Remodelling of myocardial intercalated disc protein connexin 43 causes increased susceptibility to malignant arrhythmias in ARVC/D patients.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a primary cardiomyopathy characterised by fibrofatty replacement and ventricular arrhythmias. The occurrence of malignant arrhythmias may be associated with fatty infiltration and intercalated disk remodelling, but the specific pathological remodelling pattern is not yet clear. METHODS: Twelve explanted hearts from patients diagnosed with ARVC/D according to the 2010 Task Force Criteria and pathology examination were divided into two groups with (SVT, n=6) or without (non-SVT, n=6) recurrent sustained ventricular tachycardia (SVT) before heart transplantation. Six ARVC autopsy hearts and six normal donor hearts were also collected. We evaluated the fibrofatty infiltration by Masson staining and the expression of intercalated disc proteins through immunohistochemistry staining combined with western blot using the ventricular tissue of ARVC as well as normal hearts. RESULTS: There was significant fatty replacement in the right ventricles of both the SVT and the non-SVT groups compared to normal hearts, but no significant differences were found in fibre, fatty and residual myocardium components between these two groups. Immunohistochemistry and western blot further showed disturbed distribution and significantly reduced expression of Connexin 43 (Cx43) in the SVT group (SVT vs. Normal P=0.010, SVT vs. non-SVT P=0.012). No significantly diminished expression was found in the non-SVT group. The cardiac histology of ARVC/D patients with sudden death verified by forensic pathology confirmed a similar phenomenon. CONCLUSION: Our pathology study on explanted and autopsied hearts indicates that the expression of Cx43 was significantly reduced and disturbed in distribution in ARVC/D myocardium with sustained ventricular tachycardia, but not in patients without malignant ventricular arrhythmias. This implies a correlation between Cx43 remodelling and malignant arrhythmias in ARVC/D patients.

Sudden Unexplained Nocturnal Death Syndrome in Central China (Hubei): A 16-Year Retrospective Study of Autopsy Cases.

A retrospective study was conducted at Tongji Forensic Medical Center in Hubei (TFMCH) from 1999 to 2014. Forty-nine cases of sudden unexplained nocturnal death syndrome (SUNDS) were collected. The SUNDS rate was 1.0% in the total number of cases, in which an incidence was fluctuating over the years. Interestingly, April and January, and 3:00 to 6:00 AM were the peak months and times of death. Among the decedents, farmers and migrant workers accounted for 67.3%. The syndrome predominantly attacked males in their 30s. One victim had sinus tachycardia. Thirteen victims (26.5%) were witnessed and had abnormal symptoms near death. Macroscopically, compared to sudden noncardiac deaths, the weights of brain, heart, and lungs had no statistical difference in SUNDS. Microscopically, the incidence of lung edema (45 cases, 91.8%) was significantly higher in SUNDS group than in the control group (27 cases, 55.1%). 82.9% of 35 SUNDS cases examined displayed minor histological anomalies of the cardiac conduction system (CCS), including mild or moderate fatty, fibrous or fibrofatty tissue replacement, insignificant stenosis of node artery, and punctate hemorrhage in the node area. These findings suggested that minor CCS abnormalities might be the substrates for some SUNDS deaths. Therefore, SUNDS victims might suffer ventricular fibrillation and acute cardiopulmonary failure before death. Further in-depth studies are needed to unveil the underlying mechanisms of SUNDS.

Commotio Cordis Caused by Violence in China: Epidemiological Characteristics Detected at the Tongji Forensic Medical Center.

