Genetic assessment and folate receptor autoantibodies in infantile-onset cerebral folate deficiency (CFD) syndrome.

INTRODUCTION: Cerebral folate deficiency (CFD) syndromes are defined as neuro-psychiatric conditions with low CSF folate and attributed to different causes such as autoantibodies against the folate receptor-alpha (FR) protein that can block folate transport across the choroid plexus, FOLR1 gene mutations or mitochondrial disorders. High-dose folinic acid treatment restores many neurologic deficits. STUDY AIMS AND METHODS: Among 36 patients from 33 families the infantile-onset CFD syndrome was diagnosed based on typical clinical features and low CSF folate. All parents were healthy. Three families had 2 affected siblings, while parents from 4 families were first cousins. We analysed serum FR autoantibodies and the FOLR1 and FOLR2 genes. Among three consanguineous families homozygosity mapping attempted to identify a monogenetic cause. Whole exome sequencing (WES) was performed in the fourth consanguineous family, where two siblings also suffered from polyneuropathy as an atypical finding. RESULTS: Boys (72%) outnumbered girls (28%). Most patients (89%) had serum FR autoantibodies fluctuating over 5-6 weeks. Two children had a genetic FOLR1 variant without pathological significance. Homozygosity mapping failed to detect a single autosomal recessive gene. WES revealed an autosomal recessive polynucleotide kinase 3´phosphatase (PNKP) gene abnormality in the siblings with polyneuropathy. DISCUSSION: Infantile-onset CFD was characterized by serum FR autoantibodies as its predominant pathology whereas pathogenic FOLR1 gene mutations were absent. Homozygosity mapping excluded autosomal recessive inheritance of any single responsible gene. WES in one consanguineous family identified a PNKP gene abnormality that explained the polyneuropathy and also its contribution to the infantile CFD syndrome because the PNKP gene plays a dual role in both neurodevelopment and immune-regulatory function. Further research for candidate genes predisposing to FRα-autoimmunity is suggested to include X-chromosomal and non-coding DNA regions.

Folinic acid improves verbal communication in children with autism and language impairment: a randomized double-blind placebo-controlled trial.

We sought to determine whether high-dose folinic acid improves verbal communication in children with non-syndromic autism spectrum disorder (ASD) and language impairment in a double-blind placebo control setting. Forty-eight children (mean age 7 years 4 months; 82% male) with ASD and language impairment were randomized to receive 12 weeks of high-dose folinic acid (2 mg kg-1 per day, maximum 50 mg per day; n=23) or placebo (n=25). Children were subtyped by glutathione and folate receptor-α autoantibody (FRAA) status. Improvement in verbal communication, as measured by a ability-appropriate standardized instrument, was significantly greater in participants receiving folinic acid as compared with those receiving placebo, resulting in an effect of 5.7 (1.0,10.4) standardized points with a medium-to-large effect size (Cohen's d=0.70). FRAA status was predictive of response to treatment. For FRAA-positive participants, improvement in verbal communication was significantly greater in those receiving folinic acid as compared with those receiving placebo, resulting in an effect of 7.3 (1.4,13.2) standardized points with a large effect size (Cohen's d=0.91), indicating that folinic acid treatment may be more efficacious in children with ASD who are FRAA positive. Improvements in subscales of the Vineland Adaptive Behavior Scale, the Aberrant Behavior Checklist, the Autism Symptom Questionnaire and the Behavioral Assessment System for Children were significantly greater in the folinic acid group as compared with the placebo group. There was no significant difference in adverse effects between treatment groups. Thus, in this small trial of children with non-syndromic ASD and language impairment, treatment with high-dose folinic acid for 12 weeks resulted in improvement in verbal communication as compared with placebo, particularly in those participants who were positive for FRAAs.

Thyroid dysfunction in children with autism spectrum disorder is associated with folate receptor α autoimmune disorder.

