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

Abstract

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.

Keywords

mildronate carnitine palmitoyltransferase I mitochondrial respiration cardioprotection

Introduction

l-Carnitine is a cofactor required for the transport of long-chain fatty acids (FAs) into the mitochondria and for the regulation of cellular energy metabolism.^1,2 Mildronate (3-[2,2,2-trimethylhydrazinium] propionate) is a pharmacological agent that decreases the level of l-carnitine in plasma and in heart tissue³ by acting as an inhibitor of γ-butyrobetaine (GBB) hydroxylase⁴ and as a competitive inhibitor of type 2 organic cation/carnitine transporter (OCTN2).⁵ The mildronate-induced decrease in the tissue concentration of l-carnitine causes a decrease in FA oxidation⁴ and facilitates glucose utilization,⁶ which prevents ischemia–reperfusion injury in healthy rat hearts in vitro,⁷ in vivo,⁸ and in a type 2 diabetic Goto-Kakizaki rat model.⁹ Previous studies suggest that the angio- and cardioprotective effects of mildronate depend on its ability to inhibit l-carnitine biosynthesis and thus decrease the levels of l-carnitine in plasma and heart tissue and increase in GBB concentration.^7,9,10

The present study was undertaken to delineate the importance of mildronate-induced changes in l-carnitine and GBB concentrations for the cardioprotective effect of mildronate. The administration of a combination of mildronate and l-carnitine is expected to prevent the mildronate-induced decrease in l-carnitine concentration in tissue and to maintain the mildronate treatment-induced increase in GBB concentration.¹¹ To compare the anti-infarction activity of mildronate and a combination of mildronate and l-carnitine in rats, isolated hearts were subjected to ischemia–reperfusion injury, after a 14-day treatment. We also determined the activity of carnitine palmitoyltransferase I (CPT-I), a crucial enzyme for the transport of long-chain FA into the mitochondria,¹² and the activity of carnitine acetyltransferase (CrAT), an enzyme that modulates the acetylcoenzyme A/free coenzyme A ratio.² The messenger RNA (mRNA) levels of CPT-IA and CPT-IB in heart tissues and mitochondrial respiration were determined. The effect of cytochrome c on mitochondrial respiration to evaluate the intactness of outer mitochondrial membrane (OMM)¹³ was determined. The present study for the first time indicates that the anti-infarction effect of mildronate is diminished in the presence of added l-carnitine.

Materials and Methods

Materials

Mildronate (3-[2,2,2-trimethylhydrazinium] propionate dihydrate) was obtained from JSC Grindeks (Riga, Latvia). l-Carnitine was purchased from Lonza (Basel, Switzerland). Potassium dihydrogenphosphate (KH₂PO₄), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), imidazole, taurine, 2-(N-morpholino)ethanesulfonic acid (MES), saponin, phosphocreatine, ethylene glycol tetraacetic acid (EGTA), sodium chloride, sodium hydrogenphosphate, calcium chloride dihydrate, potassium chloride, sodium bicarbonate, sodium acetate, and magnesium chloride hexahydrate were purchased from Acros Organics (Geel, Belgium). EDTA, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Igepal CA 630, pyruvic acid, malic acid, dithiothreitol, adenosine triphosphate (ATP) sodium salt, adenosine diphosphate (ADP), cytochrome c, Type IX glycogen, and amyloglucosidase were purchased from Sigma (Schnelldorf, Germany). Acetyl-CoA and palmitoyl-CoA were purchased from Larodan (Malmö, Sweden). Bovine serum albumin (BSA) was purchased from Europa Bioproducts Ltd (Cambridge, UK). Methanol and acetonitrile were purchased from Merck (Darmstadt, Germany). Sodium pentobarbital (Narkodorm-n) solution was purchased from Alvetra (Vienna, Austria). Heparin sodium was purchased from Panpharma (Fougeres, France).

Animals and Treatments

A total of 75 male Wistar rats weighing 200 to 240 g were obtained from the Laboratory of Experimental Animals, Riga Stradins University (Riga, Latvia) and housed under standard conditions (21-23°C, 12-hour light/dark cycle, relative humidity 45%-65%) with unlimited access to food (R3 diet, Lactamin AB, Kimstad, Sweden) and water. Rats were adapted to these conditions for 2 weeks prior to the treatment.