Commotio cordis (CC) is a recognized rare cause of sudden death in which an apparently minor blow to the chest causes ventricular fibrillation and cardiac arrest. CC diagnosis is still a challenge for forensic pathologists. A retrospective study of 9794 autopsy cases was conducted at the Department of Forensic Medicine, Tongji Medical College (DFM-TMC, China) from 1955 to 2014. A total of 39 cases (0.4%) were determined to be caused by CC. A male preponderance (male to female of 37:2) was found in the victims, whose age ranged from 13 to 47 years, including more than 85% individuals in their 10s and 20s. Most victims (27 cases, 69.2%) came from village. The highest rate of victims was found for middle school and college students (15 cases, 38.5%), followed by prisoners (11 cases, 28.2%), farmers (9 cases, 23.1%), workers (3 cases, 7.7%), and office staff (1 case, 2.6%). Chest blows were produced by fists (28 cases, 71.8%), feet (6 cases, 15.4%), knee (2 case, 5.1%), head (1 case, 2.6%), or objects (2 cases, 5.1%). Witness statements indicated that most victims collapsed after being impacted in the precordium. The autopsy findings were unremarkable except bruises, contusions, or subcutaneous hemorrhage in the anterior chest (13 cases), bleeding of intercostal muscles (5 cases), and disperse focal petechiae of the epicardium (11 cases). All CC cases in this study were caused by violent attacks and related to criminal processes. Correct diagnosis of CC due to violence has important implications in the judicial system.

Shear stress blunts tubuloglomerular feedback partially mediated by primary cilia and nitric oxide at the macula densa.

The present study tested whether primary cilia on macula densa serve as a flow sensor to enhance nitric oxide synthase 1 (NOS1) activity and inhibit tubuloglomerular feedback (TGF). Isolated perfused macula densa was loaded with calcein red and 4,5-diaminofluorescein diacetate to monitor cell volume and nitric oxide (NO) generation. An increase in tubular flow rate from 0 to 40 nl/min enhanced NO production by 40.0 ± 1.2%. The flow-induced NO generation was blocked by an inhibitor of NOS1 but not by inhibition of the Na/K/2Cl cotransporter or the removal of electrolytes from the perfusate. NO generation increased from 174.8 ± 21 to 276.1 ± 24 units/min in cultured MMDD1 cells when shear stress was increased from 0.5 to 5.0 dynes/cm(2). The shear stress-induced NO generation was abolished in MMDD1 cells in which the cilia were disrupted using a siRNA to ift88. Increasing the NaCl concentration of the tubular perfusate from 10 to 80 mM NaCl in the isolated perfused juxtaglomerular preparation reduced the diameter of the afferent arteriole by 3.8 ± 0.1 µm. This response was significantly blunted to 2.5 ± 0.2 µm when dextran was added to the perfusate to increase the viscosity and shear stress. Inhibition of NOS1 blocked the effect of dextran on TGF response. In vitro, the effects of raising perfusate viscosity with dextran on tubular hydraulic pressure were minimized by reducing the outflow resistance to avoid stretching of tubular cells. These results suggest that shear stress stimulates primary cilia on the macula densa to enhance NO generation and inhibit TGF responsiveness.

Expression of androgen receptor in coronary artery in the cases of sudden coronary death.

To study the expression of androgen receptor (AR) in the cases of sudden death caused by coronary heart disease (CHD) and relationship between AR and sudden coronary death (SCD) to explore the mechanism of the development of coronary atherosclerosis and provide references for the prevent and treatment of CHD and medicolegal identification of SCD. 53 cases selected from the autopsied cases in our department from 2011 to 2012 were divided into 3 groups: 18 case of SCD, including 11 males and 7 females, as experimental group, another 18 cases, including 11 males and 7 females, with CHD but died of mechanical injuries and poisoning as control group I, and 17 cases without CHD who also died of mechanical injuries and poisoning including 10 males and 7 females, as control group II. After HE-stained and immunohistochemistry-stained (SP) for the slices, the expression of AR in coronary arteries were observed and the average optical density (AOD) of positive signal in each case were detected by using Image-Pro Plus 6.0 software. We found strongly positive expression of AR in control group II, weakly positive expression in control group I and a little weaker in the experimental group. The expression of AR in coronary artery was reduced in CHD patients, which suggested that androgen may have favorable effects on the cardiovascular system.

Single nucleotide polymorphisms of ERß and coronary atherosclerotic disease in Chinese Han women.

