Anti-tumor effect of gallic acid on LL-2 lung cancer cells transplanted in mice.

We previously reported that gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol, can induce apoptosis in four kinds of human lung cancer cell lines in vitro. The present study further investigated the in vivo anti-tumor effects of orally administered gallic acid. Gallic acid reduced cell viability of LL-2 mouse lung cancer cells in vitro dose dependently, with a 50% inhibitory concentration (IC50) value of around 200 microM. C57Black mice were transplanted with LL-2 cells, and administered gallic acid (1 mg/ml in drinking water, ad libitum) and/or cisplatin (4 mg/kg i.p. injection, once a week). The average weight of the transplanted tumors, obtained at 29 days after transplantation, in the mice of control, gallic acid-treated cisplatin-treated and cisplatin plus gallic acid-treated groups was 4.02, 3.65, 3.19 and 1.72 g, respectively. The average tumor weight of the mice treated with cisplatin combined with gallic acid was significantly smaller than that of the control group (p<0.05). The amount of apoptotic cells in the tumor tissues of mice treated with gallic acid and/or cisplatin was significantly higher than those of the control mice. Combination of gallic acid and cisplatin increased the tumor cell apoptosis compared with the treatment with cisplatin alone. The present findings suggest that the combination of gallic acid with an anti-cancer drug, including cisplatin, may be an effective protocol for lung cancer therapy.

Dynamic process of apoptosis in adult rat cardiomyocytes analyzed using 48-hour videomicroscopy and electron microscopy: beating and rate are associated with the apoptotic process.

Dynamic process of apoptosis has not been elucidated in adult rat cardiomyocytes. Soluble Fas ligand (0.1 microg/ml) in the presence of actinomycin D (0.05 microg/ml) induced apoptosis in cultured adult rat cardiomyocytes, as documented by activated caspase-3, DNA fragmentation, and apoptotic ultrastructure. In the present model, we observed 60 adult cardiomyocytes with a normal rod shape under a real-time videomicroscope continuously for 48 hours. Seventeen cells (28%) were unchanged and 7 cells (12%) showed oncosis (so-called necrosis) in which no beating was evident. In the remaining 36 cells (apoptosis, 60%), a slow beating (17 +/- 3/min) was initiated 16 +/- 1 hours later. Approximately 1 hour later, the rod cells showed long-axial shortening as bone- or club-like, or square-shaped, accompanied with faster beating rates (35 +/- 7/min). In 29 cells (type A1 and A2), marked shrinkage occurred; the cellular shape became almost completely round with a smooth surface and the beating ceased 3.0 +/- 0.4 hours later. Then, smooth budding appeared 0.6 +/- 0.2 hours later. Apoptotic bodies were found in 8 cells 10 +/- 4 hours later (type A1, 13%) but not in 21 cells (type A2, 35%). In the other 7 cells (type A3, 12%), the cell surface became rough 8 +/- 3 hours later and the beating ceased. Maximal beating rate was greatest in type A1 (72 +/- 26/min) and greater in type A2 (29 +/- 5/min) than in type A3 (10 +/- 2/min). Electron microscopy confirmed apoptotic ultrastructure even in the cardiomyocytes with bone-, club-like, or square shapes, suggesting that type A3 as well as A1 and A2 is also under apoptotic process. A caspase inhibitor, zVAD.fmk, blocked beating, apoptotic morphology, and DNA fragmentation, indicating these depended on caspase activation. In the caspase-dependent apoptotic process of cultured adult cardiomyocytes, beating and the following deformity of the cellular edges were the initial signs and the rate of beating was related with the subsequent three different processes of apoptosis.

Combination of miglitol, an anti-diabetic drug, and nicorandil markedly reduces myocardial infarct size through opening the mitochondrial K(ATP) channels in rabbits.

