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. 2007 Oct;6(4):294-306.
doi: 10.1016/j.cmet.2007.09.001.

A Conserved Role for Phosphatidylinositol 3-kinase but Not Akt Signaling in Mitochondrial Adaptations That Accompany Physiological Cardiac Hypertrophy

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Free PMC article

A Conserved Role for Phosphatidylinositol 3-kinase but Not Akt Signaling in Mitochondrial Adaptations That Accompany Physiological Cardiac Hypertrophy

Brian T O'Neill et al. Cell Metab. .
Free PMC article

Abstract

Physiological cardiac hypertrophy is associated with mitochondrial adaptations that are characterized by activation of PGC-1alpha and increased fatty acid oxidative (FAO) capacity. It is widely accepted that phosphatidylinositol 3-kinase (PI3K) signaling to Akt1 is required for physiological cardiac growth. However, the signaling pathways that coordinate physiological hypertrophy and metabolic remodeling are incompletely understood. We show here that activation of PI3K is sufficient to increase myocardial FAO capacity and that inhibition of PI3K signaling prevents mitochondrial adaptations in response to physiological hypertrophic stimuli despite increased expression of PGC-1alpha. We also show that activation of the downstream kinase Akt is not required for the mitochondrial adaptations that are secondary to PI3K activation. Thus, in physiological cardiac growth, PI3K is an integrator of cellular growth and metabolic remodeling. Although PI3K signaling to Akt1 is required for cellular growth, Akt-independent pathways mediate the accompanying mitochondrial adaptations.

