The transcriptional coactivator PGC-1alpha is essential for maximal and efficient cardiac mitochondrial fatty acid oxidation and lipid homeostasis

Abstract

High-capacity mitochondrial ATP production is essential for normal function of the adult heart, and evidence is emerging that mitochondrial derangements occur in common myocardial diseases. Previous overexpression studies have shown that the inducible transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha is capable of activating postnatal cardiac myocyte mitochondrial biogenesis. Recently, we generated mice deficient in PGC-1alpha (PGC-1alpha(-/-) mice), which survive with modestly blunted postnatal cardiac growth. To determine if PGC-1alpha is essential for normal cardiac energy metabolic capacity, mitochondrial function experiments were performed on saponin-permeabilized myocardial fibers from PGC-1alpha(-/-) mice. These experiments demonstrated reduced maximal (state 3) palmitoyl-l-carnitine respiration and increased maximal (state 3) pyruvate respiration in PGC-1alpha(-/-) mice compared with PGC-1alpha(+/+) controls. ATP synthesis rates obtained during maximal (state 3) respiration in permeabilized myocardial fibers were reduced for PGC-1alpha(-/-) mice, whereas ATP produced per oxygen consumed (ATP/O), a measure of metabolic efficiency, was decreased by 58% for PGC-1alpha(-/-) fibers. Ex vivo isolated working heart experiments demonstrated that PGC-1alpha(-/-) mice exhibited lower cardiac power, reduced palmitate oxidation, and increased reliance on glucose oxidation, with the latter likely a compensatory response. (13)C NMR revealed that hearts from PGC-1alpha(-/-) mice exhibited a limited capacity to recruit triglyceride as a source for lipid oxidation during beta-adrenergic challenge. Consistent with reduced mitochondrial fatty acid oxidative enzyme gene expression, the total triglyceride content was greater in hearts of PGC-1alpha(-/-) mice relative to PGC-1alpha(+/+) following a fast. Overall, these results demonstrate that PGC-1alpha is essential for the maintenance of maximal, efficient cardiac mitochondrial fatty acid oxidation, ATP synthesis, and myocardial lipid homeostasis.

Fig. 1.

Respiration and ATP synthesis rates for permeabilized myocardial fibers and isolated mitochondria. A: respiration of saponin-permeabilized left ventricular (LV) fibers from hearts of 3.5-mo-old female peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α^+/+ and PGC-1α^−/− mice was measured as described in materials and methods. The respiration buffer contained 20 μM palmitoyl-l-carnitine (PC) and 5 mM malate for PC respiration, 10 mM pyruvate and 5 mM malate for pyruvate respiration, and 5 mM glutamate and 2 mM malate for glutamate respiration. Following measurements of basal respiration, state 3 (maximal ADP-stimulated) respiration was determined by exposing fibers to 1 mM ADP, with the subsequent determination of postoligomycin (uncoupled) respiration. The respiratory control (RC) quotient represents the following ratio: (state 3 respiration/postoligomycin respiration). Results are means ± SE; n = 10 for PC and pyruvate experiments and 6 for glutamate experiments. *P < 0.05 and **P < 0.01 compared with corresponding PGC-1α^+/+ values. B: maximal ATP synthesis rates and efficiency of ATP synthesis [ATP produced per oxygen consumed (ATP/O)] in permeabilized LV fibers prepared in parallel from the same hearts used for respiration analysis. ATP/O represents the following ratio: (state 3 ATP synthesis rate/state 3 respiration rate). Results are means ± SE; n ≥ 8 for PC and pyruvate experiments and 5 for glutamate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with corresponding PGC-1α^+/+ values. C: respiration of mitochondria isolated from combined left and right cardiac ventricles of 4-mo-old sex-matched PGC-1α^+/+ and PGC-1α^−/− mice was measured in buffer containing 5 mM succinate and 10 μM rotenone as described in materials and methods. Following the assessment of basal respiration, state 3 (maximal ADP-stimulated) respiration was determined by exposing mitochondria to 350 μM ADP with the subsequent determination of postoligomycin (uncoupled) respiration. The RC quotient represents the following ratio: (state 3 respiration/postoligomycin respiration). Results are means ± SE; n = 6. *P < 0.05 compared with corresponding PGC-1α^+/+ values.