Folate receptor α (FRα) autoantibodies (FRAAs) are prevalent in autism spectrum disorder (ASD). FRAAs disrupt folate transport across the blood-brain barrier by binding to the FRα. Thyroid dysfunction is frequently found in children with ASD. We measured blocking and binding FRAAs and thyroid-stimulating hormone (TSH), free thyroxine (T4) (FT4), total triiodothyronine (T3) (TT3), reverse T3 (rT3), thyroid-releasing hormone (TRH) and other metabolites in 87 children with ASD, 84 of whom also underwent behaviour and cognition testing and in 42 of whom FRAAs, TSH and FT4 were measured at two time points. To better understand the significance of the FRα in relation to thyroid development, we examined FRα expression on prenatal and postnatal thyroid. TSH, TT3 and rT3 were above the normal range in 7%, 33% and 51% of the participants and TRH was below the normal range in 13% of the participants. FT4 was rarely outside the normal range. TSH concentration was positively and the FT4/TSH, TT3/TSH and rT3/TSH ratios were inversely related to blocking FRAA titres. On repeated measurements, changes in TSH and FT4/TSH ratio were found to correspond to changes in blocking FRAA titres. TSH and the FT4/TSH, TT3/TSH and rT3/TSH ratios were related to irritability on the Aberrant Behavior Checklist and several scales of the Social Responsiveness Scale (SRS), whereas TT3 was associated with SRS subscales and TRH was related to Vineland Adaptive Behavior Scale subscales. The thyroid showed significant FRα expression during the early prenatal period, although expression decreased significantly in later gestation and postnatal thyroid tissue. The results of the present study suggest that thyroid dysfunction in ASD may be related to blocking FRAA. The high expression of FRα in the early foetal thyroid suggests that foetal and neonatal exposure to maternal FRAAs could affect the development of the thyroid and may contribute to the pathology in ASD.

Prevention of behavioral deficits in rats exposed to folate receptor antibodies: implication in autism.

Folate receptor alpha (FRα) autoantibodies have been associated with fetal abnormalities and cerebral folate deficiency-related developmental disorders. Over 70% of the children with autism spectrum disorders (ASD) are positive for these autoantibodies and high-dose folinic acid is beneficial in treating these children. Here we show that antibodies (Abs) to the rat FRα administered during gestation produce communication, learning and cognitive deficits in a rat model that can be prevented by folinic acid and dexamethasone. FRα Ab can trigger inflammation as well as block folate transport to the fetus and to the developing brain to produce the functional deficits. In humans, exposure to FRα autoantibodies during fetal development and infancy could contribute to brain dysfunction such as that seen in ASD and other developmental disorders. Identifying women positive for the autoantibody and treating them with high-dose folinic acid along with other interventions to lower the autoantibody titer are effective strategies that may be considered to reduce the risk of having a child with developmental deficits.

The basis for folinic acid treatment in neuro-psychiatric disorders.

Multiple factors such as genetic and extraneous causes (drugs, toxins, adverse psychological events) contribute to neuro-psychiatric conditions. In a subgroup of these disorders, systemic folate deficiency has been associated with macrocytic anemia and neuropsychiatric phenotypes. In some of these, despite normal systemic levels, folate transport to the brain is impaired in the so-called cerebral folate deficiency (CFD) syndromes presenting as developmental and psychiatric disorders. These include infantile-onset CFD syndrome, infantile autism with or without neurologic deficits, a spastic-ataxic syndrome and intractable epilepsy in young children expanding to refractory schizophrenia in adolescents, and finally treatment-resistant major depression in adults. Folate receptor alpha (FRα) autoimmunity with low CSF N(5)-methyl-tetrahydrofolate (MTHF) underlies most CFD syndromes, whereas FRα gene abnormalities and mitochondrial gene defects are rarely found. The age at which FRα antibodies of the blocking type emerge, determines the clinical phenotype. Infantile CFD syndrome and autism with neurological deficits tend to be characterized by elevated FRα antibody titers and low CSF MTHF. In contrast, in infantile autism and intractable schizophrenia, abnormal behavioral signs and symptoms may wax and wane with fluctuating FRα antibody titers over time accompanied by cycling changes in CSF folate, tetrahydrobiopterin (BH4) and neurotransmitter metabolites ranging between low and normal levels. We propose a hypothetical model explaining the pathogenesis of schizophrenia. Based on findings from clinical, genetic, spinal fluid and MRI spectroscopic studies, we discuss the neurochemical changes associated with these disorders, metabolic and regulatory pathways, synthesis and catabolism of neurotransmitters, and the impact of oxidative stress on the pathogenesis of these conditions. A diagnostic algorithm and therapeutic regimens using high dose folinic acid, corticosteroids and milk-free diet is presented which has proven to be beneficial in providing adequate folate to the brain and decreasing the FRα autoantibody titer in those positive for the antibody.