Animals were randomly separated into 3 experimental groups and given daily oral doses of water (control rats, n = 29), 100 mg/kg mildronate (n = 25) or a combination of mildronate and l-carnitine (100 and 100 mg/kg, n = 21) for 2 weeks. Animals of each experimental group were randomly assigned to either myocardial infarction or metabolic analysis.

The experimental procedures were carried out in accordance with the guidelines of the European Community and local laws and policies, and the procedures were approved by the Latvian Animal Protection Ethical Committee of the Food and Veterinary Service, Riga, Latvia. All experiments were performed in a blinded manner.

Ten animals from each group were used for the isolated heart infarction study. Another 5 animals from each group were sacrificed at the end of the 14-day treatment, and aliquots of heart tissues of these animals were frozen in liquid nitrogen and stored at −80°C for analysis (lipid profile, glycogen concentration, and concentrations of carnitine, GBB, and mildronate) at a later time. Additional aliquots of the same tissues were immediately used for the isolation of mitochondria for the determination of CPT-I and CrAT activity. Another 6 animals from each group were used for the isolation of mitochondria for the determination of mitochondrial respiration. Another 4 animals from the control and mildronate-treated groups were used to determine the ischemia-induced respiration stimulation by exogenous cytochrome c in saponin-permeabilized cardiac left ventricular fibers isolated from ischemic hearts.

Determination of l-Carnitine, GBB, and Mildronate Concentrations

Determination of l-carnitine, GBB, and mildronate concentrations in the heart homogenate and blood plasma samples was performed by ultraperformance liquid chromatography–tandem mass spectrometry using a positive ion electrospray mode, as described previously.¹⁴ l-Carnitine, GBB, and mildronate concentrations were determined in the hearts not used in the infarction experiments. Nonfasting blood samples were collected (n = 5 per group) from tail veins; samples were centrifuged (1050g) and the plasma was stored at −80°C for analysis at a later time.

Isolated Rat Heart Infarction Study and Hemodynamic Measurements

The infarction study was performed according to the Langendorff technique as described previously.⁷ In brief, rats were anesthetized with sodium pentobarbital (60 mg/kg intraperitoneally [ip]), and heparin (1 IU/g) was administered concomitantly. Hearts were excised and retrogradely perfused with oxygenated Krebs-Henseleit buffer, via the aorta at a constant pressure of 60 mm Hg. Water–ethanol mixture (1:1)-filled balloon (polyethylene wrapping film) connected to a physiological pressure transducer (ADInstruments, Chalgrove, UK) was inserted into the left ventricle, and the baseline end-diastolic pressure was set at 5 to 10 mm Hg. Heart rate (HR), left-ventricle developed pressure (LVDP), contractility, and relaxation were recorded continuously using PowerLab8/30 system from ADInstruments. Coronary flow was measured using an ultrasound flow detector system connected to PowerLab8/30. A 4-0 coated, braided silk suture (Sofsilk; Syneture, Dublin, Ireland) was passed under the left anterior descending coronary artery (LAD) and threaded through a small plastic tube to permit reversible occlusion of the coronary artery. Hearts were adapted for 20 minutes and occlusion was performed for 60 minutes by constricting threads through a plastic tube. Successful occlusion was confirmed by a decrease in coronary flow of about 40%.^9,15 Reperfusion was achieved by releasing the threads. At the end of the 150-minute reperfusion, the LAD was reoccluded and the nonrisk zone was stained with 0.1% methylene blue solution in Krebs-Henseleit buffer infused via the aortic root. Hearts were sectioned transversely from the apex to the base into 4 slices of 2-mm thickness and incubated in 1% triphenyl-tetrazolium chloride in phosphate buffer (pH 7.4, 37°C) for 10 minutes to stain viable tissue red and necrotic tissue white. Computerized planemetric analysis of the photographs of left-ventricle slices was performed using Image-Pro Plus v6.3 software to determine the area at risk (AR) and area of necrosis (AN), each expressed as a percentage of the left ventricle area. Obtained values were then used to calculate the infarct size (IS) as a percentage of the risk area, according to the formula IS = AN/AR × 100%.

