Yoshinori Koga

Multiple mitochondrial DNA deletions exist in cardiomyocytes of patients with hypertrophic or dilated cardiomyopathy

Genetic impairment was revealed in idiopathic cardiomyopathy and the responsible DNA locus was estimated. Mitochondrial DNA were amplified from autopsied cardiac specimens from three patients who died from hypertrophic or dilated cardiomyopathy by using polymerase chain reaction (PCR). By using two novel methods for PCR gene amplification, the pleioplasmic existence of multiple populations of differently deleted mitochondrial DNA in all specimens from the patients was confirmed. Mitochondrial DNA with a 7,436 bp deletion which commonly existed among the specimens was sequenced and the direct repeat at each edge of deletion was identified as (CATCAACAACCG) which was located in ATPase 6 gene and in the D-loop region. From our results mitochondrial DNA mutations could also be an important contributory factor to cardiomyopathy.

Prognosis in hypertrophic cardiomyopathy

One hundred thirty-six patients with hypertrophic cardiomyopathy were followed up for 1 to 17 years. Twenty-one patients had died, 14 of them suddenly, two from heart failure, two from cerebral embolism, and three from noncardiac causes. Life table analysis revealed that sudden death was significantly associated with young age less than 20 years (relative risk [rr] = 8.63, when compared with those greater than 40 years) and with positive Masters single two-step test (rr = 3.55). Heart failure was more frequent in patients with positive Masters single test (rr = 4.27) and with left ventricular end-diastolic pressure greater than 20 mm Hg (rr = 2.58). Atrial fibrillation, observed in 15 patients, was a poor prognostic sign, resulting in five cardiac deaths and seven heart failures. In contrast, prognosis was favorable in patients with apical hypertrophy with giant negative T wave. Thus Japanese patients with hypertrophic cardiomyopathy showed a prognosis consistent with Western patients, except for excellent outcome of apical hypertrophy.

Predictors of sudden cardiac death in hypertrophic cardiomyopathy

Patients with hypertrophic cardiomyopathy (HC) die suddenly. Proposed risk factors for sudden cardiac death (SCD) in HC are youth, a family history of SCD, syncope, and ventricular tachycardia. Hemodynamic variables have not convincingly proved to be risk factors for SCD. Therefore, this study was designed to examine predictors of SCD in a large number of patients with HC during long-term follow-up periods. The relation of studied variables (clinical, electrocardiographic, echocardiographic, hemodynamic, and exercise test findings) to SCD in 309 patients with HC who were initially diagnosed during 1971 through 1994 (mean follow-up 9.4 years) was examined by multivariate analysis. SCD occurred in 28 patients. Independent predictors of SCD were a smaller difference between peak and rest systolic blood pressure during exercise testing (p=0.006), and higher left ventricular outflow tract pressure gradient at rest (p=0.003). Exercise-related SCD occurred in 8 patients and exercise-unrelated SCD in 20 patients (mean age 28 vs 47 years, p <0.05). Thus, patients of exercise-related SCD were younger and had smaller increases in systolic blood pressure during exercise testing, whereas patients with exercise-unrelated SCD were older and had higher left ventricular outflow tract pressure gradient.

Point mutations in mitochondrial DNA in patients with hypertrophic cardiomyopathy

Recent advances suggest that mutations in nuclear DNA are involved in the etiology of autosomal dominant hypertrophic cardiomyopathy. Mitochondria have their own DNA, and mutations in mitochondrial DNA have been shown to contribute to the genesis of various diseases. In this study, we developed rapid sequencing methods with the use of a fluorescence-based sequencing system and analyzed total mitochondrial DNA of seven patients with nonautosomal dominant hypertrophic cardiomyopathy. Multiple point mutations were observed in all patients with hypertrophic cardiomyopathy, although some of them were common among the subjects examined and the others are unique to each subject. Point mutations in transfer RNA genes were observed in five of the seven patients, and point mutations that replaced conserved amino acids were also observed. These mutations may result in the impairment of mitochondrial function. According to these results, mutations in mitochondrial DNA may contribute to the genesis of some cases of nonautosomal dominant hypertrophic cardiomyopathy, and our methods may be useful for the detection of point mutations in mitochondrial DNA.

