Increased plasma concentration of trimethylamine N-oxide (TMAO), a proatherogenic metabolite, has been linked to adverse cardiovascular outcomes; however, it remains unclear whether TMAO is a biomarker or whether it induces direct detrimental cardiovascular effects. Because altered cardiac energy metabolism and mitochondrial dysfunction play crucial roles in the development of cardiovascular diseases, we hypothesized that increased TMAO concentration may alter mitochondrial energy metabolism. The aim of the present study was to determine the effects of TMAO on cardiac mitochondrial energy metabolism. Acute exposure of cardiac fibers to TMAO decreased LEAK (substrate-dependent) and OXPHOS (oxidative phosphorylation-dependent) mitochondrial respiration with pyruvate and impaired substrate flux via pyruvate dehydrogenase. The administration of TMAO at a dose of 120mg/kg for 8 weeks increased TMAO concentration in plasma and cardiac tissues 22-23 times to about 15μM and 11nmol/g, respectively. Long-term TMAO administration decreased mitochondrial LEAK state respiration with pyruvate by 30% without affecting OXPHOS state respiration. However, no significant changes in mitochondrial reactive oxygen species production were observed after acute exposure of cardiac fibers to TMAO under physiological conditions. In addition, both long-term TMAO administration and acute exposure to TMAO decreased respiration with palmitoyl-CoA indicating impaired β-oxidation. Taken together, our results demonstrate that increased TMAO concentration impairs pyruvate and fatty acid oxidation in cardiac mitochondria. Thus, the accumulation of TMAO in cardiac tissues leads to disturbances in energy metabolism that can increase the severity of cardiovascular events.

The accumulation of long-chain fatty acids (FAs) and their CoA and carnitine esters is observed in the ischaemic myocardium after acute ischaemia/reperfusion. The aim of the present study was to identify harmful FA intermediates and their detrimental mechanisms of action in mitochondria and the ischaemic myocardium. In the present study, we found that the long-chain acyl-CoA and acylcarnitine content is increased in mitochondria isolated from an ischaemic area of the myocardium. In analysing the FA derivative content, we discovered that long-chain acylcarnitines, but not acyl-CoAs, accumulate at concentrations that are harmful to mitochondria. Acylcarnitine accumulation in the mitochondrial intermembrane space is a result of increased carnitine palmitoyltransferase 1 (CPT1) and decreased carnitine palmitoyltransferase 2 (CPT2) activity in ischaemic myocardium and it leads to inhibition of oxidative phosphorylation, which in turn induces mitochondrial membrane hyperpolarization and stimulates the production of reactive oxygen species (ROS) in cardiac mitochondria. Thanks to protection mediated by acyl-CoA-binding protein (ACBP), the heart is much better guarded against the damaging effects of acyl-CoAs than against acylcarnitines. Supplementation of perfusion buffer with palmitoylcarnitine (PC) before occlusion resulted in a 2-fold increase in the acylcarnitine content of the heart and increased the infarct size (IS) by 33%. A pharmacologically induced decrease in the mitochondrial acylcarnitine content reduced the IS by 44%. Long-chain acylcarnitines are harmful FA intermediates, accumulating in ischaemic heart mitochondria and inducing inhibition of oxidative phosphorylation. Therefore, decreasing the acylcarnitine content via cardioprotective drugs may represent a novel treatment strategy.

The inhibition of gamma-butyrobetaine (GBB) hydroxylase, a key enzyme in the biosynthesis of carnitine, contributes to lay ground for the cardioprotective mechanism of action of mildronate. By inhibiting the biosynthesis of carnitine, mildronate is supposed to induce the accumulation of GBB, a substrate of GBB hydroxylase. This study describes the changes in content of carnitine and GBB in rat plasma and heart tissues during long-term (28 days) treatment of mildronate [i.p. (intraperitoneal) 100 mg/kg/daily]. Obtained data show that in concert with a decrease in carnitine concentration, the administration of mildronate caused a significant increase in GBB concentration. We detected about a 5-fold increase in GBB contents in the plasma and brain and a 7-fold increase in the heart. In addition, we tested the cardioprotective effect of mildronate in isolated rat heart infarction model after 3, 7, and 14 days of administration. We found a statistically significant decrease in necrotic area of infarcted rat hearts after 14 days of treatment with mildronate. The cardioprotective effect of mildronate correlated with an increase in GBB contents. In conclusion, our study, for the first time, provides experimental evidence that the long-term administration of mildronate not only decreases free carnitine concentration, but also causes a significant increase in GBB concentration, which correlates with the cardioprotection of mildronate.

Background and purpose:  Mildronate [3‐(2,2,2‐trimethylhydrazinium) propionate] is an anti‐ischaemic drug whose mechanism of action is based on its inhibition of L‐carnitine biosynthesis and uptake. As L‐carnitine plays a pivotal role in the balanced metabolism of fatty acids and carbohydrates, this study was carried out to investigate whether long‐term mildronate treatment could influence glucose levels and prevent diabetic complications in an experimental model of type 2 diabetes in Goto‐Kakizaki (GK) rats.Experimental approach:  GK rats were treated orally with mildronate at doses of 100 and 200 mg·kg⁻¹ daily for 8 weeks. Plasma metabolites reflecting glucose and lipids, as well as fructosamine and β‐hydroxybutyrate, were assessed. L‐carnitine concentrations were measured by ultra performance liquid chromatography with tandem mass spectrometry. An isolated rat heart ischaemia‐reperfusion model was used to investigate possible cardioprotective effects. Pain sensitivity was measured with a tail‐flick latency test.Key results:  Mildronate treatment significantly decreased L‐carnitine concentrations in rat plasma and gradually decreased both the fed‐ and fasted‐state blood glucose. Mildronate strongly inhibited fructosamine accumulation and loss of pain sensitivity and also ameliorated the enhanced contractile responsiveness of GK rat aortic rings to phenylephrine. In addition, in mildronate‐treated hearts, the necrosis zone following coronary occlusion was significantly decreased by 30%.Conclusions and implications:  These results demonstrate for the first time that in GK rats, an experimental model of type 2 diabetes, mildronate decreased L‐carnitine contents and exhibited cardioprotective effects, decreased blood glucose concentrations and prevented the loss of pain sensitivity. These findings indicate that mildronate treatment could be beneficial in diabetes patients with cardiovascular problems.