Determination of Lipid Profile and Heart Glycogen Concentration

The concentration of FAs (free FA and acyl-coenzyme A) and triglycerides in the heart tissue was determined using commercially available enzymatic kits from Wako (Neuss, Germany) and Instrumentation Laboratory (Lexington, Massachusetts) as previously described, with the exception that heart tissues were homogenized twice for 25 seconds.¹⁶ Glycogen concentration in heart tissues was determined as previously described.¹⁷ Glucose concentration was determined using a glucose determination kit from Instrumentation Laboratory. Absorption measurements were carried out using a µQuant Microplate Spectrophotometer (BioTek, Bad Friedrichshall, Germany).

Quantitative Reverse Transcriptase–Polymerase Chain Reaction Analysis of CPT-IA and CPT-IB Transcripts and Determination of Enzyme Activity

Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of CPT-IA and CPT-IB was performed as described previously.⁶ Activities of CPT-I and CrAT were measured by isolation of mitochondria from heart tissues using a method adapted from Wilcke et al.¹⁸ The heavy mitochondrial fraction was used for the experiment. The CPT-I activity was measured as previously described¹⁴ in the presence of l-carnitine concentrations that were found in the heart tissue after the treatment (Figure 1, 700 μmol/L for control, 200 μmol/L for mildronate group, and 600 μmol/L for combination group). Enzyme activity was calculated per milligram of protein. The activity of CrAT was measured as previously described,¹⁹ with the exception that l-carnitine was not added for activity measurements so that the activity of the CrAT enzyme could be measured when only mitochondrial l-carnitine is present.

Figure 1.

Effects of 14-day treatment with mildronate (100 mg/kg; M 100) or mildronate and l-carnitine (100 + 100 mg/kg; C + M) on l-carnitine and γ-butyrobetaine (GBB) concentrations. Values are represented as mean ± standard error of the mean (SEM) of 5 animals. *Indicates significant difference from the control group.

Respiration Measurements

Mitochondrial respiration was measured at 37°C using Clark-type electrode (Microelectrodes Inc, Bedford, New Hampshire) as previously described,²⁰ with some modifications as indicated below. In brief, tissues were homogenized for 25 seconds in the medium (10 mL/g tissue) containing 180 mmol/L potassium chloride (KCl), 10 mmol/L Tris-HCl, and 1 mmol/L EGTA, and pH 7.7 at 4°C. The homogenate was centrifuged for 5 minutes at 750g and then the supernatant was centrifuged again for 10 minutes at 6800g to obtain the mitochondrial pellet. The final mitochondrial pellet was resuspended in the buffer containing 180 mmol/L KCl, 20 mmol/L Tris-HCl, and pH 7.2 at 4°C. Palmitoyl-coenzyme A (40 μmol/L) and corresponding l-carnitine concentrations (Figure 1) and 5 mmol/L pyruvate/5 mmol/L malate were used for respiration measurements in the incubation medium containing 150 mmol/L KCl, 1 mmol/L magnesium chloride (MgCl₂), 10 mmol/L Tris-HCl, 5 mmol/L KH₂PO₄, and pH 7.2. Citrate synthase activity was measured as previously reported²¹ and used to standardize mitochondrial protein amount.

