Najma Latif

Role of Human Valve Interstitial Cells in Valve Calcification and Their Response to Atorvastatin

Background— Calcific aortic valve stenosis is a common disease in the elderly and is characterized by progressive calcification and fibrous thickening of the valve, but the cellular and molecular mechanisms are not fully understood. We hypothesized that human valve interstitial cells (ICs) are able to differentiate into osteoblast-like cells through the influence of defined mediators and that this process can be modulated pharmacologically. Methods and Results— To test this hypothesis, we treated primary cultures of human aortic valve ICs with osteogenic media, bone morphogenic proteins ([BMPs] BMP-2, BMP-4, and BMP-7), and tissue growth factor-β ([TGF-β] TGF-β1 and TGF-β3) for 21 days. These mediators induced osteoblast differentiation of valve ICs by significantly increasing the activity and expression of alkaline phosphatase ([ALP] P<0.001). A cytokine protein array revealed that atorvastatin treatment (100 &mgr;mol/L) of human valve ICs caused a downregulation in levels of expression of BMP-2, BMP-6, TGF-β1, and TGF-β3 after 24 hours. In addition, human valve ICs treated with atorvastatin in the presence of osteogenic media showed a significant reduction in ALP activity in comparison to cells treated with osteogenic media only (P=<0.001). This was further confirmed with immunocytochemical staining of valve ICs, whereby atorvastatin markedly reduced the expression of ALP and osteocalcin induced by osteogenic media in comparison to untreated cells. Conclusions— These findings suggest that human valve ICs are capable of osteoblastic differentiation, by potential mediators which can be pharmacologically targeted by atorvastatin.

Frequency and specificity of antiheart antibodies in patients with dilated cardiomyopathy detected using SDS-PAGE and western blotting

OBJECTIVES This study was designed to investigate the organ and disease specificity of antiheart antibodies in patients with dilated cardiomyopathy. BACKGROUND Autoimmune disease is characterized by the presence of circulating autoantibodies, and autoimmune mechanisms may play a role in the pathogenesis of dilated cardiomyopathy. METHODS An SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) procedure followed by Western blotting was used to screen serum samples for antiheart antibodies of two immunoglobulin classes, IgM and IgG, from 52 patients with dilated cardiomyopathy and 48 patients with ischemic heart disease as control subjects. Use of two-dimensional gel electrophoresis followed by Western blotting and protein sequencing enabled us to identify the protein bands against which antiheart antibodies were produced in both groups of patients. RESULTS Strong IgG antiheart antibodies against myocardial proteins, cross-reacting with skeletal muscle proteins, were detected in significantly more patients with dilated cardiomyopathy (n = 24 [46%]) than with ischemic heart disease (n = 8 [17%]) (p = 0.001). Patients with dilated cardiomyopathy showed a significantly greater frequency and reactivity of IgG antiheart antibodies against six myocardial proteins (molecular weight 30, 35, 40, 60, 85 and 200 kD) than did patients with ischemic heart disease. These were identified as myosin light chain 1, tropomyosin, actin, heat shock protein (HSP)-60, an unidentified protein and myosin heavy chain, respectively. CONCLUSIONS We detected strong IgG antiheart antibodies in significantly more patients with dilated cardiomyopathy than with ischemic heart disease. The most immunogenic band was that corresponding to HSP-60. Antibodies against HSP-60 were found in 85% and 42% of patients with dilated cardiomyopathy and ischemic heart disease, respectively, confirming our hypothesis of an immune involvement in dilated cardiomyopathy.

Upregulation of the Bcl-2 family of proteins in end stage heart failure

OBJECTIVES To elucidate the pattern of expression of four members of the Bcl-2 family of proteins and to correlate this with terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labelling (TUNEL) and DNA fragmentation. BACKGROUND Apoptosis has been implicated as a possible mechanism in the development of heart failure. However, the mechanisms involved remain unclear. METHODS We have studied the expression of four members of the Bcl-2 family that are involved in the regulation of apoptosis and analyzed DNA fragmentation as a marker of apoptosis and as a biochemical criterion to distinguish between apoptosis and necrosis in dilated cardiomyopathy (DCM), ischemic heart disease (IHD) and normal donors. RESULTS Western blot analysis and immunocytochemistry of the proapoptotic and antiapoptotic Bcl-2 proteins demonstrated significantly higher levels of all these proteins in the diseased groups compared with normal donors. Additionally, Bax was significantly higher in the IHD group compared with DCM. Terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labelling analysis demonstrated a significantly higher percentage of TUNEL-positive cells in the diseased groups compared with the control. Genomic DNA extraction of ventricular myocardial tissue showed no demonstrable DNA laddering for any of the groups. CONCLUSIONS The significant increases in the levels of the proapoptotic proteins Bak and Bax and the higher percentage of TUNEL-positive cells in both diseased groups suggests the presence of ongoing apoptosis. However, increases in the antiapoptotic proteins, Bcl-2 and Bcl-xL, suggest a possible concomitant, compensatory antiapoptotic mechanism in patients with heart failure.

