Introduction: The normal heart utilizes fatty acids (FA; by ~60-80%) and glucose (by ~20-30%) for ATP production and can switch dynamically between substrates depending on their availability. FA are imported into mitochondria via the carnitine shuttle, which entails the formation of acylcarnitine (AC) intermediates. In patients with myocardial infarction or heart failure, increased levels of long-chain ACs, such as oleoyl-AC (C18:1)- and stearoyl-AC (C18:0), predict adverse outcomes. Long-chain ACs were reported to exert an inhibitory effect on mitochondrial oxidative metabolism.Therefore, we investigated how AC C18:1 and C18:0 regulate substrate utilization and respiration in murine and human cardiac mitochondria and isolated murine cardiac myocytes.  

Results: In isolated murine cardiac mitochondria supplied with glutamate/malate (G/M), addition of 12.5 µM C18:1 in state 3 (i.e., after ADP addition) acutely stimulated respiration, whereas state 3 respiration supported by pyruvate and malate (P/M) was higher than with G/M and could not be further increased by addition of 12.5 µM C18:1. However, addition of 25 µM C18:1 inhibited respiration and dissipated membrane potential independent of the substrates used. Similarly, C18:1 concentration-dependently inhibited P/M respiration of mitochondria isolated from human left atrial appendages.Inhibition was reversible after wash-out, indicating that ACs do not irreversibly damage mitochondrial membranes. The CoA esters of AC C18:1 and C18:0 had an even more potent inhibition on state 3 respiration compared to their respective ACs, indicating that the carnitine moiety is not necessary for this effect. Addition of 25 µM AC C18:1 to murine cardiac mitochondria respiring on P/M in state 3 led to a modest decrease in NAD(P)H autofluorescence, reflecting oxidation of the NAD(P)H pool. When inhibiting upstream pyruvate dehydrogenase and α-ketoglutarate dehydrogenase with the compound CPI-613, the effects on NAD(P)H redox state were more pronounced than with ACs, and H₂O₂ emission strongly increased (presumably due to depletion of NADPH), while this was not the case with C18:1.

In isolated murine cardiac myocytes (C57BL/6N) supplied with glucose and pyruvate and loaded with Indo-1 AM (to determine cytosolic Ca²⁺) and TMRM (to determine the mitochondrial membrane potential, ΔΨ_m), C18:1 and C18:0 induced cytosolic Ca²⁺ overload and cellular arrhythmias in a concentration-dependent fashion, with a threshold between 1 and 5 µM, respectively. Cytosolic Ca²⁺ overload and arrhythmias preceded dissipation of ΔΨ_m and cell death.

Conclusions: C18:1 stimulated respiration at low to intermediate concentrations, but blocked it at higher concentrations in murine ventricular and human atrial mitochondria. The blockade in substrate utilization likely occurred downstream of the α-ketoglutarate dehydrogenase, where glutamate enters the Krebs cycle and the respiratory chain, but unlikely within the chain itself. At respiration-blocking concentrations, C18:1 and C18:0 induce cytosolic Ca²⁺ overload, arrhythmias, dissipation of ΔΨ_m and cell death in cardiac myocytes. This may contribute to adverse outcomes and arrhythmias in patients with cardiovascular diseases in whom accumulation of these ACs is observed.