In order to evaluate the integrity of OMM, rat hearts (n = 4 per group) were isolated and perfused according to the Langendorff technique as described above. The only difference was that the hearts were adapted for 20 minutes and then subjected to 60 minutes of regional ischemia without the reperfusion period (nonischemic hearts [normal] were isolated and adapted for 20 minutes). Immediately after that the bundles of cardiac fibers were isolated from left ventricular ischemic area (from left ventricle tissues in case of nonischemic [normal] hearts) and were further prepared as described previously. ¹³ Briefly, bundles of fibers were permeabilized with 50 μg/mL saponin at 4°C for 30 minutes in buffer A (20 mmol/L imidazole, 0.5 mmol/L dithiothreitol, 20 mmol/L taurine, 7.1 mmol/L MgCl₂, 50 mmol/L MES, 5 mmol/L ATP, 15 mmol/L phosphocreatine, 2.62 mmol/L CaK₂EGTA, 7.38 mmol/L K₂EGTA, pH 7.0 at 0°C). The fibers were washed twice for 10 minutes in buffer B (20 mmol/L imidazole, 0.5 mmol/L dithiothreitol, 20 mmol/L taurine, 1.61 mmol/L MgCl₂, 100 mmol/L MES, 3 mmol/L KH₂PO₄, 2.95 mmol/L CaK₂EGTA, 7.05 mmol/L K₂EGTA, and pH 7.1 at 37°C). Oxygen uptake rates of permeabilized cardiac fibers were determined at 37°C with Clark-type electrode in buffer B containing respiratory substrates (6 mmol/L pyruvate/6 mmol/L malate or 10 mmol/L succinate) and 2 mg/mL BSA. To assess the integrity of OMM, 30 μmol/L cytochrome c was added after addition of 1 mmol/L ADP and the V_{ADP+Cyt c}/V_ADP ratio was calculated.

Statistical Analysis

All data are expressed as the mean ± standard error of the mean (SEM). For statistical analyses, the 1-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test or Student t test were used. To test the hemodynamic parameters before, during, and after occlusion, the repeated measures ANOVA followed by Tukey test was used. P values less than .05 were considered statistically significant. Statistical calculations were performed using Prism 3.0 software (GraphPad, San Diego, California).

Results

Concentrations of l-Carnitine, GBB, and Mildronate

After treatment with mildronate, the l-carnitine concentration in heart tissue was decreased by 69%. Treatment with mildronate and l-carnitine combination had no significant impact on l-carnitine concentration in heart tissue as compared with control (Figure 1). Treatment with mildronate and the combination of both substances increased the GBB concentration in heart tissue by 6- and 7-fold, respectively (Figure 1). Mildronate concentration in heart tissue in the mildronate-only treated group was 3.2 times higher than in the group treated with a combination of both substances (Table 1).

Figure 2.

Effects of 14-day treatment with mildronate (100 mg/kg; M 100) or mildronate and l-carnitine (100 + 100 mg/kg; C + M) on carnitine palmitoyltransferase I (CPT-I) activity (A) or mitochondrial respiration on 40 μmol/L palmitoyl-coenzyme A (B) in the presence of l-carnitine concentrations found in the heart tissue after the treatment (700 μmol/L for control, 200 μmol/L for M 100, and 600 μmol/L for C + M group). Values are represented as mean ± standard error of the mean (SEM) of 5 or 6 animals (CPT-I activity or mitochondrial respiration). *Indicates significant difference from the control group.

Table 1.

Effects of Drug Treatment on Nonfasted Rat Tissue and Blood Plasma Parameters^a

	Control	M 100	C + M
Heart tissues
Glycogen, mg/g tissue	0.20 ± 0.11	0.13 ± 0.04	0.20 ± 0.12
FAs, μmol/g tissue	1.87 ± 0.28	1.98 ± 0.13	2.02 ± 0.26
Triglycerides, μmol/g tissue	4.0 ± 0.5	4.8 ± 1.6	3.9 ± 0.8
Mildronate, nmol/g tissue	−	980 ± 75	307 ± 15
Mitochondria
CrAT activity, mU/mg prot.	5.6 ± 1.5	5.8 ± 0.6	6.1 ± 0.7
Blood plasma
l-Carnitine, μmol/L	69 ± 3	22 ± 1^b	57 ± 2^b
GBB, μmol/L	2.0 ± 0.1	21 ± 2^b	39 ± 12^b
Mildronate, μmol/L	−	68 ± 7	19 ± 5

Abbreviations: FAs, fatty acids; SEM, standard error of the mean; CrAT, carnitine acetyltransferase; GBB, γ-butyrobetaine; M, mildronate; C + M, l-carnitine and mildronate.

^aEffects of 14-day treatment with mildronate (100 mg/kg; M 100) or mildronate and l-carnitine (100 + 100 mg/kg, C + M) on tissue and blood plasma parameters. Values are represented as mean ± SEM of 5 to 10 animals.