BACKGROUND: Growing evidence has shown that with the increase of age, the incidence of coronary atherosclerotic disease (CAD) in women increases to equal that of men. Several studies on the single nucleotide polymorphisms [SNPs] seem to provide evidence in support of the protective role estrogen receptor ß (ERß) has in reducing the risk of CAD. OBJECTIVE: To determine the association of ERß SNPs rs1256049 RsaI 1082 A > G and rs4986938 AluI 1730 G > A with coronary atherosclerotic disease in Chinese Han women. METHODS: We designed a nested case-control research, in which 120 case women and 30 control women were selected from the Forensic Medicine Department of Tongji Medical College, HUST. We isolated DNA from their lung paraffin blocks, and then screened for these two SNPs for each DNA sample. Post-statistical analysis of their genotypes and haplotypes was used to figure out the targeted association. RESULTS: We found no significant difference between the genotypes or haplotypes of the two SNPs and the risk of CAD. However, the rs4986938 heterozygote AG variant was correlated with a significantly lower risk for CAD than did homozygote GG variant in the group of less than 40 years old. Haplotype AA of the two SNPs was correlated with a higher risk for CAD in the same group. CONCLUSION: The rs4986938 AluI 1730 G > A seems to be quite involved in the genetic basis of the disease and needs more attention in future studies. Meanwhile, this very association made between CAD and the mentioned SNP seems to be affected quite a bit by age.

Identification of a novel de novo mutation associated with PRKAG2 cardiac syndrome and early onset of heart failure.

INTRODUCTION: The major structure elements of the AMP-activated protein kinase (AMPK) are α, ß, and γ sunbunits. Mutations in γ2 subunit (PRKAG2) have been associated with a cardiac syndrome including inherited ventricular preexcitation, conduction disorder and hypertrophy mimicking hypertrophic cardiomyopathy. The aim of the present study was to identify PRKAG2 syndrome among patients presenting with left ventricular hypertrophy (LVH). METHODS AND RESULTS: Nineteen unrelated subjects with unexplained LVH were clinically and genetically evaluated. Among 4 patients with bradycardia, manifestations of preexcitation were only found in a 19 year old male who also developed congestive heart failure 3 years later. Electrophysiological study of this case identified the coexistence of an AV accessory pathway and AV conduction defect. Histological analysis of his ventricular tissue isolated by biopsy confirmed excessive glycogen accumulation, prominent myofibrillar disarray and interstitial fibrosis. Direct sequencing of his DNA revealed a heterozygous mutation in PRKAG2 consisting of an A-to-G transition at nucleotide 1453 (c.1453A>G), predicting a substitution of a glutamic acid for lysine at highly-conserved residue 485 (p.Lys485Glu, K485E), which was absent in his unaffected family members and in 215 healthy controls. To assess the role of K485 in the structure and function of the protein, computational modeling calculations and conservation analyses were performed. Electrostatic calculations indicate that K485 forms a salt bridge with the conserved D248 residue in the AMPK ß subunit, which is critical for proper regulation of the enzyme, and the K485E mutant disrupts the connection. CONCLUSIONS: Our study identifies a novel de novo PRKAG2 mutation in a young, in which progression of the disease warrants close medical attention. It also underlines the importance of molecular screening of PRKAG2 gene in patients with unexplained LVH, ventricular preexcitation, conduction defect, and/or early onset of heart failure.

[Expression of fas protein of myocardium in dilated cardiomyopathy].

OBJECTIVE: To investigate Fas protein expression of the myocardium in dilated cardiomyopathy (DCM) and its relationship with occurrence of sudden death caused by DCM. METHODS: Nine autopsy cases of sudden death caused by DCM along with the heart samples were chosen from the archives in the Department of Forensic Medicine, Tongji Medical College, HUST from 1997 to 2007. Other 11 cases which died of violence and other diseases were selected as the control group. Expressions of myocardial Fas protein in the samples were quantitatively detected by immunohistochemistry and computerized imaging analysis. RESULTS: Myocardial Fas protein expression increased significantly in the DCM group. Positive color showed brown-yellow granulated or striped distribution in the longitudinal section of myocardial within the cell membrane and cytoplasm, and showed circular brown granules in the cross section of the cell membrane, while these changes were not observed in the control group though there was focal weak staining noted. Statistical significance was observed between the experimental and control groups (P = 0.002), but no statistical significance was found for the average optical density value between these two groups (P = 0.675). CONCLUSION: The expression of Fas protein increased obviously in the DCM group. Such alteration in expression quantity and distribution of myocardial Fas protein may be related to arrhythmia and heart failure in the patients with DCM.