The anti-diabetic drug miglitol, an alpha-glucosidase inhibitor, which is currently used clinically, reduces myocardial infarct size by reducing the glycogenolytic rate through inhibition of the alpha-1,6-glucosidase of glycogen-debranching enzyme in the heart. Nicorandil, a K(ATP) channel opener with a nitrate-like effect, which is also currently used clinically, also reduces the infarct size. Therefore, we hypothesized that combination of nicorandil and submaximal dose of miglitol could markedly reduce myocardial infarct size more than miglitol or nicorandil alone, and investigated the mechanism for the infarct size-reducing effect. Japanese white rabbits without collateral circulation were subjected to 30 min coronary occlusion followed by 48 h reperfusion. Pre-ischaemic treatment with submaximal dose of miglitol (5 mg kg(-1), i.v.) and nicorandil alone (100 microg kg(-1) min(-1) 5 min) moderately reduced the infarct size as a percentage of area at risk (24+/-4 and 25+/-4%, respectively), and 10 mg kg(-1) of miglitol markedly reduced the infarct size (15+/-2%) compared with the controls (42+/-2%). Combination of 5 mg kg(-1) of miglitol and nicorandil (100 microg kg(-1) min(-1) 5 min), and 10 mg kg(-1) of miglitol and nicorandil (100 microg kg(-1) min(-1) 5 min) significantly reduced the infarct size (13+/-4 and 12+/-3%, respectively) more than miglitol or nicorandil alone. Pretreatment with 5HD completely abolished the infarct size-reducing effect of 10 mg kg(-1) of miglitol alone (36+/-7%) and that of combination of 5 mg kg(-1) of miglitol and nicorandil (46+/-2%). Combination of nicorandil and submaximal dose of miglitol markedly reduced the myocardial infarct size more than miglitol or nicorandil alone. This effect was suggested to be related to the opening of mitochondrial K(ATP) channels.

Relationship between thallium-201 myocardial SPECT and findings of endomyocardial biopsy specimens in dilated cardiomyopathy.

The purpose of this study was to clarify which myocardial histological findings associated with dilated cardiomyopathy (DCM) are reflected in quantitative 201Tl myocardial SPECT. We obtained studied SPECT images from 21 patients with DCM 10 minutes and 2 hours after they received an injection of 111 MBq 201Tl at rest. We calculated the percent coefficient of variation of myocardial 201Tl counts [%CV(TI)], the washout rate (WR), standard deviation of WR [SD(WR)], extent score (ES) and severity score (SS). We used image analysis to measure % fibrosis, % myocytes, the ratio of fibrous tissue to myocyte tissue (F/My), myocyte size and standard deviation of myocyte size [SD(My)] in left ventricular endomyocardial biopsy specimens. The %CV(Tl) was correlated with % fibrosis and F/My. The ES and SS also correlated with F/My. The correlation between SD(WR) and SD(My) was significant. The present findings suggest that %CV(Tl), ES and SS of rest 201Tl SPECT reflect myocardial fibrosis and that the standard deviation of washout reflects the distribution of myocyte size.

Characterization of ultrastructure and its relation with DNA fragmentation in Fas-induced apoptosis of cultured cardiac myocytes.

The purposes of the present study were to define precisely the ultrastructural features of apoptosis in cultured cardiomyocytes and to determine whether DNA fragmentation is essential for the apoptotic morphology. When cultured neonatal murine cardiomyocytes were incubated with an agonistic anti-Fas antibody in the presence of a non-toxic amount of actinomycin D or cycloheximide, approximately 70% of them had lost their viability after 24 h. The dead cardiomyocytes showed the typical ultrastructural changes of apoptosis on transmission and scanning electron microscopy, as well as by positive in situ nick end-labelling (TUNEL), positive Taq polymerase-based in situ ligation, a DNA ladder pattern on gel electrophoresis, and an increase in the active fragment of caspase-3. According to TUNEL at the electron microscopic level, apoptotic nuclear change, cytoplasmic shrinkage, and DNA fragmentation always occurred simultaneously in apoptotic cardiomyocytes. Other ultrastructural features of apoptosis were the appearance of abundant lipid-like structures in the cytoplasm of cardiomyocytes at the early phase, and a high incidence of plasma membrane rupture and formation of apoptotic bodies at the later phase. When zinc, an inhibitor of Ca2+/Mg2+-dependent endonuclease, was added to the present model, activation of caspase-3 and an apoptotic ultrastructure were still observed in spite of the lack of DNA fragmentation, indicating that this type of myocyte death is also apoptosis. In conclusion, the typical apoptotic ultrastructure and DNA fragmentation occur simultaneously in association with caspase-3 activation in Fas-stimulated cultured cardiomyocytes. Apoptotic morphology can, however, be observed even without DNA fragmentation.

