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Dietary fish oil attenuates cardiac hypertrophy in lipotoxic cardiomyopathy due to systemic carnitine deficiency
Abstract
1,2-Diacylglycerol (DAG), a lipid second messenger that activates protein kinase C (PKC), is increased with a distinct fatty acid composition in the heart of the juvenile visceral steatosis (JVS) mouse, which develops pathological cardiac hypertrophy with lipid accumulation induced by the perturbation of fatty acid beta-oxidation due to systemic carnitine deficiency. Fish oil (FO) may exert its beneficial effects on the cardiomyopathy in JVS mice by modifying the molecular species composition of myocardial DAG. To test this hypothesis, we investigated the effects of dietary FO on the hypertrophied hearts in JVS mice.
Both control and JVS mice were fed a FO diet (containing 10% FO) or a standard diet from 4 weeks of age.
At 8 weeks of age, the ventricular-to-body weight ratio in JVS mice was 2.7-fold higher than that in controls (9.9 +/- 0.1 vs. 3.7 +/- 0.1 mg/g, P < 0.01) and was reduced by dietary FO (7.7 +/- 0.1 mg/g, P < 0.01 vs. JVS mice). In JVS mice, myocardial DAG levels were elevated by 2.3-fold with a distinct fatty acid composition with increases in C18:1n-7,9 and C18:2n-6 fatty acids compared with controls; dietary FO had no effects on the total DAG levels but significantly altered the fatty acid composition of DAG with reduction of both fatty acid species. Immunoblot analysis showed that dietary FO prevented the membrane translocation of cardiac PKCs alpha, beta2, and epsilon in JVS mice. Dietary FO did not affect the plasma and myocardial total carnitine levels in JVS mice. Furthermore, dietary FO significantly improved the progressive left ventricular dysfunction and survival rate in JVS mice.
Dietary FO may attenuate cardiac hypertrophy with improvements in cardiac function and survival in JVS mice via modification of the molecular species composition of myocardial DAG and the consequent inhibition of PKC redistribution. These results suggest the implication of the molecular species composition of DAG in the pathogenesis of lipotoxic cardiomyopathy due to perturbations of fatty acid beta-oxidation.
... A second hypothesis is that ω3-PUFAs attenuate cardiac hypertrophy (Takahashi et al., 2005; Duda et al., 2007; Ramadeen et al., 2010; Chen et al., 2011), resulting in a decrease of NHE-1 activity (Baartscheer et al., 2005). In the present study, however, the relative heart weight and cell capacitance, both indices of cardiac hypertrophy, did not differ between ω3-PUFAs and ω9-MUFAs fed rabbits or their myocytes, respectively (Figure 6). ...
... Various studies demonstrate that ω3-PUFAs suppress development of hypertrophy and HF (Takahashi et al., 2005; Duda et al., 2007; Ramadeen et al., 2010; Chen et al., 2011). Because cardiac hypertrophy leads to a decrease of the surface to volume ratio of myocytes, an increased number of NHE-1 proteins are required to maintain a normal “cytoplasmic” NHE-1 mediated acid load recovery. ...
... ω3-PUFAs affect several of these NHE-1 stimuli. They reduce diacylglycerol and PKC, activate the parasympathetic nervous system, and reduce angiotensin-converting enzyme (ACE) activity (Mohan and Das, 2001; Seung-Kim et al., 2001; Takahashi et al., 2005), although the latter is not a consistent finding (Ogawa et al., 2009). An intriguing question is why the NHE-1 activity in myocytes of ω3-PUFA fed animals is lower than in myocytes of ω9-MUFA fed animals in our model of volume- and pressure-overload, while it is not different in healthy rabbits. ...
Background: Increased consumption of omega-3 polyunsaturated fatty acids (ω3-PUFAs) from fish oil (FO) may have cardioprotective effects during ischemia/reperfusion, hypertrophy, and heart failure (HF). The cardiac Na⁺/H⁺-exchanger (NHE-1) is a key mediator for these detrimental cardiac conditions. Consequently, chronic NHE-1 inhibition appears to be a promising pharmacological tool for prevention and treatment. Acute application of the FO ω3-PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibit the NHE-1 in isolated cardiomyocytes. We studied the effects of a diet enriched with ω3-PUFAs on the NHE-1 activity in healthy rabbits and in a rabbit model of HF induced by volume- and pressure-overload. Methods: Rabbits were allocated to four groups. The first two groups consisted of healthy rabbits, which were fed either a diet containing 1.25% (w/w) FO (ω3-PUFAs), or 1.25% high-oleic sunflower oil (ω9-MUFAs) as control. The second two groups were also allocated to either a diet containing ω3-PUFAs or ω9-MUFAs, but underwent volume- and pressure-overload to induce HF. Ventricular myocytes were isolated by enzymatic dissociation and used for intracellular pH (pHi) and patch-clamp measurements. NHE-1 activity was measured in HEPES-buffered conditions as recovery rate from acidosis due to ammonium prepulses. Results: In healthy rabbits, NHE-1 activity in ω9-MUFAs and ω3-PUFAs myocytes was not significantly different. Volume- and pressure-overload in rabbits increased the NHE-1 activity in ω9-MUFAs myocytes, but not in ω3-PUFAs myocytes, resulting in a significantly lower NHE-1 activity in myocytes of ω3-PUFA fed HF rabbits. The susceptibility to induced delayed afterdepolarizations (DADs), a cellular mechanism of arrhythmias, was lower in myocytes of HF animals fed ω3-PUFAs compared to myocytes of HF animals fed ω9-MUFAs. In our rabbit HF model, the degree of hypertrophy was similar in the ω3-PUFAs group compared to the ω9-MUFAs group. Conclusion: Dietary ω3-PUFAs from FO suppress upregulation of the NHE-1 activity and lower the incidence of DADs in our rabbit model of volume- and pressure-overload.
... Membranous and cytosolic fractions of protein extracts from the myocardium were prepared as described previously [27]. All samples (20 Ag) were subjected to immunoblot analysis with the use of the antibodies against PKCh2 and PKC( (Santa Cruz Biotechnology, Santa Cruz, CA, USA), as reported previously [27]. ...
... Membranous and cytosolic fractions of protein extracts from the myocardium were prepared as described previously [27]. All samples (20 Ag) were subjected to immunoblot analysis with the use of the antibodies against PKCh2 and PKC( (Santa Cruz Biotechnology, Santa Cruz, CA, USA), as reported previously [27]. ...
... It has also been reported that the particular molecular species of DAG, rather than the total level, is related to the activation of PKC [42,43]. Recently, we have reported that dietary treatment with nÀ3 polyunsaturated fatty acids attenuated cardiac hypertrophy, altered the distinct fatty acid composition of myocardial DAG and inhibited the membrane translocation of PKC isoenzymes in JVS mice [27]. These results suggest that the distinct molecular species composition of DAG contribute to the PKC activation in JVS mice. ...
Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily and are key regulators of fatty acid oxidation (FAO) in the heart. Systemic carnitine deficiency (SCD) causes disorders of FAO and induces hypertrophic cardiomyopathy with lipid accumulation. We hypothesized that activation of PPARalpha by fenofibrate, a PPARalpha agonist, in addition to conventional L-carnitine supplementation may exert beneficial effects on the lipotoxic cardiomyopathy in juvenile visceral steatosis (JVS) mouse, a murine model of SCD.
Both wild-type (WT) and JVS mice were fed a normal chow, 0.2% fenofibrate containing chow (FE), a 0.1% L-carnitine containing chow (CA) or a 0.1% L-carnitine + 0.2% fenofibrate containing chow (CA + FE) from 4 weeks of age. Four to 8 animals per group were used for each experiment and 9 to 11 animals per group were used for survival analysis.
At 8 weeks of age, JVS mice exhibited marked ventricular hypertrophy, which was more attenuated by CA + FE than by CA or FE alone. CA + FE markedly reduced the high plasma and myocardial triglyceride levels and increased the low myocardial ATP content to control levels in JVS mice. In JVS mice, myocardial 1,2-diacylglycerol (DAG) was significantly increased and showed a distinct fatty acid composition with elevation of 18:1(n-7,9) and 18:2(n-6) fatty acids compared with that in WT mice. CA + FE significantly altered the fatty acid composition of DAG and inhibited the membrane translocation of cardiac protein kinase C beta2 in JVS mice. Furthermore, CA + FE prevented the progressive left ventricular dysfunction and dramatically improved the survival rate in JVS mice (survival rate at 400 days after birth: 89 vs. 0%, P < 0.0001).
PPARalpha activation, in addition to l-carnitine supplementation, may rescue the detrimental lipotoxic cardiomyopathy in SCD by improving cardiac energy and lipid metabolism as well as systemic lipid metabolism.
... The study demonstrated that the administration of L-carnitine restored left-ventricular tissue free-carnitine levels, prevented myocardial stiffening and pulmonary congestion, and improved survival in the hypertensive HFpEF model, which confirmed our hypothesis. The associations between decreased blood free-carnitine levels and reduced ejection fraction and the L-carnitine-induced improvement of ejection fraction have been reported in patients suffering primary systemic carnitine deficiency and [20,[43][44][45][46][47]. The current study expanded these previous studies by demonstrating that L-carnitine was effective in the treatment of heart failure even if ejection fraction was preserved, and the L-carnitine administration did not change left-ventricular endocardial and midwall fractional shortening in the HFpEF model. ...
... Free-carnitine is transported into organs through OCTN2 [56]. Spontaneous mutation of OCTN2 gene in primary systemic carnitine deficiency causes the decrease in plasma and tissue levels of free-carnitine [43,45]. Therefore, the decrease in plasma free-carnitine levels at HFpEF stage of age 20 weeks may be explained by the increased urinary free-carnitine excretion in association with the down-regulated renal OCTN2 expression. ...
Prognosis of heart failure with preserved ejection fraction (HFpEF) remains poor because of unknown pathophysiology and unestablished therapeutic strategy. This study aimed to identify a potential therapeutic intervention for HFpEF through metabolomics-based analysis.
Metabolomics with capillary electrophoresis time-of-flight mass spectrometry was performed using plasma of Dahl salt-sensitive rats fed high-salt diet, a model of hypertensive HFpEF, and showed decreased free-carnitine levels. Reassessment with enzymatic cycling method revealed the decreased plasma and left-ventricular free-carnitine levels in the HFpEF model. Urinary free-carnitine excretion was increased, and the expression of organic cation/carnitine transporter 2, which transports free-carnitine into cells, was down-regulated in the left ventricle (LV) and kidney in the HFpEF model. L-Carnitine was administered to the hypertensive HFpEF model. L-Carnitine treatment restored left-ventricular free-carnitine levels, attenuated left-ventricular fibrosis and stiffening, prevented pulmonary congestion, and improved survival in the HFpEF model independent of the antihypertensive effects, accompanied with increased expression of fatty acid desaturase (FADS) 1/2, rate-limiting enzymes in forming arachidonic acid, and enhanced production of arachidonic acid, a precursor of prostacyclin, and prostacyclin in the LV. In cultured cardiac fibroblasts, L-carnitine attenuated the angiotensin II-induced collagen production with increased FADS1/2 expression and enhanced production of arachidonic acid and prostacyclin. L-Carnitine-induced increase of arachidonic acid was canceled by knock-down of FADS1 or FADS2 in cultured cardiac fibroblasts. Serum free-carnitine levels were decreased in HFpEF patients.
L-carnitine supplementation attenuates cardiac fibrosis by increasing prostacyclin production through arachidonic acid pathway, and may be a promising therapeutic option for HFpEF.
... Dietary supplementation with ω-3 PUFA has multiple effect on cardiac biochemistry and function that were not evaluated in the present study. Takahashi et al observed that supplementation with fish oil attenuated hypertrophic cardiomyopathy in mice with carnitine deficiency [29]. Fish oil alters the diacylglycerol composition in the heart and prevented activation of protein kinase C (PKC) isoforms α, β 2 , and ε by reducing their translocation from the cytosol to the plasma membrane. ...
... Fish oil alters the diacylglycerol composition in the heart and prevented activation of protein kinase C (PKC) isoforms α, β 2 , and ε by reducing their translocation from the cytosol to the plasma membrane. Since chronic PKC activation has been linked to LVH and heart failure [30], the consumption of ω-3 PUFA could reduce the risk for heart failure [29] by suppressing PKC activity. In addition, ω-3 PUFA supplementation affect membrane composition and ion permeability, and could potential improve Ca2+ uptake into the sarcoplasmic reticulum and optimize the ability of the mitochondria to generate ATP [31,32]. ...
Epidemiological studies suggest that consumption of omega-3 polyunsaturated fatty acids (omega-3 PUFA) decreases the risk of heart failure. We assessed the effects of dietary supplementation with omega-3 PUFA from fish oil on the response of the left ventricle (LV) to arterial pressure overload.
Male Wistar rats were fed a standard chow or a omega-3 PUFA-supplemented diet. After 1 week rats underwent abdominal aortic banding or sham surgery (n=9-12/group). LV function was assessed by echocardiography after 8 weeks. In addition, we studied the effect of omega-3 PUFA on the cardioprotective adipocyte-derived hormone adiponectin, which may alter the pro-growth serine-threonine kinase Akt.
Banding increased LV mass to a greater extent with the standard chow (31%) than with omega-3 PUFA (18%). LV end diastolic and systolic volumes were increased by 19% and 105% with standard chow, respectively, but were unchanged with omega-3 PUFA. The expression of adiponectin was up-regulated in adipose tissue, and the plasma adiponectin concentration was significantly elevated. Treatment with omega-3 PUFA increased total Akt protein expression in the heart, but decreased the fraction of Akt in the active phosphorylated form, and thus did not alter the amount of active phospho-Akt.
Dietary supplementation with omega-3 PUFA attenuated pressure overload-induced LV dysfunction, which was associated with elevated plasma adiponectin.
... Furthermore, EPA and DHA can improve mitochondrial function, impairment of which has been demonstrated in the pathogenesis of cardiac hypertrophy [28,48]. It has been also suggested that fish oil rich in omega-3 fatty acids could prevent cardiac hypertrophy through DAG-PKC pathway [41]. Moreover, Siddiqui et al. [38] reported that the attenuating effects of DHA on cardiac hypertrophy are mediated through inhibition of down-stream kinases of the ERK pathway. ...
Study aim : In this study, we evaluated the effects of acute and chronic exercise on the plasma FAs and their association with cardiac hypertrophy indices.
Material and methods : In this pilot study, 15 sedentary and 15 athlete women underwent acute and long-term water aerobic exercise and their plasma FA levels and a number of electrocardiographic parameters, such as left ventricular end-diastolic diameter index (LVEDDI), left ventricular ejection fraction (LVEF), left ventricular mass index (LVMI), and wall thickness were evaluated before and after the exercise program.
Results : The acute exercise significantly increased palmitic and oleic acid levels in non-athletes and stearic acid in both groups. However, the same type of exercise decreased linoleic acid only in non-athlete women (p < 0.05). The water aerobics training caused a significant decrease in the levels of palmitic, stearic, and arachidonic acid, SFA/UFA, and ω3/ ω6 ratios and also an increase in α-Linolenic acid and MUFA in non-athletes. We found positive and negative correlations between LVEF with ω3 and SFA/UFA ratio in both groups, respectively. In the non-athlete group, the ω3/ω6 ratio showed negative correlations with LVMI and LVEDDI.