A case of arrhythmogenic right ventricular cardiomyopathy without arrhythmias.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by recurrent coma, ventricular tachycardias and the replacement of the myocardium with fatty and fibrous tissue. We described a 42-year-old female patient without clinical arrhythmias which was diagnosed as ARVC by magnetic resonance imaging (MRI), but the transvenous endomyocardial biopsy was not specific. The patient received heart transplantation due to her refractory heart failure and the pathology of explanted heart demonstrated typical replacement of fatty and fibrous tissue and piles of infiltrated lymphocytes in myocardial tissue. It is concluded that ARVC might not have any arrhythmias and inflammatory process may be involved in the mechanism of ARVC. Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/6573514507145351.

Andrographolide induces cell cycle arrest and apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes.

The pseudo-tumoral expansion of fibroblast-like synoviocytes is a hallmark of rheumatoid arthritis (RA), and targeting rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) may have therapeutic potentials in this disease. Andrographolide, a diterpenoid compound isolated from the herb Andrographis paniculata, has been reported to have potent anti-inflammatory activity. In the present study, we aimed to investigate the effects of andrographolide on human RAFLSs and the underlying molecular mechanism(s). RAFLSs were isolated from patients with RA and treated with or without various concentrations (i.e., 10, 20, and 30 µM) of andrographolide for 48 h. 3-[4,5-Dimethyl-2-yl]-2,5-diphenyl tetrazolium bromide assay revealed that andrographolide treatment decreased the proliferation of RAFLSs in a dose-dependent manner. Cell cycle analysis using propidium iodide (PI) staining showed a G0/G1 cell cycle arrest in andrographolide-treated RAFLSs. Immunoblotting analysis of key cell cycle regulators demonstrated that andrographolide treatment caused a dose-dependent increase in the expression of cell-cycle inhibitors p21 and p27 and a concomitant reduction of cyclin-dependent kinase 4. Exposure to andrographolide-induced apoptosis of RAFLSs measured by annexin V/PI double staining, which was coupled with promotion of cytochrome C release from mitochondria and activation of caspase-3. Moreover, andrographolide-treated RAFLSs displayed a significant decrease in the Bcl-2/Bax ratio compared to untreated cells. In conclusion, our data demonstrate that andrographolide exerts anti-growth and pro-apoptotic effects on RAFLSs, thus may have therapeutic potential for the treatment of RA.

[Toll-like receptor 4 expression in the coronary atherosclerotic plaques of patients died from sudden cardiac death].

OBJECTIVE: To investigate the Toll like receptor 4 (TLR4) expression in the coronary atherosclerotic plaques in patients died from sudden cardiac death (SCD) or non SCD. METHODS: Autopsied coronary artery samples from 75 patients died from SCD (n = 28), non-SCD (n = 28) or non-CHD (n = 19) were examined and the R value (positive cells' areas/scanning areas) and A value (average optical density) of TLR4 expression in the coronary arteries were detected qualitatively by the immunohistochemistry (SABC method) and image analysis technologies. RESULTS: SCD group: 13 (46.4%) cases showed strong positive expression of TLR4; 11 (39.3%), positive expression; 4 (14.3%), weak positive expression. CHD group: 8 (28.6%) cases showed weak positive expression; 17 (60.7%), very weak positive expression; 3 (10.7%), no positive expression. There was no positive expression of TLR4 in non-CHD samples. A (1.140 +/- 0.101) and R value (0.0269 +/- 0.0027) in SCD group were significantly higher than in non-SCD and control groups (all P < 0.01). A value was siginificantly higher in CHD group (0.719 +/- 0.205) than that in control group (0.481 +/- 0.033, P < 0.05) while R value (0.0085 +/- 0.0007, 0.0046 +/- 0.0004) was similar between the groups (P > 0.05). CONCLUSION: The increased positive expressive of TLR4 in the atherosclerotic plaque can be regarded as an important pathological marker of SCD.