Folinic acid treatment for schizophrenia associated with folate receptor autoantibodies.

BACKGROUND: Auto-antibodies against folate receptor alpha (FRα) at the choroid plexus that block N(5)-methyltetrahydrofolate (MTHF) transfer to the brain were identified in catatonic schizophrenia. Acoustic hallucinations disappeared following folinic acid treatment. Folate transport to the CNS prevents homocysteine accumulation and delivers one-carbon units for methyl-transfer reactions and synthesis of purines. The guanosine derivative tetrahydrobiopterin acts as common co-factor for the enzymes producing dopamine, serotonin and nitric oxide. METHODS: Our study selected patients with schizophrenia unresponsive to conventional treatment. Serum from these patients with normal plasma homocysteine, folate and vitamin B12 was tested for FR autoantibodies of the blocking type on serial samples each week. Spinal fluid was analyzed for MTHF and the metabolites of pterins, dopamine and serotonin. The clinical response to folinic acid treatment was evaluated. RESULTS: Fifteen of 18 patients (83.3%) had positive serum FR auto-antibodies compared to only 1 in 30 controls (3.3%) (χ(2)=21.6; p<0.0001). FRα antibody titers in patients fluctuated over time varying between negative and high titers, modulating folate flux to the CNS, which explained low CSF folate values in 6 and normal values in 7 patients. The mean±SD for CSF MTHF was diminished compared to previously established controls (t-test: 3.90; p=0.0002). A positive linear correlation existed between CSF MTHF and biopterin levels. CSF dopamine and serotonin metabolites were low or in the lower normal range. Administration of folinic acid (0.3-1mg/kg/day) to 7 participating patients during at least six months resulted in clinical improvement. CONCLUSION: Assessment of FR auto-antibodies in serum is recommended for schizophrenic patients. Clinical negative or positive symptoms are speculated to be influenced by the level and evolution of FRα antibody titers which determine folate flux to the brain with up- or down-regulation of brain folate intermediates linked to metabolic processes affecting homocysteine levels, synthesis of tetrahydrobiopterin and neurotransmitters. Folinic acid intervention appears to stabilize the disease process.

Cerebral folate receptor autoantibodies in autism spectrum disorder.

