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Carnitines are involved in mitochondrial transport of fatty acids and are of critical importance for maintaining normal mitochondrial function. This review summarizes recent experimental and clinical studies showing that mitochondrial dysfunction secondary to a disruption of carnitine homeostasis may play a role in decreased NO signaling and the development of endothelial dysfunction. Future challenges include development of agents that can positively modulate L-carnitine homeostasis which may have high therapeutic potential.
L-carnitine is a ubiquitously occurring trimethylated amino acid that plays an important role in the transport of long chain fatty acids across the inner mitochondrial membrane . Carnitines exist either as free carnitines or as acylcarnitines (Figure 1 A). The acylcarnitines are products of the reaction in which acyl moieties are transferred to carnitine from acyl-CoA. These acyl groups vary in length from short chain (acetyl) to long chain (palmitoyl).
This reaction is catalyzed by a family of enzymes known as acyltransferases. These enzymes differ on the basis of the structural specificity of the acyl group and their sub-cellular localization. Carnitine deficiency or abnormalities in the carnitine acyltransferase systems results in a reduced β-oxidation of fatty acids and therefore, reduced energy (ATP) production. Recent studies suggest that mitochondrial dysfunction, secondary to a disruption of carnitine homeostasis, may also play a role in the loss of nitric oxide (NO) signaling and the development of endothelial dysfunction associated with a variety of cardiovascular diseases .
Carnitine in humans is derived from diet and de novo biosynthesis using lysine and methionine. The main dietary sources of carnitine are red meat, fish, and dairy products which can supply 2 to 12 μmols/day/kg of body weight, whereas 1–2 μmols of carnitine is endogenously synthesized . The main sites of L-carnitine synthesis are the kidney, liver and brain. After synthesis, carnitine is transported through the circulation and is then taken up by other tissues through active transport systems. In the kidney, carnitine and butyrobetaine are reabsorbed efficiently to minimize the urinary loss. Sodium dependent cationic transporter (OCTN2)  is the primary known transporter responsible both for transport of carnitine to other tissues and reabsorption in kidney.
Carnitine biosynthesis is a multi-step process (Figure 1 B). Trimethyl-lysine residues are the primary precursors for carnitine biosynthesis, which are hydroxylated on the third carbon by the enzyme trimethyl-lysine deoxygenase (TMLD) in mitochondria of liver, kidney, heart, muscle and brain . The product of this reaction is 3-hydroxy-trimethyl-lysine which is converted to 4-trimethylaminobutyraldehyde and glycine by a reaction catalyzed by 3-hydroxy-trimethyl-lysine aldolase. 4-trimethylaminobutyraldehyde is then dehydrogenated by 4-trimethylaminobutanal dehydrogenase (TMABA-DH) results in the formation of 4-trimethylammoniobutanoate, also known as butyrobetaine. Finally, butyrobetaine is hydroxylated on the third carbon by butyrobetaine dioxygenase (BBD) to form L-carnitine.
It is well established that carnitines play an important role as carriers of activated fatty acids across the inner mitochondrial membrane and are essential for energy production through fatty acid metabolism. Carnitines are also involved in the removal of accumulated toxic fatty acyl-CoA metabolites, and help in buffering the balance between free and acyl-CoA. Carnitine is present in the form of either free carnitine (nonesterified molecule; FC), or acylcarnitines (esterified form; AC). Thus, the AC/FC ratio is a measure of acylated carnitines versus the free carnitines. L-carnitine deficiency can raise the AC/FC ratio resulting in a form of functional carnitine deficiency. Other factors such as enzymatic alterations in carnitine metabolism can also result in higher levels of acylated carnitines and hence an elevated AC/FC ratio. Thus, a low AC/FC ratio suggests healthy mitochondria whereas a high AC/FC ratio suggests a decreased mitochondrial capacity for energy production.