^bIndicates significant difference from the control group.

As shown in Table 1 mildronate treatment significantly decreased the concentration of l-carnitine in blood plasma, and a 3-fold decrease in l-carnitine concentration was achieved after 14 days of mildronate treatment as compared to control. Treatment with a combination of both substances decreased l-carnitine concentration by only 20% after 14 days of treatment. The GBB concentration in blood plasma was significantly increased in all the treatment groups. Treatment with mildronate or the combination of both substances increased GBB concentration by up to 10- and 20-fold, respectively. The blood plasma mildronate concentration in the mildronate-only treated group was 3.6 times higher than in the group treated with a combination of both substances (Table 1).

Enzyme Activity, Mitochondrial Respiration Measurements, and RT-PCR Analysis of CPT-IA and CPT-IB

The activity of CPT-I was significantly decreased by 26% in the mildronate group when compared to control (Figure 2A). After treatment with mildronate, the mitochondrial respiration on palmitoyl-coenzyme A was significantly decreased by ∼27% (Figure 2B). Treatment with mildronate did not have any significant impact on mitochondrial respiration on pyruvate/malate when compared to control (data not shown). The activity of CrAT was similar in all groups (Table 1).

The cytochrome c effect was significantly higher in the left ventricular fibers isolated from ischemic hearts. Mildronate treatment significantly diminished the ischemia-induced respiration stimulation by exogenous cytochrome c when both pyruvate/malate and succinate were used as respiratory substrates (Figure 3).

Figure 3.

The effect of cytochrome c on respiration of saponin-permeabilized cardiac ventricular fibers isolated from normoxic or ischemic hearts from control or mildronate treated (100 mg/kg, 14 days, M 100) animals. Values are represented as mean ± standard error of the mean (SEM) of 4 animals. #Indicates significant difference from the respective ischemic control group. *Indicates significant difference from the respective normal (nonischemic) control group.

No significant differences were observed in the mRNA levels of CPT-IA and CPT-IB in any of the treated groups when compared to control (data not shown).

Cardioprotective Effects During Isolated Rat Heart Infarction

The anti-infarction effect of mildronate was investigated in an isolated rat heart infarction model. Values for the AR were similar in hearts of all experimental groups, and the AR in all groups was about 40% of the area of the left ventricle (Figure 4A). Mildronate significantly reduced IS by 38% as compared to the control group (Figure 4B). In contrary, mildronate and l-carnitine combination had no significant impact on IS.

Figure 4.

Effects of 14-day treatment with mildronate (100 mg/kg; M 100) or mildronate and l-carnitine (100 + 100 mg/kg; C + M) on values of the area at risk as percentage of the left ventricle area (A) and infarct size as percentage of the area at risk (B). Values are represented as mean ± standard error of the mean (SEM) of 8 to 10 animals. *Indicates significant difference from the control group.

Hemodynamic Parameters

Results of the hemodynamic measurements are shown in Table 2. No significant differences were observed in the basal HR, LVDP, coronary flow, contractility/relaxation (dP/dt maximum and minimum), or the cardiac workload (HR × LVDP) in any of the treatment groups when compared to the control group. Similarly, no significant differences in hemodynamic parameters were observed during the occlusion procedure as compared to the control group. Meanwhile, during the occlusion procedure, coronary flow was decreased by an average of 39%, LVDP was decreased by an average of 33%, and cardiac workload (HR × LVDP) was decreased by an average of 36% for all experimental groups as compared to the values observed prior to occlusion.

Table 2.