Cerebral folate deficiency (CFD) syndrome is a neurodevelopmental disorder typically caused by folate receptor autoantibodies (FRAs) that interfere with folate transport across the blood-brain barrier. Autism spectrum disorders (ASDs) and improvements in ASD symptoms with leucovorin (folinic acid) treatment have been reported in some children with CFD. In children with ASD, the prevalence of FRAs and the response to leucovorin in FRA-positive children has not been systematically investigated. In this study, serum FRA concentrations were measured in 93 children with ASD and a high prevalence (75.3%) of FRAs was found. In 16 children, the concentration of blocking FRA significantly correlated with cerebrospinal fluid 5-methyltetrahydrofolate concentrations, which were below the normative mean in every case. Children with FRAs were treated with oral leucovorin calcium (2 mg kg(-1) per day; maximum 50 mg per day). Treatment response was measured and compared with a wait-list control group. Compared with controls, significantly higher improvement ratings were observed in treated children over a mean period of 4 months in verbal communication, receptive and expressive language, attention and stereotypical behavior. Approximately one-third of treated children demonstrated moderate to much improvement. The incidence of adverse effects was low. This study suggests that FRAs may be important in ASD and that FRA-positive children with ASD may benefit from leucovorin calcium treatment. Given these results, empirical treatment with leucovorin calcium may be a reasonable and non-invasive approach in FRA-positive children with ASD. Additional studies of folate receptor autoimmunity and leucovorin calcium treatment in children with ASD are warranted.

Transcobalamin II receptor polymorphisms are associated with increased risk for neural tube defects.

OBJECTIVE: Women who have low cobalamin (vitamin B(12)) levels are at increased risk for having children with neural tube defects (NTDs). The transcobalamin II receptor (TCblR) mediates uptake of cobalamin into cells. Inherited variants in the TCblR gene as NTD risk factors were evaluated. METHODS: Case-control and family-based tests of association were used to screen common variation in TCblR as genetic risk factors for NTDs in a large Irish group. A confirmatory group of NTD triads was used to test positive findings. RESULTS: 2 tightly linked variants associated with NTDs in a recessive model were found: TCblR rs2336573 (G220R; p(corr)=0.0080, corrected for multiple hypothesis testing) and TCblR rs9426 (p(corr)=0.0279). These variants were also associated with NTDs in a family-based test before multiple test correction (log-linear analysis of a recessive model: rs2336573 (G220R; RR=6.59, p=0.0037) and rs9426 (RR=6.71, p=0.0035)). A copy number variant distal to TCblR and two previously unreported exonic insertion-deletion polymorphisms were described. CONCLUSIONS: TCblR rs2336573 (G220R) and TCblR rs9426 represent a significant risk factor in NTD cases in the Irish population. The homozygous risk genotype was not detected in nearly 1000 controls, indicating that this NTD risk factor may be of low frequency and high penetrance. 9 other variants are in perfect linkage disequilibrium with the associated single nucleotide polymorphisms. Additional work is required to identify the disease-causing variant. Our data suggest that variation in TCblR plays a role in NTD risk and that these variants may modulate cobalamin metabolism.

Cerebral folate deficiency and CNS inflammatory markers in Alpers disease.

We describe a 3.5-year-old female with Alpers disease with a POLG genotype of p.A467T/p.G848S and with a lethal outcome. Laboratory investigation revealed elevated CSF neopterin, IL-6, IL-8, IFN-gamma, reduced CSF 5-methyltetrahydrofolate (5MTHF), and increased serum as well as CSF folate receptor blocking autoantibodies. Treatment with oral Leucovorine (5-formyl-tetrahydrofolate) was initiated at 0.25mg/kg bid, and later increased to 4mg/kg bid. Under treatment CSF levels of 5MTHF, seizure frequency and communicative abilities improved. Over a time span of 17months, CSF levels of IL-6 and IFN-gamma decreased, levels of folate receptor blocking autoantibodies continued to raise, whereas CSF IL-8 remained elevated 1500-fold above normal. The child died without apparent stress at the age of 5.5years. Alpers disease, a neurodegenerative disease usually presents in the first years of life as a progressive encephalopathy with multifocal myoclonic seizures, developmental regression, cortical blindness and early death. The underlying genetic defect has been attributed to mutations of the catalytic subunit of the mitochondrial DNA polymerase-gamma leading to an organ-specific mitochondrial DNA depletion syndrome with reduced activity of respiratory chain enzyme complexes in the brain and the liver. A curative therapy is not available. This case report of Alpers disease provides new insights into the pathophysiology of Alpers disease, where mitochondrial dysfunction in conjunction with inflammatory cytokines and blocking folate receptor autoantibodies may lead to a secondary cerebral folate deficiency syndrome. The treatment of the latter provides relief to the patient without stopping the underlying disease.