[Endomyocardial biopsy: procedure and pathology].

Endomyocardial biopsy is one of the examinations seeking for the etiologies of myocardial diseases, different from the others evaluating physiological or functional states. The biopsy is most powerful for monitoring rejection of the transplanted hearts and anthracycline-induced cardiotoxicity. It can make a differential diagnosis between myocardial diseases based on the specific histological findings. Although the method is well established, the indication should be carefully selected, and attentions should be paid to prevent the possible complications. Besides histological approaches at light and electron microscopic levels, biochemical, pharmacological and immunological approaches on the biopsies will promise further understanding of the pathophysiological mechanisms underlying cardiac diseases.

N-methyl-1-deoxynojirimycin (MOR-14), an alpha-glucosidase inhibitor, markedly improves postischemic left ventricular dysfunction.

We examined whether pharmacological inhibition of glycogenolysis by N-methyl-1-deoxynojirimycin (MOR-14), a new compound which reduces the glycogenolytic rate by inhibiting the alpha-1,6-glucosidase activity of the glycogen-debranching enzyme, can protect the heart against postischemic left ventricular dysfunction. The hearts of male Sprague-Dawley rats were excised, and perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. The hearts were paced at 320 beats/min except during the ischemia. Left ventricular developed pressure (LVDP, mmHg), +/-dP/dt (mmHg/s), and coronary flow (ml/min) were continuously monitored. All hearts were perfused for a total of 120 min including a 30-min preischemic period followed by a 30-min episode of global ischemia and 60 min reperfusion. with or without 0.5 or 2 mM of MOR-14 during the 30-min preischemic period or the first 30 min of reperfusion. In another series of experiments, the myocardial content of glycogen and lactate was measured during the 30-min episode of ischemia in groups treated with and without 2mM of MOR-14. Preischemic but not postischemic treatment with MOR-14 significantly improved LVDP and +/-dP/dt without altering coronary flow during reperfusion in a dose-dependent manner. MOR-14 significantly preserved the glycogen content and significantly attenuated the lactate accumulation during the 30-min episode of ischemia. Preischemic treatment with MOR-14 is protective against postischemic left ventricular dysfunction through the inhibition of glycogenolysis in the isolated rat heart.

Induction of apoptosis by gallic acid in lung cancer cells.

The apoptosis-inducing effect of gallic acid (3,4,5-trihydroxybenzoic acid) was investigated in four human lung cancer cell lines, SBC-3 (small cell carcinoma), EBC-1 (squamous cell carcinoma), A549 (adenocarcinoma) and SBC-3/CDDP (cisplatin-resistant subclone of SBC-3). Gallic acid induced apoptosis in a dose-dependent manner as evidenced by analyses of DNA fragmentation, changes in cell morphology and loss of viability. Fifty percent inhibitory concentration (IC50) values of gallic acid on the cell viability of SBC-3, EBC-1 and A549 were around 10, 20 and 60 microg/ml, respectively. The IC50 value for SBC-3/CDDP cells was almost the same as that of SBC-3, suggesting that susceptibility of cells to gallic acid-induced apoptosis is not altered by the acquisition of cisplatin resistance. The apoptotic process was effectively triggered by 30 min exposure to gallic acid. A caspase inhibitor and alpha-tocopherol effectively prevented the gallic acid-induced apoptosis, indicating the involvememt of caspase activation and oxidative processes during the course of apoptosis in gallic acid-treated cancer cells. These findings suggest the possible applicability of gallic acid in lung cancer therapy, especially to circumvent resistance to anti-cancer drugs.