Conclusions : The study indicated that the 12-week exercise by sedentary women could make their plasma FAs composition similar to athlete women. Moreover, the plasma FA levels were associated with cardiac hypertrophy indices, showing the importance of FAs in physiological hypertrophy.
... This study did not support the notion that diets high in SFAs would result in cardiac hypertrophy and dysfunction. Fish oil-based or n-3 polyunsaturated fatty acids-based diets have been previously shown to be protective against cardiac hypertrophy, remodeling, and contractile dysfunction in other animal models indicating that the source of fat could have a significant impact on cardiac function [46,47]. ...
Purpose:
High calorie intake leads to obesity, a global socio-economic and health problem, reaching epidemic proportion in children and adolescents. Saturated and monounsaturated fatty acids from animal (lard) fat are major components of the western-pattern diet and its regular consumption leads to obesity, a risk factor for cardiovascular disease. However, no clear evidence exists whether consumption of diet rich in saturated (SFAs) and monounsaturated (MUFAs) fatty acids has detrimental effects on cardiac structure and energetics primarily due to excessive calories. We, therefore, sought to determine the impact of high calories versus fat content in diet on cardiac structure and mitochondrial energetics.
Methods:
Six-week-old C57BL/6J mice were fed with high calorie, high lard fat-based diet (60% fat, HFD), high-calorie and low lard fat-based diet (10% fat, LFD), and lower-calorie and fat diet (standard chow, 12% fat, SCD) for 10 weeks.
Results:
The HFD- and LFD-fed mice had higher body weight, ventricular mass and thickness of posterior and septal wall with increased cardiomyocytes diameter compared to the SCD-fed mice. These changes were associated with a reduction in the mitochondrial oxidative phosphorylation (OXPHOS) complexes I and III activity compared to the SCD-fed mice without significant differences between the HFD- and LFD-fed animals. The HFD-fed animals had higher level of malondialdehyde (MDA) than LFD and SCD-fed mice.
Conclusions:
We assume that changes in cardiac morphology and selective reduction of the OXPHOS complexes activity observed in the HFD- and LFD-fed mice might be related to excessive calories with additional effect of fat content on oxidative stress.
... In the absence of an underlying pathology, short durations of dietary intervention (3-8 wk) were found to have no effect on baseline cardiac contractile function measured in vivo and ex vivo (porcine and rodent) (13,20), whereas chronic diet treatment (24 mo) improved baseline ejection fraction in marmoset monkeys (31). Conversely, 2 wk of fish oil supplementation was sufficient to improve baseline ejection fraction of mice with cardiomyopathy induced by systemic carnitine deficiency (45). Cardiac contractile performance (ex vivo) following ischemia has been reported to be improved or not changed in hearts of animals fed a fish oil supplemented diet compared with a control diet (41,42,48). ...
A definitive understanding of the role of dietary lipids in determining cardio-protection (or cardio-detriment) has been elusive. Randomized trial findings have been variable and sex- specificity of dietary interventions has not been determined. In this investigation the sex-selective cardiac functional effects of three diets enriched by omega-3 or omega-6 polyunsaturated fatty acids (PUFA) or enriched to an equivalent extent in saturated fatty acid components were examined in rats after an 8 week treatment period. In females the myocardial membrane omega-6:omega-3 PUFA ratio was two-fold higher than male in the omega-6 diet replacement group. In diets specified to be high in omega-3 PUFA or in saturated fat, this sex difference was not apparent. Isolated cardiomyocyte and heart Langendorff perfusion experiments were performed, and molecular measures of cell viability assessed. Under basal conditions the contractile performance of omega-6 fed female cardiomyocytes and hearts was reduced compared with males. Omega-6 fed females exhibited impaired systolic resilience after ischemic insult. This response was associated with increased post ischemia necrotic cell damage evaluated by coronary lactate dehydrogenase during reperfusion in omega-6 fed females. Cardiac and myocyte functional parameters were not different between omega-3 and saturated fat dietary groups and within these groups there were no discernible sex differences. Our data provide evidence at both cardiac and cardiomyocyte level that dietary saturated fatty acid intake replacement with an omega-6 (but not omega-3) enriched diet has selective adverse cardiac effect in females. This finding has potential relevance in relation to women, cardiac risk and dietary management.
... Numerous studies have reported beneficial effects and/or associations of dietary n-3 FA, from both ALA, and DHA and EPA on atherosclerosis and its risk factors (Rosenberg, 2002;Mori et al., 1997;Zaloga et al., 2006;Baylin et al, 2003;Takahashi et al., 2005). Furthermore, epidemiological studies have revealed that both dietary ALA and EPA/DHA intake are positively associated with reduced CVD risk (Djousse et al., 2001;Hu et al., 1999, Hu et al., 2002Albert et al., 1998;Daviglus et al., 1997, Thies et al., 2003. ...
... Dietary supplementation with FO normalized levels of EPA-DHA in membrane DAG and reduced saturated FA and arachidonic acid concentrations. We did not see reductions in total levels of acyl CoA, TG or ceramides, a finding similar to that observed with FO treatment of carnitine deficient cardiomyopathy (24). In some models (29,30) including streptozotocin-induced diabetic rats (31), FO-preserved myocardial contractility was associated with reduced DAG and TG accumulation. ...
: Fish oil (FO) supplementation may improve cardiac function in some patients with heart failure, especially those with diabetes. To determine why this occurs, we studied the effects of FO in mice with heart failure either due to transgenic expression of the lipid uptake protein acyl CoA synthetase 1 (ACS1) or overexpression of the transcription factor peroxisomal proliferator-activated receptor (PPAR) γ via the cardiac-specific myosin heavy chain (MHC) promoter. ACS1 mice and control littermates were fed 3 diets containing low-dose or high-dose FO or nonpurified diet (NPD) for 6 weeks. MHC-PPARγ mice were fed low-dose FO or NPD. Compared with control mice fed with NPD, ACS1, and MHC-PPARγ, mice fed with NPD had reduced cardiac function and survival with cardiac fibrosis. In contrast, ACS1 mice fed with high-dose FO had better cardiac function, survival, and less myocardial fibrosis. FO increased eicosapentaenoic and docosahexaenoic acids and reduced saturated fatty acids in cardiac diacylglycerols. This was associated with reduced protein kinase C alpha and beta activation. In contrast, low-dose FO reduced MHC-PPARγ mice survival with no change in protein kinase C activation or cardiac function. Thus, dietary FO reverses fibrosis and improves cardiac function and survival of ACS1 mice but does not benefit all forms of lipid-mediated cardiomyopathy.
... Some investigations suggest beneficial effects of n23 FAs on hemodynamics (31), left ventricular indexes (32), and inflammation (33). For example, fish oil has been shown to inhibit natriuretic peptide production (34) and to alter the diacylglycerol composition in the heart and prevents activation of protein kinase C (35,36); chronic activation of protein kinase C has been related to left ventricular hypertrophy and HF (37). Canola oil, a major dietary source of ALA, was shown to reduce the incidence of ventricular fibrillation in rats (38). ...
Data on the relation of plasma and dietary omega-3 (n-3) fatty acids (FAs) with heart failure (HF) risk have been inconsistent.
We evaluated the relation of n-3 FAs with HF in US male physicians.
We used nested case-control (n = 1572) and prospective cohort study designs (n = 19,097). Plasma phospholipid n-3 FAs were measured by using gas chromatography, and food-frequency questionnaires were used to assess dietary n-3 FAs and fish intake. Incident HF was ascertained via annual follow-up questionnaires and validated in a subsample.
The mean age was 58.7 y at blood collection. In a multivariable model, plasma α-linolenic acid (ALA) was associated with a lower risk of HF in a nonlinear fashion (P-quadratic trend = 0.02), and the lowest OR was observed in quintile 4 (0.66; 95% CI: 0.47, 0.94). Plasma EPA and DHA were not associated with HF, whereas plasma docosapentaenoic acid (DPA) showed a nonlinear inverse relation with HF for quintile 2 (OR: 0.55; 95% CI: 0.39, 0.79). Dietary marine n-3 FAs showed a trend toward a lower risk of HF in quintile 4 (HR: 0.81; 95% CI: 0.64, 1.02) and a nonlinear pattern across quintiles. Fish intake was associated with a lower risk of HF, with RRs of ∼0.70 for all categories of fish consumption greater than one serving per month.
Our data are consistent with an inverse and nonlinear relation of plasma phospholipid ALA and DPA, but not EPA or DHA, with HF risk. Fish consumption greater than once per month was associated with a lower HF risk.
... Whereas C18:1n-7 may have some protective effects at normal physiological concentrations, it is possible that higher levels may adversely affect biologically active proteins in myocardial membranes, potentially contributing to the progression of HF. Thus, enhanced myocardial expression of C18:1n-7, an elongated product of palmitoleic acid, has been reported in hypertrophic cardiomyopathy in mice with carnitine deficiency [22], possibly promoting harmful effects. Moreover, Lemaitre et al. [23] recently found that high levels of C18:1n-7 were associated with sudden cardiac death, independent of low levels of EPA and DHA. ...
Free fatty acids (FFAs) are the major energy sources of the heart, and fatty acids (FAs) are active components of biological membranes. Data indicate that levels of FAs and their composition may influence myocardial function and inflammation. The aim of this study was to investigate whether total levels and composition of FAs and FFAs in plasma are altered in clinical heart failure (HF) and whether any alterations in these parameters are correlated with the severity of HF.
Plasma from 183 patients with stable HF was compared with plasma from 44 healthy control subjects.
Our main findings are as follows: (i) patients with HF had decreased levels of several lipid parameters and increased levels of FFAs in plasma, compared with controls, which were significantly correlated with clinical disease severity. (ii) Patients with HF also had a decreased proportion in the plasma of several n-3 polyunsaturated FAs, an increased proportion of several monounsaturated FAs, and a decreased proportion of some readily oxidized long-chain saturated FAs. (iii) These changes in FA composition were significantly associated with functional class, impaired cardiac function (i.e., decreased cardiac index and increased plasma N-terminal pro-B-type natriuretic peptide levels) and enhanced systemic inflammation (i.e., increased high-sensitivity C-reactive protein levels). (iv) Low levels of C20:4n-3 (eicosatetraenoic acid) and in particular high levels of C18:1n-7 (vaccenic acid) were significantly associated with total mortality in this HF population.
Our data demonstrate that patients with HF are characterized by a certain FA phenotype and may support a link between disturbed FA composition and the progression of HF.
... The filtrate was assayed for total carnitine (including acylcarnitine and free carnitine) using the enzymatic cycling method (Total carnitine 'Kainos' , Kainos Laboratories, Tokyo, Japan). 22 Light and Electron Microscopy BAT samples for examination by light microscopy were fixed in 10% phosphate-buffered formalin (pH 7.4), processed into wax blocks, sectioned (4 mm thick) and stained with hema-toxylin and eosin. Tissue fragments of BAT for electron microscopy were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. ...
The juvenile visceral steatosis (JVS) mouse is a mutant strain with an inherited systemic carnitine deficiency. Mice of this strain show clinical signs attributable to impaired heat production and disturbed energy production. Brown adipose tissue (BAT) is the primary site of non-shivering thermogenesis in the presence of uncoupling protein-1 (UCP-1) in rodents and humans, especially in infants. To investigate the possible cause of impaired heat production in BAT, we studied the morphological features, carnitine concentration, and UCP-1 production of BAT in JVS mice. The effect of carnitine administration on these parameters was also examined. JVS mice aged 5 or 10 days (60 each) and age-matched control mice were used in this study, along with 10-day-old JVS mice treated subcutaneously with L-carnitine once a day between postpartum days 5 and 10. JVS mice showed lower body temperatures and lower concentrations of carnitine in BAT. Morphologically, BAT cells in JVS mice contained large lipid vacuoles and small mitochondria, similar to those present in white adipose tissue cells. In addition, UCP-1 mRNA and protein expression levels were significantly reduced in JVS as compared with control mice. Carnitine treatment resulted in significant increases in body temperature and carnitine concentrations in BAT, together with the recovery of normal morphological features. UCP-1 mRNA and protein expression levels were also significantly increased. These findings strongly suggest that carnitine is essential for maintaining the function and morphology of BAT.
... n-3 PUFA may attenuate cardiac hypertrophy through different mechanisms. In vivo experiments in mice indicate that dietary fish oil supplementation have inhibitory effects on cardiac hypertrophy by inhibiting Ras -Raf-1extracellular regulated kinase 1 and 2 -p90 ribosomal s6 kinase pathaway (201), and possibly via modification of the molecular composition of myocardial 1,2-diacylglicerol and the consequent inhibition of protein kinase C (PKC) redistribution (213) . ...
Over the past 20 years, there has been significant progress in our knowledge of the pathophysiology of heart failure (HF) with consequent considerable development of both pharmacological and non pharmacological approaches. Despite improved therapeutic strategies, HF still remains burdensome in terms of mortality, quality of life, and hospitalization costs. A new and promising medical treatment to improve survival in HF patients stems from the recent results of the Italian study, Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardico-Heart Failure (GISSI-HF). GISSI-HF was a randomized, large scale, double-blind, placebo-controlled trial showing that n-3 PUFA (850-882 mg/d) reduced mortality and admission to the hospital for cardiovascular reasons in patients with chronic heart failure (HF) who were already receiving recommended therapies. The clinical benefit observed in GISSI-HF seemed to be mediated prominently by the antiarrhythmic effects of n-3 PUFA, though an effect on mechanisms related to HF progression cannot be excluded. This article presents the results of GISSI-HF study and reviews the previous clinical evidence on n-3 PUFA and risk of heart failure and discusses in depth the potential mechanisms through which n-3 PUFA treatment can improve clinical outcome in HF patients.
... Studies specifically examining the role of diets rich in polyunsaturated FA (PUFA) in the hearts of rats found that a diet high in fish oil ω-3 FA prevented cardiac remodeling and dysfunction following aortic banding-induced pressure overload [80]. Additionally, in mice with pathological cardiac hypertrophy and lipid accumulation resulting from systemic carnitine deficiency, fish oil containing diets reversed LV dysfunction through a proposed mechanism of altered diacylglycerol (DAG) composition and protein kinase c (PKC) redistribution [82]. However, diets rich in ω-6 PUFA, fed to diabetic rats resulted in increased cardiac necrosis [83]. ...
Obesity and insulin resistance are associated with ectopic lipid deposition in multiple tissues, including the heart. Excess lipid may be stored as triglycerides, but are also shunted into non-oxidative pathways that disrupt normal cellular signaling leading to organ dysfunction and in some cases apoptosis, a process termed lipotoxicity. Various pathophysiological mechanisms have been proposed to lead to lipotoxic tissue injury, which might vary by cell type. Specific mechanisms by which lipotoxicity alter cardiac structure and function are incompletely understood, but are beginning to be elucidated. This review will focus on mechanisms that have been proposed to lead to lipotoxic injury in the heart and will review the state of knowledge regarding potential causes and correlates of increased myocardial lipid content in animal models and humans. We will seek to highlight those areas where additional research is warranted.
