Preservation of Acyl Coenzyme A Attenuates Pathological and Metabolic Cardiac Remodeling Through Selective Lipid Trafficking

Abstract

Background: Metabolic remodeling in heart failure contributes to dysfunctional lipid trafficking and lipotoxicity. Acyl coenzyme A synthetase-1 (ACSL1) facilitates long-chain fatty acid (LCFA) uptake and activation with coenzyme A (CoA), mediating the fate of LCFA. The authors tested whether cardiac ACSL1 overexpression aids LCFA oxidation and reduces lipotoxicity under pathological stress of transverse aortic constriction (TAC).

Methods: Mice with cardiac restricted ACSL1 overexpression (MHC-ACSL1) underwent TAC or sham surgery followed by serial in vivo echocardiography for 14 weeks. At the decompensated stage of hypertrophy, isolated hearts were perfused with ¹³C LCFA during dynamic-mode ¹³C nuclear magnetic resonance followed by in vitro nuclear magnetic resonance and mass spectrometry analysis to assess intramyocardial lipid trafficking. In parallel, acyl CoA was measured in tissue obtained from heart failure patients pre- and postleft ventricular device implantation plus matched controls.

Results: TAC-induced cardiac hypertrophy and dysfunction was mitigated in MHC-ACSL1 hearts compared with nontransgenic hearts. At 14 weeks, TAC increased heart weight to tibia length by 46% in nontransgenic mice, but only 26% in MHC-ACSL1 mice, whereas ACSL1 mice retained greater ejection fraction (ACSL1 TAC: 65.8±7.5%; nontransgenic TAC: 45.9±7.3) and improvement in diastolic E/E'. Functional improvements were mediated by ACSL1 changes to cardiac LCFA trafficking. ACSL1 accelerated LCFA uptake, preventing C16 acyl CoA loss post-TAC. Long-chain acyl CoA was similarly reduced in human failing myocardium and restored to control levels by mechanical unloading. ACSL1 trafficked LCFA into ceramides without normalizing the reduced triglyceride storage in TAC. ACSL1 prevented de novo synthesis of cardiotoxic C16- and C24-, and C24:1 ceramides and increased potentially cardioprotective C20- and C22-ceramides post-TAC. ACLS1 overexpression activated AMP activated protein kinase at baseline, but during TAC, prevented the reduced LCFA oxidation in hypertrophic hearts and normalized energy state (phosphocreatine:ATP) and consequently, AMP activated protein kinase activation.

Conclusions: This is the first demonstration of reduced acyl CoA in failing hearts of humans and mice, and suggests possible mechanisms for maintaining mitochondrial oxidative energy metabolism by restoring long-chain acyl CoA through ASCL1 activation and mechanical unloading. By mitigating cardiac lipotoxicity, via redirected LCFA trafficking to ceramides, and restoring acyl CoA, ACSL1 delayed progressive cardiac remodeling and failure.

Keywords: acyl coenzyme A; ceramides; coenzyme A ligases; fatty acids; heart failure.

Figure 1.. Transverse aortic constriction (TAC)-induced cardiac hypertrophy is mitigated by ACSL1 overexpression.

(A) Western blot results (B) and densitometry quantification normalized to Calseq loading control relative to NTG sham group (n=5). Cardiac ACSL1 protein expression is unaffected by TAC. (C) Heart weight:tibia length increased in NTG TAC mice and MHC-ACSL1 TAC mice in response to 14 weeks of TAC, though the relative increase in hypertrophy vs shams was attenuated in MHC-ACSL1 TAC (n = 13–17). *p<0.05, ***p<0.001, ****p<0.0001. (D) LV posterior wall thickness in diastole, from echocardiography. At 14 weeks TAC, only NTG TAC mice had increased LV wall thickness at diastole vs respective shams (n = 3–7 mice at each time point). **p<0.01 vs NTG TAC week 14, ‡p<0.01 vs NTG TAC week 4; vs MHC-ACSL1 TAC week 4: @@ p<0.01.

Figure 2.. ACSL1 protects against declines in cardiac function during 14 weeks of TAC.

(A) Factional shortening (FS) was higher in MHC-ACSL1 TAC vs NTG TAC mice after 14 weeks of TAC. Further, FS declined in NTG TAC mice through the 14 weeks, whereas week 14 FS was similar to week 1 baseline in MHC-ACSL1 TAC mice. Though, FS was not completely normalized in MHC-ACSL1 TAC vs respective shams. (B) Increased left ventricle internal diameter (LVEDD) at end-systole, indicating impaired blood ejection, was present in NTG TAC but not MHC-ACSL1 TAC hearts from 14 weeks of TAC (C) LVEDD at end-diastole was similar in all groups consistent with TAC modeling early decompensated hypertrophy (n = 4–7 mice at each time point for 2A-C). (D) Week 14 E/A ratio was elevated in NTG TAC vs NTG sham and MHC-ACSL1 TAC vs MHC-ACSL1 sham, respectively. However, the increase over time from week 1 to week 14 in E/A was significant only in NTG TAC and not MHC-ACSL1 TAC. (E) The E/E’ ratio over 14 weeks increased relative to week 1 baseline only in NTG TAC and not MHC-ACSL1 TAC. ACSL1 provided moderate protection of diastolic function. (n = 3–7 mice at each time point for 2D-F). Comparisons denoted vs NTG week 14: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; vs NTG TAC week 4: †p<0.05; vs MHC-ACSL1 TAC week 4: @ p<0.05; vs MHC-ACSL1 TAC week 14: S p<0.05, § p<0.01.

