Reinis Vilskersts

Mildronate, an inhibitor of carnitine biosynthesis, induces an increase in gamma-butyrobetaine contents and cardioprotection in isolated rat heart infarction.

Abstract: 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.

Protective effects of mildronate in an experimental model of type 2 diabetes in Goto-Kakizaki rats

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.

Mildronate decreases carnitine availability and up-regulates glucose uptake and related gene expression in the mouse heart

AIMS l-carnitine has been shown to play a central role in both fat and carbohydrate metabolisms. This study investigated whether acute and long-term treatments with an l-carnitine biosynthesis inhibitor, mildronate (3-(2,2,2-trimethylhydrazinium) propionate), modulate glucose uptake. MAIN METHODS The effects of acute and long-term administration of mildronate at a dose of 200 mg/kg (i.p. daily for 20 days) were tested in mouse blood plasma and heart. KEY FINDINGS Acute administration of mildronate in vivo, or in vitro administration with perfusion buffer in isolated heart experiments, did not induce any effects on glucose blood concentration and uptake in the heart. Mildronate long-term treatment significantly decreased carnitine concentration in plasma and heart tissues, as well as increased the rate of insulin-stimulated glucose uptake by 35% and the expression of glucose transporter 4, hexokinase II, and insulin receptor proteins in mouse hearts. In addition, expression of both carnitine palmitoyltransferases IA and IB were significantly increased. Mildronate long-term treatment statistically significantly decreased fed state blood glucose from 6+/-0.2 to 5+/-0.1 mM, but did not affect plasma insulin and C-peptide levels. SIGNIFICANCE Our experiments demonstrate for the first time that long-term mildronate treatment decreases carnitine content in the mouse heart and leads to increased glucose uptake and glucose metabolism-related gene expression.

The heart is better protected against myocardial infarction in the fed state compared to the fasted state

OBJECTIVE A variety of calorie restriction diets and fasting regimens are popular among overweight people. However, starvation could result in unexpected cardiovascular effects. Therefore, it is necessary to evaluate the short-term effects of diets on cardiovascular function, energy metabolism and potential risk of heart damage in case of myocardial infarction. The objective of the present study was to investigate whether the increased level of glucose oxidation or reduction of fatty acid (FA) load in the fed state provides the basis for protection against myocardial infarction in an experimental rat model of ischemia-reperfusion. MATERIALS/METHODS We tested the effects of the availability of energy substrates and their metabolites on the heart functionality and energy metabolism under normoxic and ischemia-reperfusion conditions. RESULTS In a fasted state, the heart draws energy exclusively from FAs, whereas in a fed state, higher concentration of circulating insulin ensures a partial switch to glucose oxidation, while the load of FA on heart and mitochondria is reduced. Herein, we demonstrate that ischemic damage in hearts isolated from Wistar rats and diabetic Goto-Kakizaki rats is significantly lower in the fed state compared to the fasted state. CONCLUSIONS Present findings indicate that postprandial or fed-state physiology, which is characterised by insulin-activated glucose and lactate utilisation, is protective against myocardial infarction. Energy metabolism pattern in the heart is determined by insulin signalling and the availability of FAs. Overall, our study suggests that even overnight fasting could provoke and aggravate cardiovascular events and high-risk cardiovascular patients should avoid prolonged fasting periods.

Mildronate, a Regulator of Energy Metabolism, Reduces Atherosclerosis in apoE/LDLR–/– Mice

Background/Aims: Mildronate, an inhibitor of L-carnitine biosynthesis and transport, is used in clinics as a modulator of cellular energy metabolism and is a cardioprotective drug. L-Carnitine is a pivotal molecule in fatty acid oxidation pathways and its regulation in vasculature might be a promising approach for antiatherosclerotic treatment. This study was performed to evaluate the effects of mildronate treatment on the progression of atherosclerosis and the content of L-carnitine in the vascular wall. Methods: ApoE/LDLR–/– mice received mildronate at doses of 30 and 100 mg/kg for 4 months. Lipid profile was measured in plasma and atherosclerotic lesions were analyzed in whole aorta and aortic sinus. L-Carnitine concentration was assessed in rat aortic tissues after 2 weeks of treatment with mildronate at a dose of 100 mg/kg. Results: The chronic treatment with mildronate at a dose of 100 mg/kg significantly reduced the size of atherosclerotic plaques in the aortic roots and in the whole aorta, and slightly decreased the free cholesterol level. In addition, mildronate treatment decreased L-carnitine concentration in rat aortic tissues. Conclusions: Long-term mildronate treatment decreases L-carnitine content in aortic tissues and attenuates the development of atherosclerosis in apoE/LDLR–/– mice.

The Cardioprotective Effect of Mildronate is Diminished After Co-Treatment With l-Carnitine

Mildronate, an inhibitor of l-carnitine biosynthesis and uptake, is a cardioprotective drug whose mechanism of action is thought to rely on the changes in concentration of l-carnitine in heart tissue. In the present study, we compared the cardioprotective effect of mildronate (100 mg/kg) and a combination of mildronate and l-carnitine (100 + 100 mg/kg) administered for 14 days with respect to the observed changes in l-carnitine level and carnitine palmitoyltransferase I (CPT-I)-dependent fatty acid metabolism in the heart tissues. Concentrations of l-carnitine and its precursor γ-butyrobetaine (GBB) were measured by ultraperformance liquid chromatography with tandem mass spectrometry. In addition, mitochondrial respiration, activity of CPT-I, and expression of CPT-IA/B messenger RNA (mRNA) were measured. Isolated rat hearts were subjected to ischemia–reperfusion injury. Administration of mildronate induced a 69% decrease in l-carnitine concentration and a 6-fold increase in GBB concentration in the heart tissue as well as a 27% decrease in CPT-I-dependent mitochondrial respiration on palmitoyl-coenzyme A. In addition, mildronate treatment induced a significant reduction in infarct size and also diminished the ischemia-induced respiration stimulation by exogenous cytochrome c. Treatment with a combination had no significant impact on l-carnitine concentration, CPT-I-dependent mitochondrial respiration, and infarct size. Our results demonstrated that the mildronate-induced decrease in l-carnitine concentration, concomitant decrease in fatty acid transport, and maintenance of the intactness of outer mitochondrial membrane in heart mitochondria are the key mechanisms of action for the anti-infarction activity of mildronate.

