Ribonucleic acid extraction from archival formalin fixed paraffin embedded myocardial tissues for gene expression and pathogen detection.

INTRODUCTION: Archival tissue samples preserved in formalin are a great source of treasure for biomedical research and diagnostics. Formalin, though is a good preservative, causes the modification of nucleic acid limiting the application of fixed tissues. The present study evaluated three methods of RNA extraction for constitutive gene expression and pathogen detection. MATERIAL AND METHODS: Sixteen archival formalin-fixed paraffin-embedded (FFPE) myocardial tissues were subjected to RNA extraction by Trizol, SDS, and RNeasy FFPE kit followed by RT-PCR and Taqman Real-Time PCR to study the expression of housekeeping genes. RESULTS: RNA was extracted from all 16 myocardial tissues (100%) by RNeasy FFPE kit, as compared to 14/16 by Trizol and 8/10 by SDS methods. The expression of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was observed in RNA extracted by RNeasy FFPE kit and Trizol. High yield of RNA was obtained by RNeasy FFPE kit than Trizol (P = 0.002) and SDS(P = 0.012). Of the three methods, RNeasy FFPE kit was evaluated for Enterovirus RNA detection in 16 other histopathologically confirmed FFPE tissues of dilated cardiomyopathy (DCM) cases and Enterovirus genome was detected in 4/16 (25%) FFPE tissues of DCM cases. The enteroviral sequences of the viral isolates revealed 99% homology with Human coxsackievirus B5. CONCLUSION: The Qiagen RNeasy FFPE kit resulted in significantly high reproducibility of RNA from FFPE myocardial tissues, which are suitable for amplification by Taq-Man Real-Time and RT-PCR. Thus, the results show that these FFPE tissues can be used for gene expression, pathogen detection, and epidemiological studies.

Therapeutic potential of Terminalia arjuna in cardiovascular disorders.

The bark of the tree Terminalia arjuna (Roxb.) is widely used in Indian medicine (Ayurveda) for various cardiovascular ailments. The bark has been reported to contain several bioactive compounds. Many experimental studies have reported its antioxidant, anti-ischemic, antihypertensive, and antihypertrophic effects, which have relevance to its therapeutic potential in cardiovascular diseases in humans. Several clinical studies have reported its efficacy mostly in patients with ischemic heart disease, hypertension, and heart failure. However, a major shortcoming in all these experimental and clinical studies is the absence of phytochemical standardization of the extracts. In addition, many clinical studies are poor in terms of design and methods used for generating safety data. This review discusses how to address all these issues for a scientific validation of this medicinal plant.

Proteomic analysis of circulating human monocytes in coronary artery disease.

Monocytes play an important role in inflammation and atherosclerosis; however, the molecular details underlying these diverse functions are not completely understood. Proteomic analysis of monocytes can provide new insights into their biological role in coronary artery disease (CAD). Twenty angiographically confirmed male, CAD patients (≥50% stenosis) attending cardiology clinic of Nehru Hospital, PGIMER, Chandigarh, and who were not receiving any lipid lowering therapy and 20 TMT negative subjects who served as controls were enrolled in the study. Circulating monocytes isolated from overnight fasting blood samples were analyzed by 2D gel electrophoresis (pH 4-7), and differentially expressed protein spots were subjected to mass spectrometry and identification of proteins. We observed 333 ± 40 protein spots in monocytes from patients and 312 ± 20 in controls; out of which 63 protein spots showed altered intensity in CAD patients. Thirteen spots showed fivefold increased and two protein spots showed fivefold decreased expression in CAD group as compared to control group, respectively. Two proteins showing decreased expression in monocytes from CAD patients were identified as: (i) glutathione transferase and (ii) heat shock protein 70 KDa. Proteins showing increased expression in CAD patients were identified as: (i) vimentin, (ii) mannose binding lectin receptor protein, and (iii) S100A8 calcium-binding protein. The results of our study offer identification of several proteins in monocytes which can provide new perspectives in role of monocytes in pathogenesis of atherosclerosis.

Common variants of HMGCR, CETP, APOAI, ABCB1, CYP3A4, and CYP7A1 genes as predictors of lipid-lowering response to atorvastatin therapy.

There is interindividual variation in lipid-lowering response to statins. The objective of this study was to investigate whether common variation in genes involved in lipid and statin metabolism modify the effect of statins on serum total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and high-density lipoprotein-cholesterol concentration in coronary artery disease (CAD) patients. We studied the association between 18 single-nucleotide polymorphisms (SNPs) in six genes (HMGCR, CETP, APOAI, ABCB1, CYP3A4, CYP7A1) in response to atorvastatin therapy (20 mg/day) in 265 newly diagnosed CAD patients using multivariable adjusted general linear regression. Variant alleles of ABCB1 (-41A/G), HMGCR SNP29 G/T, rs5908A/G, rs12916C/T, and CYP7A1-204A/C polymorphisms were significantly associated with attenuated LDL-C reduction and variant alleles of CETP TaqI, -629C/A, and APOAI PstI polymorphisms were associated with higher increase in high-density lipoprotein-cholesterol. A three-loci interaction model consisting of CYP7A1rs892871AA/APOAIPstIP1P1/HMGCR rs12916CT was a better predictor for LDL-C lowering, when compared with single polymorphisms analysis on statin response. Variant genotypes of APOAI -2500C/T, CETP 405I/V, and ABCB1 3435C/T showed higher risk of myocardial infarction events (p < 0.05) in a 1-year follow-up of CAD patients. These results suggest that SNPs in lipid and statin pathway genes are associated with reduced LDL-C lowering by statins and identify individuals who may be resistant to maximal LDL-C lowering by statins.