Cardiac Ankyrin Repeat Protein Gene (ANKRD1) Mutations in Hypertrophic Cardiomyopathy

OBJECTIVESnThe purpose of this study was to explore a novel disease gene for hypertrophic cardiomyopathy (HCM) and to evaluate functional alterations caused by mutations.nnnBACKGROUNDnMutations in genes encoding myofilaments or Z-disc proteins of the cardiac sarcomere cause HCM, but the disease-causing mutations can be found in one-half of the patients, indicating that novel HCM-susceptibility genes await discovery. We studied a candidate gene, ankyrin repeat domain 1 (ANKRD1), encoding for the cardiac ankyrin repeat protein (CARP) that is a Z-disc component interacting with N2A domain of titin/connectin and N-terminal domain of myopalladin.nnnMETHODSnWe analyzed 384 HCM patients for mutations in ANKRD1 and in the N2A domain of titin/connectin gene (TTN). Interaction of CARP with titin/connectin or myopalladin was investigated using coimmunoprecipitation assay to demonstrate the functional alteration caused by ANKRD1 or TTN mutations. Functional abnormalities caused by the ANKRD1 mutations were also examined at the cellular level in neonatal rat cardiomyocytes.nnnRESULTSnThree ANKRD1 missense mutations, Pro52Ala, Thr123Met, and Ile280Val, were found in 3 patients. All mutations increased binding of CARP to both titin/connectin and myopalladin. In addition, TTN mutations, Arg8500His, and Arg8604Gln in the N2A domain were found in 2 patients, and these mutations increased binding of titin/connectin to CARP. Myc-tagged CARP showed that the mutations resulted in abnormal localization of CARP in cardiomyocytes.nnnCONCLUSIONSnCARP abnormalities may be involved in the pathogenesis of HCM.

Chronic l-Arginine Administration Attenuates Cardiac Hypertrophy in Spontaneously Hypertensive Rats

Nitric oxide inhibits proliferation and migration of vascular smooth muscle cells and contractility of cardiomyocytes in vitro. In spontaneously hypertensive rats (SHR), evidence suggests intrinsic abnormalities of the L-arginine-nitric oxide axis, such as low cGMP-dependent protein kinase in the heart and abnormal L-arginine metabolism. To investigate the in vivo effect of L-arginine on cardiac hypertrophy, 30 SHR and 30 Wistar-Kyoto rats (WKY) were randomly grouped to receive L-arginine (7.5 g/L in drinking water) or vehicle for 12 weeks. L-Arginine treatment did not affect body weight or arterial pressure in either strain. In vehicle-treated animals, the heart/body weight ratio was significantly higher in SHR than in WKY (P < .01). L-Arginine treatment decreased the heart/body weight ratio in SHR (P < .05) but did not affect it in WKY. Expression of skeletal alpha-actin mRNA, known to be expressed in the hypertrophied myocardium, was attenuated in L-arginine-treated SHR compared with vehicle-treated SHR. Cardiac cGMP content and nitrate/nitrite content were less in SHR than WKY. L-Arginine treatment increased these levels only in SHR, suggesting enhanced nitric oxide production. Thus, chronic L-arginine administration attenuated cardiac hypertrophy independently of blood pressure and increased myocardial content of cGMP and nitrate/nitrite. Our results suggest that abnormality of the cardiac L-arginine-nitric oxide axis may play an important role in the pathogenesis of cardiac hypertrophy in SHR.

A Myosin Missense Mutation, Not A Null Allele, Causes Familial Hypertrophic Cardiomyopathy

BACKGROUNDnHypertrophic cardiomyopathy (HCM) is characterized by myocardial hypertrophy of unknown etiology. Missense mutations of the cardiac beta-myosin-heavy-chain (beta-MHC) gene that may be responsible for cardiac hypertrophy have been detected in patients with HCM. On the other hand, gross structural abnormalities in the cardiac beta-MHC gene, ie, an alpha/beta hybrid gene and partial deletion of the gene, have also been reported. The direct correlation between gross abnormalities and development of HCM is not well understood.nnnMETHODS AND RESULTSnWe analyzed the structure of the cardiac beta-MHC gene from patients with HCM by using polymerase chain reaction-DNA conformation polymorphism analysis and found two sequence variations in exons 3 and 22 in one patient. These sequence variations at codon 54 (exon 3; nonsense mutation) and codon 870 (exon 22; Arg-to-His mutation) were identified by direct sequencing and dot-blot hybridization with allele-specific oligonucleotide probes. Relatives of this patient were examined for the mutations. It was revealed that the missense mutation was inherited from the affected father and the nonsense mutation from the unaffected grandmother through the unaffected mother. In addition, the missense mutation was also found in seven other patients from two other unrelated multiplex HCM families.nnnCONCLUSIONSnThe Arg870His mutation was suggested to cause HCM. In contrast, the gene with the nonsense mutation would encode for a cardiac beta-MHC protein of only 53 amino acid residues, which may be too short to be incorporated into the thick filament assembly of cardiac myosin chains and showed no dominant phenotype of heart disease. This is the first report of a nonsense mutation in the human cardiac beta-MHC gene.