Apoptosis and overexpression of bax protein and bax mRNA in smooth muscle cells within intimal hyperplasia of human radial arteries : analysis with arteriovenous fistulas used for hemodialysis.

There is a type of arteriosclerosis with remodeling of middle-size arteries in which intimal hyperplasia of smooth muscle cells (SMCs) plays the main role, and there are few macrophages, T lymphocytes, and foam cells. It is unknown whether apoptosis and the expression of Bax, an inducer of apoptosis, are increased according to the progression of this type of human arteriosclerosis, which is different from so-called atherosclerosis. Bax heterodimerizes with Bcl-2, an inhibitor of apoptosis, and the ratio of Bax to Bcl-2 determines cellular apoptosis or survival. Thus, we investigated apoptosis and the expressions of Bax, bax mRNA, and Bcl-2 in human arteriovenous (AV) fistulas used for hemodialysis, a representative of arteriosclerosis of the aforementioned type. The material was 20 radial arteries obtained from 20 patients with chronic renal failure undergoing AV shunt surgery. SMCs, macrophages, and T lymphocytes were immunohistochemically identified at the light microscopic (LM) level. Apoptosis was detected by in situ terminal deoxynucleotidyl transferase (TdT)-mediated digoxigenin-dUTP nick end labeling (TUNEL) at both the LM and electron microscopic (EM) level. Cell proliferating activity was estimated by proliferating cell nuclear antigen (PCNA). Bax and Bcl-2 were detected by immunohistochemistry and Western blot analysis. Expression of bax mRNA was detected by in situ hybridization. LM TUNEL-positive cells in both the intima and media were significantly increased according to the percent stenosis of the vessels. EM analysis revealed that ultrastructures of apoptotic SMCs were seen in both synthetic and contractile phenotypes. Their frequency of occurrence in the intima and media were greater in those vessels with >50% stenosis than in those with <50% stenosis (5.2+/-0.7% versus 1.0+/-0.3% in the intima and 2. 1+/-0.5% versus 0.2+/-0.1% in the media). The proportion of apoptotic SMCs with ruptured plasma membranes was greater than that of apoptotic SMCs with intact membranes in the intima of the former (4.1+/-0.6% versus 1.1+/-0.1%). Only those SMCs with apoptotic ultrastructures had TUNEL-positive nuclei with moderate or marked accumulation of immunogold particles at the EM level. However, ultrastructures of oncosis (primary necrosis) were not observed. Immunohistochemical analyses showed significant positive correlations between percent stenosis of vessels and the percentage of either PCNA-positive intimal cells or Bax-positive areas in the intima and media. Bcl-2-positive cells were not observed in the intima but mainly in the outer media. The percentage of Bcl-2-positive medial cells was definitely decreased at an early stage after formation of the AV fistula but did not change according to the duration of hemodialysis or the progression of arteriosclerosis. Western blot analysis of Bax or Bcl-2 and in situ hybridization of bax mRNA confirmed the immunohistochemical data. Thus, regulation of cellularity in intimal hyperplasia of SMCs in human arteriosclerosis with remodeling is mediated by proliferation and apoptosis but not oncosis. The apoptosis is probably induced by an increase in the Bax to Bcl-2 ratio.

Fifteen-year follow-up of a heterozygous Fabry's disease patient associated with pre-excitation syndrome.