... 100 More importantly, an untreated carnitine defi ciency may adversely impact cardiac function and is associated with sudden death [101][102][103][104][105][106][107] and dilated cardiomyopathy. [108][109][110] Carnitine supplementation will prevent such complications and is warranted once a carnitine defi ciency is identifi ed. Carnitine also plays a critical role in fatty acid transport into the mitochondria and may contribute to abnormal fatty acid metabolism. ...
Verbal apraxia is a neurologically based motor planning speech disorder of unknown etiology common in autism spectrum disorders. Vitamin E deficiency causes symptoms that overlap those of verbal apraxia. Polyunsaturated fatty acids in the cell membrane are vulnerable to lipid peroxidation and early destruction if vitamin E is not readily available, potentially leading to neurological sequelae. Inflammation of the gastrointestinal (GI) tract and malabsorption of nutrients such as vitamin E and carnitine may contribute to neurological abnormalities. The goal of this investigation was to characterize symptoms and metabolic anomalies of a subset of children with verbal apraxia who may respond to nutritional interventions.
A total of 187 children with verbal apraxia received vitamin E + polyunsaturated fatty acid supplementation. A celiac panel, fat-soluble vitamin test, and carnitine level were obtained in patients having blood analyzed.
A common clinical phenotype of male predominance, autism, sensory issues, low muscle tone, coordination difficulties, food allergy, and GI symptoms emerged. In all, 181 families (97%) reported dramatic improvements in a number of areas including speech, imitation, coordination, eye contact, behavior, sensory issues, and development of pain sensation. Plasma vitamin E levels varied in children tested; however, pretreatment levels did not reflect clinical response. Low carnitine (20/26), high antigliadin antibodies (15/21), gluten-sensitivity HLA alleles (10/10), and zinc (2/2) and vitamin D deficiencies (4/7) were common abnormalities. Fat malabsorption was identified in 8 of 11 boys screened.
We characterize a novel apraxia phenotype that responds to polyunsaturated fatty acids and vitamin E. The association of carnitine deficiency, gluten sensitivity/food allergy, and fat malabsorption with the apraxia phenotype suggests that a comprehensive metabolic workup is warranted. Appropriate screening may identify a subgroup of children with a previously unrecognized syndrome of allergy, apraxia, and malabsorption who are responsive to nutritional interventions in addition to traditional speech and occupational therapy. Controlled trials in apraxia and autism spectrum disorders are warranted.
... 12,[112][113][114] 7. v-3PUFA in animal models of HF Experimental studies also support and help in the understanding of the favourable effect of EPA þ DHA on HF. Takahashi et al. 115 found that dietary supplementation with fish oil attenuated cardiac hypertrophy with improved cardiac function and prolonged life in mice with genetic systemic carnitine deficiency. These mice develop HF secondary to the inability to oxidize fatty acids. ...
Heart failure (HF) is a complex clinical syndrome with multiple aetiologies. Current treatment options can slow the progression to HF, but overall the prognosis remains poor. Clinical studies suggest that high dietary intake of the omega-3 polyunsaturated fatty acids (omega-3PUFA) found in fish oils (eicosapentaenoic and docosahexaenoic acids) may lower the incidence of HF, and that supplementation with pharmacological doses prolongs event-free survival in patients with established HF. The mechanisms for these potential benefits are complex and not well defined. It is well established that fish oil supplementation lowers plasma triglyceride levels, and more recent work demonstrates anti-inflammatory effects, including reduced circulating levels of inflammatory cytokines and arachidonic acid-derived eicosanoids, and elevated plasma adiponectin. In animal studies, fish oil favourably alters cardiac mitochondrial function. All of these effects may work to prevent the development and progression of HF. The omega-3PUFA found in plant sources, alpha-linolenic acid, may also be protective in HF; however, the evidence is not as compelling as for fish oil. This review summarizes the evidence related to use of omega-3PUFA supplementation as a potential treatment for HF and discusses possible mechanisms of action. In general, there is growing evidence that supplementation with omega-3PUFA positively impacts established pathophysiological targets in HF and has potential therapeutic utility for HF patients.
... In this issue of Cardiovascular Research, Takahashi et al. present the interesting finding that dietary supplementation with fish oil attenuates the development of hypertrophic cardiomyopathy and prolongs life in mice with carnitine deficiency [2]. Moreover, they found that ingestion of fish oil differentially altered the myocardial concentrations of various diacylglycerols and prevented diacylglyerol-induced activation of protein kinase C (PKC) isoforms a, h 2 , and ( by reducing their translocation from the cytosol to the plasma membrane. ...
See article by Takahashi et al. [2] (pages 213–223) in this issue.
There are a myriad of structural and biochemical abnormalities associated with the failing heart, yet the hallmark of this syndrome is the inability to effectively transfer the chemical energy from foodstuffs to the mechanical energy of left ventricular ejection against aortic pressure. The causes and consequences of dysfunctional energy metabolism in heart failure are poorly understood; nevertheless, there is growing evidence to suggest that alterations in energy substrate metabolism contribute to cardiac hypertrophy, left ventricular remodeling, and systolic dysfunction. Present medical therapies for heart failure act via suppression of neurohormonal activation (e.g. β-adrenergic receptor antagonists, angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists, and aldosterone receptor antagonists), reducing volume overload (diuretics), or hemodynamic symptoms (inotropic agents). Despite optimal treatment with current drugs, most patients continue to deteriorate, and the prognosis remains poor; thus, additional effective therapies are needed that act independent of …
*Corresponding author. Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4970, USA. Tel.: +1 216 368 5585; fax: +1 216 368 3952. Email address: wcs4{at}case.edu
... Increased PUFA intake is associated with a reduction of reactive oxygen species and an increase in antioxidants during hypertension [187][188][189]. Dietary fish oils can prevent LVH in transgenic mice with a carnitine transporter mutation, which was attributed to modified diacylglycerol composition and resultant inhibition of PKC activation [190]. Siddiqui et al. demonstrated that n-3 polyunsaturated lipid docosahexaenoic acid (DHA) prevents cardiac hypertrophy by inhibition of the Ras-Raf1-Erk1/2-p90 rsk signaling pathway in a phenylephrine-induced hypertrophy model [191]. ...
Currently, a high carbohydrate/low fat diet is recommended for patients with hypertension; however, the potentially important role that the composition of dietary fat and carbohydrate plays in hypertension and the development of pathological left ventricular hypertrophy (LVH) has not been well characterized. Recent studies demonstrate that LVH can also be triggered by activation of insulin signaling pathways, altered adipokine levels, or the activity of peroxisome proliferator-activated receptors (PPARs), suggesting that metabolic alterations play a role in the pathophysiology of LVH. Hypertensive patients with high plasma insulin or metabolic syndrome have a greater occurrence of LVH, which could be due to insulin activation of the serine-threonine kinase Akt and its downstream targets in the heart, resulting in cellular hypertrophy. PPARs also activate cardiac gene expression and growth and are stimulated by fatty acids and consumption of a high fat diet. Dietary intake of fats and carbohydrate and the resultant effects of plasma insulin, adipokine, and lipid concentrations may affect cardiomyocyte size and function, particularly in the setting of chronic hypertension. This review discusses potential mechanisms by which dietary carbohydrates and fats ca affect cardiac growth, metabolism, and function, mainly in the context of pressure overload-induced LVH.
Regulation of cardiac fatty acid metabolism is central to the development of cardiac hypertrophy and heart failure. We investigated the effects of select fatty acids on the expression of genes involved in immediate early as well as inflammatory and hypertrophic responses in adult rat cardiomyocytes. Cardiac remodeling begins with upregulation of immediate early genes for c-fos and c-jun, followed by upregulation of inflammatory genes for nuclear factor kappa B (NF-κB) and nuclear factor of activated T-cells (NFAT). At later stages, genes involved in hypertrophic responses, such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), are upregulated. Adult rat cardiomyocytes were treated with palmitic acid, a saturated fatty acid; oleic acid, a monounsaturated fatty acid; linoleic acid, a polyunsaturated fatty acid belonging to the n-6 class; and docosahexaenoic acid, a polyunsaturated fatty acid belonging to the n-3 class. Linoleic acid produced a greater increase in the mRNA expression of c-fos, c-jun, NF-κB, NFAT3, ANP, and BNP relative to palmitic acid and oleic acid. In contrast, docosahexaenoic acid caused a decrease in the expression of genes involved in cardiac hypertrophy. Our findings suggest that linoleic acid may be a potent inducer of genes involved in cardiac hypertrophy, whereas docosahexaenoic acid may be protective against the cardiomyocyte hypertrophic response.
Background & aims:
While marine omega-3 fatty acids have been associated with a lower mortality in heart failure patients, data on omega-3 and incident heart failure are inconsistent. We systematically reviewed the evidence on the association of omega-3 fatty acids and fish intake with the incidence of heart failure in this meta-analysis.
Methods:
We identified relevant studies by searching MEDLINE and EMBASE databases up to August 31, 2011 without restrictions and by reviewing reference lists from retrieved articles.
Results:
A total of 176,441 subjects and 5480 incident cases of heart failure from 7 prospective studies were included in this analysis. Using random effect model, the pooled relative risk for heart failure comparing the highest to lowest category of fish intake was 0.85 (95% CI; 0.73-0.99), p = 0.04; corresponding value for marine omega-3 fatty acids was 0.86 (0.74-1.00), p = 0.05. There was no evidence for heterogeneity across studies of fish consumption (I(2) = 8%). In contrast, there was modest heterogeneity for omega-3 fatty acid analysis (I(2) = 44%). Lastly, there was no evidence for publication bias.
Conclusions:
This meta-analysis is consistent with a lower risk of heart failure with intake of marine omega-3 fatty acids. These observational findings should be confirmed in a large randomized trial.
Despite aggressive pharmacotherapy, heart failure is still clinical problem. Current therapies improve clinical symptoms and slow progression to heart failure, but overall the prognosis remains poor. Evidence from epidemiological, clinical and experimental studies indicates a beneficial role of the omega-3 polyunsaturated fatty acids (omega-3 PUFA) found in fish oils in the prevention and management of heart failure. Although the mechanisms is still unclear, clinical and animals studies indicate that the benefits of omega-3 PUFA may be attributed to a number of distinct biological effects on lipoprotein metabolism, inflammation response and mitochondrial function. This review summarise the data related to use of omega-3 PUFA supplementation as a potential treatment for heart failure and discussed possible mechanism of action.
Regular fish or fish oil intake is associated with a low incidence of heart failure clinically, and fish oil-induced reduction in cardiac remodelling seen in hypertrophy models may contribute. We investigated whether improved cardiac energy efficiency in non-hypertrophied hearts translates into attenuation of cardiac dysfunction in hypertrophied hearts. Male Wistar rats (n 33) at 8 weeks of age were sham-operated or subjected to abdominal aortic stenosis to produce pressure-overload cardiac hypertrophy. Starting 3 weeks post-operatively to follow initiation of hypertrophy, rats were fed a diet containing 10 % olive oil (control) or 5 % fish oil (ROPUFA® 30 (17 % EPA, 10 % DHA))+5 % olive oil (FO diet). At 15 weeks post-operatively, ventricular haemodynamics and oxygen consumption were evaluated in the blood-perfused, isolated working heart. Resting and maximally stimulated cardiac output and external work were >60 % depressed in hypertrophied control hearts but this was prevented by FO feeding, without attenuating hypertrophy. Cardiac energy efficiency was lower in hypertrophy, but greater in FO hearts for any given cardiac mass. Coronary blood flow, restricted in hypertrophied control hearts, increased with increasing work in hypertrophied FO hearts, revealing a significant coronary vasodilator reserve. Pronounced cardiac dysfunction in hypertrophied hearts across low and high workloads, indicative of heart failure, was attenuated by FO feeding in association with membrane incorporation of n-3 PUFA, principally DHA. Dietary fish oil may offer a new approach to balancing the high oxygen demand and haemodynamic requirements of the failing hypertrophied heart independently of attenuating hypertrophy.
Since omega-3 polyunsaturated fatty acids (n-3 PUFAs) can alter ventricular myocyte calcium handling, these fatty acids could adversely affect cardiac contractile function, particularly following myocardial infarction. Therefore, 4 wk after myocardial infarction, dogs were randomly assigned to either placebo (corn oil, 1 g/day, n = 16) or n-3 PUFAs supplement [docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) ethyl esters; 1, 2, or 4 g/day; n = 7, 8, and 12, respectively] groups. In vivo, ventricular function was evaluated by echocardiography before and after 3 mo of treatment. At the end of the 3-mo period, hearts were removed and in vitro function was evaluated using right ventricular trabeculae and isolated left ventricular myocytes. The treatment elicited significant (P < 0.0001) dose-dependent increases (16.4-fold increase with 4 g/day) in left ventricular tissue and red blood cell n-3 PUFA levels (EPA + DHA, placebo, 0.42 +/- 0.04; 1 g/day, 3.02 +/- 0.23; 2 g/day, 3.63 +/- 0.17; and 4 g/day, 6.97 +/- 0.33%). Regardless of the dose, n-3 PUFA treatment did not alter ventricular function in the intact animal (e.g., 4 g/day, fractional shortening: pre, 42.9 +/- 1.6 vs. post, 40.1 +/- 1.7%; placebo: pre, 39.2 +/- 1.3 vs. post, 38.4 +/- 1.6%). The developed force per cross-sectional area, changes in length- and frequency-dependent behavior in contractile force, and the inotropic response to beta-adrenoceptor activation were also similar for trabeculae obtained from placebo- or n-3 PUFA-treated dogs. Finally, calcium currents and calcium transients were the same in myocytes from n-3 PUFA- and placebo-treated dogs. Thus dietary n-3 PUFAs did not adversely alter either in vitro or in vivo ventricular contractile function in dogs with healed infarctions.
Diabetes mellitus is estimated to affect approximately 7% of the populations living a western lifestyle. Of the multiple etiologies associated with diabetes, heart failure is the most common cause of death. A specific type of heart disease called diabetic cardiomyopathy is thought to be partially responsible. At this time, no one specific treatment is available for diabetic cardiomyopathy due to the wide variety of possible complex molecular changes, including metabolic disturbances, myocardial fibrosis, LV hypertrophy, and increased ROS production. Abnormal copper metabolism in diabetes has been proposed to form part of the pathway that leads to diabetic cardiomyopathy. Our group have shown that treatment with the copper (CuII) chelator, triethylenetetramine, ameliorates the effects of diabetes on the heart at both the functional and molecular level. This thesis aimed to further these studies by increasing our understanding of the mechanism of triethylenetetramine action on the diabetic heart. This was primarily achieved through the use of microarray technology but included the use of a range of molecular experimental techniques. During this investigation it was determined that the most suitable microarray platform for our studies was the Affymetrix GeneChip® system. Using this system we identified more than 1600 gene changes associated with diabetes in the left ventricle wall. A disproportionate number of significant messenger RNA transcript changes were associated with the mitochondria and further investigation of these genes revealed changes associated with perturbed lipid metabolism and increased oxidative stress. A second study investigated the molecular mechanisms underpinning improved cardiac function in the left ventricle of the heart from diabetic and sham animals treated with triethylenetetramine. There was an observed decrease in diabetic cardiac tissue triglyceride towards normal, possibly through improvement of the structure and stability of the mitochondria. Only a small number of changes in gene expression were detected after triethylenetetramine treatment using microarray technology, and none were detected using real time-quantitative PCR. The final aim of this thesis was to understand the absorption and excretion of triethylenetetramine by both sham and diabetic animals. Our study found differences in the ability of diabetic animals to absorb and metabolise triethylenetetramine compared to sham animals. Also, the length of exposure was found to be an influencing factor in triethylenetetramine metabolism.