[Pathomorphologic study of the fibrous cap and fatty core of coronary arthrosclerotic plaque in sudden coronary death].

OBJECTIVE: The differences in the thickness of fibrous cap and the percentage of fatty core of the coronary atherosclerotic plaques between sudden coronary death (SCD) group and the control group were investigated. METHODS: Sixty-four autopsy cases were divided into SCD and control groups. Samples were taken from the most severely damaged portions of the coronary atherosclerotic plaques, sectioned, stained with HE, and the percentage of examined by light microscopy for morphologic changes and structural alternations. Image analysis system was adopted to compare the thickness of fibrous cap and percentage of fatty core in the whole plaque between the two groups, and allthe data were analyzed and calculated with SPSS 11.5 statistic software. RESULTS: There were 15 grade III and 21 grade IV atherosclerotic cases found in the SCD group, while there were 16 and 12 found in the control group, respectively. Although no significant differences on the severity of atherosclerosis were found between the two groups (P > 0.05), there were significant differences on the thickness of the fibrous cap and the percentage of fatty core found between the two groups (P < 0.01). CONCLUSION: Our study indicates that there are significant differences in the thickness of fibrous cap and the percentage of fatty core in atherosclerosis plaques between the SCD group and the control group. These observed differences may be helpful for morphological diagnosis of SCD.

[Recent advances in venous air embolism].

Air embolism, a life-threatening complication of medical procedure, is a frequently encountered challenge in the forensic practices. It can be easily missed due to ignorance by forensic examiner or it could be difficult to be identified due to prolonged storage of the cadaver. This article reviews the etiological factors, pathophysiological changes, clinical manifestation, diagnosis, and the medicolegal identification of air embolism. The authors suggest that modern imaging techniques including echocardiogram, computer tomography, and magnetic resonance imaging play animportant role in the clinical diagnosis and forensic identification of air embolism.

Sudden death due to arrhythmogenic right ventricular cardiomyopathy: Two case reports.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a kind of primary myocardial disease characterized by the regional or global replacement of right ventricular myocardium by fatty and fibrolipomatous tissues. The ARVC, usually presenting with different clinical manifestations and pathological changes, were mainly seen in young men and is one of the main causes of sudden death in the young. Here two autopsied cases of Chinese men aged 30 and 23 years old who appeared healthy but died suddenly while at work are reported respectively. One of the victims had extensive and severe pathological changes in his heart involving the left ventricular wall as well as the ventricular septum and the right atrium. Not only was there a global fatty and fibrolipomatous tissue replacement of the right ventricular myocardia, but also mild sarcoplasmic coagulation in the myocardium and focal lymphocytic infiltration in the myocardial interstitium of the right ventricular wall. In addition, slight atherosclerosis of the coronary artery and intimal thickening of the sino-atrial node were observed. It is believed that there are no marked differences in the pathological changes of ARVC between Chinese patients and patients from western countries. The etiology and pathogenesis of ARVC could not be explained by a single cause or factor and they are probably related to various congenital and acquired causes or factors.

[Monocyte chemotactic protein-1 expression in coronary atherosclerosis plaque of sudden coronary death patients].

OBJECTIVE: To investigate the expression of monocyte chemotactic protein 1 (MCP-1) in coronary atherosclerosis plaque of sudden coronary death (SCD) patients and the relationship between MCP-1 expression and SCD. METHODS: Autopsy heart samples (n = 90) collected during 2001 - 2003 were divided to SCD group (n = 36) and 2 control groups: control group I, non-SCD CHD (n = 28), control group II, non-cardiac death (n = 26). The immuno-histochemistry SABC techniques (R, positive MCP-1 cell area/totl area) and computerized images analysis (A) were performed to detect the expression of MCP-1 in different groups. RESULTS: R and A in plaques are significant higher in SCD group than control group I and II (0.1264 +/- 0.013 vs 0.0269 +/- 0.0110 and 0.0267 +/- 0.0100, P = 0.04), (0.4534 +/- 0.083 vs 0.2303 +/- 0.040 and 0.2158 +/- 0.0400, P = 0.00), and similar between control group I and control group II. CONCLUSIONS: MCP-1 expression is increased in coronary atherosclerosis plaque of SCD patients.