A 47-year-old woman with heterozygous Fabry's disease with pre-excitation syndrome has been followed up for 15 years. Diagnosis was confirmed by the typical electron microscopic feature of the endomyocardial specimen and a decreased plasma alpha-galactosidase activity. As the disease progressed, the interventricular septum thickened from 11 to 17 mm as measured by echocardiography, while the AH interval was prolonged from 80 to 140 msec. In Fabry's disease, the PR interval has been reported to be variable from short PR to AV block. Therefore, this case may be helpful to understand the time course in the AV conduction abnormalities with the progression of Fabry's disease.

Mode and role of cell death during progression of atherosclerotic lesions in hypercholesterolemic rabbits.

Two cell types, macrophages and smooth muscle cells (SMCs), play important roles in the development of atherosclerotic lesions. Both contribute to the formation of the lesions not only by their presence but also by taking in or releasing extracellular substrates during life and at death. The present study aimed to elucidate their turnover, focusing on the detailed description of the modes of death in each cell type, and the roles of their death in the progression from early into advanced atherosclerotic lesions. Ascending aortas were obtained from New Zealand white male rabbits fed a diet with 1% cholesterol for 3 months (3-M group, n= 6) and 6 months (6-M group, n = 6). They were histologically examined, and the cell death was checked by in situ nick end-labeling (TUNEL), using a Taq polymerase-based in situ ligation assay with/without combination of immunohistochemistry, electron microscopy (EM), and TUNEL at the EM level. Intimal hyperplasia and luminal stenosis advanced with increased dietary interval, and the aortic intima of the 3-M group consisted of histological types I-III atherosclerotic lesions, whereas that of the 6-M group included types III-V. Along with the progression, the cellular population decreased, but the area of fibrosis increased. The percentage area of macrophages declined (from 60% +/- 5% to 23% +/- 2%), but that of SMCs increased (from 5% +/- 1% to 10% +/- 2%). The positive cells for in situ ligation were less frequent in the 6-M group (0.05% +/- 0.01%) than in the 3-M group (0.2% +/- 0.04%), which was due to a decrease in SMCs positive for in situ ligation. The frequency of TUNEL-positive cells was higher than that of in situ ligation-positive cells in both groups, suggesting that cell death involved not only apoptosis but also oncosis. This was confirmed using EM: cell death occurred via both apoptosis and oncosis. EM-TUNEL positively labeled not only apoptotic but also some oncotic nuclei. Death of macrophages and SMCs involves both apoptosis and oncosis in the aortic intima of hypercholesterolemic rabbits. Decline in the dying rate of SMCs might be associated with the formation of SMC-rich and collagen-rich lesions in the late advanced stage of atherosclerosis, although such a cause-effect relationship is to be further confirmed.

Expression and distribution of brain natriuretic peptide in human right atria.

OBJECTIVES: We investigated expression of brain natriuretic peptide (BNP) as well as atrial natriuretic peptide (ANP) and their genes in human right atria. Their relations with atrial pressure were also examined. BACKGROUND: The BNP plays a roll in electrolyte-fluid homeostasis such as ANP. The tissue level is reported to be elevated in the failing ventricles. However, expression and transmural distribution of BNP in the atria remain unclear. METHODS: Expression of ANP and BNP was immunohistochemically investigated in the right atrial (RA) specimens from 21 patients who had undergone cardiac surgery. The mRNA of specimens were quantitatively measured by Northern blot analysis and also evaluated by in situ hybridization. In addition, plasma levels of ANP and BNP were measured in the patients. RESULTS: The BNP immunoreactivity was diffusely seen in RA tissue of patients with mean RA pressure (mRAP) of 5 mm Hg or more, but it was noted only in the subendocardial half of the atria of those with mRAP less than 5 mm Hg. There was a significant correlation between the incidence of BNP-positive myocytes and mRAP (r = 0.850, p < 0.0001). Conversely, ANP-positive myocytes were found diffusely in all cases. In Northern blot analysis, the mRNAs levels of ANP and BNP in the atrial tissue were positively correlated with the mRAP (ANP, p = 0.775, p < 0.005 and BNP, p = 0.771, p < 0.005). In situ hybridization confirmed these findings. The mRNA levels were significantly correlated to each other (r = 0.845, p < 0.0002). Plasma ANP and BNP levels were elevated in the patients compared with that in controls; however, none were significantly correlated with the mRAP. CONCLUSIONS: Expression of BNP and BNP mRNA is augmented in the atria with increased pressure, and distributed predominantly in the subendocardial side. The level of BNP mRNA was well correlated with that of ANP mRNA. Thus, these two genes might be commonly regulated in response to atrial pressure.

