Masaru Ichida

Differential Regulation of Exonic Regulatory Elements for Muscle-specific Alternative Splicing during Myogenesis and Cardiogenesis

Muscle-specific isoform of the mitochondrial ATP synthase γ subunit (F1γ) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492–8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998)Biochem. Biophys. Res. Commun. 251, 603–608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F1γ gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F1γ wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F1γ Pu-del and F1γ Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pu-del mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F1γ Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F1γ Pu-rich minigene mice revealed that the F1γ Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F1γ pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.

Genes of Human ATP Synthase: Their Roles in Physiology and Aging

The reaction of ATP synthase (F0F1) is the final step in oxidative phosphorylation (OXPHOS). Although OXPHOS has been studied extensively in bacteria, no tissue-specific functions nor bioenergetic disease, such as mitochondrial encephalomyopathy and aging occur in these organisms. Recent developments of the Human Genome Project will become an important factor in the study of mammalian bioenergetics. To elucidate the physiological roles of human F0F1, genes encoding the subunits of F0F1 were sequenced, and their expression in human cells was analyzed. The following results were obtained: A. The roles of the residues in F0F1 are not only to transform the energy of the electrochemical potential (ΔμH+) across the membrane, but also to respond rapidly to the changes in the energy demand by regulating the intramolecular rotation of F0F1 with the ΔμH+ and the inhibitors of the ATPase. B. The roles of the control regions of the F0F1 genes, are to coordinate both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) depending on the energy demand of the cells, especially in muscle. C. The cause of the age-dependent decline of ATP synthesis has been attributed to the accumulation of mutations in mtDNA. However, the involvement of nDNA in the decline is also important because of telomere shortening in somatic cells, and age-dependent mtDNA expression analyzed with ρ° cells (cells without mtDNA).

Clinical significance of left ventricular apical aneurysms in hypertrophic cardiomyopathy patients: The role of diagnostic electrocardiography

OBJECTIVES Some patients with hypertrophic cardiomyopathy (HCM) develop left ventricular apical aneurysm, leading to serious cardiovascular complications. The aims of this study were to identify the incidence and clinical course of HCM patients with apical aneurysms in Japan, and to evaluate the role of electrocardiography (ECG) as a screening test to detect apical aneurysms in HCM patients. METHODS AND RESULTS In a retrospective, single center analysis of a population of 247 HCM patients, 21 patients (8.5%) had left ventricular apical aneurysms. Their mean age was 60 ± 14 years (range: 23-77 years) at study entry. Over 4.7 ± 3.3 years of follow-up, 10 patients (47.6%) experienced an adverse clinical event (annual event rate: 10.1%/y), including five implantable cardioverter-defibrillator (ICD) implantations for ventricular tachycardia/ventricular fibrillation (VT/VF), an appropriate discharge of ICD for VT/VF, and four nonfatal thromboembolic strokes. Two patients developed systolic dysfunction (ejection fraction <50%). No sudden cardiac death or progressive heart failure was detected. Fourteen patients showed ST-segment elevation (≥ 1 mm) in V3 through V5 of ECG. In four patients, progression of the ST-segment elevation was recognized. When the ST-segment elevation was used to identify apical aneurysms in HCM patients, the sensitivity was 66.7%, and the specificity was 98.7%. CONCLUSION Apical aneurysms in HCM patients in Japan are not rare, and are associated with serious cardiovascular complications. The early diagnosis of apical aneurysms can be achieved by serial ECG.

An anti-ulcer drug, geranylgeranylacetone, suppresses inducible nitric oxide synthase in cultured vascular smooth muscle cells.

Objective Geranylgeranylacetone (GGA) is commonly used as an anti-ulcer drug. If GGA affects inducible nitric oxide synthase (iNOS) in the vascular tissue, it could influence disease progression in coronary arteries. We investigated the effects of the anti-ulcer drug GGA on iNOS activity in vascular smooth muscle cells. Methods We measured the production of nitrite, a stable metabolite of nitric oxide, in cultured rat vascular smooth muscle cells with the Griess reagent. iNOS protein and mRNA expressions were assayed by western blotting and northern blotting, respectively. The levels of nuclear factor (NF)-κB proteins in nuclear extracts were analyzed by gel retardation assay. Heat shock protein 70, a cytoprotective molecule, was evaluated by western blotting. Results Incubation of cultures with interleukin-1β for 24 h caused a significant increase in nitrite generation. Interleukin-1β-induced nitrite production by vascular smooth muscle cells was significantly suppressed by GGA in a dose-dependent manner. GGA-suppressed nitrite production was accompanied by decreased iNOS mRNA and protein accumulations. GGA by itself did not modulate the basal level of nitrite production. Interleukin-1β induced NF-κB activation in vascular smooth muscle cells, and the addition of GGA further inhibited this NF-κB activation. GGA itself induced heat shock protein 70 expression in a dose-dependent manner. Conclusion These findings demonstrated that GGA suppresses iNOS expression in cytokine-stimulated cultured vascular smooth muscle cells partially through the suppression of NF-κB activation, suggesting that GGA may modulate the pathophysiology of cardiovascular diseases including atherosclerosis. In addition, this effect may be associated with heat shock protein 70 production by GGA.