Gene Profiling Changes in Cytoskeletal Proteins During Clinical Recovery After Left Ventricular–Assist Device Support

Background—After left ventricular–assist device (LVAD) support, a proportion of patients recover sufficient ventricular function to enable explantation of the device. The exact molecular mechanisms involved in myocardial recovery remain unknown. Cytoskeletal proteins are essential for the structure and function of the cardiac myocyte and might play a major role. Methods and Results—A total of 15 patients with nonischemic cardiomyopathy who required LVAD implantation were studied; 6 recovered sufficiently to allow explantation of the device compared with 9 who did not recover and required transplantation. LV myocardial samples were collected at implantation and explantation/transplantation. Affymetrix microarray analysis was performed on the paired samples and analyzed with reference to sarcomeric and nonsarcomeric cytoskeletal proteins. In the recovery group, of the nonsarcomeric proteins, lamin A/C increased 1.5-fold (P<0.05) and spectrin 1.6-fold (P<0.05) between the times of implantation and explantation. Integrins β1, β6, and α7 decreased 1.7-fold (P<0.05), 2.4-fold (P<0.05), and 1.5-fold (P<0.05), respectively, but integrins α5 and β5 increased 2.3-fold (P<0.01) and 1.2-fold (P<0.01) at explantation. The following sarcomeric proteins changed in the recovered group only: β-actin increased 1.4-fold (P<0.05); α-tropomyosin, 1.3-fold (P<0.05); α1-actinin, 1.8-fold (P<0.01); and α-filamin A, 1.6-fold (P<0.05). Both troponin T3 and α2-actinin decreased by 1.6-fold at the time of explantation (P<0.05). Vinculin decreased 1.7-fold (P=0.001) in the recovered group but increased by 1.7-fold (P<0.05) in the nonrecovered group. Vinculin protein levels decreased 4.1-fold in the recovered group. Conclusions—Myocardial recovery was associated with a specific pattern of changes in sarcomeric, nonsarcomeric, and membrane-associated proteins, which could have important implications in understanding the mechanisms involved.

Elevated p53 expression is associated with dysregulation of the ubiquitin-proteasome system in dilated cardiomyopathy

AIMS The molecular mechanisms that regulate cardiomyocyte apoptosis and their role in human heart failure (HF) are uncertain. Expression of the apoptosis regulator p53 is governed by minute double minute 2 (MDM2), an E3 enzyme that targets p53 for ubiquitination and proteasomal processing, and by the deubiquitinating enzyme, herpesvirus-associated ubiquitin-specific protease (HAUSP), which rescues p53 by removing ubiquitin chains from it. Here, we examined whether elevated expression of p53 was associated with dysregulation of ubiquitin-proteasome system (UPS) components and activation of downstream effectors of apoptosis in human dilated cardiomyopathy (DCM). METHODS AND RESULTS Left ventricular myocardial samples were obtained from patients with DCM (n = 12) or from non-failing (donor) hearts (n = 17). Western blotting and immunohistochemistry revealed that DCM tissues contained elevated levels of p53 and its regulators MDM2 and HAUSP (all P < 0.01) compared with non-failing hearts. DCM tissues also contained elevated levels of polyubiquitinated proteins and possessed enhanced 20S-proteasome chymotrypsin-like activities (P < 0.04) as measured in vitro using a fluorogenic substrate. DCM tissues contained activated caspases-9 and -3 (P < 0.001) and reduced expression of the caspase substrate PARP-1 (P < 0.05). Western blotting and immunohistochemistry revealed that DCM tissues contained elevated expression levels of caspase-3-activated DNAse (CAD; P < 0.001), which is a key effector of DNA fragmentation in apoptosis and also contained elevated expression of a potent inhibitor of CAD (ICAD-S; P < 0.01). CONCLUSION Expression of p53 in human DCM is associated with dysregulation of UPS components, which are known to regulate p53 stability. Elevated p53 expression and caspase activation in DCM was not associated with activation of both CAD and its inhibitor, ICAD-S. Our findings are consistent with the concept that apoptosis may be interrupted and therefore potentially reversible in human HF.