Mitochondrial complex I encephalomyopathy and cerebral 5-methyltetrahydrofolate deficiency.

Folate transport to the brain depends on ATP-driven folate receptor-mediated transport across choroid plexus epithelial cells. Failure of ATP production in Kearns-Sayre syndrome syndrome provides one explanation for the finding of low spinal fluid (CSF) 5-methyltetrahydrofolate (5MTHF) levels in this condition. Therefore, we suspect the presence of reduced folate transport across the blood-spinal fluid barrier in other mitochondrial encephalopathies. In the present patient with mitochondrial complex I encephalomyopathy a low 5-methyltetrahydrofolate level was found in the CSF. Serum folate receptor autoantibodies were negative and could not explain the low spinal fluid folate levels. The epileptic seizures did not respond to primidone monotherapy, but addition of ubiquinone-10 and radical scavengers reduced seizure frequency. Add-on treatment with folinic acid led to partial clinical improvement including full control of epilepsy, followed by marked recovery from demyelination of the brainstem, thalamus, basal ganglia and white matter. Cerebral folate deficiency is not only present in Kearns-Sayre syndrome but may also be secondary to the failure of mitochondrial ATP production in other mitochondrial encephalopathies. Treatment with folinic acid in addition to supplementation with radical scavengers and cofactors of deficient respiratory enzymes can result in partial clinical improvement and reversal of abnormal myelination patterns on neuro-imaging.

Folate receptor autoantibodies and spinal fluid 5-methyltetrahydrofolate deficiency in Rett syndrome.

Rett syndrome was associated with low cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5MTHF) in 42-50% of European patients whereas approximately 93% of the patients from North-America had a normal CSF 5MTHF status. We determined the CSF folate status in Rett patients living in North- and South-Western Europe and measured serum folate receptor (FR) autoantibodies of the blocking type to explain the reduced folate transport across the choroid plexus. Irrespective of their MECP2 genotype and despite normal plasma folate values, 14 of 33 Rett patients (42%) had low CSF folate levels. Blocking FR autoantibodies were found in 8 of the Rett patients (24%), 6 of whom had low CSF folate levels. FR autoimmunity was primarily found within the group of Rett patients with low CSF folate status with a higher incidence in North-Western Europe. In Rett patients from North-America 74 of 76 girls had higher folate values in both serum and CSF than European patients. The food folate fortification in North-America may account for the higher folate levels and may prevent CFD in these Rett patients. FR autoimmunity occurred predominantly in Rett patients from North-Western Europe and may contribute to cerebral folate deficiency (CFD).

Antizyme and antizyme inhibitor activities influence cellular responses to polyamine analogs.

Close structural analogs of spermidine and spermine, polyamine mimetics, are potential chemotheraputic agents as they depress cellular polyamines required for tumor growth. Specific mimetic analogs stimulate synthesis of the regulatory protein antizyme (AZ), which not only inactivates the initial enzyme in polyamine biosynthesis but also inhibits cellular uptake of polyamines. The role of AZ induction in influencing cellular uptake of representative analogs was investigated using three analogs produced by Cellgate Inc., CGC-11047, CGC-11102, and CGC-11144, which exhibit markedly distinct AZ-inducing potential. An inverse correlation was noted between the AZ-inducing activity of a compound and the steady-state levels accumulated in cells. As some tumor cells over express AZI as a means of enhancing the polyamines required for aggressive growth, analog sensitivity was examined in transgenic CHO cells expressing exogenous antizyme inhibitor protein (AZI). Although AZI over expression increased cell sensitivity to analogs, the degree of this affect varied with the analog used.

Unusual aspects of the polyamine transport system affect the design of strategies for use of polyamine analogues in chemotherapy.