The effects of dietary supplementation with fat of different fatty acid profile and chronic intermittent hypoxia (CIH) on the fatty acid composition of serum and heart lipids were analysed. Adult male Wistar rats were fed a standard non-fat diet enriched with 10 % of lard, fish oil (n-3 PUFA) or maize oil (n-6 PUFA) for 10 weeks. After 4 weeks on the diets, each group was divided in two subgroups, either exposed to CIH in a barochamber (7000 m, twenty-five exposures) or kept at normoxia. In normoxic rats, the fish oil diet increased the level of conjugated dienes. The n-6:n-3 PUFA ratio in serum TAG, phospholipids (PL), cholesteryl esters (CE) and heart TAG, PL and diacylglycerols (DAG) followed the ratio in the fed diet (in the sequence maize oil>lard>fish oil). In heart TAG, PL and DAG, 20 : 4n-6 and 18 : 2n-6 were replaced by 22 : 6n-3 in the fish oil group. The main fatty acid in CE was 20 : 4n-6 in the lard and maize oil groups whereas in the fish oil group, half of 20 : 4n-6 was replaced by 20 : 5n-3. CIH further increased 20 : 5n-3 in CE in the fish oil group. CIH decreased the n-6:n-3 PUFA ratio in serum CE, heart TAG, PL and DAG in all dietary groups and stimulated the activity of catalase in the maize and fish oil groups. In conclusion, PUFA diets and CIH, both interventions considered to be cardioprotective, distinctly modified the fatty acid profile in serum and heart lipids with specific effects on conjugated diene production and catalase activity.
Clinically and experimentally, a case for omega-3 polyunsaturated fatty acid (PUFA) cardioprotection in females has not been clearly established. The goal of this study was to investigate whether dietary omega-3 PUFA supplementation could provide ischemic protection in female mice with an underlying genetic predisposition to cardiac hypertrophy. Mature female transgenic mice (TG) with cardiac-specific overexpression of angiotensinogen that develop normotensive cardiac hypertrophy and littermate wild-type (WT) mice were fed a fish oil-derived diet (FO) or PUFA-matched control diet (CTR) for 4 wk. Myocardial membrane lipids, ex vivo cardiac performance (intraventricular balloon) after global no-flow ischemia and reperfusion (15/30 min), and reperfusion arrhythmia incidence were assessed. FO diet suppressed cardiac growth by 5% and 10% in WT and TG, respectively (P < 0.001). The extent of mechanical recovery [rate-pressure product (RPP) = beats/min x mmHg] of FO-fed WT and TG hearts was similar (50 +/- 7% vs. 45 +/- 12%, 30 min reperfusion), and this was not significantly different from CTR-fed WT or TG. To evaluate whether systemic estrogen was masking a protective effect of the FO diet, the responses of ovariectomized (OVX) WT and TG mice to FO dietary intervention were assessed. The extent of mechanical recovery of FO-fed OVX WT and TG (RPP, 50 +/- 4% vs. 64 +/- 8%) was not enhanced compared with CTR-fed mice (RPP, 60 +/- 11% vs. 80 +/- 8%, P = 0.335). Dietary FO did not suppress the incidence of reperfusion arrhythmias in WT or TG hearts (ovary-intact mice or OVX). Our findings indicate a lack of cardioprotective effect of dietary FO in females, determined by assessment of mechanical and arrhythmic activity postischemia in a murine ex vivo heart model.
We examined the development of cardiac hypertrophy in juvenile visceral steatosis (JVS) mice, a model of systemic carnitine
deficiency, by varying the amount of lipid in the diet. Cardiac hypertrophy was markedly attenuated by decreasing soy bean
oil (SBO) from 5% (w/w) to 1%. Triglyceride contents of the ventricles of JVS mice fed 1% SBO were significantly lower than
in JVS mice fed 5% SBO. The addition of medium-chain triglycerides metabolically utilized by JVS mice did not affect the development
of cardiac hypertrophy. On the other hand, the mRNA levels of atrial natriuretic peptide and skeletal α-actin, which are related
to cardiac hypertrophy, were also attenuated by decreasing lipid in the diet. Adenylate energy charge and creatine phosphate
in the heart of JVS mice at the early stage of hypertrophy were not significantly different from control mice given the same
laboratory chow (4.6% of lipid). Although urinary prostaglandin F2α levels were found to be increased in JVS mice at 15 days of age when they developed cardiac hypertrophy, administration of
aspirin was not efficacious. We, therefore, propose that the proportion of lipid in the diet is important in the development
of cardiac hypertrophy in carnitine-deficient JVS mice, and that this is not related to prostaglandin formation.
Fish oils rich in n-3 fatty acids reduce serum triglyceride levels. This well known effect has been shown to be caused by decreased very low-density lipoprotein triglyceride secretion rates in kinetic studies in humans. Animal studies have explored the biochemical mechanisms underlying this effect. Triglyceride synthesis could be reduced by n-3 fatty acids in three general ways: reduced substrate (i.e. fatty acids) availability, which could be secondary to increase in beta-oxidation, decreased free fatty acids delivery to the liver, decreased hepatic fatty acids synthesis; increased phospholipid synthesis; or decreased activity of triglyceride-synthesizing enzymes (diacylgylcerol acyltranferase or phosphatidic acid phosphohydrolase).
Rarely were experimental conditions used in rat studies physiologically relevant to the human situation in which 1.2% energy as n-3 fatty acids lowers serum triglyceride levels. Nevertheless, the most consistent effect of n-3 fatty acids feeding in rats is to decrease lipogenesis. Increased beta-oxidation was frequently, but not consistently, reported with similar numbers of studies reporting increased mitochondrial compared with peroxisomal oxidation. Inhibition of triglyceride-synthesizing enzymes was only occasionally noted.
As the vast majority of studies fed unphysiologically high doses of n-3 fatty acids, these findings in rats must be considered tentative, and the mechanism by which n-3 fatty acids reduce triglyceride levels in humans remains speculative.
Carnitine is an essential cofactor for the oxidation of fatty acid in the mitochondria and an efficient therapeutics for primary carnitine deficiency. We herein analyzed the prolonged effects of carnitine on the reduced locomotor activity and energy metabolism of fasted carnitine-deficient juvenile visceral steatosis (jvs(-/-)) mice. We found that a single carnitine administration to 24-h fasted jvs(-/-) mice in the morning increased both the locomotor activity and oxygen consumption at night not only on the same day, but also on the next day, when the carnitine levels in the blood and tissues were already as low as at the original carnitine-deficient state. We also found that fat utilization for energy production significantly increased under fasting even in jvs(-/-) mice and was stimulated in the carnitine-administrated fasted jvs(-/-) mice at night, in comparison to that observed in the saline-administered jvs(-/-) mice, at least for 2 days even under the low plasma and tissue carnitine levels. These results suggest that the low tissue carnitine levels are therefore not the sole rate-limiting factor of general fatty acid oxidation in carnitine-deficient jvs(-/-) mice.
We examined the influence of dietary fatty acid (FA) classes on the expression of protein kinase C (PKC) delta and epsilon in relation to the cardioprotective effects of chronic intermittent hypoxia (CIH). Adult male Wistar rats were fed a nonfat diet enriched with 10% lard (saturated FA [SFA]), fish oil (n-3 polyunsaturated FA [n-3 PUFA]), or corn oil (n-6 PUFA) for 10 weeks. After 4 weeks on the diet, each group was divided into two subgroups that were either exposed to CIH in a barochamber (7000 m, 8 hrs/ day) or kept at normoxia for an additional 5-6 weeks. A FA phospholipid profile and Western blot analysis of PKC were performed in left ventricles. Infarct size was assessed in anesthetized animals subjected to 20-min coronary artery occlusion and 3-hr reperfusion. CIH decreased the n-6/n-3 PUFA ratio in all groups by 23% independently of the initial value set by various diets. The combination of n-3 diet and CIH had a stronger antiarrhythmic effect during reperfusion than the n-3 diet alone; this effect was less pronounced in rats fed the n-6 diet. The normoxic n-6 group exhibited smaller infarctions (by 22%) than the n-3 group. CIH decreased the infarct size in n-3 and SFA groups (by 20% and 23%, respectively) but not in the n-6 group. Unlike PKC epsilon, the abundance of PKC delta in the myocardial particulate fraction was increased by CIH except for the n-6 group. Myocardial infarct size was negatively correlated (r=- 0.79) with the abundance of PKC delta in the particulate fraction. We conclude that lipid diets modify the infarct size-limiting effect of CIH by a mechanism that involves the PKC delta-dependent pathway.
Primary systemic carnitine deficiency is an autosomal recessive disorder caused by a decreased renal reabsorption of carnitine because of mutations of the carnitine transporter OCTN2 gene, and hypertrophic cardiomyopathy is a common clinical feature of homozygotes. Although heterozygotes for OCTN2 mutations are generally healthy with normal cardiac performance, heterozygotes may be at risk for cardiomyopathy in the presence of additional risk factors, such as hypertension. To test this hypothesis, we investigated the effects of surgically induced pressure overload on the hearts of heterozygous mutants of a murine model of OCTN2 mutation, juvenile visceral steatosis mouse (jvs/+). Eleven-week-old jvs/+ mice and age-matched wild-type mice were used. At baseline, there were no differences in physical characteristics between wild-type and jvs/+ mice. However, plasma and myocardial total carnitine levels in jvs/+ mice were lower than in wild-type mice. Both wild-type and jvs/+ mice were subjected to ascending aortic constriction with or without 1% l-carnitine supplementation for 4 weeks. At 4 weeks after ascending aortic constriction, jvs/+ mice showed an exaggeration of cardiac hypertrophy and pulmonary congestion, further increased gene expression of atrial natriuretic peptide in the left ventricles, further deterioration of left ventricular fractional shortening, reduced myocardial phosphocreatine:adenosine triphosphate ratio, and increased mortality compared with wild-type mice; l-carnitine supplementation prevented these changes in jvs/+ mice subjected to ascending aortic constriction. In conclusion, cardiomyopathy and heart failure with energy depletion may be induced by pressure overload in heterozygotes for OCTN2 mutations and could be prevented by l-carnitine supplementation.
Long-chain n-3 polyunsaturated fatty acids (PUFAs) are selectively incorporated into cardiac cell membranes from the diet in a dose-related manner. Regular intake can slow the heart rate, reduce myocardial oxygen consumption, and increase coronary reserve. These properties contribute to preconditioning-like effects of resistance to myocardial ischaemic damage and improved post-ischaemic recovery. These effects can be demonstrated in isolated hearts independently of the effects of n-3 PUFAs on neural or blood parameters. The enrichment of myocardial membranes with n-3 PUFA also reduces vulnerability to cardiac arrhythmias, particularly ventricular fibrillation during myocardial ischaemia and reperfusion, and attenuates heart failure and cardiac hypertrophy. n-3 PUFA concentrations can increase from 7% to 15% in the myocardial membranes of rats (mainly in the form of docosahexaenoic acid [22: 6 n-3]) with dietary intakes of only 0.3% fish oil, equivalent to two meals of salmon per week in the human diet. Dietary fish oil produces changes in cardiac function that might contribute to cardiovascular health benefits in humans and does so by modifying cardiac membranes within a dose range achievable in the human diet.
Epidemiological evidence from Greenland Eskimos and Japanese fishing villages suggests that eating fish oil and marine animals can prevent coronary heart disease. Dietary studies from various laboratories have similarly indicated that regular fish oil intake affects several humoral and cellular factors involved in atherogenesis and may prevent atherosclerosis, arrhythmia, thrombosis, cardiac hypertrophy and sudden cardiac death. The beneficial effects of fish oil are attributed to their n-3 polyunsaturated fatty acid (PUFA; also known as omega-3 fatty acids) content, particularly eicosapentaenoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3). Dietary supplementation of DHA and EPA influences the fatty acid composition of plasma phospholipids that, in turn, may affect cardiac cell functions in vivo. Recent studies have demonstrated that long-chain omega-3 fatty acids may exert beneficial effects by affecting a wide variety of cellular signaling mechanisms. Pathways involved in calcium homeostasis in the heart may be of particular importance. L-type calcium channels, the Na+-Ca2+ exchanger and mobilization of calcium from intracellular stores are the most obvious key signaling pathways affecting the cardiovascular system; however, recent studies now suggest that other signaling pathways involving activation of phospholipases, synthesis of eicosanoids, regulation of receptor-associated enzymes and protein kinases also play very important roles in mediating n-3 PUFA effects on cardiovascular health. This review is therefore focused on the molecular targets and signaling pathways that are regulated by n-3 PUFAs in relation to their cardioprotective effects.
The dramatic increase in the prevalence of obesity and its strong association with cardiovascular disease have resulted in unprecedented interest in understanding the effects of obesity on the cardiovascular system. A consistent, but puzzling clinical observation is that obesity confers an increased susceptibility to the development of cardiac disease, while at the same time affording protection against subsequent mortality (termed the obesity paradox). In this review we focus on evidence available from human and animal model studies and summarize the ways in which obesity can influence structure and function of the heart. We also review current hypotheses regarding mechanisms linking obesity and various aspects of cardiac remodeling. There is currently great interest in the role of adipokines, factors secreted from adipose tissue, and their role in the numerous cardiovascular complications of obesity. Here we focus on the role of leptin and the emerging promise of adiponectin as a cardioprotective agent. The challenge of understanding the association between obesity and heart failure is complicated by the multifaceted interplay between various hemodynamic, metabolic, and other physiological factors that ultimately impact the myocardium. Furthermore, the end result of obesity-associated changes in the myocardial structure and function may vary at distinct stages in the progression of remodeling, may depend on the individual pathophysiology of heart failure, and may even remain undetected for decades before clinical manifestation. Here we summarize our current knowledge of this complex yet intriguing topic.