Adipose tissue: friend or foe?

The perception of adipose tissue has changed considerably with the dramatic increase in the incidence of obesity and obesity-related comorbidities over the past 3 decades. Excess fat is no longer associated with wealth, but is instead recognized as a risk factor for many diseases. Adipose tissue is increasingly being identified as a vital, complex endocrine organ, and not simply as a fat store. Not all fat is created equal—regional, developmental, structural, and functional variations exist. Epicardial adipose tissue is a metabolically active organ producing a number of factors that modulate cardiac structure and function. The global epidemic of obesity and metabolic syndrome imposes a major disease burden, particularly of cardiovascular disease. In this Review, we describe the various types of adipose tissue—their developmental biology, differentiation, cell heterogeneity, and functional characteristics. We discuss the link between adipose tissue and inflammation, the signaling factors released by adipose tissue, as well as cardiac adiposity and its relevance to cardiovascular diseases. Finally, we review the myocardial regenerative potential of adipose-tissue-derived stem cells. We believe that a thorough understanding of adipose tissue is of great clinical value.

Activation of apoptotic and inflammatory pathways in dysfunctional donor hearts.

Background. Myocardial dysfunction is common after brain death, but the mechanisms remain unclear. Apoptosis is tightly regulated by enzymes termed the caspases. We have investigated the caspases involved in the terminal part of the apoptotic pathway in dysfunctional (nontransplanted) donor hearts and their relation to inflammatory markers and compared them to hearts with good ventricular function (transplanted donors). Methods. Thirty-one donor hearts assessed for transplantation were examined. Western blotting was used to measure pro-caspase-9, caspase-3, DFF45, the activated nuclease CPAN and poly (ADP-ribose) polymerase, a DNA repair enzyme inactivated by caspase-3. Caspase-3 activity was also measured. Histologic and immunocytochemical analysis for HLA Class II and Real Time polymerase chain reaction for tumor necrosis factor-&agr; and interleukin 6 were performed to detect inflammatory activation. Results. Cleaved caspase-9 was higher (5.53±0.6 vs. 3.64±0.4 O.D. units, P <0.01) in nontransplanted compared with transplanted donors and there was a trend for higher pro-caspase-9 (5.20±1.0 vs. 4.22±0.4 O.D. units, P =NS). Levels of pro-caspase-3 were higher in nontransplanted (9.66±0.5 vs. 5.15±0.5 O.D. units, P <0.00001) donors and cleavage products of caspase-3 were elevated in 14 of 14 nontransplanted and 2 of 17 transplanted donors. Intact DFF-45 (8.94±0.36 vs. 6.14±0.30 O.D. units, P <0.000005), its spliced product (2.38±0.35 vs. 0.4±0.21 O.D. units, P =0.0001) and the nuclease caspase-activated nuclease (2.01±0.3 vs. 0.66±0.16 OD units, P =0.001) were higher in nontransplanted donors. The caspase-3 substrate poly (ADP-ribose) polymerase was higher in nontransplanted (1.16±0.13 vs. 0.61±0.22 O.D. units, P =0.57) donors. Conclusions. The caspases are elevated in dysfunctional donor hearts compared with hearts with good ventricular function with a possible link to inflammatory activation supporting the concept that brain death causes inflammatory activation which can lead to apoptosis with a possible important effect on function.

Heat Stress Contributes to the Enhancement of Cardiac Mitochondrial Complex Activity

