 g for WT vs. 27.0 ± 3.0 for CP (not significant). This technique allowed real-time measurement of the rate of label incorporation from pyruvate into alanine (A), bicarbonate (B), or lactate (C). *P < 0.05 and ***P < 0.01.