For sixteen years, the American institute of Nutrition Rodent Diets, AIN-76 and AIN-76A, have been used extensively around the world. Because of numerous nutritional and technical problems encountered with the diet during this period, it was revised. Two new formulations were derived: AIN-93G for growth, pregnancy and lactation, and AIN-93M for adult maintenance. Some major differences in the new formulation of AIN-93G compared with AIN-76A are as follows: 7 g soybean oil/100 g diet was substituted for 5 g corn oil/ 100 g diet to increase the amount of linolenic acid; cornstarch was substituted for sucrose; the amount of phosphorus was reduced to help eliminate the problem of kidney calcification in female rats; L-cystine was substituted for DL-methionine as the amino acid supplement for casein, known to be deficient in the sulfur amino acids; manganese concentration was lowered to one-fifth the amount in the old diet; the amounts of vitamin E, vitamin K and vitamin B-12 were increased; and molybdenum, silicon, fluoride, nickel, boron, lithium and vanadium were added to the mineral mix. For the AIN-93M maintenance diet, the amount of fat was lowered to 40 g/kg diet from 70 g/kg diet, and the amount of casein to 140 g/kg from 200 g/kg in the AIN-93G diet. Because of a better balance of essential nutrients, the AIN-93 diets may prove to be a better choice than AIN-76A for long-term as well as short-term studies with laboratory rodents.
Primary systemic carnitine deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. SCD has also been linked to sudden infant death syndrome. Membrane-physiological studies have suggested a defect of the carnitine transport system in the plasma membrane in SCD patients and in the mouse model, juvenile visceral steatosis. Although the responsible loci have been mapped in both human and mouse, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2. Our observation that OCTN2 has the ability to transport carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5. Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.
Epidemiological and animal-based investigations have indicated that the development of skin cancer is in part associated with poor dietary practices. Lipid content and subsequently the derived fatty acid composition of the diet are believed to play a major role in the development of tumorigenesis. Omega 3 (ω3) fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can effectively reduce the risk of skin cancer whereas omega 6 (ω6) fatty acids such as arachidonic acid (AA) reportedly promote risk. To investigate the effects of fatty acids on tumorigenesis, we performed experiments to examine the effects of the ω3 fatty acids EPA and DHA and of the ω6 fatty acid AA on phorbol 12-tetradecanoate 13-acetate (TPA)-induced or epidermal growth factor (EGF)-induced transcription activator protein 1 (AP-1) transactivation and on the subsequent cellular transformation in a mouse epidermal JB6 cell model. DHA treatment resulted in marked inhibition of TPA- and EGF-induced cell transformation by inhibiting AP-1 transactivation. EPA treatment also inhibited TPA-induced AP-1 transactivation and cell transformation but had no effect on EGF-induced transformation. AA treatment had no effect on either TPA- or EGF-induced AP-1 transactivation or transformation, but did abrogate the inhibitory effects of DHA on TPA- or EGF-induced AP-1 transactivation and cell transformation in a dose-dependent manner. The results of this study demonstrate that the inhibitory effects of ω3 fatty acids on tumorigenesis are more significant for DHA than for EPA and are related to an inhibition of AP-1. Similarly, because AA abrogates the beneficial effects of DHA, the dietary ratio of ω6 to ω3 fatty acids may be a significant factor in mediating tumor development.
Stimulation of protein kinase C (PKC) activity in lipid vesicles in vitro was achieved by pure molecular species of diacylglycerol (DAG), specifically 1-stearoyl-2-acyl-sn-glycerol substituted with 2-arachidonoyl,2-eicosapentaenoyl or 2-docosahexaenoyl (SAG, SEG, and SDG, respectively). PKC activity was measured in lipid vesicles containing 30 mol% 1-palmitoyl-2-oleoyl-sn-glycerol-3-phospho-L-serine (POPS), 68-70 mol% 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), and 0-2 mol% DAG in the presence of 20 microM calcium. Our results demonstrate that amplification of PKC activity differs significantly among these molecular species of DAG. In particular, SDG at 0.5 mol% is more potent in increasing PKC activity than is dioleoylglycerol (DOG), SEG, or SAG, and SAG and SDG at 1.0 and 2.0 mol% have similar potencies which are greater than those of DOG or SEG. These findings demonstrate that sn-2 substitutions in DAG by specific n-3 and n-6 polyunsaturated fatty acids increase the potency of DAG to stimulate PKC activity in vitro.
Increased cardiovascular mortality occurs in diabetic patients with or without coronary artery disease and is attributed to the presence of diabetic cardiomyopathy. One potential mechanism is hyperglycemia that has been reported to activate protein kinase C (PKC), preferentially the beta isoform, which has been associated with the development of micro- and macrovascular pathologies in diabetes mellitus. To establish that the activation of the PKCbeta isoform can cause cardiac dysfunctions, we have established lines of transgenic mice with the specific overexpression of PKCbeta2 isoform in the myocardium. These mice overexpressed the PKCbeta2 isoform transgene by 2- to 10-fold as measured by mRNA, and proteins exhibited left ventricular hypertrophy, cardiac myocyte necrosis, multifocal fibrosis, and decreased left ventricular performance without vascular lesions. The severity of the phenotypes exhibited gene dose-dependence. Up-regulation of mRNAs for fetal type myosin heavy chain, atrial natriuretic factor, c-fos, transforming growth factor, and collagens was also observed. Moreover, treatment with a PKCbeta-specific inhibitor resulted in functional and histological improvement. These findings have firmly established that the activation of the PKCbeta2 isoform can cause specific cardiac cellular and functional changes leading to cardiomyopathy of diabetic or nondiabetic etiology.
Protein kinase C (PKC) activation in the heart has been linked to a hypertrophic phenotype and to processes that influence contractile function. To establish whether PKC activation is sufficient to induce an abnormal phenotype, PKCbeta was conditionally expressed in cardiomyocytes of transgenic mice. Transgene expression in adults caused mild and progressive ventricular hypertrophy associated with impaired diastolic relaxation, whereas expression in newborns caused sudden death associated with marked abnormalities in the regulation of intracellular calcium. Thus, the PKC signaling pathway in cardiocytes has different effects depending on the timing of expression and, in the adult, is sufficient to induce pathologic hypertrophy.
Recent data suggest that omega-3 fatty acids may be effective in epilepsy, cardiovascular disorders, arthritis, and as mood stabilizers for bipolar disorder; however, the mechanism of action of these compounds is unknown. Based on earlier studies implicating omega-3 fatty acids as inhibitors of protein kinase C activity in intact cells, we hypothesized that omega-3 fatty acids may act through direct inhibition of second messenger-regulated kinases and sought to determine whether the omega-3 double bond might uniquely confer pharmacologic efficacy and potency for fatty acids of this type. In our studies we observed that omega-3 fatty acids inhibited the in vitro activities of cAMP-dependent protein kinase, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and the mitogen-activated protein kinase (MAPK). Our results with a series of long-chain fatty acid structural homologs suggest an important role for the omega-3 double bond in conferring inhibitory efficacy. To assess whether omega-3 fatty acids were capable of inhibiting protein kinases in living neurons, we evaluated their effect on signal transduction pathways in the hippocampus. We found that omega-3 fatty acids could prevent serotonin receptor-induced MAPK activation in hippocampal slice preparations. In addition, we evaluated the effect of omega-3 fatty acids on hippocampal long-term potentiation, a form of synaptic plasticity known to be dependent on protein kinase activation. We observed that omega-3 fatty acids blocked long-term potentiation induction without inhibiting basal synaptic transmission. Overall, our results from both in vitro and live cell preparations suggest that inhibition of second messenger-regulated protein kinases is one locus of action of omega-3 fatty acids.
Studies in human and rodent models have shown that activation of protein kinase C-beta (PKC-beta) is associated with the development of pathological hypertrophy, suggesting that ablation of the PKC-beta pathway might prevent or reverse cardiac hypertrophy. To explore this, we studied mice with targeted disruption of the PKC-beta gene (knockout, KO). There were no detectable differences in expression or distribution of other PKC isoforms between the KO and control hearts as determined by Western blot analysis. Baseline hemodynamics were measured using a closed-chest preparation and there were no differences in heart rate and arterial or left ventricular pressure. Mice were subjected to two independent hypertrophic stimuli: phenylephrine (Phe) at 20 mg x kg(-1) x day(-1) sq infusion for 3 days, and aortic banding (AoB) for 7 days. KO animals demonstrated an increase in heart weight-to-body weight ratio (Phe, 4.3 +/- 0.6 to 6.1 +/- 0.4; AoB, 4.0 +/- 0.1 to 5.8 +/- 0.7) as well as ventricular upregulation of atrial natriuretic factor mRNA analogous to those seen in control animals. These results demonstrate that PKC-beta expression is not necessary for the development of cardiac hypertrophy nor does its absence attenuate the hypertrophic response.
Preliminary clinical data indicate that omega-3 fatty acids may be effective mood stabilizers for patients with bipolar disorder. Both lithium and valproic acid are known to inhibit protein kinase C (PKC) activity after subchronic administration in cell culture and in vivo. The current study was undertaken to determine the effects of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on protein kinase C phosphotransferase activity in vitro. Various concentrations of DHA, EPA, and arachidonic acid (AA) were incubated with the catalytic domain of protein kinase C beta from rat brain. Protein kinase C activity was measured by quantifying incorporation of (32)P-PO(4) into a synthetic peptide substrate. Both DHA and EPA, as well as the combination of DHA and EPA, inhibited PKC activity at concentrations as low as 10 micromol l(-1). In contrast, arachidonic acid had no effect on PKC activity. Thus, PKC represents a potential site of action of omega-3 fatty acids in their effects on the treatment of bipolar disorder.
Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported. Here, deltaPKC-selective activator and inhibitor peptides were designed rationally, based on molecular modeling and structural homology analyses. Together with previously identified activator and inhibitor peptides of epsilonPKC, deltaPKC peptides were used to identify cardiac functions of these isozymes. In isolated cardiomyocytes, perfused hearts, and transgenic mice, deltaPKC and epsilonPKC had opposing actions on protection from ischemia-induced damage. Specifically, activation of epsilonPKC caused cardioprotection whereas activation of deltaPKC increased damage induced by ischemia in vitro and in vivo. In contrast, deltaPKC and epsilonPKC caused identical nonpathological cardiac hypertrophy; activation of either isozyme caused nonpathological hypertrophy of the heart. These results demonstrate that two related PKC isozymes have both parallel and opposing effects in the heart, indicating the danger in the use of therapeutics with nonselective isozyme inhibitors and activators. Moreover, reduction in cardiac damage caused by ischemia by perfusion of selective regulator peptides of PKC through the coronary arteries constitutes a major step toward developing a therapeutic agent for acute cardiac ischemia.
Protein kinase C (PKC) epsilon and PKCdelta translocation in neonatal rat ventricular myocytes (NRVMs) is accompanied by subsequent activation of the ERK, JNK, and p38(MAPK) cascades; however, it is not known if either or both novel PKCs are necessary for their downstream activation. Use of PKC inhibitors to answer this question is complicated by a lack of isoenzyme specificity, and the fact that many PKC inhibitors stimulate JNK and p38(MAPK) activity. Therefore, replication-defective adenoviruses (Advs) encoding constitutively active (ca) mutants of PKCepsilon and PKCdelta were used to test if either or both of these PKCs are sufficient to activate ERKs, JNKs, and/or p38(MAPK) in NRVMs. Adv-caPKCepsilon infection (1 to 25 multiplicities of viral infection (MOI); 4 to 48 hours) increased total PKCepsilon levels in a time- and dose-dependent manner, with maximal expression observed 8 hours after Adv infection. Adv-caPKCepsilon induced a time- and dose-dependent increase in phosphorylated p42 and p44 ERKs, as compared with a control Adv encoding beta-galactosidase (Adv-nebetagal). Maximal ERK phosphorylation occurred 8 hours after Adv infection. In contrast, JNK was only minimally activated, and p38(MAPK) was relatively unaffected. Adv-caPKCdelta infection (1 to 25 MOI, 4 to 48 hours) increased total PKCdelta levels in a similar fashion. Adv-caPKCdelta (5 MOI) induced a 29-fold increase in phosphorylated p54 JNK, and a 15-fold increase in phosphorylated p38(MAPK) 24 hours after Adv infection. In contrast, p42 and p44 ERK were only minimally activated. Whereas neither Adv induced NRVM hypertrophy, Adv-caPKCdelta, but not Adv-caPKCepsilon, induced NRVM apoptosis. We conclude that the novel PKCs differentially regulate MAPK cascades and apoptosis in an isoenzyme-specific and time-dependent manner.
This study was conducted on human Jurkat T cell lines to elucidate the role of EPA and DHA, n-3 PUFA, in the modulation of two mitogen-activated protein (MAP) kinases, that is, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). The n-3 PUFA alone failed to induce phosphorylation of ERK1/ERK2. We stimulated the MAP kinase pathway with anti-CD3 antibodies and phorbol 12-myristate 13-acetate (PMA), which act upstream of the MAP kinase (MAPK)/ERK kinase (MEK) as U0126, an MEK inhibitor, abolished the actions of these two agents on MAP kinase activation. EPA and DHA diminished the PMA- and anti-CD3-induced phosphorylation of ERK1/ERK2 in Jurkat T cells. In the present study, PMA acts mainly via protein kinase C (PKC) whereas anti-CD3 antibodies act via PKC-dependent and -independent mechanisms. Furthermore, DHA and EPA inhibited PMA-stimulated PKC enzyme activity. EPA and DHA also significantly curtailed PMA- and ionomycin-stimulated T cell blastogenesis. Together these results suggest that EPA and DHA modulate ERK1/ERK2 activation upstream of MEK via PKC-dependent and -independent pathways and that these actions may be implicated in n-3 PUFA-induced immunosuppression.
—Denys, A., A. Hichami, and N. A. Khan. Eicosapentaenoic acid and docosahexaenoic acid modulate MAP kinase (ERK1/ERK2) signaling in human T cells. J. Lipid Res. 2001. 42: 2015–2020.
Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKC alpha, beta II, delta, and epsilon (only wild-type zeta) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKC alpha, beta II, delta, and epsilon revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKC alpha, but not betaI I, delta, epsilon, or zeta induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [(3)H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKC alpha, beta II, delta, and epsilon revealed a necessary role for PKC alpha as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKC epsilon reduced cellular viability. A mechanism whereby PKC alpha might regulate hypertrophy was suggested by the observations that wild-type PKC alpha induced extracellular signal-regulated kinase1/2 (ERK1/2), that dominant negative PKC alpha inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKC alpha-induced hypertrophic growth. These results implicate PKC alpha as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.
Increasing evidence demonstrates that protein kinase C betaII (PKCbetaII) promotes colon carcinogenesis. We previously reported that colonic PKCbetaII is induced during colon carcinogenesis in rodents and humans, and that elevated expression of PKCbetaII in the colon of transgenic mice enhances colon carcinogenesis. Here, we demonstrate that PKCbetaII represses transforming growth factor beta receptor type II (TGFbetaRII) expression and reduces sensitivity to TGF-beta-mediated growth inhibition in intestinal epithelial cells. Transgenic PKCbetaII mice exhibit hyperproliferation, enhanced colon carcinogenesis, and marked repression of TGFbetaRII expression. Chemopreventive dietary omega-3 fatty acids inhibit colonic PKCbetaII activity in vivo and block PKCbetaII-mediated hyperproliferation, enhanced carcinogenesis, and repression of TGFbetaRII expression in the colonic epithelium of transgenic PKCbetaII mice. These data indicate that dietary omega-3 fatty acids prevent colon cancer, at least in part, through inhibition of colonic PKCbetaII signaling and restoration of TGF-beta responsiveness.