Pharmacological effects of meldonium: Biochemical mechanisms and biomarkers of cardiometabolic activity.

Meldonium (mildronate; 3-(2,2,2-trimethylhydrazinium)propionate; THP; MET-88) is a clinically used cardioprotective drug, which mechanism of action is based on the regulation of energy metabolism pathways through l-carnitine lowering effect. l-Carnitine biosynthesis enzyme γ-butyrobetaine hydroxylase and carnitine/organic cation transporter type 2 (OCTN2) are the main known drug targets of meldonium, and through inhibition of these activities meldonium induces adaptive changes in the cellular energy homeostasis. Since l-carnitine is involved in the metabolism of fatty acids, the decline in its levels stimulates glucose metabolism and decreases concentrations of l-carnitine related metabolites, such as long-chain acylcarnitines and trimethylamine-N-oxide. Here, we briefly reviewed the pharmacological effects and mechanisms of meldonium in treatment of heart failure, myocardial infarction, arrhythmia, atherosclerosis and diabetes.

Effects of long-term mildronate treatment on cardiac and liver functions in rats.

Mildronate is a cardioprotective drug that improves cardiac function during ischaemia and functions by lowering l-carnitine concentration in body tissues and modulating myocardial energy metabolism. The aim of the present study was to characterise cardiovascular function and liver condition after long-term mildronate treatment in rats. In addition, changes in the plasma lipid profile, along with changes in the concentration of mildronate, l-carnitine and gamma-butyrobetaine were monitored in the rat tissues. Wistar rats were perorally treated daily with a mildronate dose of either 100, 200 or 400 mg/kg for 4, 8 or 12 weeks. The l-carnitine-lowering effect of mildronate was dose-dependent. However, the carnitine levels reached a plateau after about four weeks of treatment. During the additional weeks of treatment, the carnitine levels were not considerably changed. The obtained results provide evidence that even a high dose of mildronate does not alter cardiovascular parameters and the function of isolated rat hearts. Furthermore, the histological evaluation of liver tissue cryosections and measurement of biochemical markers of hepatic toxicity showed that all the measured values were within the normal reference range. Our results provide evidence that long-term mildronate administration induces significant changes in carnitine homeostasis, but it is not associated with cardiac impairment or disturbances in liver function.

Administration of L-carnitine and mildronate improves endothelial function and decreases mortality in hypertensive Dahl rats.

Hypertension is a well established risk factor for the development of cardiovascular diseases and increased mortality. This study was performed to investigate the effects of the administration of L-carnitine or mildronate, an inhibitor of L-carnitine biosynthesis, or their combination on the development of hypertension-related complications in Dahl salt-sensitive (DS) rats fed with a high salt diet. Male DS rats were fed laboratory chow containing 8% NaCl from 7 weeks of age. Experimental animals were divided into five groups and treated for 8 weeks with vehicle (water; n = 10), L-carnitine (100 mg/kg, n = 10), mildronate (100 mg/kg, n = 10) or a combination of L-carnitine and mildronate at the doses above (n = 10). During the experiment, control group animals continued to consume a diet with normal salt content. Administration of the combination significantly improved the survival rate for 50% of the population. None of the tested compounds or their combination influenced high salt intake-induced hypertension, while treatment with mildronate and the combination for 8 weeks significantly decreased resting heart rate by 12% and 10%, respectively. Feeding with high salt diet had no influence on systolic function of the heart, but it induced thickening of the ventricular walls and development of heart hypertrophy that was not improved by the administration of tested compounds. In addition, administration of the combination attenuated the development of endothelial dysfunction in isolated aortic rings. In conclusion, our results demonstrate that treatment with a combination of L-carnitine and mildronate is protective against hypertension-induced complications in an experimental model of salt-induced hypertension.

The anti-inflammatory and antinociceptive effects of NF-κB inhibitory guanidine derivative ME10092

The guanidine compound ME10092 (1-(3,4-dimethoxy-2-chlorobenzylideneamino)-guanidine) is known to possess anti-radical and anti-ischemic activity but its molecular targets have not been identified. This study investigated whether ME10092 regulates the nuclear factor kappa B (NF-kappaB)-mediated signal transduction in vivo. The effect of ME10092 treatment (1-100 pmol/mouse) on nuclear translocation of NF-kappaB, activation of expression of inflammatory mediators and production of nitric oxide were measured in the lipopolysaccharide (LPS)-induced brain inflammation model in mice in vivo. The antinociceptive activity of ME10092 was tested in the formalin-induced paw licking test. ME10092 dose-dependently inhibited LPS-induced nuclear translocation of NF-kappaB, transcription of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Electron paramagnetic resonance measurements showed that ME10092 inhibited the LPS-induced increase in nitric oxide content in mouse brain tissue in a dose-dependent manner. In the formalin-induced paw licking test, ME10092 (at the dose of 3mg/kg, p.o. twice daily for eight days) significantly reduced nociceptive response. In conclusion, above results indicate that ME10092 inhibits NF-kappaB activation and suppresses the up-regulation of inflammatory mediators in experimental models in vivo.