One strategy for inhibiting tumour cell growth is the use of polyamine mimetics to depress endogenous polyamine levels and, ideally, obstruct critical polyamine-requiring reactions. Such polyamine analogues make very unusual drugs, in that extremely high intracellular concentrations are required for growth inhibition or cytotoxicity. Cells exposed to even sub-micromolar concentrations of such analogues can achieve effective intracellular levels because these compounds are incorporated by the very aggressive polyamine uptake system. Once incorporated to these levels, many of these analogues induce the synthesis of a regulatory protein, antizyme, which inhibits both polyamine synthesis and the transporter they used to enter the cell. Thus this feedback system allows steady-state maintenance of effective cellular doses of such analogues. Accordingly, effective cellular levels of polyamine analogues are generally inversely related to their capacity to induce antizyme. Antizyme activity is down-regulated by interaction with several binding partners, most notably antizyme inhibitor, and at least a few tumour tissues exhibit deficiencies in antizyme expression. Our studies explore the role of antizyme induction by several polyamine analogues in their physiological response and the possibility that cell-to-cell differences in antizyme expression may contribute to variable sensitivities to these agents.

Folate receptor autoimmunity and cerebral folate deficiency in low-functioning autism with neurological deficits.

Reduced folate transport to the CNS was identified in two autism spectrum disorders, i.e., Rett syndrome and infantile low-functioning autism with neurological abnormalities. Twenty-five patients with early-onset low-functioning autism with or without neurological deficits, were evaluated for serum folate, cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5MTHF), and serum FR autoantibodies of the blocking type to determine the significance of folate receptor (FR) autoantibodies with respect to folate transport across the blood-CSF barrier. In spite of normal serum folate, CSF 5MTHF was low in 23 of 25 patients. The reduced CSF folate in 19 of these 23 patients could be explained by serum FR autoantibodies blocking the folate binding site of the membrane-attached FR on the choroid epithelial cells. Oral folinic acid supplements led to normal CSF 5MTHF and partial or complete clinical recovery after 12 months. Serum FR autoimmunity appears to represent an important factor in the pathogenesis of reduced folate transport to the nervous system among children with early-onset low-functioning autism associated with or without neurological deficits. Early detection of FR autoantibodies may be a key factor in the prevention and therapeutic intervention among this subgroup of patients with autism.

Time course of lysophosphatidylcholine release from ischemic human myocardium parallels the time course of early ischemic ventricular arrhythmia.

BACKGROUND: We determined the kinetics of the release of lysophosphatidylcholine (LPC) into the coronary sinus of patients undergoing stress tests after coronary artery bypass grafting. The kinetics were consistent with a role for this amphiphile in the pathogenesis of ischemic ventricular arrhythmia, a major cause of sudden death. METHODS: Stress testing was initiated in the operating suite by pacing at a rate of 160 beats/min for 2 min. Ischemia was then induced by clamping the bypass grafts to the anterior wall for a maximal time of 4 min. RESULTS: The pacing procedure induced a prompt but reversible increase in coronary sinus LPC concentration from a baseline of 60.9 +/- 2.5 to 83.8 +/- 5.0 mumol/l via pacing alone, and a further increase to 101.8 +/- 6.7 mumol/l when the grafts were clamped for 2 min (P < 0.01). Six minutes after the cessation of pacing, LPC concentration returned to 67.5 +/- 4.4 mumol/l. CONCLUSIONS: These results demonstrate that severe myocardial ischemia is an agonist for rapid release of LPC from the myocardium. Kinetics of this release paralleled the time-course of early onset of electrophysiologic changes in isolated myocytes and perfused heart preparations in vitro. These results indicate that LPC may have an important role in the pathogenesis of ischemic ventricular arrhythmia in patients.

Lysophosphatidylcholine accumulation in ischemic human myocardium.