Myocardial Cx43 expression in the cases of sudden death due to dilated cardiomyopathy.

PURPOSE: Probing into myocardial connexin (Cx) 43 expression in the cases of sudden death due to dilated cardiomyopathy (DCM) and relationship between Cx43 expression and sudden death. METHOD: Myocardial Cx43 was detected with immunohistochemical staining in the cases of 11 sudden death caused by DCM and 14 cases of control group who died of violent reasons and other diseases, which were autopsied in our department from 1997 to 2003. RESULT: Of 11 cases of DCM, there were 10 men and 1 woman with ranging in age from 7 to 49 years old (x (37.8) years old for 9 adult cases). Of 14 cases in the control group, there were 10 men and 4 women with ranging in age from 11 to 53 years old (x (29.9) years old for 11 adult cases). Myocardial Cx43 expression was obviously decreased in DCM group. Positive dyeing spots were different in size, distribution, color and disparity, some of them were distributed in the form of particle. Obvious change had not been observed in the cases of control group or with only slight changes in coloring degree and expressive area. The quantitative data showed that there was significant difference between two groups (p=0.0075) about Cx43 expressive area, but there was no difference between the left and right ventricles (p>0.05) in each group itself. And there was not difference between the two groups about average optical density of expression. CONCLUSION: Myocardial Cx43 expression is obviously reduced in the patients with DCM who die suddenly. The alteration of quantity and distribution of myocardial Cx43 expression is probably related to sudden death of the patients with DCM.

[Progression of the manner of cardiomyocyte death and its potential significance in forensic medicine].

The manner of cell death is a hotspot of medical researchers. Apoptosis and necrosis were considered as two manners of cell death in the past. But recently a new manner of cell death--oncosis is gradually accepted by the pathologists. Oncosis is different from apoptosis in morphologic, mechanism and the role in cardiovascular diseases. In this paper, the progression of the research about manner of the cardiomyocyte death and its significance in forensic medicine in recent years was reviewed.

[The advance of protection for hazard factor during autopsy].

Recently, the special characteristics of work with SARS require particular attention to the facilities, equipment, policies and procedures involved. In fact, an autopsy also subject prosectors and others to a wide variety of hazards, including bloodborne, aerosolized pathogens and others (for example SARS). Forensic pathologists and other persons in close proximity to an autopsy need personal protective equipment, fourthemore, laboratory procedure and facility design principles of biosafety should be established for the protection of all personnal involved in the work.

[Gap junctions, connexins and sudden death caused by coronary heart disease].

Gap junctions construct hydrophilic trans-membrane channels which adjust the intercellular communication of chemistry and electricity. In the heart, individual cardiac myocytes are linked by gap junctions. These junctions form low resistance pathways along which the electrical impulse flows rapidly and repeatedly between all the myocardium, ensuring their synchronous contraction. In recent years, some researchers have found that connexins, the protein molecules of gap junction channels, are reduced in number or redistributed from intercalated disks (ID) to lateral cell borders in a variety of cardiac disease, especially in ischemic heart disease. The gap junction remodeling is considered to be arrhythmogenic. These findings will lead us to a new realm in the diagnostic of sudden death caused by coronary heart disease.

Determination of free salicylic acid in chewing aspirin tablets by HPLC.

OBJECTIVE: To establish a HPLC method for determining the content of free salicylic acid in chewing aspirin tablets. METHOD: The determination was conducted on a HPLC column (C(18), 150 mm x 4.6 mm x 5 microm) with methanol-water-glacial acetic acid (8.0 5.5 1.0) as the mobile phase and the detection wavelength of 302 nm. RESULTS: The calibration curve was linear within the concentration range of 2.65 to 31.77 microg/ml (r=0.999 97) of salicylic acid. The average recovery rate was 100.21% with relative standard deviation of 0.53% (n=6). CONCLUSION: HPLC is quick and accurate of determining the content of free salicylic acid for chewing aspirin tablets.