Carnitine acyltransferases reversibly transfer the acyl group from acyl-CoA to carnitine. These can be divided into 3 types: carnitine palmitoyl transferase (CPT), carnitine octanoyltransferase (COT), and carnitine acetyltransferase (CrAT), based upon their specificity to acyl groups and their cellular localization. CrAT uses short-chain acyl groups (C1-C4) as substrate, whereas COT uses both medium and long-chain fatty acids (C5-C12) having a preference for medium chain acyl-CoA. CPT transfers medium and long chain acyl-CoA (>C12) to carnitine with a preference for long chain acyl-CoA, at least at physiological concentrations of carnitines. The CPT system is an important component of the “carnitine shuttle” which facilitates the transport of long-chain fatty acids from cytosol into the mitochondrial matrix, the site of β-oxidation (Figure 2). This transport system consists of carnitine palmitoyl transferase 1 (CPT1) localized in the outer mitochondrial membrane, the carnitine-acylcarnitine translocase (CACT), an integral inner membrane protein, and carnitine palmitoyltransferase 2 (CPT2) localized on the inner mitochondrial membrane . CPT1 conjugates carnitine with long chain fatty acids, carnitine acylcarnitine translocase (CACT) transfers the acylcarnitine across the inner plasma membrane and CPT2 conjugates the fatty acid back to Coenzyme A for subsequent β-oxidation. CPT-1 has different isoforms with tissue specific expression, CPT-1A (liver), CPT-1B (muscle), and CPT-1C (brain) . Finally, carnitine acetyl transferase (CrAT), which resides in the matrix, is able to reconvert the short- and medium chain acyl-CoAs into acylcarnitines using intra-mitochondrial carnitine. Decreased CrAT activity leads to increased levels of acyl-CoA, which leads to inhibition of multiple enzymatic processes involved in oxidative metabolism.
Carnitine transport is mediated by a family of organic cation transporters (OCTs). OCTs play an important role in the intracellular carnitine homeostasis. Three main members of this family are OCTN1, OCTN2, and OCTN3. OCTN1 is a lower-affinity mitochondrial carnitine transporter. It is a pH-dependent and sodium-independent multispecific organic cation carrier  which may be energized by the proton antiport mechanism in renal epithelial cells . Newly synthesized L-carnitine is also transported through the circulation and effectively absorbed by other tissues by active sodium-dependent transport. OCTN2 is the sodium-dependent organic cation transporter  and this is the most important controlling factor for carnitine pools in the plasma membrane . It also mediates cellular uptake and absorption by the kidneys of both carnitine as well as its precursor, 4-trimethylaminobutyric acid (butyrobetaine, BB) . OCTN2 has a Km of 2–6 μM for carnitine and is expressed in a variety of tissues including muscle, heart, kidney, and fibroblasts [12,13]. OCTN3 is a mammalian peroxisomal membrane carnitine transporter which is uniquely involved in intracellular carnitine-dependent transport . It is expressed exclusively in testes, kidney, and intestine and has the highest specificity for carnitine .
Inborn errors of metabolism are one of the largest groups of genetic diseases that lead to cardiomyopathy which has been associated with high morbidity and mortality. Inborn errors of metabolism mainly include diseases such as fatty acid oxidation defects, mitochondrial dysfunctions, organic acidurias, as well as lysosomal and glycogen storage disorders. In the last few years mouse models have been developed to study the inborn errors of fatty acid oxidation with phenotypes representative of the human diseases (as shown in Table 1). These diseases will be discussed briefly below.