[Participation of apoptosis in renal amyloidosis].

Renal amyloidosis shows symptoms of renal dysfunction due to the deposition of amyloid protein in the kidney. Recently, it was reported that apoptosis plays an important role in the pathogenesis of type-2 diabetes mellitus and Alzheimer's disease of which amyloid deposition is seen in the tissue. We investigated whether or not apoptosis and related factors are observed in renal amyloidosis. In situ nick end labeling (TUNEL) was performed in seven autopsied renal tissues with primary and secondary amyloidosis and 10 autopsied renal tissues without renal disease as the control. The number of TUNEL-positive cells was significantly increased in both the glomeruli and tubulus of the kidney with amyloidosis than in the control. Electron microscopic analysis was performed on one biopsied renal tissue with amyloidosis and six biopsied renal tissues with minor abnormalities as the control. Typical apoptotic cells were observed only in the former. Bax product, an inducer of apoptosis, and Bcl-2 protein, an inhibitor of apoptosis, were examined immunohistochemically in the seven autopsied renal tissues with amyloidosis and 10 autopsied control tissues. Bax was overexpressed in the tubulus and glomeruli of subjects with renal amyloidosis, compared to the normal controls. However, Bcl-2 protein was not detected in the glomeruli in any of the subjects examined. These results indicate that apoptotic cells are increased in number in renal amyloidosis and Bax overexpression may play an important role in this increase.

Expression of atrial and brain natriuretic peptides and their genes in hearts of patients with cardiac amyloidosis.

OBJECTIVES: We investigated the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) and their genes in the hearts of patients with cardiac amyloidosis and those with isolated atrial amyloidosis. BACKGROUND: The expression of ANP and BNP is augmented in the ventricles of failing or hypertrophied hearts, or both. The expression of ANP and BNP in the ventricles of hearts with cardiac amyloidosis, which is hemodynamically similar to restrictive cardiomyopathy, is not yet known. ANP is the precursor protein of isolated atrial amyloid. METHODS: We analyzed the immunohistocytochemical localizations of ANP and BNP as well as the expression of their mRNAs by in situ hybridization in the myocardium and measured the plasma levels of ANP and BNP in patients with cardiac amyloidosis. RESULTS: Four of the five right and all six left ventricular endomyocardial biopsy specimens obtained from six patients with cardiac amyloidosis were immunohistochemically positive for both ANP and BNP; none of the biopsy specimens from eight normal subjects were positive for ANP or BNP. All four of the right atria obtained at operation showed positive immunoreactions for both peptides. Electron microscopy identified specific secretory granules in ventricular myocytes of the patients with cardiac amyloidosis, but not in ventricular myocytes from the normal control subjects. Double immunocytochemical analysis revealed the co-localization of ANP and BNP in the same granules and that isolated atrial amyloid fibrils were immunoreactive for ANP and BNP, whereas ventricular amyloid fibrils were negative for both peptides. Both ANP mRNA and BNP mRNA were expressed in the ventricles of the patients with cardiac amyloidosis but not in the normal ventricles. The autopsy study of four patients with cardiac amyloidosis revealed an almost transmural distribution of ANP and BNP, with predominance in the endocardial side. Plasma BNP levels in the patients were markedly elevated ([mean +/- SD] 1,165.1+/-561.2 pg/ml) compared with those in the control subjects (8.9+/-6.0 pg/ml, p < 0.05). CONCLUSIONS: Expression of ANP and BNP and their genes was augmented in the ventricular myocytes of the patients with cardiac amyloidosis. Both regional mechanical stress by amyloid deposits and hemodynamic stress by diastolic dysfunction may be responsible for the expression of the peptides in patients with cardiac amyloidosis.