Effects of Drug Treatment on Isolated Heart Parameters^a

	Heart Rate, BPM	Coronary Flow, mL/min	LVDP, mm Hg	dP/dt max, mm Hg/s	dP/dt min, mm Hg/s	Heart Rate × LVDP
Basal heart parameters
Control	247 ± 14	7.6 ± 0.8	74 ± 14	1845 ± 162	−1300 ± 127	17 219 ± 2315
M 100	237 ± 13	7.8 ± 0.7	72 ± 13	1749 ± 143	−1279 ± 137	17 189 ± 3517
C + M	246 ± 9	7.2 ± 0.6	59 ± 11	1456 ± 175	−1020 ± 130	14 939 ± 3182
Heart parameters during ischemia
Control	229 ± 10	4.8 ± 0.7^b	47 ± 9^b	1111 ± 143^b,c	−829 ± 105^b,c	11 118 ± 2651^b,c
M 100	223 ± 14	5.0 ± 0.7^b	50 ± 11^b	1250 ± 159^b	−924 ± 107^b	11 074 ± 2275^b
C + M	235 ± 7	4.3 ± 0.5^b	41 ± 9^b	990 ± 108^b,c	−719 ± 83^b,c	9808 ± 2271^b,c
Heart parameters during reperfusion
Control	231 ± 14	5.8 ± 1.0	65 ± 8	1739 ± 159	−1178 ± 88	14 945 ± 2095
M 100	240 ± 12	6.0 ± 0.8	59 ± 9	1611 ± 150	−1121 ± 88	14 234 ± 2228
C+M	252 ± 14	5.0 ± 0.7	52 ± 7	1423 ± 163	−964 ± 90	13 513 ± 2223

Abbreviations: BPM, beats per minute; dP/dt, contractility/relaxation; LVDP, left-ventricle developed pressure; SEM, standard error of the mean; M, mildronate; C + M, l-carnitine and mildronate.

^aEffects of 14-day treatment with mildronate (100 mg/kg; M 100) or mildronate and l-carnitine (100 + 100 mg/kg; C + M) on isolated heart parameters. Values are represented as mean ± SEM of 9 to 10 animals.

^bIndicates a significant difference from the same group before occlusion.

^cIndicates a significant difference from the same group during reperfusion.

In the reperfusion stage, the coronary flow recovered to 70% to 75% and the other hemodynamic parameters recovered to about 90% of their basal levels, but no significant differences were observed between the treated groups and the control.

Lipid Profile, Glucose Concentration, and Glycogen Concentration

The glycogen, FAs, and triglyceride concentrations in the heart tissues of nonfasted rats are shown in Table 1. No significant differences were observed in the tissue lipid profiles or glycogen concentration values for any of the treated groups when compared to the control group.

Discussion

The results of the present study, performed in an isolated rat heart ischemia–reperfusion injury model, provide evidence that the pivotal factor for the development of the anti-infarction activity of mildronate in heart tissue is a decreased l-carnitine concentration. For the first time it was shown that prevention of mildronate-induced decrease in l-carnitine concentration diminishes the anti-infarction effect of mildronate. Treatment with mildronate resulted in a marked decrease in l-carnitine concentration in heart tissues (Figure 1) as well as a significant decrease in IS (Figure 4B) as compared to control. Treatment with a combination of mildronate and l-carnitine diminished both the mildronate-induced decrease in l-carnitine tissue concentration (Figure 1) and the cardioprotective effect of mildronate (Figure 4B). Changes in the blood plasma concentration of l-carnitine (Table 1) resembled changes observed in the heart tissue. Treatment with mildronate did not have significant effects on hemodynamic parameters either before or during ischemia–reperfusion (Table 2). Similarly, treatment with the combination of mildronate and l-carnitine did not influence the hemodynamic parameters when compared to control, indicating that the observed effects on IS are not related to changes in cardiac workload.

Mildronate treatment significantly diminished the ischemia-induced respiration stimulation by exogenous cytochrome c (Figure 3). These results indicate that the cardioprotective effect of mildronate could be attributed to the maintenance of the intactness of OMM which is expected to protect against ischemia-impaired ATP synthesis^22,23 and improve the survival prognosis of myocardium. The results of the present study (Figure 3) point to the maintained functionality of OMM as a possible mechanism behind the preserved ATP levels in mildronate-treated rat hearts in ischemia-related conditions reported in previous studies.^24,25

Treatment with mildronate alone or with the combination of mildronate and l-carnitine induced an increase in the heart tissue concentration of GBB (Figure 1). Mildronate inhibits GBB hydroxylase⁴ and thus prevents the conversion of GBB into l-carnitine. Additional increase in the heart tissue concentration of GBB after l-carnitine cotreatment might be related to the conversion of l-carnitine into GBB due to the endogenous flora of the gastrointestinal tract.²⁶ It has been hypothesized that the antiischemic mechanism of action of mildronate could result from the accumulation of GBB.⁷ Present data (Figure 1) indicate that the increase in GBB concentration in heart tissue cannot be associated with the cardioprotective effects of mildronate because the highest GBB content was observed in the combination treatment group, in which the treatment did not reduce IS (Figure 4).