Fig. 2.

Alterations in substrate utilization and cardiac hydraulic work in isolated working hearts from PGC-1α^−/− mice. A: an isolated mouse working heart perfusion was performed with hearts from 7- to 8-mo-old sex-matched male and female mice (as described in materials and methods). Results for cardiac hydraulic work are means ± SE; n ≥ 6. *P < 0.05 compared with corresponding PGC-1α^+/+ values. B: to determine palmitate and glucose oxidation rates, trace amounts of [³H]palmitate (0.1 μCi/ml) and [U-¹⁴C]glucose (0.1 μCi/ml) were used in the isolated working heart perfusate. Results represent mean oxidation rates per gram ventricular dry weight ± SE; n ≥ 5. *P < 0.05 compared with corresponding PGC-1α^+/+ values.

Fig. 3.

Altered mitochondrial cristal density in PGC-1α^−/− cardiac ventricles. Representative electron micrographs of the cardiac LV apex obtained from normally fed 2-mo-old PGC-1α^+/+ and PGC-1α^−/− female mice are shown (representative of n ≥ 6 comparisons). Scale bars are shown to the right for the low (×30,000; top) and high (×120,000, bottom) magnifications.

Fig. 4.

Assessment of cardiac ventricular neutral lipid and triglyceride (TAG) content. Representative histological sections stained with oil red O of the cardiac left ventricle from 4-mo-old PGC-1α^+/+ and PGC-1α^−/− mice following a 24-h fast are shown (left). The red droplets are indicative of neutral lipid accumulation. Mean cardiac ventricular TAG levels (right) were determined by two-dimensional electrospray ionization mass spectrometric (ESI/MS) analysis performed on myocardial lipid extracts from both fed and 24-h fasted PGC-1α^+/+ and PGC-1α^−/− mice (n ≥ 5). For the fast, 4-mo-old female mice were individually housed on wood chip bedding. Results are means ± SE. *P < 0.0001 compared with fasted PGC-1α^+/+ values.

Fig. 5.

Two-dimensional ESI/MS fingerprint and quantitation of TAG molecular species of cardiac ventricular lipid extracts from fasted PGC-1α^+/+ and PGC-1α^−/− mice. A and B: two-dimensional ESI/MS analyses were performed on cardiac ventricular lipid extracts from 4-mo-old female PGC-1α^+/+ (A) and PGC-1α^−/− (B) mice following a 24-h fast as described in materials and methods. The top spectra depict the relative intensity of each TAG species compared with the T17:1 internal standard (IS) peak for TAGs. Below the relative intensity TAG spectrum, the subsequent horizontal rows depict the detailed analyses of TAG molecular species that contain the specific fatty acyl chain noted along the left axis. For example, the “NL 256.2 (16:0)” row indicates all the TAG molecular species containing at least one 16:0 fatty acyl chain. C: quantitation by ESI/MS of the relative abundance of fatty acyl species in TAG of cardiac ventricular lipid extracts from 24-h fasted PGC-1α^+/+ and PGC-1α^−/− mice. Results are means ± SE; n ≥ 5. *P < 0.05 and **P < 0.01 compared with fasted PGC-1α^+/+ values. D: quantitation by ESI/MS of representative increased TAG species in cardiac ventricular lipid extracts from 24-h fasted PGC-1α^+/+ and PGC-1α^−/− mice. The increased TAG species in hearts of fasted PGC-1α^−/− mice relative to PGC-1α^+/+ mice included 16:0/16:0/16:0 TAG, 16:0/16:0/18:1 TAG, and 16:0/18:1/18:2 TAG. Results are means ± SE; n ≥ 5. *P < 0.05, **P < 0.01, and ***P < 0.0001 compared with corresponding PGC-1α^+/+ values.