Disruption of mitochondrial function is acknowledged as a critical event in a number of pathological conditions, including hypoxia-ischemic injuries, stroke, and diabetes. The carnitine acyltransferase pathway has been shown to be of critical importance for maintaining normal mitochondrial function. Fatty acids are transported via carnitine into mitochondria for their subsequent oxidation to generate ATP. Carnitines also remove the acyl groups from the mitochondria as acylcarnitines. Increase in free fatty acid levels can induce mitochondrial dysfunction resulting in cell death and/or enhanced secondary generation of reactive oxygen species. These effects can be attenuated with L-carnitine treatment . In addition, L-carnitine can suppress the palmitoyl-CoA induced dysfunction, membrane permeability transition (MPT), and cytochrome c release of isolated mitochondria as well as reduce the mitochondrial swelling and depolarization induced by long chain fatty acids (LCFAs) and palmitoyl-CoA through a protective mechanism thought to occur in and around the mitochondrial membrane . L-carnitine also suppresses oleic acid-mediated MPT by accelerating β-oxidation . Studies have also shown that carnitine has a protective effect both on mitochondria and in whole cells by inhibiting free fatty acid-induced mitochondrial membrane damage and/or its secondary effects [23,25]. The protective effects of carnitine and related metabolites have been postulated to be due to improved energy metabolism and the inhibition of electron leakage from mitochondrial electron transport systems . Administration of carnitine has also been shown to decrease free fatty acids in serum and tissues and prevent tissue injury in juvenile visceral steatosis (JVS) mice that lack a carnitine transporter . Mitochondria are also emerging as important signaling transducers in the apoptotic pathway in a variety of disease states. Previous studies have shown a close relationship between carnitine and mitochondria. Cultured neonatal rat cardiac myocytes were studied to assess mitochondrial alterations during apoptosis. It was shown that CPT1 inhibition, ceramide accumulation, and complex III inhibition were downstream events in cardiac apoptosis mediated by long-chain fatty acids, such as palmitate . L-carnitine has also been shown to inhibit mitochondria-dependent apoptosis both in vivo and in vitro [29,30].
The role of mitochondria in cardiovascular pathologies is being extensively scrutinized. In cardiovascular diseases, there is increased production of reactive oxygen species (ROS). This leads to endothelial dysfunction and impaired endothelium dependent vasodilation by reducing the bioavailability of nitric oxide (NO). Studies on the mechanism(s) underlying the effects of L-carnitine in cardiovascular diseases have demonstrated that the chronic administration of L-carnitine can reduce blood pressure and attenuate the inflammatory process associated with arterial hypertension . Carnitine treatment has also been shown to increase post-ischemic blood flow in patients with peripheral vascular disease, indicative of an improvement in functional circulatory reserve . L-carnitine has been shown to limit ischemia reperfusion injury by preventing long-chain acyl-CoA accumulation and subsequent production of ROS by damaged mitochondria. L-carnitine also improves repair mechanisms for oxidative-induced damage to membrane phospholipids and reduces the ischemia-induced apoptosis and the consequent remodeling of the left ventricle . L-carnitine restores endothelial dysfunction present in spontaneously hypertensive rats (SHR) and has the ability to increase the release of the vasodilator PGI2 and enhance the production of TXA2 in normotensive rats. An acute L-carnitine treatment to SHR was also able to restore endothelium-dependent relaxations of aortic rings .
NO production and availability plays a major role in determining endothelial cell functions. Decreases in endothelial nitric oxide synthase (eNOS) expression and/or activity leading to decreased bioavailable NO generation has been linked to endothelial dysfunction and vascular remodeling. We have observed reduced mitochondrial function as measured by increased lactate/pyruvate ratios, increased uncoupling protein-2 (UCP-2) and decreased mitochondrial superoxide dismutase (SOD-2) protein levels in a lamb model of pulmonary hypertension . Further, the mitochondrial dysfunction was associated with decrease in interaction of HSP90 with eNOS, which correlated with a progressive decrease in relative eNOS activity, and increased NOS-dependent superoxide levels . Thus, L-carnitine supplementation may help to maintain eNOS-HSP90 interactions, NO signaling, and normal endothelial function. Finally, GPLC (a novel form of carnitine) supplementation with exercise has been shown to elevate NO levels in human subjects. Besides, decreasing oxidative stress, GPLC supplementation was associated with normalization/enhancement of circulating NO levels .