Long-term treatment with mildronate induces preconditioning-like effects by causing changes in FA and glucose metabolism-related gene expression. Thus, treatment with mildronate at doses of 200 mg/kg⁶ and 800 mg/kg^27,28 induces a compensatory increase in CPT-I protein and mRNA expression as well as an increase in maximal CPT-I enzyme activity. Similarly, increased expression of glucose metabolism-related genes has been observed in heart tissue.⁶ In the present study, due to the use of a lower dose of mildronate (100 mg/kg), we did not observe significant changes in mRNA expression of CPT-I. Activity of CPT-I in heart tissue was significantly reduced by 26% and mitochondrial respiration on palmitoyl-coenzyme A was significantly reduced by 27%, as measured in the presence of 200 μmol/L l-carnitine found in heart tissue (Figure 1) after the mildronate treatment. Meanwhile, reduction in heart concentration of l-carnitine induced by mildronate treatment does not affect CrAT activity (Table 1), thus providing experimental evidence for the unchanged CoA/acetyl-CoA ratio.^1,2 Thus, at least 60% to 70% reduction in heart concentration of l-carnitine (Figure 1) is necessary to reduce CPT-I-dependent long-chain FA transport and oxidation (Figure 2A, B) with subsequent changes in metabolic pathways and reduction in IS. In comparison, after treatment with l-carnitine in combination with mildronate, l-carnitine concentration and CPT-I activity were similar to control levels (Figures 1 and 2A) and, as a consequence, IS was not reduced (Figure 4B).

Mildronate concentrations in the hearts of the mildronate-only treated rats were about 3 times higher than those in the hearts of rats treated with a combination of mildronate and l-carnitine (Table 1). However, this could not explain the difference in the anti-infarction effects between the 2 treatment groups, as we have shown that the mildronate content reaches ∼600 nmol/g of heart tissue only after 3 days of treatment with mildronate at a dose of 100 mg/kg; no cardioprotection could be observed at that time point in our previous study.⁷ Therefore, it can be concluded that changes in the concentration of mildronate in the heart do not induce acute cardioprotective properties.

It is well documented that decrease in FA oxidation and increase in glucose oxidation are associated with protection and improvement in the functional recovery of the heart during ischemia–reperfusion.^29,30 Biochemical consequences of decreased l-carnitine concentration could be similar to that of increased concentration of malonyl-CoA, which is described as a potent endogenous inhibitor of cardiac FA oxidation, secondary to inhibition of CPT-I.^31,32 The results of the present study provide additional evidence that the l-carnitine homeostasis is a valuable target for the regulation of energy metabolism pathways and treatment of cardiovascular diseases. In addition, our results suggest that the therapeutic efficiency of mildronate depends on the mildronate treatment-induced decrease in l-carnitine concentration. Our finding is of clinical importance, especially given the fact that the meat (the main dietary source of l-carnitine³³) consumption can significantly affect mildronate-induced changes in l-carnitine concentration in the blood plasma of healthy volunteers.³⁴ Altogether, these findings suggest that the therapeutic efficiency of mildronate could be monitored by following changes in the blood plasma concentration of l-carnitine.

In conclusion, the decrease in l-carnitine concentration and concomitant decrease in FA transport are the main mechanisms of cardioprotective action of mildronate.

Footnotes

The work reported was done at the Latvian Institute of Organic Synthesis.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The European Social Foundation agreement No. 2009/0147/1DP/1.1.2.1.2/09/IPIA/VIAA/009; The European Regional Development Fund grant No. 2DP/2.1.1.1.0/10/APIA/VIAA/063; the Latvian State Research Program grant No. 2010.10-4/VPP-4.

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