Dietary fat supply to failing hearts determines dynamic lipid signaling for nuclear receptor activation and oxidation of stored triglyceride

Abstract

Background: Intramyocardial triglyceride (TG) turnover is reduced in pressure-overloaded, failing hearts, limiting the availability of this rich source of long-chain fatty acids for mitochondrial β-oxidation and nuclear receptor activation. This study explored 2 major dietary fats, palmitate and oleate, in supporting endogenous TG dynamics and peroxisome proliferator-activated receptor-α activation in sham-operated (SHAM) and hypertrophied (transverse aortic constriction [TAC]) rat hearts.

Methods and results: Isolated SHAM and TAC hearts were provided media containing carbohydrate with either (13)C-palmitate or (13)C-oleate for dynamic (13)C nuclear magnetic resonance spectroscopy and end point liquid chromatography/mass spectrometry of TG dynamics. With palmitate, TAC hearts contained 48% less TG versus SHAM (P=0.0003), whereas oleate maintained elevated TG in TAC, similar to SHAM. TG turnover in TAC was greatly reduced with palmitate (TAC, 46.7±12.2 nmol/g dry weight per min; SHAM, 84.3±4.9; P=0.0212), as was β-oxidation of TG. Oleate elevated TG turnover in both TAC (140.4±11.2) and SHAM (143.9±15.6), restoring TG oxidation in TAC. Peroxisome proliferator-activated receptor-α target gene transcripts were reduced by 70% in TAC with palmitate, whereas oleate induced normal transcript levels. Additionally, mRNA levels for peroxisome proliferator-activated receptor-γ-coactivator-1α and peroxisome proliferator-activated receptor-γ-coactivator-1β in TAC hearts were maintained by oleate. With these metabolic effects, oleate also supported a 25% improvement in contractility over palmitate with TAC (P=0.0202).

Conclusions: The findings link reduced intracellular lipid storage dynamics to impaired peroxisome proliferator-activated receptor-α signaling and contractility in diseased hearts, consistent with a rate-dependent lipolytic activation of peroxisome proliferator-activated receptor-α. In decompensated hearts, oleate may serve as a beneficial energy substrate versus palmitate by upregulating TG dynamics and nuclear receptor signaling.

Keywords: fatty acids; genes; hypertrophy; lipids; metabolism.

Figure 1

Contractile function of sham-operated (SHAM) and hypertrophic (TAC) isolated perfused hearts. TAC induced an increase in heart weight (A), heart weight-to-body weight ratio (B), and heart weight-to-tibia length ratio (C) 12 weeks after surgery. Left ventricular (LV) function (D–G). Rate-pressure product (RPP) (D), an index of cardiac work output, and left ventricular developed pressure (LVDP) (E) were similarly reduced compared to SHAM in TAC hearts metabolizing oleate or palmitate. Oleate maintained LV contractility and relaxation (+ and −dP/dt, respectively) in TAC hearts, while TAC hearts metabolizing palmitate demonstrated impaired contractility and relaxation both reduced versus SHAM and TAC oleate groups (F,G). White bar, SHAM; black bar, TAC. (n=13 for all groups). Error bars indicate mean ± SEM. *P<0.05 versus SHAM, †P<0.05 versus TAC Oleate.

Figure 2

Incorporation rates of ¹³C-oleate and ¹³C-palmitate into triglyceride (TG). (A) Representative, selected ¹³C NMR spectra (from bottom to top, 2 min acquisition each) from an sham-operated (SHAM) heart perfused with ¹³C-oleate. Signal at chemical shift 30.5 parts per million (ppm) reflects the ¹³C-enriched methylene (-CH₂-) groups as ¹³C-enriched long-chain fatty acid (LCFA) is esterified into TG. Signal at chemical shifts 56, 34.6, and 28.3 ppm reflects ¹³C enrichment of glutamate at the 2-, 4-, and 3-carbon positions. (B) TG enrichment profiles reflect incorporation of ¹³C-enriched LCFA throughout perfusion. White circle, SHAM oleate; black circle, TAC oleate; white square, SHAM palmitate; black square, TAC palmitate. (n=6 for all groups). Error bars indicate mean ± SEM.

Figure 3

Myocardial triglyceride (TG) turnover and content, acyl intermediates, and related protein levels of enzymes for TG dynamics. (A) Oleate supported elevated turnover in normal (SHAM) hearts and attenuated the drop in turnover in hypertrophied (TAC) hearts versus palmitate (n=6 for each group). (B) Palmitate failed to maintain normal levels of TG in TAC hearts, while oleate supported normal TG similar SHAM hearts (n=6 for each group). (C) Oleate induced greater ¹³C enrichment of TG than palmitate in both SHAM and TAC (n=6 for each group). (D) TAC hearts supplied palmitate contained lower DAG, versus SHAM (n=3 for each group). (E) Palmitate elevated C16 ceramide in TAC hearts, versus SHAM Palmitate and TAC Oleate (n=5 for each group). (F–H) Western Blot analysis of rate-limiting enzymes of TG synthesis, diglyceride acyltransferase 1 (DGAT1), and hydrolysis, adipose triglyceride lipase (ATGL) from whole tissue lysate. Calsequestrin (CALSEQ) served as a loading control (n=3 for each group; P=0.14 versus all other groups). (I) Mean time constants characterizing the saturable exponential phase of ¹³C TG enrichment (n=6 for each group). (J) Western Blot analysis of CD36 from isolated total cardiac membranes. Na⁺/K⁺ ATPase served as a loading control (n=3 for each group). White bar, SHAM; black bar, TAC. Error bars indicate mean ± SEM. *P<0.05, versus SHAM Oleate; †P<0.05, versus TAC Oleate; ‡P<0.05, versus SHAM Palmitate; “n.s.”, not statistically significant.

