Reactivation of fatty acid oxidation by medium chain fatty acid prevents myocyte hypertrophy in H9c2 cell line.

Metabolic shift is an important contributory factor for progression of hypertension-induced left ventricular hypertrophy into cardiac failure. Under hypertrophic conditions, heart switches its substrate preference from fatty acid to glucose. Prolonged dependence on glucose for energy production has adverse cardiovascular consequences. It was reported earlier that reactivation of fatty acid metabolism with medium chain triglycerides ameliorated cardiac hypertrophy, oxidative stress and energy level in spontaneously hypertensive rat. However, the molecular mechanism mediating the beneficial effect of medium chain triglycerides remained elusive. It was hypothesized that reduction of cardiomyocyte hypertrophy by medium chain fatty acid (MCFA) is mediated by modulation of signaling pathways over expressed in cardiac hypertrophy. The protective effect of medium chain fatty acid (MCFA) was evaluated in cellular model of myocyte hypertrophy. H9c2 cells were stimulated with Arginine vasopressin (AVP) for the induction of hypertrophy. Cell volume and secretion of brain natriuretic peptide (BNP) were used for assessment of cardiomyocyte hypertrophy. Cells were pretreated with MCFA (Caprylic acid) and metabolic modulation was assessed from the expression of medium-chain acyl-CoA dehydrogenase (MCAD), cluster of differentiation-36 (CD36) and peroxisome proliferator-activated receptor (PPAR)-α mRNA. The signaling molecules modified by MCFA was evaluated from protein expression of mitogen activated protein kinases (MAPK: ERK1/2, p38 and JNK) and Calcineurin A. Pretreatment with MCFA stimulated fatty acid metabolism in hypertrophic H9c2, with concomitant reduction of cell volume and BNP secretion. MCFA reduced activated ERK1/2, JNK and calicineurin A expression mediated by AVP. In conclusion, the beneficial effect of MCFA is possibly mediated by stimulation of fatty acid metabolism and modulation of MAPK and Calcineurin A.

Metabolic Modulation by Medium-Chain Triglycerides Reduces Oxidative Stress and Ameliorates CD36-Mediated Cardiac Remodeling in Spontaneously Hypertensive Rat in the Initial and Established Stages of Hypertrophy.

BACKGROUND: Left ventricular hypertrophy (LVH) is characterized by a decrease in oxidation of long-chain fatty acids, possibly mediated by reduced expression of the cell-surface protein cluster of differentiation 36 (CD36). Spontaneously hypertensive rats (SHRs) were therefore supplemented with medium-chain triglycerides (MCT), a substrate that bypasses CD36, based on the assumption that the metabolic modulation will ameliorate ventricular remodeling. METHODS: The diet of 2-month-old and 6-month-old SHRs was supplemented with 5% MCT (Tricaprylin), for 4 months. Metabolic modulation was assessed by mRNA expression of peroxisome proliferator-activated receptor α and medium-chain acyl-CoA dehydrogenase. Blood pressure was measured noninvasively. LVH was assessed with the use of hypertrophy index, cardiomyocyte cross-sectional area, mRNA expression of B-type natriuretic peptide, cardiac fibrosis, and calcineurin-A levels. Oxidative stress indicators (cardiac malondialdehyde, protein carbonyl, and 3-nitrotyrosine levels), myocardial energy level (ATP, phosphocreatine), and lipid profile were determined. RESULTS: Supplementation of MCT stimulated fatty acid oxidation in animals of both age groups, reduced hypertrophy and oxidative stress along with the maintenance of energy level. Blood pressure, body weight, and lipid profile were unaffected by the treatment. CONCLUSIONS: The results indicate that modulation of myocardial fatty acid metabolism by MCT prevents progressive cardiac remodeling in SHRs, possibly by maintenance of energy level and decrease in oxidative stress.

Mitoprotective antioxidant EUK-134 stimulates fatty acid oxidation and prevents hypertrophy in H9C2 cells.

Oxidative stress is an important contributory factor for the development of cardiovascular diseases like hypertension-induced hypertrophy. Mitochondrion is the major source of reactive oxygen species. Hence, protecting mitochondria from oxidative damage can be an effective therapeutic strategy for the prevention of hypertensive heart disease. Conventional antioxidants are not likely to be cardioprotective, as they cannot protect mitochondria from oxidative damage. EUK-134 is a salen-manganese complex with superoxide dismutase and catalase activity. The possible role of EUK-134, a mitoprotective antioxidant, in the prevention of hypertrophy of H9C2 cells was examined. The cells were stimulated with phenylephrine (50 µM), and hypertrophy was assessed based on cell volume and expression of brain natriuretic peptide and calcineurin. Enhanced myocardial lipid peroxidation and protein carbonyl content, accompanied by nuclear factor-kappa B gene expression, confirmed the presence of oxidative stress in hypertrophic cells. Metabolic shift was evident from reduction in the expression of medium-chain acyl-CoA dehydrogenase. Mitochondrial oxidative stress was confirmed by the reduced expression of mitochondria-specific antioxidant peroxiredoxin-3 and enhanced mitochondrial superoxide production. Compromised mitochondrial function was apparent from reduced mitochondrial membrane potential. Pretreatment with EUK-134 (10 µM) was effective in the prevention of hypertrophic changes in H9C2 cells, reduction of oxidative stress, and prevention of metabolic shift. EUK-134 treatment improved the oxidative status of mitochondria and reversed hypertrophy-induced reduction of mitochondrial membrane potential. Supplementation with EUK-134 is therefore identified as a novel approach to attenuate cardiac hypertrophy and lends scope for the development of EUK-134 as a therapeutic agent in the management of human cardiovascular disease.