Free oxygen radicals contribute to platelet aggregation and cyclic flow variations in stenosed and endothelium-injured canine coronary arteries

OBJECTIVESnThe purpose of this study was to test the hypothesis that free oxygen radicals contribute to platelet aggregation and cyclic flow variations in stenosed and endothelium-injured coronary arteries.nnnBACKGROUNDnAlthough free oxygen radicals, such as superoxide anion and hydrogen peroxide, have been shown to alter platelet function in vitro, the potential role of free oxygen radicals has not been fully described in an in vivo model of coronary artery thrombosis.nnnMETHODSnCyclic flow variations were produced in dogs by an external constrictor placed at the site of the left anterior descending coronary artery with injured endothelium. Blood flow in this artery was monitored by a pulsed Doppler flow probe. If cyclic flow variations were observed during postoperative days, dogs intravenously received superoxide dismutase plus catalase. In anesthetized dogs that did not develop an episode of cyclic flow variations, the effect of intracoronary infusion of xanthine plus xanthine oxidase or hydrogen peroxide on arterial blood flow velocity was studied. In platelet studies, the effect of free oxygen radicals and radical scavengers on platelet aggregation was examined.nnnRESULTSnIn conscious dogs with cyclic flow variations, superoxide dismutase plus catalase significantly reduced cyclic flow variations (n = 7), whereas saline infusion had no effect (n = 7). The infusion of xanthine plus xanthine oxidase or hydrogen peroxide significantly induced cyclic flow variations in four of six dogs or in five of seven dogs, respectively. In vitro platelet studies showed that xanthine plus xanthine oxidase or hydrogen peroxide significantly enhanced platelet aggregation, and superoxide dismutase or catalase significantly inhibited such aggregation.nnnCONCLUSIONSnReduction of free radical formation decreases platelet aggregation and may eliminate cyclic flow variations, whereas promotion of free radical generation enhances platelet aggregation and may induce cyclic flow variations. Thus, free oxygen radicals are an important mediator in this model.

Cyclic flow variations in a conscious dog model of coronary artery stenoses and endothelial injury correlate with acute ischemic heart disease syndromes in humans

OBJECTIVESnThe purpose of this study was to test the hypothesis that episodes of cyclic flow variations (CFVs) in conscious dogs with coronary stenoses and endothelial injury correlate with acute ischemic heart disease syndromes in humans.nnnBACKGROUNDnAlthough the canine model with CFVs has proved to be a useful model of coronary thrombosis, whether CFVs progress to these syndromes has not been clearly described.nnnMETHODSnCyclic flow variations were produced by an external constrictor placed at the site of the left anterior descending coronary artery with injured endothelium. Blood flow in this artery and 24-h Holter electrocardiogram (ECG) were recorded during the 1st 5 postoperative days.nnnRESULTSnOf 41 dogs that underwent the initial operative procedure successfully, 29 developed an episode of CFVs. In five dogs in which CFVs persisted throughout the monitoring period, the left anterior descending coronary artery flow decreased until day 3 and thereafter increased through day 5. Transient coronary occlusion during CFVs induced ST segment changes that returned to baseline after reflow. In 12 dogs, CFVs progressed to persistent coronary occlusion, and histologic examination revealed thrombus formation at the stenotic site and evidence of myocardial infarction. Four of these 12 dogs died suddenly of ventricular arrhythmias during persistent coronary occlusion; another 5 dogs died of reperfusion arrhythmias during CFVs with no evidence of myocardial infarction.nnnCONCLUSIONSnConscious dogs with CFVs closely correlated with clinical acute ischemic heart disease syndromes, suggesting them to be a useful model for investigating the complex mechanisms of cellular interactions in the pathogenesis of these syndromes.

Neutrophil activation after percutaneous transluminal coronary angioplasty

We investigated whether percutaneous transluminal coronary angioplasty (PTCA) would induce neutrophil activation in patients with coronary artery disease. Blood samples were taken from the coronary sinus in 14 patients who underwent PTCA and in 9 control subjects who underwent coronary arteriography (CAG). Flow cytometry was used to measure membrane surface expression of beta 2 integrin (CD11b) and the generation of hydrogen peroxide in neutrophils after ex vivo phorbol myristate acetate stimulation by 2,7-dichlorofluorescein. Neutrophil elastase was measured by an immunoenzymatic method. Surface expression of CD11b increased significantly, approximately twofold, after PTCA but not after CAG. Mean fluorescence intensity of 2,7-dichlorofluorescein in stimulated neutrophils decreased significantly after PTCA, suggesting a previous in vivo activation, but not after CAG. Neutrophil elastase increased significantly after PTCA but not after CAG. These data indicate that PTCA induces neutrophil activation and suggest that neutrophils may contribute to the ischemic injury.