Perimortem cesarean section or perimortem cesarean supracervical hysterectomy

The pregnant uterus may prevent successful cardiopulmonary resuscitation (CPR). In CPR for pregnant women, the large gravid uterus decreases venous return from the inferior vena cava, and obstructs blood flow through the abdominal aorta. Delivery of the baby, cesarean section, empties the uterus and relieves the aortocaval compression, increasing the chance for successful CPR. Current recommendations are that perimortem cesarean section (PCS) should be performed to save not only the infant’s, but also mother’s life [1, 2]. CS, really? At 38 weeks, a 31-year-old primiparous woman with controlled diabetes mellitus underwent labor induction with oxytocin infusion without success, with the fetal membranes remaining intact. One hour after stopping oxytocin, an emergent call from the toilet of the delivery room where she was found in cardiopulmonary arrest (CPA). CPR was immediately started; however, spontaneous circulation did not return within 4 min. Thus, based on The American Heart Association (AHA) 2010 Guidelines [2, 3], we decided to perform PCS. PCS was performed, yielding a 2,754 g infant with an Apgar score of 2/4 (1/5 min), with cord arterial blood pH 6.64 and BE -21.0 mEq/L. The infant was delivered 36 min after CPA. We had no experience of CPA for pregnant women and also had no system of ‘‘do PCS at the delivery room’’ at that time [3]. Only central surgery unit, which located in some distances from the delivery unit, was available for PCS. That was the reason why 36 min was required for the delivery of the infant. Because of the circulatory collapse, blood sample was not obtained, and disseminated intravascular coagulation (DIC) before PCS was not estimated. After delivering the baby, cardiac contractions and spontaneous respiration resumed with blood pressure (BP) 50/30 mmHg and heart rate (HR) 100 bpm. The uterus was atonic; however, without bleeding possibly due to hypotension. Brain computed tomography (CT) revealed no remarkable findings at that time. Echocardiography revealed normal cardiac structure and movement, rejecting the possibility of pulmonary embolism or myocardial infarction. Considering that the patient had shock with subsequent DIC and uterine atony, amniotic fluid embolism (AFE) should also be considered. Non-ruptured membranes did not preclude AFE. Serum markers for AFE, sialyl Tn and zinc-coproporphyrin [4], were not measured. AFE could not be completely ruled out. The next day, CT revealed a large brain stem infarction. Thus, we considered that CPA was caused by brain stem infarction. CT soon after the CPA did not reveal it, which may not preclude this diagnosis: CT findings of brain infarction usually become evident later. After reestablishment of circulation (BP 110/50 mmHg, HR 100 bpm), atonic bleeding (approximately 5,100 mL) occurred and DIC manifested: hemoglobin 3.3 g/dL, platelet 3.9 9 10 lL, fibrinogen 25 mg/dL, fibrin degradation product 2,494 lg/mL, and D-dimer 1,006 lg/mL. Due to an unresuscitatable cause, hysterectomy was not performed. Uterine compression suture [5] or transarterial S. Matsubara (&) R. Usui T. Watanabe M. Imayoshi Department of Obstetrics and Gynecology, Jichi Medical University, 3311-1 Shimotsuke, Tochigi 329-0498, Japan e-mail: matsushi@jichi.ac.jp

Left ventricular apical and atrial appendage thrombus with hypertrophic cardiomyopathy in sinus rhythm : a case report

A 47-year-old female with a history of untreated hypertension and diabetes mellitus was referred because of a left ventricular echocardiographic mass with congestive heart failure. At the time of admission, she had already had a cardio-embolic stroke with loss of recent memory and slight paralysis of the right upper arm. It was difficult to distinguish between thrombus and tumor. However, her clinical condition required surgical resection as soon as possible. We performed cardiac CT to evaluate the coronary arteries and to scan the mass at the left ventricular apex. This CT evaluation revealed another mass at the left atrial appendage. Thus, these two masses were highly suggestive of thrombi. Subemergency surgical resection of the two masses and a part of the myocardium at the left ventricular apex was successfully performed. The pathological results showed that both the mass in the left ventricular apex and the mass in the left atrial appendage were thrombi, and the myocardial disarray confirmed the echocardiographic diagnosis as hypertrophic cardiomyopathy.

Development of Acute Pericarditis Associated with New-onset Rheumatoid Arthritis in a Diabetic Patient with Renal Impairment: The Elusive Nature of Uremia

Uremic patients may have a variety of organ involvement, however, the precise causality may be impossible to determine in some cases because the symptoms of uremia are also associated with other diseases. With an emphasis on the elusive nature of uremia, we herein describe a 53-year-old man with preexisting renal impairment who developed acute pericarditis with deterioration of his renal function. Hemodialysis was immediately initiated on the presumption of uremia, however, articular symptoms emerged approximately a month later and led to a final diagnosis of rheumatoid arthritis, followed by successful withdrawal of hemodialysis.

Effectiveness of bromocriptine treatment in a patient with peripartum cardiomyopathy

Peripartum cardiomyopathy (PPCM) is a rare but potentially life-threatening disorder that occurs in late pregnancy or the early puerperium despite optimal medical therapy. Recently, oxidative stress-mediated generation of antiangiogenic and proapoptotic 16-kDa prolactin, and subsequent impaired cardiac microvascularization have been related to PPCM. In turn, prolactin blockade with bromocriptine has been proven successful in preventing the onset of PPCM in mice and in patients at high risk for the disease. Here, we report the efficacy of bromocriptine for treatment of a patient with PPCM.