Decreased myocardial expression of dystrophin and titin mRNA and protein in dilated cardiomyopathy: possibly an adverse effect of TNF-alpha.

BACKGROUND AND AIMS: While the molecular basis of dilated cardiomyopathy (DCM) remains uncertain, concrete evidence is emerging that sarcomeric and cytoskeleton gene expression of myocardium isolated from failing versus non-failing patients differ dramatically. The central aim to this work was to find out the possible role of dystrophin and titin along with the TNF-alpha in the pathogenesis of cardiomyopathy. PATIENTS AND METHODS: mRNA levels and protein expression of a cytoskeletal protein, dystrophin and a sarcomeric protein, titin in endomyocardial biopsies of DCM patients were examined using RT-PCR and immunohistochemistry, respectively. Further, we examined the effect of TNF-alpha on myocardial expression of titin and dystrophin in vitro in rat cardiac myoblast cell line (H9c2). RESULTS: We observed significantly decreased mRNA and protein levels of dystrophin and titin in endomyocardial biopsy of DCM patients as compared to control group. The decreased levels of these proteins correlated with the severity of the disease. Plasma levels of both TNF-alpha and its soluble receptors TNFR1 and TNFR2 were found to be significantly higher in patients as compared to control group. Treatment of H9c2 cells with TNF-alpha resulted in a dose- and time-dependent decrease in mRNA levels of dystrophin and titin. Pretreatment of these cells with MG132, an inhibitor of nuclear factor kappa B (NF-kappaB) pathway, abolished TNF-alpha-induced reduction in mRNA levels of dystrophin and titin. CONCLUSION: Our results suggest that reduced expression of dystrophin and titin is associated with the pathophysiology of DCM, and TNF-alpha may modulate the expression of these proteins via NF-kappaB pathway.

ECG changes of hyperkalemia during paced rhythm.

Paced rhythms can mask ECG changes of several conditions. ECG changes due to hyperkalemia during paced rhythm have not been well described. We report a patient with isolated noncompaction of left ventricle with left ventricular dysfunction and complete heart block on a permanent pacemaker who developed hyperkalemia. Typical ECG changes of hyperkalemia including widening of QRS complex and sine waves were seen even during paced rhythm that reverted with correction of hyperkalemia.

A combination of proatherogenic single-nucleotide polymorphisms is associated with increased risk of coronary artery disease and myocardial infarction in Asian Indians.

Common single-nucleotide polymorphisms (SNPs) in genes of lipid metabolism modestly influence plasma low-density lipoprotein cholesterol (LDL-C) and risk of coronary artery disease (CAD). We evaluated a panel of LDL-C-modulating SNPs for potential association with risk of CAD in Asian Indians. Fifteen SNPs of CETP, ABCB1, APOAI, CYP7A1, and HMGCR genes were genotyped in 265 CAD patients and 150 controls of North Indian origin. A proatherogenic genotype score was formulated based on number of alleles associated with LDL-C and was evaluated for association with risk of CAD. We observed 12 SNPs from CETP, APOAI, ABCB1, CYP7A1, and HMGCR genes to be associated with baseline LDL-C and high-density lipoprotein cholesterol levels and increased risk of CAD (p < 0.05). Co-occurrence of three or more risk alleles (proartherogenic genotype score >or=3) was associated with increased risk of CAD and myocardial infarction. Analysis of epistatic interactions revealed CETPTaqIB1B1/405II/APOAI-75GA to be best model of CAD risk prediction in our population. Our study highlights synergistic association of multiple SNPs of lipid pathway with LDL-C levels and risk of CAD, and indicates that co-occurrence of proatherogenic risk alleles may provide incremental information about CAD risk beyond lipid concentrations.

Genetic and clinical profile of Indian patients of idiopathic restrictive cardiomyopathy with and without hypertrophy.