Luminal narrowing of coronary capillaries in human hypertrophic hearts: an ultrastructural morphometrical study using endomyocardial biopsy specimens.

BACKGROUND: Abnormal microcirculation has been suggested in hearts with pathological conditions, particularly in hypertrophic hearts, even in the presence of normal epicardial coronary arteries. However, the morphology of coronary capillaries has not been well investigated in those hearts. METHODS: Ultrastructural morphometry of the capillaries in 47 endomyocardial biopsy specimens taken from 30 patients was performed. PATIENTS: Six patients had hypertrophic cardiomyopathy with dilated cardiomyopathy-like features (DCM-like HCM), six had HCM, six had DCM, five had postmyocarditis, and seven were normal subjects. RESULTS: The short axial diameters of capillaries were similar among the groups. Abnormal stenosis of more than 90% luminal narrowing was found in 31% of capillaries of the DCM-like HCM group, 16% of the HCM group, 13% of the DCM group, 11% of the postmyocarditis group, and 2% of the normal subjects. Mean (SD) stenosis of the lumen was most severe in DCM-like HCM (78(8)%), and more severe in HCM (67(9)%), DCM (66(8)%), and postmyocarditis (68(4)%) than normal subjects (56(8)%). The mean cross sectional areas of capillaries were similar among the groups; however, the endothelial cellular area was significantly (p < 0.05) greater in DCM-like HCM (24.2 (8.2) microns2) than in normal subjects (14.7 (1.8) microns2), indicating that capillary narrowing was due to the increased volume of capillary endothelial cells. The endothelial cells of the stenosed capillaries showed severely oedematous changes of the cytoplasm wholely or partially, but the cytoplasmic organelles and nuclei appeared intact. CONCLUSION: Narrowing of the coronary capillaries may be of pathophysiological significance in microcirculatory abnormality in hypertrophic hearts, particularly in patients with DCM-like HCM.

Intranuclear mitochondria in human myocardial cells.

Intranuclear mitochondria have occasionally been reported in various cells including cardiac myocytes, but the incidence and details including the specific mechanism(s) whereby mitochondria exist in the nuclear matrix are unknown. We studied the ultrastructure of a total of 91 consecutive endomyocardial biopsy specimens obtained from 50 patients with various cardiac diseases. Intranuclear mitochondria were found in three patients: two with hypertrophic cardiomyopathy and one with chronic myocarditis. Their myocytes were markedly hypertrophic and the shapes of the nuclei were bizarre. Specimens from these patients were further studied on serial sections, and some of the nuclei with mitochondria showed ruptured nuclear envelope. Intranuclear mitochondria were found in at least 6% of the patients with cardiac diseases (3% of the specimens examined), and a novel mechanism of the pathogenesis was documented: communication between the nuclear matrix and cytoplasm through the rupture in the nuclear envelope. The nuclear rupture might be simply due to a laboratory artifact. On the other hand, it might imply some pathophysiological significances in the degeneration of myocytes in cardiac diseases.

[Sarcoidosis in a patient with autoimmune hemolytic anemia].