In recent years, L-carnitine has become more prevalent in therapies aimed at improving mitochondrial energy metabolism and has been shown to be beneficial in cardiovascular pathologies. The therapeutic potential of L-carnitine in the management of diseases such as peripheral vascular diseases, congestive heart failure, angina, and anthracycline-induced cardiotoxicity has been frequently reported [35,36]. Carnitine availability becomes a limiting step for β-oxidation in certain physiological and pathological diseases, and carnitine supplementation enhances fatty acid metabolism in the mitochondria, restoring normal mitochondrial function by maintaining the equilibrium between acyl-CoA and free CoA . L-carnitine has also been shown to be an effective cardioprotective agent in different models of experimental ischemia. The supplementation of the myocardium with carnitine resulted in an increased tissue carnitine content, a stimulation of pyruvate oxidation, lessening of the severity of ischemic injury, and improvement in the recovery of heart function during reperfusion . L-carnitine supplementation has been shown to improve CoA recycling and increase the mitochondrial-free CoA levels by shuttling the short-chain acyl groups from within mitochondria to the cytosol. A multi-central study with 472 patients was conducted to evaluate the effects of L-carnitine administration on long-term left ventricular dilation in patients with acute anterior myocardial infarction. Carnitine treatment attenuated left ventricular dilation and resulted in smaller left ventricular volumes . Some clinical studies have also suggested that L-carnitine can be employed as a treatment drug in patients with essential hypertension and diabetes . In a recently published study involving newborn screening for MCAD deficiency medium chain length acylcarnitines, octanoylcarnitine (C8) and decanoylcarnitine (C10) were measured on newborn screening blood spot cards . Out of 121,000 live births, 17 newborns had C8 values above the screening cut-off of 0.38 μmol/L. Ten newborns had elevated C8 on repeat cards and were investigated further. Both C8 and C8/C10 ratios remained abnormal in all confirmed MCAD cases. Upon frequent feeding and carnitine supplementation, none of the patients had metabolic crises or adverse outcomes. Another study in patients undergoing hemodialysis has shown that L-carnitine supplementation decreases the left ventricular mass . The cardiac morphology and function of 10 patients given 10 mg/kg of L-carnitine orally, immediately after hemodialysis sessions, 3 times per week for a 12-month period were compared with 10 untreated control patients. Therapy resulted in an increase in serum-free carnitine levels from 28.4 ± 4.7 to 58.5 ± 12.1 micromol/L and the regression of left ventricular hypertrophy, even in those with normal systolic function.
L-carnitine supplementation in CACT deficiency has also shown therapeutic benefits by improving the acylcarnitine profile and preventing further attacks of hypoglycemia and arrhythmia [43–45]. Although only a study with 10 CACT deficiency patients suffering from cardiomyopathy, therapy with L-carnitine resulted in improvement in cardiac function. The effect of carnitine supplementation on endothelial dysfunction in hypertension has also been studied in spontaneously hypertensive rats (SHR). L-carnitine was able to restore endothelium-dependent relaxations of aortic rings from SHR and reduce ROS as well as increase NO participation in endothelium-dependent relaxations in SHR . Further, a recently published study in rats has shown nutritional supplementation with L-carnitine and moderate physical exercise could improve the mitochondrial oxidative metabolism and subsequently limit the side-effects of aging . L-carnitine supplementation has been shown to influence the influx of fatty acids into the mitochondria, alter lipid accumulation in the skeletal muscle , and also increase the oxidation of dietary fatty acids in healthy humans [48,49].
There is a great need for therapies to provide more effective treatments for cardiovascular disorders. Carnitines have an essential role in the regulation of mitochondrial function and recent studies both in animal models and human subjects have emphasized the importance of mitochondrial function in regulating NO signaling. Thus, as appears likely, the disruption of carnitine metabolism is an important contributing factor in the development of endothelial dysfunction, then preventing the disruption of carnitine homeostasis may attenuate, at least in part, the endothelial injury associated with cardiovascular disease. Therefore, we propose that the development of agents that can positively modulate L-carnitine homeostasis may have high therapeutic potential.
This research was supported in part by grants HL60190, HL67841, HL72121, HL70061, HD039110, and HD057406 all from the National Institutes of Health to SMB, and by a grant from the Fondation Leducq to SMB.
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