Ligand specific variation in cardiac response to stimulation of peroxisome proliferator-activated receptor-alpha in spontaneously hypertensive rat.

Left ventricular hypertrophy (LVH) is an independent risk factor for cardiac failure. Reduction of LVH has beneficial effects on the heart. LVH is associated with shift in energy substrate preference from fatty acid to glucose, mediated by down regulation of peroxisome proliferator-activated receptor-alpha (PPAR-α). As long-term dependence on glucose can promote adverse cardiac remodeling, it was hypothesized that, prevention of metabolic shift by averting down regulation of PPAR-α can reduce cardiac remodeling in spontaneously hypertensive rat (SHR). Cardiac response to stimulation of PPAR-α presumably depends on the type of ligand used. Therefore, the study was carried out in SHR, using two different PPAR-α ligands. SHR were treated with either fenofibrate (100 mg/kg/day) or medium-chain triglyceride (MCT) Tricaprylin (5% of diet) for 4 months. Expression of PPAR-α and medium-chain acylCoA dehydrogenase served as markers, for stimulation of PPAR-α. Both ligands stimulated PPAR-α. Decrease of blood pressure was observed only with fenofibrate. LVH was assessed from heart-weight/body weight ratio, histology and brain natriuretic peptide expression. As oxidative stress is linked with hypertrophy, serum and cardiac malondialdehyde and cardiac 3-nitrotyrosine levels were determined. Compared to untreated SHR, LVH and oxidative stress were lower on supplementation with MCT, but higher on treatment with fenofibrate. The observations indicate that reduction of blood pressure is not essentially accompanied by reduction of LVH, and that, progressive cardiac remodeling can be prevented with decrease in oxidative stress. Contrary to the notion that reactivation of PPAR-α is detrimental; the study substantiates that cardiac response to stimulation of PPAR-α is ligand specific.

Reactivation of peroxisome proliferator-activated receptor alpha in spontaneously hypertensive rat: age-associated paradoxical effect on the heart.

Prevention of left ventricular hypertrophy remains a challenge in the prevention of hypertension-induced adverse cardiac remodeling. Cardiac hypertrophy is associated with a shift in energy metabolism from predominantly fatty acid to glucose with a corresponding reduction in the expression of fatty acid oxidation enzyme genes. Although initially adaptive, the metabolic switch seems to be detrimental in the long run. This study was taken up with the objective of examining whether the stimulation of fatty acid oxidation by the activation of peroxisome proliferator-activated receptor alpha (PPARα), a key regulator of fatty acid metabolism, can prevent cardiac hypertrophy. Fenofibrate was used as the PPARα agonist. Spontaneously hypertensive rats (SHRs) in the initial stages of hypertrophy (2 months) and those with established hypertrophy (6 months) were treated with fenofibrate (100 mg·kg·d for 60 days). Cluster of differentiation 36 (CD36)-responsible for myocardial fatty acid uptake, carnitine palmitoyl transferase 1ß-a mitochondrial transporter protein and medium chain acyl-Co-A dehydrogenase-a key enzyme in beta oxidation of fatty acids were selected as indicators of fatty acid metabolism. Hypertrophy was apparent at 2 months and metabolic shift at 4 months of age in SHRs. The treatment prevented cardiac remodeling in young animals but aggravated hypertrophy in older animals. Hypertrophy showed a positive association with malondialdehyde levels and cardiac NF-κB gene expression, signifying the role of oxidative stress in the mediation of hypertrophy. Expression of carnitine palmitoyl transferase 1ß and medium chain acyl-Co-A dehydrogenase was upregulated on treatment. However, CD36 showed an age-dependent variation on treatment, with no change in expression in young rats and downregulation in older animals. It is inferred that the stimulation of PPARα before the initiation of metabolic remodeling may prevent cardiac hypertrophy, but reactivation after the metabolic adaptation aggravates hypertrophy. Whether the downregulation of CD36 is mediated by decreased substrate availability remains to be explored. Age-dependent paradoxical effect on the heart in response to fenofibrate, used as a lipid-lowering drug, can have therapeutic implications.