Both idiopathic restrictive cardiomyopathy (IRCM) and hypertrophic cardiomyopathy (HCM) are part of the same disease spectrum and are due to sarcomeric gene mutations. A patient with restrictive physiology without left ventricular hypertrophy (LVH) would be diagnosed as IRCM, while one with LVH would be diagnosed as HCM with restrictive physiology. We studied a group of patients with restrictive physiology for mutations in beta-myosin heavy chain (MYH7) and troponin I (TNNI3) gene. Consecutive probands in the HCM and IRCM cohort over a 4-year period were considered for this study. These included 10 IRCM and 102 HCM patients. All were Asian Indians. Among the 17 patients who had restrictive physiology 10 were IRCM patients and seven were HCM patients. Of the HCM patients, seven (6.9%) had restrictive physiology. Mean age of these 17 patients was 40.1 +/- 19.2 years (range: 15-67 ), six (35.3%) were males. Maximal left ventricular wall thickness of the seven HCM probands was 20.7 +/- 5.2 mm (range: 16-31), while it was normal in the IRCM probands. Ten probands (58.8%) were in NYHA class III or IV. Seven patients (41.2%) had atrial fibrillation. All the probands were screened for mutations in selected exons of MYH7 and TNNI3 genes. One IRCM patient was found to have p.Arg721Lys mutation in the MYH7 gene. She died due to progressive congestive cardiac failure at the age of 47 years. One HCM proband with a maximal left ventricular wall thickness of 17 mm had p.Arg192His mutation in the TNNI3 gene. She had features consistent with restrictive physiology. Her father and sister had died of restrictive cardiomyopathy. IRCM and HCM with restrictive physiology, both are part of the clinical expression of MYH7 and TNNI3 mutations and lead to worse clinical onset and progression of the disease.

Unusual cause of rapidly progressive right-sided heart failure: aortic sinus of Valsalva aneurysm causing ball valve obstruction of the tricuspid valve.

A rare presentation with rapidly progressive right heart failure due to tricuspid inflow obstruction (simulating right-sided valvular heart disease) caused by a non-coronary cusp sinus of Valsalva aneurysm with small perforation is reported. The aneurysm was causing ball valve obstruction at the tricuspid valve, leading to dilated right atrium and back pressure changes which were relieved after successful aneurysectomy.

Circulating proinflammatory cytokines and N-terminal pro-brain natriuretic peptide significantly decrease with recovery of left ventricular function in patients with dilated cardiomyopathy.

BACKGROUND: Increased levels of TNF-alpha, IL-6, their soluble receptors, and NT-proBNP have been observed in patients with dilated cardiomyopathy (DCM). In the present study, we assessed the possible involvement of proinflammatory cytokines and their soluble receptors with and without recovery of LV function in DCM patients. METHODS AND RESULTS: Forty patients with DCM were enrolled and divided into two groups: Group I consisted of DCM patients (n = 30) whose left ventricular ejection fraction (LVEF) had not recovered on follow up and Group II comprised DCM patients (n = 10) whose LVEF had recovered. Ten healthy subjects were included as controls (Group III). TNF-alpha, IL-6,TNFR1, TNFRII, gp130, and NT-proBNP levels were significantly increased in Group I and were significantly lower in patients with LVEF recovery as compared to those without recovery of LVEF (P < 0.05). CONCLUSION: Circulating TNF-alpha, IL-6, and NT-proBNP appear to correlate with the LV function recovery of patients with DCM and could be used as prognostic biomarkers.

Familial conduction system disease associated with hypertrophic cardiomyopathy.

A large family with a new phenotype of conduction system disease including heart blocks and preexcitation associated with hypertrophic cardiomyopathy and transmitted in an autosomal dominant pattern is described.

Protection against acute adriamycin-induced cardiotoxicity by garlic: role of endogenous antioxidants and inhibition of TNF-alpha expression.

BACKGROUND: Oxidative stress is the major etiopathological factor in adriamycin-induced cardiotoxicity. Relatively low amounts of endogenous antioxidant makes the heart vulnerable to oxidative stress-induced damage. Chronic oral administration of garlic has been reported to enhance the endogenous antioxidants of heart. We hypothesized that garlic-induced enhanced cardiac antioxidants may offer protection against acute adriamycin-induced cardiotoxicity. RESULTS: Rats were either administered freshly prepared garlic homogenate (250 and 500 mg/kg daily, orally, for 30 days) or probucol (cumulative dose, 120 mg/kg body weight divided in 12, i.p. over a period of 30 days) or double distilled water (vehicle), followed by a single dose of adriamycin (30 mg/kg i.p.). In the adriamycin group, increased oxidative stress was evidenced by a significant increase in myocardial TBARS (thiobarbituric acid reactive substances) and decrease in myocardial SOD (superoxide dismutase), catalase and GPx (glutathione peroxidase) activity. Histopathological studies showed focal as well as subendocardial myocytolysis with infiltration of macrophages, lymphocytes and edema. Immunocytochemistry showed marked expression of TNF-alpha (tumor necrosis factor-alpha) in the myocardium. Increase in myocardial TBARS and decrease in endogenous antioxidants by adriamycin was prevented significantly in the garlic treated rat hearts, which was comparable to the probucol-treated group. Histopathological evidence of protection was also evident in both garlic-treated and probucol-treated groups. Probucol, 250 mg/kg and 500 mg/kg of garlic reduced adriamycin induced TNF-alpha expression in the myocardium and was associated with reduced myocyte injury. CONCLUSIONS: It is concluded that chronic garlic administration prevents acute adriamycin-induced cardiotoxicity and decreases myocardial TNF-alpha expression.