The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-alpha as a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-alpha into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-alpha activity affects dephosphorylation of the sarcoplasmic reticulum Ca(2+) ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca(2+) loading and the Ca(2+) transient. PKC-alpha directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-alpha on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-alpha functions as a nodal integrator of cardiac contractility by sensing intracellular Ca(2+) and signal transduction events, which can profoundly affect propensity toward heart failure.
Protein kinase C (PKC) isoenzymes play a critical role in cardiomyocyte hypertrophy. At least three different phorbol ester-sensitive PKC isoenzymes are expressed in neonatal rat ventricular myocytes (NRVMs): PKC-α, -δ, and -ε. Using replication-defective adenoviruses (AdVs) that express wild-type (WT) and dominant-negative (DN) PKC-α together with phorbol myristate acetate (PMA), which is a hypertrophic agonist and activator of all three PKC isoenzymes, we studied the role of PKC-α in signaling-specific aspects of the hypertrophic phenotype. PMA induced nuclear translocation of endogenous and AdV-WT PKC-α in NRVMs. WT PKC-α overexpression increased protein synthesis and the protein-to-DNA (P/D) ratio but did not affect cell surface area (CSA) or cell shape compared with uninfected or control AdV β-galactosidase (AdV βgal)-infected cells. PMA-treated uninfected cells displayed increased protein synthesis, P/D ratio, and CSA and elongated morphology. PMA did not further enhance protein synthesis or P/D ratio in AdV-WT PKC-α-infected cells. To assess the requirement of PKC-α for these PMA-induced changes, AdV-DN PKC-α or AdV βgal-infected NRVMs were stimulated with PMA. Without PMA, AdV-DN PKC-α had no effects on protein synthesis, P/D ratio, CSA, or shape vs. AdV βgal-infected NRVMs. PMA increased protein synthesis, P/D ratio, and CSA in AdV βgal-infected cells, but these parameters were significantly reduced in PMA-stimulated AdV-DN PKC-α-infected NRVMs. Overexpression of DN PKC-α enhanced PMA-induced cell elongation. Neither WT PKC-α nor DN PKC-α affected atrial natriuretic factor gene expression. Insulin-like growth factor-1 also induced nuclear translocation of endogenous PKC-α. PMA but not WT PKC-α overexpression induced ERK1/2 activation. However, AdV-DN PKC-α partially blocked PMA-induced ERK activation. Thus PKC-α is necessary for certain aspects of PMA-induced NRVM hypertrophy.
To date, the proximal molecular targets through which dietary n-3 polyunsaturated fatty acids (PUFA) suppress the inflammatory process have not been elucidated. Because cholesterol and sphingolipid-enriched rafts have been proposed as platforms for compartmentalizing dynamically regulated signaling assemblies at the plasma membrane, we determined the in vivo effects of fish oil and highly purified docosahexaenoic acid (DHA; 22:6n-3) on T cell microdomain lipid composition and the membrane subdomain distribution of signal-transducing molecules (protein kinase C (PKC)theta;, linker for activation of T cells, and Fas/CD95), before and after stimulation. Mice were fed diets containing 5 g/100 g corn oil (control), 4 g/100 g fish oil (contains a mixture of n-3 PUFA) plus 1 g/100 g corn oil, or 4 g/100 g corn oil plus 1 g/100 g DHA ethyl ester for 14 days. Dietary n-3 PUFA were incorporated into splenic T cell lipid raft and soluble membrane phospholipids, resulting in a 30% reduction in raft sphingomyelin content. In addition, polyclonal activation-induced colocalization of PKCtheta; with lipid rafts was reduced by n-3 PUFA feeding. With respect to PKCtheta; effector pathway signaling, both AP-1 and NF-kappaB activation, IL-2 secretion, and lymphoproliferation were inhibited by fish oil feeding. Similar results were obtained when purified DHA was fed. These data demonstrate for the first time that dietary DHA alters T cell membrane microdomain composition and suppresses the PKCtheta; signaling axis.
Since the first AHA Science Advisory “Fish Consumption, Fish Oil, Lipids, and Coronary Heart Disease,”1 important new findings, including evidence from randomized controlled trials (RCTs), have been reported about the beneficial effects of omega-3 (or n-3) fatty acids on cardiovascular disease (CVD) in patients with preexisting CVD as well as in healthy individuals.2 New information about how omega-3 fatty acids affect cardiac function (including antiarrhythmic effects), hemodynamics (cardiac mechanics), and arterial endothelial function have helped clarify potential mechanisms of action. The present Statement will address distinctions between plant-derived (α-linolenic acid, C18:3n-3) and marine-derived (eicosapentaenoic acid, C20:5n-3 [EPA] and docosahexaenoic acid, C22:6n-3 [DHA]) omega-3 fatty acids. (Unless otherwise noted, the term omega-3 fatty acids will refer to the latter.) Evidence from epidemiological studies and RCTs will be reviewed, and recommendations reflecting the current state of knowledge will be made with regard to both fish consumption and omega-3 fatty acid (plant- and marine-derived) supplementation. This will be done in the context of recent guidance issued by the US Environmental Protection Agency and the Food and Drug Administration (FDA) about the presence of environmental contaminants in certain species of fish.
### Coronary Heart Disease
As reviewed by Stone,1 three prospective epidemiological studies within populations reported that men who ate at least some fish weekly had a lower coronary heart disease (CHD) mortality rate than that of men who ate none.3–6⇓⇓⇓ More recent evidence that fish consumption favorably affects CHD mortality, especially nonsudden death from myocardial infarction (MI), has been reported in a 30-year follow-up of the Chicago Western Electric Study.7 Men who consumed 35 g or more of fish daily compared with those who consumed none had a relative risk of death from CHD of 0.62 and a relative risk of nonsudden death from MI of 0.33. In an …
Background
Eicosapentaenoic acid is a fish oil fatty acid that has been shown to decrease blood pressure (BP) in humans. The mechanism by which this fatty acid produces this effect is unknown. Angiotensin II increases BP by inducing vasoconstriction of vascular smooth muscle cells, an event that is mediated by an increase of intracellular calcium and an increase of protein kinase C activity.
Methods
We determined the effects of eicosapentaenoic acid on angiotensin II-induced calcium signaling, and protein kinase C activity in cultured rat aortic smooth muscle cells. Incorporation of eicosapentaenoic acid into cell phospholipids was determined by gas chromatography/mass spectrometry. Intracellular calcium concentration was determined using fura-2, and protein kinase C activity was assessed by an ELISA assay using a phospho-specific antiserum for protein kinase C substrates.
Results
We found that eicosapentaenoic acid was incorporated into cell phospholipids within 20 min. Eicosapentaenoic acid (10 or 25 μmol/L) did not alter basal intracellular calcium concentration, but decreased the peak response to 100 nmol/L angiotensin II. Eicosapentaenoic acid also decreased the amount of calcium released by thapsigargin, a drug that releases calcium from the sarcoplasmic reticulum, and decreased cation influx after angiotensin II stimulation. Angiotensin II stimulated phosphorylation of protein kinase C substrates. Preincubation of cells with 10 or 25 μmol/L eicosapentaenoic acid significantly inhibited this phosphorylation.
Conclusions
Our results demonstrate that acute incorporation of eicosapentaenoic acid into vascular smooth muscle cell phospholipids inhibits intracellular calcium mobilization and protein kinase C activation. These are potential mechanisms by which eicosapentaenoic acid reduces vasoconstriction.
Time for primary review 34 days.
Currently the precise biochemical pathogenesis of cardiac hypertrophy remains unclear. A great deal of investigation has been directed toward the examination of various signal transduction pathways that are thought to be involved in stimulating this process. In particular, the signaling pathways that use heterotrimeric G protein-coupled receptors have been the focus for many investigations. This review will focus on the function of the Gαq family of proteins and its downstream effectors during cardiac hypertrophy. Hypertrophy is the final common pathway for a variety of insults to the normal cardiovascular system. Many mechanical and hormonal stimuli such as hypertension, valve disorders, and ischemic events, can produce a hypertrophic response. These pathologic stimuli cause an increase in the workload placed upon the heart resulting in hypertrophy and remodeling that has been observed at the myocyte [1] and the gross anatomical level. Current thought suggests that the hypertrophic response is a compensatory mechanism that allows normal cardiac function against a gradient of increasing workload. If the pathologic stimulus is sufficiently intense or prolonged, a period of failure characterized by impaired function, dilation of the left ventricle, and pulmonary congestion ensues.
Recently, experiments using cardiac specific transgenesis to investigate G protein-mediated mechanisms of cardiac hypertrophy have yielded novel information that may give us cause to re-evaluate our traditional stratification of hypertrophic states [2]. It is important to evaluate these results along with other conventional and transgenic models so that we can develop a more comprehensive understanding of this complex disease.
Heterotrimeric G protein-coupled receptors serve to convey an extracellular biochemical signal to intracellular effectors. These receptors are heptahelical structures with extracellular, transmembrane, and intracellular domains coupled to specific G proteins which are comprised of three (α, β, γ) subunits. When the G protein complex is in …
* Corresponding author. Tel.: 1-216-844-3293; fax: 1-216-844-3145 raw19{at}po.cwru.edu
Objective: The juvenile visceral steatosis (JVS) mouse, a murine model of systemic carnitine deficiency, shows a disorder of fatty acid oxidation and develops cardiac hypertrophy associated with lipid accumulation. Recently, α-tocopherol was shown to decrease 1,2-diacylglycerol (DAG) levels. We investigated the involvement of DAG in cardiac hypertrophy due to energy metabolism disorder by evaluating the effects of α-tocopherol administration on the hearts of JVS mice. Methods: Both JVS and control mice were fed a high α-tocopherol diet or a standard diet from 4 to 8 weeks of age. Myocardial DAG levels and fatty acid composition were assessed at 8 weeks of age. Results: The ventricular to body weight ratio in the JVS mice was significantly higher than that in the control mice [11.2±0.1 (mean±S.E.M.) versus 3.8±0.1 mg/g, P<0.01], and was reduced by α-tocopherol treatment (9.7±0.2 mg/g, P<0.01 versus JVS mice). However, echocardiographic analysis showed the exaggeration of left ventricular dilatation in the α-tocopherol treated JVS mice (P<0.01 versus JVS mice). The myocardial thiobarbituric-acid-reactive substance level was not affected by α-tocopherol treatment. The myocardial DAG level was 2.5-fold higher in the JVS mice compared with that in the control mice (2004±136 versus 806±36 ng/mg dry weight, P<0.01) with a significant increase in 18:1 and 18:2 fatty acids. α-Tocopherol treatment reduced myocardial DAG levels in the JVS mice (1443±49 ng/mg dry weight, P<0.01 versus JVS mice) without any alteration of the fatty acid composition. Conclusions: α-Tocopherol treatment may partially reduce cardiac hypertrophy but it may also depress cardiac function in the JVS mice by decreasing the myocardial DAG level. An increase in DAG might be involved in the development of cardiac hypertrophy and in the maintenance of cardiac function in energy metabolism disorder of the heart. © 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.
Several extracellular agents and stress stimuli, such as tumour necrosis factor alpha, chemotherapeutic agents and heat, cause ceramide accumulation. They do this by regulating enzymes involved in its metabolism. Ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, cell-cycle arrest and cell senescence.
Ceramides and 1,2-diacylglycerol have been demonstrated in intracellular signaling pathways. A method of simultaneous mass
determination of ceramides and 1,2-diacylglycerol in tissues was developed using the latroscan which combines thin layer chromatography
and flame ionization detection (TLC/FID) techniques. Because of relatively low amounts of these components in tissues, the
fraction of nonpolar lipids, which included ceramides and glycerides, was eluted with chloroform/acetone mixture (3∶1, vol/vol)
through a silicic acid column to eliminate the polar phospholipids. Development of Chromarods was carried out using three
solvent systems in a four-step development technique. The relationship of the peak area ratio to weight ratio compared with
cholesteryl acetate added as an internal standard was linear. The amount of ceramides increased with incubation of rat heart
homogenate and human erythrocyte membranes in the presence of sphingomyelinase (E.C. 3.1.4.12). The latroscan TLC/FID system
provided a quick and reliable assessment of ceramides and 1,2-diacylglycerol.
Cardiac hypertrophy is a well known response to increased hemodynamic load. Mechanical stress is considered to be the trigger inducing a growth response in the overloaded myocardium. Furthermore, mechanical stress induces the release of growth-promoting factors, such as angiotensin II, endothelin-1, and transforming growth factor-β, which provide a second line of growth induction. In this review, we will focus on the primary effects of mechanical stress: how mechanical stress may be sensed, and which signal transduction pathways may couple mechanical stress to modulation of gene expression, and to increased protein synthesis. Mechanical stress may be coupled to intracellular signals that are responsible for the hypertrophic response via integrins and the cytoskeleton or via sarcolemmal proteins, such as phospholipases, ion channels and ion exchangers. The signal transduction pathways that may be involved belong to two groups: (1) the mitogen-activated protein kinases (MAPK) pathway; and (2) the janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. The MAPK pathway can be subdivided into the extracellular-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK), and the 38-kDa MAPK (p38 MAPK) pathway. Alternatively, the stress signal may be directly submitted to the nucleus via the cytoskeleton without the involvement of signal transduction pathways. Finally, by promoting an increase in intracellular Ca2+ concentration stretch may stimulate the calcium/calmodulin-dependent phosphatase calcineurin, a novel hypertrophic signalling pathway.
Cultured neonatal cardiac myocytes have been utilized as a model for the study of the role of fatty acids in the alpha 1-adrenoceptor mediated phosphatidylinositol turnover. Experiments were started 24 h after seeding, when there was a confluent monolayer of beating cardiomyocytes. The cells were incubated for 3-4 days in sera containing culture medium with (1) no additives or (2) a mixture of 107 microM 18:0 and 18:1n-9, or (3) only 214 microM 18:2n-6 or (4) 214 microM 20:5n-3. No differences in the cellular content of the various phospholipid classes among the different groups of fatty acid treated cells were found. The predicted elevations of 18:1n-9, 18:2n-6 and 20:5n-3 associated with a partial depletion of 20:4n-6 were confirmed in all phospholipid classes, except for sphingomyelin. The mol% of 18:0, 18:2n-6, 20:4n-6 and 20:5n-3 in the phosphatidylinositol fraction were respectively 39, 4, 30 and 0.6 for the control treated cells, 34, 3, 15 and 0 for 18:0/18:1n-9 treated cells, 40, 17, 24 and 0.2 for the 18:2n-6 treated cells and 41, 3, 13 and 21 for the 20:5n-3 treated cells. Apart from the observed reductions in the basal rates, the phenylephrine (30 microM) stimulated production of inositolphosphates was reduced by 51% and 71%, respectively in the 18:2n-6 and 20:5n-3 treated cardiomyocytes. The basal rate of inositolphosphate formation was 37% increased in the 18:0/18:1n-9 treated cells. The [3H]-inositol incorporation into phosphatidylinositol 4,5-bisphosphate was only slightly reduced by 18:2n-6 and 20:5n-3 treatments (respectively 12 and 28% compared to control treated cells). Prolonged (30 min) alpha 1-adrenergic stimulation did not affect the contents and fatty acid profiles of any class of phospholipid, not even phosphatidylinositol. In conclusion, variations in the polyunsaturated fatty acid composition of membrane phospholipids do affect the basal and the alpha 1-adrenoceptor stimulated rate of phosphatidylinositol-4,5-bisphosphate hydrolysis. The reducing effects of 18:2n-6 and 20:5n-3 treatment on the rate of inositolphosphate production may be partially ascribed to altered levels of phosphatidyl-inositol 4,5-bisphosphate.