Figures

Figure 1
Figure 1
Activation of PI3K increases mitochondrial fatty acid (FA) metabolism in the heart. Phosphorylation of PI3K and Akt targets in caPI3K hearts (A). Palmitate oxidation (B), glycolysis (C), and glucose oxidation (D) in isolated working hearts from 5–7 week-old caPI3K and control mice. Mitochondrial respiration rates in saponin-permeabilized cardiac fibers exposed to 5 mM glutamate/2 mM malate (E) or 20 μM palmitoyl-carnitine/5 mM malate (PC) (F) as substrate. ATP synthesis rates (G) with PC in cardiac fibers. 3′Hydroxyacyl-CoA dehydrogenase (HADH) (H) and citrate synthase (CS) (I) enzymatic activity rates in whole heart homogenates. CS activity in isolated mitochondria (J) from caPI3K and control mice (n=4–8 per group; * p<0.05, ** p<0.01 vs. WT).
Figure 2
Figure 2
Inhibition of PI3K decreases mitochondrial FA metabolism in the heart. Mitochondrial respiration rates in saponin-permeabilized cardiac fibers from 5–7 week-old dnPI3K and control mice exposed to glutamate/malate (A) or palmitoyl-carnitine (PC) (B) as substrate. ATP synthesis rates (C) with PC in cardiac fibers. HADH (D) and CS (E) activity rates in whole heart homogenates (n=5–8 per group; * p<0.05, ** p<0.01 vs. WT).
Figure 3
Figure 3
Inhibition of PI3K prevents cardiac hypertrophy and mitochondrial adaptations in response to exercise training. Western blot analysis (A) and densitometric ratios (B) of phosphorylated targets of PI3K and Akt in hearts from wildtype sedentary (WT sed), wildtype exercise-trained (WT ex), dnPI3K sedentary (dnPI3K sed) and dnPI3K-exercise trained (dnPI3K ex) mice. Heart weight to body weight (HW/BW) ratio in exercise-trained dnPI3K and control mice (C). Mitochondrial respiration (D) and ATP synthesis (E) with palmitoyl-carnitine in cardiac fibers from wildtype and dnPI3K sedentary and exercise-trained mice. (n=4–6 per group; * p<0.05, ** p<0.01 vs. WT sed).
Figure 4
Figure 4
mRNA quantification of FA oxidation enzymes and oxidative phosphorylation (OXPHOS) complex subunits in hearts from caPI3K and dnPI3K mice (expressed as fold change vs. WT). (A) mRNA levels in hearts from caPI3K mice, (n=8 per group). (B) mRNA levels in hearts from dnPI3K mice, (n=12 per group). (C) mRNA levels (fold change vs. sedentary wildtype (WT Sed)) in hearts from 12-week-old exercise trained wildtype (WT Ex), sedentary dnPI3K (dnPI3K Sed), and exercise-trained dnPI3K (dnPI3K Ex) mice, (n=4–6 per group). MCAD - medium chain acyl-CoA dehydrogenase (AD); LCAD - long chain AD; VLCAD - very long chain AD; CPT1-β - carnitine palmitoyl transferase 1, muscle isoform; CPT2 - carnitine palmitoyl transferase 2; Hadh α/β - 3′hydroxyacyl-CoA dehydrogenase α or β subunit; Ndufa9 - NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 9; Ndufv1 – NADH dehydrogenase (ubiquinone) flavoprotein 1; Uqcrc1 - ubiquinol-cytochrome c reductase core protein 1; Cox4i1 – cytochrome c oxidase, subunit IV isoform 1; Cox5b - cytochrome c oxidase, subunit Vb. (*p<0.05, ** p<0.01 vs. WT or WT Sed)
Figure 5
Figure 5
Expression of transcriptional regulators of FA oxidation enzymes and OXPHOS complex subunits in hearts from caPI3K and dnPI3K mice. (A) mRNA levels in hearts from caPI3K mice vs. WT expression. (n=8 per group). (B) mRNA levels in hearts from dnPI3K mice vs. WT. (n=12 per group). (C) mRNA levels in hearts from exercise-trained wildtype (WT Ex), sedentary dnPI3K (dnPI3K Sed), and exercise-trained dnPI3K (dnPI3K Ex) mice vs. sedentary wildtype (WT Sed) (n=4–6 per group). (D) Western analysis for PGC-1α in nuclear enriched fractions from caPI3K, dnPI3K, WT Ex, and control hearts. PGC-1α expression in brown adipose tissue (BAT) from cold exposed WT mice was used as an antibody control (NS = non-specific band for loading control). PPARα - peroxisome proliferator-activated receptor α; PGC-1α and -1β - PPARγ coactivator 1α and 1β; ERRα – estrogen related receptor α. (*p<0.05, ** p<0.01 vs. WT or WT Sed).
Figure 6
Figure 6
Activation of Akt in the heart reduces mitochondrial function, and isoform specific deletion of Akt1 or Akt2 or cardiac specific overexpression of a kinase-dead Akt1 (kdAkt) does not prevent exercise-induced increases in mitochondrial FAO capacity in the heart. Mitochondrial respiration (A) and ATP production (B) with palmitoyl-carnitine (PC) in cardiac fibers from 5–7 week-old caAkt and control mice. HADH and CS activities (C) and (D) in whole heart homogenates. CS activity in isolated mitochondria (E). Western analysis for phosphorylation of Akt (Ser473) in hearts from sedentary (Sed) and exercise-trained (Ex) Akt1 −/−, Akt2 −/−, kdAkt, and control mice (F). Densitometric ratio of phospho-Akt to total Akt is shown under each sample (Phospho/total). Mitochondrial respiration with PC in cardiac fibers (G), heart weight to tibia length (HW/TL) ratio (H), and CS activity in skeletal muscle (I) from sedentary and exercise-trained Akt1 −/−, Akt2 −/−, kdAkt, and control mice. (n=4–6 per group; * p<0.05, ** p<0.01 vs. WT; † p<0.05 vs. sedentary control).
Figure 7
Figure 7
Expression of a kinase-dead Akt (kdAkt) does not impair PI3K-mediated mitochondrial adaptations despite attenuation of cardiac hypertrophy and inhibition of downstream Akt signaling. (A) Heart weight to body weight (HW/BW) ratio of double transgenic (DTG) and control mice (n=12–20 per group). (B) Phosphorylation of PI3K and Akt targets. State 3 of mitochondrial respiration (C) and ATP production (D) with palmitoyl-carnitine (PC) in cardiac fibers (n=5–9 per group). HADH and CS activities (E) and (F) in whole heart homogenates (n=5–9 per group). (G) Phosphorylation of Akt and PKCλ/ζ targets in neonatal rat cardiomyocytes (NRCM) in the presence or absence of insulin (100nM)/IGF-1 (10nM) and Akt inhibitor (Akt I) or PKCλ/ζ inhibitor (PKCλ/ζ I). Loading control for p-MARKS is Ponceau stained membrane. (H) Total CS activity in NRCM treated with Growth Factor Free (GF Free) media or insulin/IGF-1 media plus Akt or PKCλ/ζ inhibitors. (*p<0.05, ** p<0.01 vs. WT; ‡ p<0.05 vs. caPI3K; § p<0.01 vs. GF Free control; † p<0.05 vs. all other groups (GF Free) or vs. no inhibitor or Akt I following Insulin/IGF-1).

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