A positive association between cardiomyocyte volume and serum malondialdehyde levels.

Cardiac hypertrophy is the first visible sign of cardiac remodeling. Oxidative stress is implicated in the etiopathogenesis of cardiac hypertrophy. In vitro studies have shown that exposure of cardiomyocytes to free radical generators induce cell hypertrophy. However, there are no studies to show that in vivo redox status can influence cardiomyocyte growth. Blood samples were collected from healthy volunteers and serum lipid peroxidation was determined as a measure of oxidative stress. Cardiac myocytes cultured from newborn rat were exposed to serum samples. A significant correlation was observed between serum lipid peroxidation and cardiomyocyte volume, indicating that in vivo oxidative stress can act as an important co-factor in mediating the hypertrophic response. This experimental system also envisages a novel approach to identify patients prone to left ventricular remodeling and identification of humoral factors mediating the changes.

Magnesium deficiency augments myocardial response to reactive oxygen species.

Magnesium (Mg) deficiency and oxidative stress are independently implicated in the etiopathogenesis of various cardiovascular disorders. This study was undertaken to examine the hypothesis that Mg deficiency augments the myocardial response to oxidative stress. Electrically stimulated rat papillary muscle was used for recording the contractile variation. Biochemical variables of energy metabolism (adenosine triphosphate (ATP) and creatine phosphate) and markers of tissue injury (lactate dehydrogenase (LDH) release and lipidperoxidation), which can affect myocardial contractility, were assayed in Langendorff-perfused rat hearts. Hydrogen peroxide (100 micromol/L) was used as the source of reactive oxygen species. The negative inotropic response to H2O2 was significantly higher in Mg deficiency (0.48 mmol Mg/L) than in Mg sufficiency (1.2 mmol Mg/L). Low Mg levels did not affect ATP levels or tissue lipid peroxidation. However, H2O2 induced a decrease in ATP; enhanced lipid peroxidation and the release of LDH were augmented by Mg deficiency. Increased lipid peroxidation associated with a decrease in available energy might be responsible for the augmentation of the negative inotropic response to H2O2 in Mg deficiency. The observations from this study validate the hypothesis that myocardial response to oxidative stress is augmented by Mg deficiency. This observation has significance in ischemia-reperfusion injury, where Mg deficiency can have an additive effect on the debilitating consequences.

Reduction of perifusate magnesium alters inotropic response of papillary muscle to ion channel modulators.

Magnesium has a significant role in the regulation of ion transport. Marginal deficiency of Mg can therefore affect myocardial excitability and contractility. This study was taken up with the objective of examining the inotropic response of the myocardium to variation in extracellular [Mg]o and identifying the ion channels and pumps mediating the inotropic changes. Electrically stimulated rat papillary muscle was used as the experimental model and mechanical changes were recorded using a physiograph. Channel specific antagonists were used to identify the channels mediating the functional changes. Diastolic Ca2+ levels were determined in isolated myocytes by the ratiometric method using the fluorescent indicator Fura2-AM. A negative association was observed between the level of [Mg]o and force of contraction, with a peak at 0.48 mM Mg. The force of contraction in Mg deficient medium (0.48 mM) was 158% of control (1.2 mM Mg) (p < 0.001). Inotropic response to the L-type channel antagonist (verapamil-1 microm) and NaK ATPase inhibitor (Ouabain-0.3 mM) was augmented in Mg deficiency (p < 0.005), indicating activation of the channel and the pump. The response to T-type channel inhibitor (NiCl2-40 microM) was attenuated in Mg deficiency (p < 0.05). The response to the sarcoplasmic reticular Ca pump inhibitor (caffeine-10 mM) and the SR Ca2+ release channel inhibitor (ryanodine-1 microM) were not significantly affected by Mg deficiency. Diastolic level of Ca2+ increased with a decrease in Mg (p < 0.05). The observations of the study lead to the conclusion that the positive inotropic response in Mg deficiency is mediated by an increase in basal Ca2+ combined with Ca-induced-Ca release consequent to Ca2+ influx through L-type Ca channel. Variation in sensitivity to Ca channel blockers and NaK ATPase inhibitor in Mg deficiency can have pharmacological implications.

Genetic liability to epilepsy in Kerala State, India.