Hyperthermic stress is known to protect against myocardial dysfunction after ischemia-reperfusion injury. It is unclear however, what energetic mechanisms are affected by the molecular adaptation to heat stress. We hypothesized that mild hyperthermic stress can increase mitochondrial respiratory enzyme activity, affording protection to mitochondrial energetics during prolonged cardiac preservation for transplantation. Rat hearts were excised after heat-stress or sham treatment and subjected to cold cardioplegic arrest and ischemia followed by reperfusion in an ex vivo perfusion system. Cardiac function, mitochondrial respiratory, and complex activities were assessed before and after ischemia. Heat shock protein (Hsp 32, 60, and 72) expression was increased in heat-stressed hearts. This was associated with increased mitochondrial complex activities in heat-stress versus sham-treated groups for complex I-V. During reperfusion, higher complex activities and respiratory control ratios were observed in heat-stressed versus sham-treated groups. Recovery of ventricular function was improved in heat-stressed hearts. Furthermore, mitochondria in reperfused heat-stressed myocardium exhibited intact membranes with packed, parallel, lamellar cristae, whereas in sham-treated myocardium, mitochondria were severely disrupted. This study provides the first evidence of heat-stress-mediated enhancement of mitochondrial energetic capacity. This is associated with increased tolerance to ischemia-reperfusion injury. Protection by heat stress against myocardial dysfunction may be partially due to enhancement of mitochondrial energetics.

Demonstration by western blotting of antiheart antibodies before and after cardiac transplantation.

Western blotting has been used to detect antiheart antibodies in two groups of patients: two who required retransplantation for hyperacute rejection, and 22 consecutive patients, whose serum was tested at monthly intervals for three months following transplantation. Pretransplant and posttransplant serum samples were tested for IgM and IgG reactivity against the patients own heart and donor heart. In all patients the pretransplant lymphocytic crossmatch had been negative. In the two patients requiring retransplantation, both had multiple bands of strong IgM and IgG against their own heart prior to transplantation as well as antibodies against the donor heart. The study of 22 consecutive patients revealed that (1) the presence of strong antibody prior to transplantation is associated with unusually severe or frequent rejection episodes, (2) 20/22 patients made antiheart antibody following transplantation, but in 12 patients it was IgM only, and (3) most of the antiheart antibodies made posttransplant were not specific for the donor heart. Comparison of Western blotting with immunofluorescent detection of antibodies on frozen sections revealed that the Western blotting procedure is more sensitive and results are easier to interpret.

Relative induction of heat shock protein in coronary endothelial cells and cardiomyocytes: Implications for myocardial protection

OBJECTIVES Induction of the 70 kd heat shock protein in the heart is known to exert a protective effect against postischemic mechanical and endothelial dysfunction. However, the exact site of induction and the mechanisms involved remain unknown. The aim of this study was to investigate the relative capacity of endothelial and myocardial cells to express the 70 kd heat shock protein in response to heat stress, as well as their significance. METHODS (1) Postischemic recovery of cardiac mechanical and endothelial function was studied in isolated rat hearts with and without endothelial denudation with saponin. (2) Semiquantitative determination of induction of 70 kd heat shock protein by Western immunoblotting was performed in the whole cardiac homogenate, in isolated cardiac myocytes, and in coronary endothelial cells. (3) Immunocytochemistry was used to visualize the distribution of induction of 70 kd heat shock protein in both cell types. RESULTS Postischemic recovery (percent preischemic value +/- standard error of the mean) of cardiac output in hearts from heat-stressed animals was significantly improved (66.7 +/- 6.9 vs 44.5 +/- 4.5 in the control group, p < 0.01). In heat-stressed hearts treated with saponin no improvement in the recovery of cardiac output was noted (44.7 +/- 6.9 in heat-stressed hearts vs 38.0 +/- 4.0 in heat-stressed, saponin-treated hearts, p = not significant). Endothelial function (as assessed by the vasodilatory response to the endothelium-dependent vasodilator 5-hydroxytryptamine) improved from 31.0 +/- 5.2 in the control group to 65.8 +/- 7.1 in heat-stressed hearts (p < 0.02 vs control) and dropped to -1.9 +/- 3.8 in heat-stressed hearts treated with saponin. Immunocytochemistry showed that only sections of hearts from heat-treated rats showed a strong specific reaction with heat shock protein antibody. The positive staining was seen in endothelial cells. Induction of 70 kd heat shock protein content in the whole cardiac homogenate from heat-stressed rats as measured by Western immunoblotting was 5.2 +/- 1.9 (vs 0.0 in non-heat-stressed rats, p < 0.0001) and dropped to 0.0 in heat-stressed hearts treated with saponin. The tentative amount of 70 kd heat shock protein was 18.1 +/- 7.8 in isolated endothelial cells from heat-stressed hearts and 2.3 +/- 2.3 in isolated cardiac myocytes (p < 0.01 vs endothelial cells). CONCLUSIONS Coronary endothelial cells are the main site of induction of 70 kd heat shock protein in the heart and appear to contribute to the protective effects of heat stress on the recovery of mechanical and endothelial function.