Figure 3.. ACSL1 overexpression sustains myocardial bioenergetic reserve and long-chain fatty acid oxidation to mitochondrial acetyl CoA after pathological stress from TAC.

Representative 31P NMR spectra during isolated heart perfusion from (A) NTG sham, (B) NTG TAC, (C) MHC-ACSL1 sham, and (D) MHC-ACSL1 TAC. with corresponding PCr:ATP for that spectrum. PCr, α-, β-, and γ-ATP phosphate nuclei, and inorganic phosphate (Pi, both intra- and extracellular) are denoted. Elevated Pi signal in sham hearts results from higher buffer volume surrounding the smaller heart vs larger TAC hearts. (E) Mean PCr:ATP. TAC decreased PCr:ATP in NTG TAC hearts, but not in MHC-ACSL1 TAC hearts (n = 6–10). (F) The fractional contribution of ¹³C palmitate to acetyl CoA (Fc) decreased in NTG TAC hearts, but ACSL1 prevented this decrease (n = 6–7). **p<0.01, ***p<0.001.

Figure 4.. ACSL1 overexpression does not restore depleted myocardial TG stores and has only a moderate effect on TG turnover in TAC hearts.

(A) TG content of intramyocardial TG. Note TAC decreased TG pool TG content to the same magnitude in both NTG and ACSL1 hearts compared to shams. (B) Turnover rates of intramyocardial TG pool. Turnover rates were significantly reduced with TAC in only NTG TAC vs sham. However, turnover rates in MHC-ACSL1 TAC hearts did not reach the levels of NTG sham hearts (n = 5–7). *p<0.05; **p<0.01.

Figure 5.. ACSL1-accelerated uptake kinetics and incorporation rates of ¹³C LCFA into myocardial triglyceride (TG) are unaffected by TAC.

(A) Representative dynamic-mode ¹³C NMR of an individual isolated perfused heart. Each spectrum is signal-averaged over 2 minutes. (B) Incorporation of ¹³C palmitate in TG across the entire ¹³C perfusion period. (C) Early, saturable component from (B) normalized to overall level of enrichment. (D) The time constant (τ) of the exponential phase was lower in ACSL1 hearts, indicating faster LCFA trans-sarcolemmal uptake kinetics. TAC did not affect τ and MHC-ACSL1 TAC hearts maintained a low τ (n = 4–8). *p<0.05 and **p<0.01.

Figure 6.. Myocardial acyl CoA species generated from exogenous fatty acids are depleted in hypertrophy and heart failure and restored by mechanical unloading in humans and ACSL1 in mice.

(A) Heart failure patients had lower myocardial acyl CoA compared to non-diseased controls that was restored to healthy control levels by LVAD-mechanical unloading. (B) Analysis of myocardial acyl CoA species in patients revealed all long-chain acyl CoA to be depleted in failing hearts and C18:2 and C18:1 are restored by LVADs (n-values for 6A-C: control = 5, pre-LVAD and post-LVAD = 10). (C) Total myocardial acyl CoA (¹²C and ¹³C species) increased in response to ACSL1 overexpression in sham and TAC. (D) Acyl CoA species (¹²C and ¹³C species) distribution. TAC decreased C16 acyl CoA content. Note ACSL1 overexpression restored depleted C16 species in TAC hearts. (E) ¹³C acyl CoA content. ¹³C acyl CoA increased in both ACSL1 groups. (F) The ¹³C acyl CoA species distribution. The ¹³C16 acyl CoA decreased in response to TAC and was restored by ACSL1 overexpression. (G) ¹³C enrichment of acyl CoA was unaffected (n = 4–5 for 6C-G). **p<0.01, ***

Figure 7.. Activation of AMPK by phosphorylation is influenced by ACSL1 expression and TAC.

(A) Western blot results (B) and densitometry quantification normalized to Calseq loading control relative to NTG sham group (n=3). **p<0.01, †p=0.052, ‡p=0.097.

Figure 8.. Myocardial ceramide profile is negatively altered by TAC and improved with ACSL1 overexpression.

(A) Total myocardial ceramide species (¹²C and ¹³C) in perfused hearts. Ceramide increased in NTG TAC vs sham hearts, but not in MHC-ACSL1 TAC vs sham. (B) Ceramide species distribution according to R-chain length. Note, while total ceramide was elevated in NTG TAC, MHC-ACSL1 TAC, and MHC-ACSL1 sham, ceramide profile was profoundly different. C16, C24:1, and C24 ceramides increased in NTG TAC, but not MHC-ACSL1 TAC. C20 and C22 ceramides were elevated in both ACSL1 groups vs corresponding NTG groups. (C) ¹³C was detected in all ceramide components of C16 ceramide, demonstrating de novo synthesis from ¹³C palmitate. (D) Total ¹³C16 ceramide content (n= 4–7). *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001.