Hydrolysis of inositol phospholipids by phospholipase C is initiated by either receptor stimulation or opening of Ca2+ channels.
This was once thought to be the sole mechanism to produce the diacylglycerol that links extracellular signals to intracellular
events through activation of protein kinase C. It is becoming clear that agonist-induced hydrolysis of other membrane phospholipids,
particularly choline phospholipids, by phospholipase D and phospholipase A2 may also take part in cell signaling. The products
of hydrolysis of these phospholipids may enhance and prolong the activation of protein kinase C. Such prolonged activation
of protein kinase C is essential for long-term cellular responses such as cell proliferation and differentiation.
The fatty acid pattern of phosphatidylinositol and other inositol phospholipids is reported to be predominantly 1-stearoyl, 2-arachidonyl. However, literature does not report data about the effect of a modification of this fatty acid composition on the production and acidic pattern of the diacylglycerol (DAG) formed during phosphoinositide hydrolysis. Culturing cardiomyocytes in a docosahexaenoic acid supplemented medium, we obtained an homogeneous cell population whose phospholipid fatty acid pattern was strongly different from control cells, and which produced, after alpha 1-adrenergic stimulation with phenylephrine, an higher amount of DAG. This DAG was different from control DAG in fatty acid composition, too. This structurally different DAG could be responsible for a different activation pattern of protein kinase C.
We analyzed carnitine profiles in C3H-H-2 degrees strain of mouse associated with fatty liver, hyperammonemia and hypoglycemia (Koizumi et al., 1988). Carnitine levels in serum, liver and muscle of mouse with fatty liver were markedly decreased in comparison with those of control mouse (littermates without fatty liver). This is a useful animal model to analyze the role of carnitine in lipid, amino acid and carbohydrate metabolism.
The effects of 5, 10 and 20% dietary menhaden oil (MO) on the composition of heart lipid classes and fatty acids were studied. Male Sprague-Dawley rats were fed ad libitum 0, 5, 10 and 20% MO for 3 wk. The heart phosphoglyceride content and composition and cholesterol were unchanged by dietary MO. A nonlinear dose-response relationship was observed between dietary MO levels and fatty acid compositional changes. Cardiolipin, choline (PC), ethanolamine (PE) and serine/inositol (PS/PI) phosphoglycerides showed an incorporation of n-3 fatty acids, eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3), between the control and 5% MO group, a plateau between the 5 and 10% MO groups and a further increase at the 20% MO level. The initial reduction in 20:4n-6 content remained unchanged as dietary MO increased except in PE where a further reduction was found at the 20% MO level. Dietary MO did not significantly change the 20:4n-6 content in neutral lipids. Linoleic acid content was most resistant to dietary MO removal. The level of 18:2n-6 was significantly lowered in heart PC when rats were fed 10% MO. No significant differences were found in PS/PI. In PE and NL significant differences occurred only when rats were fed 20% MO. The significant fatty acid modifications of heart lipid and PL found between the control and lowest level of dietary MO (5%) suggest that dietary fish oil supplementation in human diets may not be required for this effect.
Carnitine was detected at the beginning of this century, but it was nearly forgotten among biochemists until its importance in fatty acid metabolism was established 50 years later. In the last 30 years, interest in the metabolism and functions of carnitine has steadily increased. Carnitine is synthesized in most eucaryotic organisms, although a few insects (and most likely some newborn animals) require it as a nutritional factor (vitamin BT). Carnitine biosynthesis is initiated by methylation of lysine. The trimethyllysine formed is subsequently converted to butyrobetaine in all tissues; the butyrobetaine is finally hydroxylated to carnitine in the liver and, in some animals, in the kidneys (see Fig. 1). It is released from these tissues and is then actively taken up by all other tissues. The turnover of carnitine in the body is slow, and the regulation of its synthesis is still incompletely understood. Microorganisms (e.g., in the intestine) can metabolize carnitine to trimethylamine, dehydrocarnitine (beta-keto-gamma-trimethylaminobutyric acid), betaine, and possibly to trimethylaminoacetone. In some insects carnitine can be converted to methylcholine, presumably with trimethylaminoacetone as an intermediate (see Fig. 3). In mammals the unphysiological isomer (+) carnitine is converted to trimethylaminoacetone. The natural isomer (-)carnitine is excreted unchanged in the urine, and it is still uncertain if it is degraded in mammalian tissues at all (Fig. 2). The only firmly established function of carnitine is its function as a carrier of activated fatty acids and activated acetate across the inner mitochondrial membrane. Two acyl-CoA:carnitine acyltransferases with overlapping chain-length specificities have been isolated: one acetyltransferase taking part in the transport of acetyl and short-chain acyl groups and one palmitoyltransferase taking part in the transport of long-chain acyl groups. An additional octanoyltransferase has been isolated from liver peroxisomes. Although a carnitine translocase that allows carnitine and acylcarnitine to penetrate the inner mitochondrial membrane has been deduced from functional studies (see Fig. 5), this translocase has not been isolated as a protein separate from the acyltransferases. Carnitine acetyltransferase and carnitine octanoyltransferase are also found in the peroxisomes. In these organelles the enzymes may be important in the transfer of acyl groups, which are produced by the peroxisomal beta-oxidation enzymes, to the mitochondria for oxidation in the citric acid cycle. The carnitine-dependent transport of activated fatty acids across the mitochondrial membrane is a regulated process. Malonyl-CoA inh
Endocardial fibroelastosis is a cardiomyopathy of unknown origin that affects infants and young children. It is characterized by poor myocardial contractility with hypertrophy and dilatation of the heart, especially of the left ventricle. At autopsy, a milky-white thickening of the endocardium is found. Because similar endocardial thickening is seen in congenital cardiac anomalies complicated by subendocardial ischemia (e.g., aortic atresia), it has been suggested that endocardial fibroelastosis may be related to myocardial hypoxia. Familial cases suggest that the disease may result from a genetically transmitted abnmormality affecting cardiac metabolism. The cardiac tissue in congestive cardiomyopathy contains increased numbers of mitochondria, often with cristolysis and structural distortion. The activity of mitochondrial enzymes in such tissue is reduced, suggesting impaired aerobic metabolism. We studied a family in which cardiomyopathy developed in four of five children, three of whom died suddenly and were found to have endocardial fibroelastosis at autopsy. Examination of the surviving affected child and autopsy of her brother showed that both had severe plasma and tissue carnitine deficiency - an entity associated with impaired mitochondrial oxidation of long-chain fatty acids. Treatment of the affected child with oral L-carnitine (3 g per day) markedly improved myocardial function and reduced the cardiomegaly. Skeletal-muscle carnitine increased, but after six months of treatment, the level was still below normal. Inherited systemic carnitine deficiency may be one of the treatable causes of familial cardiomyopathy.
The variable phenotypic expression and broad spectrum of genetic causes of known inherited cardiomyopathies indicate that physicians should consider genetic causes in patients with cardiomyopathy, even if a review of the family history does not reveal evidence of inheritance. Furthermore, as exemplified by the known forms of inherited cardiomyopathy described here, factors such as age, coexisting illnesses, diet, and additional genetic loci may play a part in determining the expression of the cardiomyopathy phenotype. Accordingly, a mutation may be necessary but not sufficient for the expression of cardiomyopathy in many patients. As the molecular genetic basis of cardiomyopathy is clarified, some intriguing possibilities and questions emerge. For example, do heritable genetic alterations in energy- producing enzymes, which reduce mitochondrial efficiency, determine the severity of cardiomyopathy due to chronic intermittent ischemia? Is the apparent genetic susceptibility to hypertensive cardiomyopathy due to heritable differences in contractile proteins? The increasing evidence of genetic causes of cardiomyopathy points out the need to consider these questions. The delineation of the molecular basis of inherited cardiomyopathies has led to a better understanding of pathogenetic mechanisms. As animal models of cardiomyopathy are developed, involving the ablation of the causative genes or overexpression of dominant mutant proteins, the mechanisms involved in the pathogenesis of these diseases in vivo can be elucidated. The emerging evidence demonstrates that cardiomyopathies in children and young adults are often due to genetic causes. It is an exciting time to observe the definition of the causes of these previously idiopathic disorders.
We have reported the clinical and biochemical findings in juvenile visceral steatosis (jvs) mice with systemic carnitine deficiency. This paper is the first report about cardiomyopathy in jvs mice. Adult jvs mice (at the age of 2-3 months) show cardiac hypertrophy which is caused by enlargement of the cardiac muscle cell associated with increases of non-collagen protein and DNA content. Carnitine administration (2 mg/head, twice a day, from 1 month of age) significantly suppresses the cardiac hypertrophy, showing that carnitine deficiency plays an important role in the development of the cardiac hypertrophy. The discovery of cardiac hypertrophy in carnitine-deficient jvs mice will lead to clarification of the pathophysiology of cardiomyopathy in systemic carnitine deficiency in human beings.
Protein kinases play important roles in intracellular signalling pathways in probably all cells. In the heart, they are involved in the regulation of ion handling, contractility, fuel metabolism and growth. In this review, we discuss the consequences of activation of protein kinases known to be expressed in the heart. We concentrate principally on the following: cyclic AMP-dependent protein kinase, protein kinase C, mitogen-activated protein kinase, Ca2+/calmodulin-dependent protein kinases and pyruvate dehydrogenase kinase.
Fatty acids (FAs) and their derivatives are essential cellular metabolites whose concentrations must be closely regulated. This implies that regulatory circuits exist which can sense changes in FA levels. Indeed, the peroxisome proliferator-activated receptor alpha (PPARalpha) regulates lipid homeostasis and is transcriptionally activated by a variety of lipid-like compounds. It remains unclear as to how these structurally diverse compounds can activate a single receptor. We have developed a novel conformation-based assay that screens activators for their ability to bind to PPARalpha/delta and induce DNA binding. We show here that specific FAs, eicosanoids, and hypolipidemic drugs are ligands for PPARalpha or PPARdelta. Because altered FA levels are associated with obesity, atherosclerosis, hypertension, and diabetes, PPARs may serve as molecular sensors that are central to the development and treatment of these metabolic disorders.
The juvenile visceral steatosis (JVS) mouse exhibits hereditary systemic carnitine deficiency and develops cardiac hypertrophy. The aim of this study was to clarify the characteristics of cardiac hypertrophy in the JVS mouse. Total carnitine content in IVS mouse heart was about 10% of that of control mouse heart at 4 and 8 weeks of age. The heart weight/body weight ratio was bigger in JVS mice than that in control mice at 2 weeks of age, and this difference in ratio increased with age. The wall areas of both ventricles and septum in JVS mice were larger than those of the control mice at 2 and 8 weeks. The myocyte diameter in both ventricular walls and septum in JVS mice was longer than that of the control mice. On electron microscopy, the percent of mitochondria in the myocyte was 66% in JVS mice, and 37% in control mice. The percent of lipid fraction in JVS mice was six-fold higher than that in control mice. Total content of adenine nucleotides in JVS mouse heart was about 60% of that in control mouse heart. Adenylate energy charge in JVS mouse heart was 63 and 45% of that in the control mouse heart at 4 and 8 weeks, respectively. Overall, the cardiac enlargement observed in this animal model could be accounted for by a proportional increase in the myocyte diameter in the ventricles and septum, accompanied by an increase in mitochondria. Furthermore, this cellular growth is associated with decreases in the levels of ATP and ADP, and adenylate energy charge.
The aim was to investigate the consequences of simultaneous stimulation of phospholipase C and D by agonists for the molecular species composition of 1,2-diacylglycerol and phospholipids in cardiomyocytes.
Serum-free cultured neonatal rat cardiomyocytes were stimulated by endothelin-1, phenylephrine or phorbolester. The molecular species of 1,2-diacylglycerol (in mol%) and those derived from phosphatidylcholine and phosphatidylinositol were analyzed by high-performance liquid chromatography and their absolute total concentration (nmol per dish) by gas-liquid chromatography. Phospholipids were labelled with [14C]glycerol or double-labelled with [14C]16:0 and [3H]20:4n6 for measurements of respectively, the amount of or relative rate of label incorporation into 1,2-diacylglycerol.
The major molecular species of 1,2-diacylglycerol in unstimulated cells was found to be 18:0/20:4 (57 mol%). The same species was observed predominantly in phosphatidylinositol (73 mol% compared to 11 mol% in phosphatidylcholine). A significant decrease (about 10 mol%) was found for the 18:0/20:4 species of 1,2-diacylglycerol during stimulation (10-40 min) with endothelin-1 or phorbolester, but not phenylephrine. The results of the double-labelling experiments were consistent with the latter finding: the ratio [3H]20:4 over [14C]16:0 in 1,2-diacylglycerol decreased from 1.70 in the control to 1.40 during 10-min endothelin-1 or phorbolester stimulation, but not during phenylephrine stimulation. The [14C]glycerol incorporation into 1,2-diacylglycerol remained relatively constant under agonist-stimulated conditions as did the total concentration of 1,2-diacylglycerol.
1,2-Diacylglycerol present in unstimulated cardiomyocytes is likely derived from phosphatidylinositol. During stimulation with endothelin-1 and phorbolester, but not phenylephrine, phosphatidylcholine becomes an increasingly important source for 1,2-diacylglycerol due to sustained activation of phospholipase D. The 1,2-diacylglycerol level remains relatively constant during agonist stimulation which strongly indicates that particular molecular species of 1,2-diacylglycerol more than its total concentration determine the activation of protein kinase C isoenzymes.
Several extracellular agents and stress stimuli, such as tumour necrosis factor alpha, chemotherapeutic agents and heat, cause ceramide accumulation. They do this by regulating enzymes involved in its metabolism. Ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, cell-cycle arrest and cell senescence.
Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (epsilonV1) and translocation activator (psiepsilonRACK [receptors for activated C kinase]) specifically targeting PKCepsilon. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCepsilon signaling. As expected for a PKCepsilon RACK binding peptide, confocal microscopy showed that epsilonV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCepsilon by epsilonV1 at lower expression levels upregulated alpha-skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, epsilonV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCepsilon translocation with psiepsilonRACK resulted in selectively increased beta myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCepsilon signaling in normal postnatal maturational myocardial development and suggest the potential for PKCepsilon activators to stimulate "physiological" cardiomyocyte growth.