Figure 4

Expression of peroxisome proliferator-activated receptor-α (PPAR-α) target genes and diglyceride acyltransferase 1 (DGAT1), adipose triglyceride lipase (ATGL), and PPAR-ɣ coactivator-1α (PGC-1α) and PGC-1β genes in perfused sham-operated (SHAM) and hypertrophied (TAC) heart tissue. (A,B) Western blot analysis detected decreased protein levels of PPAR-α in TAC hearts (n=3 for each group). (C) Transcript levels of select PPAR-α target genes (Cpt1b, Pdhk4, Acadm) were decreased in TAC hearts metabolizing palmitate. mRNA levels of genes encoding DGAT1 (Dgat1), ATGL (Atgl), PGC-1α (Ppargc1a) and PGC-1β (Ppargc1b) were reduced in TAC hearts metabolizing palmitate, while oleate maintained transcript levels in TAC hearts. (n=5 for each group) White bar, SHAM oleate; black bar, TAC oleate; light gray bar, SHAM palmitate; dark gray bar, TAC palmitate. Error bars indicate mean ± SEM. *P<0.05 versus all other groups, ** P<0.01 versus all other groups.

Figure 5

Expression of peroxisome proliferator-activated receptor-α (PPAR-α) target genes and diglyceride acyltransferase 1 (DGAT1), adipose triglyceride lipase (ATGL), and PPAR-ɣ coactivator-1α (PGC-1α) and PGC-1β genes in sham-operated (SHAM) and hypertrophied (TAC) hearts perfused with a 1:1 mix of oleate and palmitate. (A) Perfusing TAC hearts with 1:1 mix of oleate:palmitate supported transcript levels of Cpt1b, Pdhk4, Acadm and Dgat1 that were mid-range of values observed in TAC oleate and TAC palmitate (see Figure 4C), while levels Atgl, Ppargc1a, and Ppargc1b were the same as SHAM (n=4 for SHAM Mix, white bar; n=5 for TAC Mix, black bar). (B) TG content in TAC hearts perfused with oleate (white bar), oleate:palmitate mix (gray bar), and palmitate (black bar) (n=5–6 for each group). *P<0.05 versus SHAM Mix, **P<0.01 versus SHAM Mix. Error bars indicate mean ± SEM.

Figure 6

Tricarboxylic acid (TCA) cycle flux and substrate contribution to oxidative ATP production. (A) TCA cycle flux, as determined from kinetic analysis of ¹³C enrichment of glutamate. (n=5 for each group). (B) In vitro ¹³C NMR spectra obtained from heart tissue acid extracts provided fractional contribution of each substrate to acetyl Co-A formation and mitochondrial ATP production. Carbohydrate oxidation (white bar) includes exogenous glucose, lactate, and endogenous glycogen; exogenous long-chain fatty acid (LCFA) oxidation (black bar); oxidation of endogenous triglyceride (TG, gray bar). Elevated TG turnover by oleate corresponded with increased contribution of TG to oxidative ATP production in both sham-operated (SHAM) and hypertrophied (TAC) hearts (n=3 for each group). (C–E) Western Blot analysis of rate-limiting enzyme carnitine palmitoyltransferase 1 (CPT1) catalyzing entry of activated LCFA-CoA into mitochondria for β-oxidation. Muscle isoform CPT1b was unchanged, while CPT1a was increased in TAC groups (n=3 for each group). White bar, SHAM; black bar, TAC. Error bars indicate mean ± SEM. *P<0.05 versus SHAM, †P<0.01 versus SHAM Palmitate, ‡P<0.01 versus TAC Palmitate.

Figure 7

Summary scheme depicting the proposed rate-dependence of peroxisome proliferator-activated receptor-α (PPAR-α) activation and triglyceride (TG) oxidation on TG turnover. (A) In normal hearts, oleate supports a faster rate of TG turnover than palmitate, resulting in increased oxidation of TG by mitochondria. At baseline, either substrate supports sufficient TG turnover to maintain PPAR-α signaling. (B) In failing hearts, TG turnover supported by palmitate is reduced; TG becomes unavailable as a source of substrate for PPAR-α activation and mitochondrial ATP production. Oleate maintains normal TG content and turnover in failing hearts, with normal PPAR-α target gene expression and oxidation of TG.