BACKGROUND: Familial clustering is common in epilepsies, but pedigree patterns suggest a multi-factorial inheritance. Genetic liability for multi-factorial inheritance is population specific and such data are not available for the population of Kerala or other states in south India. OBJECTIVES: In this study, we have attempted to determine the genetic liability to epilepsy based on an adult population of this state. MATERIAL AND METHODS: Pedigrees were recorded for probands who reported to the Kerala Registry of Epilepsy and Pregnancy. In order to obtain a genetically matched sample for comparison and estimation of empiric risks, we have used the family history of the spouse except when the spouse was proband's relative. The ILAE criteria were followed for diagnosis and classification of epilepsy. RESULTS: Data were collected on 18,419 family members of 505 probands with epilepsy (82 men and 423 women) and 10,231 family members of spouses (control). The frequency of epilepsy in first and second-degree relatives of the spouses was comparable to the population frequency (0.5%), justifying the use of this sample as control. Positive family history was observed in 22.2% of probands and 8.24% of controls (Odd's Ratio 3.2, 95% Confidence Interval 2.12-4.73). An affected first-degree relative was observed in 7.5% of probands. The corresponding figure for GE, LRE and other epileptic syndromes were 10.2%, 5.8% and 5.12%, respectively. The segregation ratio for Juvenile Myoclonic Epilepsy (JME) (1:19) was higher than that for other types of Generalized Epilepsy (GE) (1:24) and Localization Related Epilepsy (LRE) (1:52). Prevalence of epilepsy among the first-degree relatives (1.96%) was greater than the square root of the population frequency (0.51%) and was higher than that for second-degree (1.24%) and third-degree (0.64%) relatives for the probands. Probands had higher parental consanguinity (13.07%) compared to controls (6.64%). The above factors support a complex inheritance. Genetic liability to epilepsy (heritability) is greater for GE (0.6) and significantly higher for JME (0.7) compared to LRE (0.4). A limitation of this study is that the inferences are based on a predominantly adult female proband sample but no gender specific differences were identified. CONCLUSIONS: The observations of this study indicate complex inheritance and the liability values are useful for genetic counseling in the local population. Further studies involving more individuals from younger age group and male gender are envisaged.

Negative inotropic response to cerium in ventricular papillary muscle is mediated by reactive oxygen species.

This study was performed with the objective of assessing the mechanical response of the myocardium to different levels of cerium and delineation of the mechanism underlying the mediation of the functional changes. Rat ventricular papillary muscle was used as the experimental model. Isolated papillary muscles were exposed to different concentrations of CeCl3 and the force of contraction was measured using a force transducer. Experiments have revealed that the negative inotropic response to CeCl3 was proportional to its concentration. The inotropic changes were found to be completely reversible at concentrations < or =5 microM, and partially reversible at higher concentrations. Neutralization of cerium-induced inotropic changes by the superoxide anion scavenger superoxide dismutase (SOD) at concentrations < or =5 microM indicates that the mechanical changes are mediated by reactive oxygen species. At higher concentrations of Ce3+, SOD partially reversed the contractile changes. The beneficial effect of SOD was seen only if the muscles were pretreated with the scavenger prior to the addition of cerium chloride.

Alteration of myocardial mechanics in marginal magnesium deficiency.

Magnesium has attracted attention as an essential element with diverse roles in the regulation of cardiac contraction. Chronic suboptimal intake of the element results in hypomagnesaemia. Experimental and clinical studies indicate the possibility of a marginal decrease in myocardial magnesium compared to those with sufficient intake. Reduction in extracellular magnesium affects myocardial excitability and contractility predominantly, by modulation of the levels of other ions that have an influence on cardiac mechanics. Majority of the in vitro experiments in isolated ventricular tissue or myocytes record an inverse relation between Mg concentration and inotropic response, mediated probably by enhanced influx of Ca2+ promoting sarcoplasmic reticular Ca2+ release. Paradoxically myocardial contractility is usually compromised in animals on Mg deficient diet or on perfusion of whole heart with low Mg (< 0.5 mM) buffer. In the whole animal or organ, magnesium deficiency induced coronary vasospasm, defective energy metabolism and excessive free radical generation may be important variables acting in concert or independently to affect myocardial function. Electrical excitability is enhanced in magnesium deficiency, and arrhythmic changes are presumed to be mediated by disturbance in K+ homeostasis. Magnesium deficiency has not received the attention it deserves probably due to absence of clinical symptoms. Magnesium deficiency concomitant with stress may be of clinical significance, leading to arrhythmic, hemodynamic and ischaemic changes in the heart. Chronic magnesium deficiency is accompanied by increased free radical generation. Free radicals are known to influence myocardial excitability and contractility. Physiologic and pathologic stress also promotes free radical generation. The additive action of free radical generation in magnesium deficiency and any form of stress may be one of the reasons for enhanced sensitivity to stress in magnesium deficiency. Clinical and experimental data on the cardiac consequences of marginal magnesium deficiency being limited, a number of factors need experimental validation. For example--the extent of change in total and ionized magnesium in the serum and heart, mechanical response of the myocardium to decrease of total and ionized magnesium in the intra- and extracellular milieu; the extent of free radical generation in magnesium deficiency and the cardiac consequence; and also the additive effect of magnesium deficiency and different forms of stress.