A 65-year-old woman was admitted to our hospital because of severe anemia. A skin biopsy was done in January 1994 and sarcoidosis was diagnosed. Diffuse reticular shadows were seen in both lung fields on a chest X-ray film and mediastinal lymph node swelling was seen on a chest CT scan. She was followed as an outpatient and was not treated. She suddenly experienced vertigo and general fatigue in March 1995. Laboratory findings on admission were as follows: Hb 6.2 g/dl, MCV 115.9 fl, Ret 198%, LDH 732 IU/L, I-Bil 1.9 mg/dl, and Coombs' test was positive. Autoimmune hemolytic anemia was diagnosed, and she was treated with prednisolone (1 mg/kg). As of the time of this writing, she has no relapse of hemolytic anemia though prednisolone was discontinued 6 months ago.

Cardiomyopathy associated with Graves' disease.

Cardiovascular changes associated with Graves' disease are generally considered to be secondary to the increased levels of thyroid hormone. We describe a case of Graves' disease in a 25-year-old man, who developed cardiomyopathy with severe heart failure. Pathological examination of the myocardial biopsies showed fibroblast infiltration and degenerative changes. After the cardiomyopathy subsided the patient developed a goitre and signs of hyperthyroidism, followed by Graves' ophthalmopathy, which was treated successfully with a combination of high-dose corticosteroids and orbital radiotherapy. These findings suggested a common pathogenesis for the cardiomyopathy and ophthalmopathy, and prompted us to investigate the expression of TSH receptor (TSH-R) in human heart. TSH-R mRNA was identified in human heart using the reverse transcriptasepolymerase chain reaction (RT-PCR) and DNA sequencing. Taken together, these data suggest that autoimmunity against the TSH-R might contribute to both the cardiomyopathy and ophthalmopathy in similar cases of Graves' disease.

Metastasizing uterine leiomyoma. A case with cardiac and pulmonary metastasis.

A case of histologically benign cardiac and pulmonary metastases from a uterine leiomyoma in a 45-year-old woman is reported. The solitary cardiac tumor consisted of five lobules with a stalk attached to the anterior papillary muscle of the right ventricle, which occupied the right ventricular cavity and almost completely obstructed the pulmonary main trunk in the systolic cycle. Multiple small nodules were found throughout both lungs. The cardiac tumor was resected at open heart surgery and open lung biopsy of the pulmonary lesion was simultaneously performed. Both of them were histologically identical to the apparently histologically benign uterine leiomyoma which had been resected five years previously. Based on the clinical and laboratory findings, it is postulated that antegrade metastases via the venous system resulted in the cardiac metastasis from the uterine leiomyoma and the secondary pulmonary metastasis from the cardiac tumor. In contrast to the light microscopic findings, ultrastructural examination suggested the possibility of malignancy associated with the presence of immature smooth muscle cells. This is the first reported case of a so-called benign metastasizing leiomyoma occurring in the heart.

Venticular expression of atrial and brain natriuretic peptides in patients with myocarditis.

We analyzed the ventricular expression of atrial and brain natriuretic peptides (ANP and BNP) in patients with myocarditis. Immunohistochemical analysis of endomyocardial biopsy specimens showed ANP and BNP immunoreactivity in the early myocarditis group (ANP in 4/10 and BNP in 3/10) and the late myocarditis group (ANP and BNP in 4/10), but not in the controls (0/8). Hemodynamic parameters, such as left ventricular volume indexes, pulmonary capillary wedge pressure, and left ventricular end-diastolic pressure, were higher and the cardiac index and ejection fraction were lower in the patients positive for ANP and/or BNP. Histological changes, including myocyte size, cellular necrosis, inflammatory infiltrates and fibrosis, were more severe in the immunohistochemically positive biopsy specimens. In the autopsy hearts, ANP- or BNP-positive myocytes were noted in the chronic myocarditis or postmyocarditis group, but not in acute fulminant myocarditis or in normal controls. Both ANP and BNP were predominantly localized in the residual myocytes edging the myocardial lesions, and also in the myocytes just beneath the left ventricular endocardium. This study demonstrated augmented ventricular ANP and BNP expressions in patients with myocarditis, and suggested that regional stress is important in the augmentation of these peptides as well as hemodynamic stress.