To test the hypothesis that activation of the protein kinase C (PKC) epsilon isoform leads to cardiac hypertrophy without failure, we studied transgenic mice with cardiac-specific overexpression of a constitutively active mutant of the PKCepsilon isoform driven by an alpha-myosin heavy chain promoter. In transgenic mice, the protein level of PKCepsilon in heart tissue was increased 9-fold. There was a 6-fold increase of the membrane/cytosol ratio, and PKC activity in the membrane fraction was 4.2-fold compared with wild-type mice. The heart weight was increased by 28%, and upregulation of the mRNA for beta-myosin heavy chain and alpha-skeletal actin was observed in transgenic mouse hearts. Echocardiography demonstrated increased anterior and posterior wall thickness with normal left ventricular function and dimensions, indicating concentric cardiac hypertrophy. Isolated cardiomyocyte mechanical function was slightly decreased, and Ca(2+) signals were markedly depressed in transgenic mice, suggesting that myofilament sensitivity to Ca(2+) was increased. No differences were observed in either the levels of cardiac Ca(2+)-handling proteins or the degree of cardiac regulatory protein phosphorylation between wild-type and transgenic mice. Unlike mice with PKCbeta(2) overexpression, transgenic mice with cardiac-specific overexpression of the active PKCepsilon mutant demonstrated concentric hypertrophy with normal in vivo cardiac function. Thus, PKC isoforms may play differential functional roles in cardiac hypertrophy and failure.
We synthesized diacylglycerols (DAGs) containing omega-6 or omega-3 polyunsaturated fatty acids [i.e., 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDG), and 1-stearoyl-2-eicosapentaenoyl-sn-glycerol (SEG)] and assessed their efficiency on activation of conventional (alpha, beta I, gamma) and novel (epsilon, delta) protein kinase C (PKC). SAG exerted significantly higher stimulatory effects than SDG and SEG on activation of PKC alpha and PKC delta. Activation of PKC beta I by SEG and SDG was higher than that by SAG. Activation of PKC gamma did not differ significantly among DAG molecular species. Addition of SAG to assays containing SEG and SDG exerted additive effects on activation of alpha and epsilon, but not on beta I and gamma, isoforms of PKC. SDG- and SEG-induced activation of PKC delta was significantly curtailed by the addition of SAG. Three DAG species significantly curtailed the PMA-induced activation of beta Iota, gamma, and delta, but not of alpha and epsilon, isoforms of PKC. Our study demonstrates for the first time that in vitro activation of different PKC isoenzymes vary in response to different DAG species, and one can envisage that this differential regulation may be responsible for their in vivo effects on target organs.
Dietary fish oil potentiates the susceptibility of cellular membranes to lipid peroxidation, although it is also known to have beneficial effects on the development of cardiovascular diseases.
The effects of dietary fish oil against doxorubicin-induced cardiomyopathy, in which free radicals and lipid peroxidation are involved, were investigated in rats.
Sprague-Dawley rats (100 g) were fed a standard diet or a high fish oil diet (containing 10% fish oil) throughout the experimental period. Four weeks after starting each diet, experimental rats were treated with doxorubicin (cumulative dose 15 mg/kg) or vehicle (0.28 M dextrose solution). After three weeks of doxorubicin treatment, the cardiac performance, myocardial lipid peroxidation and myocardial vitamin E level were assessed.
Compared with control rats, doxorubicin-treated rats showed a significantly increased mortality rate (P<0.05), and significantly decreased systolic blood pressure and left ventricular fractional shortening (P<0.01). The myocardial thiobarbituric acid-reactive substance level was significantly higher in doxorubicin-treated rats than in control rats (P<0.01), while the myocardial vitamin E level was significantly lower (P<0.05). Dietary fish oil enhanced the myocardial lipid peroxidation caused by doxorubicin, which was associated with a further decrease in myocardial vitamin E level. As a result, the rats treated with both doxorubicin and the high fish oil diet showed the highest mortality rate and the lowest cardiac performance of all the experimental groups.
Dietary fish oil may reduce antioxidant defences and accelerate susceptibility of the myocardium to lipid peroxidation in rats under doxorubicin treatment. This may partly explain why dietary fish oil does not prevent doxorubicin-induced cardiomyopathy.
Myocardial ischemia-reperfusion activates the Na(+)/H(+) exchanger, which induces arrhythmias, cell damage, and eventually cell death. Inhibition of the exchanger reduces cell damage and lowers the incidence of arrhythmias after ischemia-reperfusion. The omega-3 polyunsaturated fatty acids (PUFAs) are also known to be cardioprotective and antiarrhythmic during ischemia-reperfusion challenge. Some of the action of PUFAs may occur via inhibition of the Na(+)/H(+) exchanger. The purpose of our study was to determine the capacity for selected PUFAs to alter cardiac sarcolemmal (SL) Na(+)/H(+) exchange. Cardiac membranes highly enriched in SL vesicles were exposed to 10-100 microM eicosapentanoic acid (EPA) or docosahexanoic acid (DHA). H(+)-dependent (22)Na(+) uptake was inhibited by 30-50% after treatment with > or =50 microM EPA or > or =25 microM DHA. This was a specific effect of these PUFAs, because 50 microM linoleic acid or linolenic acid had no significant effect on Na(+)/H(+) exchange. The SL vesicles did not exhibit an increase in passive Na(+) efflux after PUFA treatment. In conclusion, EPA and DHA can potently inhibit cardiac SL Na(+)/H(+) exchange at physiologically relevant concentrations. This may explain, in part, their known cardioprotective effects and antiarrhythmic actions during ischemia-reperfusion.
To delineate the in vivo cardiac functions requiring normal δ protein kinase C (PKC) activity, we pursued loss-of-function through transgenic expression of a δPKC-specific translocation inhibitor protein fragment, δV1, in mouse hearts. Initial results using the mouse α-myosin heavy chain (αMHC) promoter resulted in a lethal heart failure phenotype. Viable δV1 mice were therefore obtained using novel attenuated mutant αMHC promoters lacking one or the other thyroid response element (TRE-1 and -2). In transgenic mouse hearts, δV1 decorated cytoskeletal elements and inhibited ischemia-induced δPKC translocation. At high levels, δV1 expression was uniformly lethal, with depressed cardiac contractile function, increased expression of fetal cardiac genes, and formation of intracardiomyocyte protein aggregates. Ultrastructural and immunoconfocal analyses of these aggregates revealed focal cytoskeletal disruptions and localized concentrations of desmin and αB-crystallin. In individual cardiomyocytes, cytoskeletal abnormalities correlated with impaired contractile function. Whereas desmin and αB-crystallin protein were increased ≈4-fold in δV1 hearts, combined overexpression of these proteins at these levels was not sufficient to cause any detectable cardiac pathology. At low levels, δV1 expression conferred striking resistance to postischemic dysfunction, with no measurable effects on basal cardiac structure, function, or gene expression. Intermediate expression of δV1 conferred modest basal contractile depression with less ischemic protection, associated with abnormal cardiac gene expression, and a histological picture of infrequent cardiomyocyte cytoskeletal deformities. These results validate an approach of δPKC inhibition to protect against myocardial ischemia, but indicate that there is a threshold level of δPKC activation that is necessary to maintain normal cardiomyocyte cytoskeletal integrity.
The juvenile visceral steatosis (JVS) mouse, a genetic model of systemic carnitine deficiency resulting from carnitine transport mutation, develops cardiac hypertrophy. We determined two putative lipid messengers, 1,2-diacylglycerol (DAG) and ceramide, in JVS and carnitine palmitoyltransferase-I (CPT-I) inhibitor etomoxir-treated mice because these lipids function as co-messengers in the myocardium via modification of protein kinase C activity.
JVS mice were evaluated at 4 and 8 weeks of age. The effect of long-term etomoxir treatment (45 mg/day) (ET) on mice was investigated in control mice from 4 to 8 weeks of age. As a model of inhibited cardiac hypertrophy, carnitine-treated JVS (CT) mice were produced. Myocardial DAG and ceramide levels and their fatty acid composition were measured.
The heart/body weight ratio increased by 100% in JVS mice compared with that in controls, while that of CT mice was normalized in comparison with controls at 8 weeks of age. DAG markedly increased in both JVS and ET mice compared with that in controls (1,677+/-84, 1,258+/-49, and 585+/-58 ng/dry wt, respectively; P<0.01 for controls versus JVS or ET mice), whereas it was decreased significantly in CT mice compared with that in JVS mice (1,066+/-54 ng/dry wt, P<0.01). Furthermore, the fatty acid composition of DAG was similar in JVS and ET mice; in particular, 18:1 and 18:2 were significantly elevated in the myocardium (P<0.01 versus controls). On the other hand, that of DAG in CT mice was similar to that of the control group. In contrast, no difference was observed in myocardial ceramide levels among the groups.
Pharmacological intervention with etomoxir mimics changes in the lipid second messenger characteristic of genetic JVS mice. The results suggest that the increases in distinct DAG species might be involved in the pathogenesis of cardiac hypertrophy as a result of disorder of fatty acid transport.
Omega-3 fatty acids have been shown in epidemiological and clinical trials to reduce the incidence of CVD. Large-scale epidemiological studies suggest that individuals at risk for CHD benefit from the consumption of plant- and marine-derived omega-3 fatty acids, although the ideal intakes presently are unclear. Evidence from prospective secondary prevention studies suggests that EPA+DHA supplementation ranging from 0.5 to 1.8 g/d (either as fatty fish or supplements) significantly reduces subsequent cardiac and all-cause mortality. For α-linolenic acid, total intakes of ≈1.5 to 3 g/d seem to be beneficial. Collectively, these data are supportive of the recommendation made by the AHA Dietary Guidelines to include at least two servings of fish per week (particularly fatty fish). In addition, the data support inclusion of vegetable oils (eg, soybean, canola, walnut, flaxseed) and food sources (eg, walnuts, flaxseeds) high in α-linolenic acid in a healthy diet for the general population (Table 5). The fish recommendation must be balanced with concerns about environmental pollutants, in particular PCB and methylmercury, described in state and federal advisories. Consumption of a variety of fish is recommended to minimize any potentially adverse effects due to environmental pollutants and, at the same time, achieve desired CVD health outcomes. RCTs have demonstrated that omega-3 fatty acid supplements can reduce cardiac events (eg, death, nonfatal MI, nonfatal stroke) and decrease progression of atherosclerosis in coronary patients. However, additional studies are needed to confirm and further define the health benefits of omega-3 fatty acid supplements for both primary and secondary prevention. For example, placebo-controlled, double-blind RCTs are needed to document both the safety and efficacy of omega-3 fatty acid supplements in both high-risk patients (eg, patients with type 2 diabetes, dyslipidemia, and hypertension, and smokers) and coronary patients on drug therapy. Mechanistic studies on their apparent effects on sudden death are also needed. A dietary (ie, food-based) approach to increasing omega-3 fatty acid intake is preferable. Still, for patients with coronary artery disease, the dose of omega-3 (≈1 g/d) may be greater than what can readily be achieved through diet alone (Table 5). These individuals, in consultation with their physician, could consider supplements for CHD risk reduction. Supplements also could be a component of the medical management of hypertriglyceridemia, a setting in which even larger doses (2 to 4 g/d) are required (Table 5). The availability of high-quality omega-3 fatty acid supplements, free of contaminants, is an important prerequisite to their extensive use.
The protein kinase C (PKC) family is implicated in cardiac hypertrophy, contractile failure, and beta-adrenergic receptor (betaAR) dysfunction. Herein, we describe the effects of gain- and loss-of-PKCalpha function using transgenic expression of conventional PKC isoform translocation modifiers. In contrast to previously studied PKC isoforms, activation of PKCalpha failed to induce cardiac hypertrophy, but instead caused betaAR insensitivity and ventricular dysfunction. PKCalpha inhibition had opposite effects. Because PKCalpha is upregulated in human and experimental cardiac hypertrophy and failure, its effects were also assessed in the context of the Galphaq overexpression model (in which PKCalpha is transcriptionally upregulated). Normalization (inhibition) of PKCalpha activity in Galpha(q) hearts improved systolic and diastolic function, whereas further activation of PKCalpha caused a lethal restrictive cardiomyopathy with marked interstitial fibrosis. These results define pathological roles for PKCalpha as a negative regulator of ventricular systolic and diastolic function.
Many of the cardiovascular benefits of fish oil result from the antiarrhythmic actions of the n-3 polyunsaturated lipids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The beneficial effects of DHA/EPA in patients with coronary artery disease and myocardial infarction may also result from modulation of the myocardial hypertrophic response. Hypertrophy was assessed in neonatal cardiomyocytes exposed to phenylephrine (PE) by measuring cell surface area, total protein synthesis ((14)C leucine incorporation), and the organization of sarcomeric alpha-actinin and by monitoring expression of atrial natriuretic factor (ANF). We report that PE induced a twofold increase in cell surface area and protein synthesis in cardiomyocytes. The hypertrophied cardiomyocytes also exhibited increased expression of ANF in perinuclear regions and organization of sarcomeric alpha-actinin into classical z-bands. Treatment of cardiomyocytes with 5 microM DHA effectively prevented PE-induced hypertrophy as shown by inhibition of surface area expansion and protein synthesis, inhibition of ANF expression, and prevention of alpha-actinin organization into z-bands. DHA treatment prevented PE-induced activation of Ras and Raf-1 kinase. The upstream inhibition of Ras --> Raf-1 effectively prevented translocation and nuclear localization of phosphorylated extracellularly regulated kinase 1 and 2 (Erk1/2). These effects consequently led to inhibition of nuclear translocation, and hence, activation of the downstream signaling enzyme p90 ribosomal S6 kinase (p90(rsk)). These results indicate that PE-induced cardiac hypertrophy can be minimized by DHA. Our results suggest that inhibition of Ras --> Raf-1 --> Erk1/2 --> p90(rsk) --> hypertrophy is one possible pathway by which DHA can inhibit cardiac hypertrophy. In vivo studies are needed to confirm these in vitro effects of DHA.
In two non-consanguineous Hungarian Roma (Gypsy) children who presented with cardiomyopathy and decreased plasma carnitine levels, we identified homozygous deletion of 17081C of the SLC22A5 gene that results in a frameshift at R282D and leads ultimately to a premature stop codon (V295X) in the OCTN2 carnitine transporter. Carnitine treatment resulted in dramatic improvement of the cardiac symptoms, echocardiographic, and EKG findings in both cases. Family investigations revealed four sudden deaths, two of them corresponded to the classic SIDS phenotype. In postmortem tissue specimens available from three of them we could verify the homozygous mutation. In liver tissue reserved from two patients lipid droplet vacuolization could be observed; the lipid vacuoles were located mainly in the peripherolobular regions of the acini. In the heart tissue signs of generalized hypertrophy and lipid vacuoles were seen predominantly in the subendocardial areas in both cases; some aggregates of smaller lipid vacuoles were separated, apparently by membranes. Review of all OCTN2 deficiency cases reported so far revealed that this is the first presentation of histopathology in classic familial sudden infant death syndrome (SIDS) with an established SLC22A5 mutation. In addition to the two affected homozygous cardiomyopathic children and three homozygous sudden death patients, the genetic analysis in 25 relatives showed 14 carriers. The mutant gene derived from five non-consanguineous grandparents, each of them having 6-14 brothers and sisters. This alone suggests a wide ancestral spread of the mutation in certain Roma subpopulations.