Lysophosphatidylcholine accumulates in the coronary sinus during pacing-induced myocardial ischemia in humans. This amphiphile accelerates Ca++ flux leading to cell injury in cultured cardiac myocytes, but it is not known whether lysophosphatidylcholine accumulation is injurious to human myocardium. In this study, we measured lysophosphatidylcholine in normal human myocardium obtained during cardiac surgery and exposed to ischemic conditions in vitro. Total lysophosphatidylcholine concentration (sum of lysophosphatidylcholine remaining in tissue and lysophosphatidylcholine released into the buffer) increased from 0.73 +/- 0.08 nmol/mg protein at baseline to 1.83 +/- 0.45 nmol/mg protein after 5 minutes of ischemia (p < 0.001), and was associated with evidence of cell injury (26% depletion of tissue lactate dehydrogenase). Significant lysophosphatidylcholine release into the incubation buffer (0.41 +/- 0.11 nmol/mg protein) also occurred after 5 minutes of ischemia. In contrast, there was no lysophosphatidylcholine accumulation or release and no lactate dehydrogenase depletion in oxygenated and perfused controls. Attenuation of lysophosphatidylcholine accumulation by incubation with lysophospholipase did not prevent cell injury. Lysoplasmalogen was not detected in ischemic tissue. We conclude that lysophosphatidylcholine accumulation is a marker of myocardial ischemia in humans.

Coronary sinus lysophosphatidylcholine accumulation during rapid atrial pacing.

The purpose of this study was to determine whether lysophosphatidylcholine (LPC) release from the myocardium could be detected in patients with pacing-induced ischemia. We measured LPC levels in plasma obtained from the coronary sinus in 20 patients undergoing diagnostic atrial pacing stress tests. In 14 patients with pacing-induced ischemia, coronary sinus LPC concentration rose from 69.1 +/- 5.3 microM at baseline to 101.7 +/- 9.0 microM at 4 minutes and to 178.0 +/- 18.0 microM at peak pacing (p less than 0.01). LPC did not increase significantly in 6 nonischemic patients (from 60.8 +/- 7.8 microM at baseline to 70.2 +/- 7.6 microM at peak pacing, difference not significant). LPC did not increase in mixed venous or arterial blood from ischemic patients. Thus, coronary sinus LPC accumulation may be an early marker of ischemia.

Potentiation of the depressant effects of lysophosphatidylcholine on contractile properties of cultured cardiac myocytes by acidosis and superoxide radical.

Lysophosphatidylcholine (LPC) accumulates in the heart during myocardial ischemia. This amphiphile accelerates Ca++ flux in cardiac myocytes and may mediate ischemic cell injury. In the present study, we evaluated the effects of LPC on the contractility of cultured neonatal rat heart cells. We also investigated the interactions between LPC and other prominent features of the ischemic milieu, acidosis, and superoxide radical. A photo-optical technique was used to measure the maximum velocities of shortening and relaxation (dS/dt and dR/dt) of cultured cells superfused with 0.1 to 100 mumol/L LPC. LPC, at all concentrations, initially increased dS/dt. After 1 minute, however, dS/dt decreased in a concentration-dependent manner in cells superfused with greater than 20 mumol/L LPC. The effect of LPC on relaxation was also dependent on LPC concentration. dR/dt increased at less than 40 mumol/L LPC but decreased at greater than or equal to 60 mumol/L LPC. Acidosis markedly potentiated LPC-mediated depression in dS/dt and dR/dt. In contrast, superoxide dismutase entirely prevented LPC-mediated depression of contractility. We conclude that whereas brief exposure to LPC stimulates contractility, prolonged exposure to greater than 40 mumol/L LPC depresses dS/dt and dR/dt in cultured myocytes. The depressant effects of LPC on contractility are potentiated by acidosis and superoxide radical. We postulate that LPC accumulation in the myocardium contributes to ischemia-mediated contractile dysfunction.