Thomas Quentin

Telethonin Deficiency Is Associated With Maladaptation to Biomechanical Stress in the Mammalian Heart

Rationale: Telethonin (also known as titin-cap or t-cap) is a 19-kDa Z-disk protein with a unique &bgr;-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonins in vivo function. Objective: Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation. Methods and Results: By using a variety of different genetically altered animal models and biophysical experiments we show that contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin–titin cross-links via &agr;-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the proapoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis (“mechanoptosis”). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect that may contribute to enhanced rates of apoptosis found in these hearts. Conclusions: Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect that may also play a role in human heart failure.

Heat Shock Protein 70 Is Able to Prevent Heat Shock-Induced Resistance of Target Cells to CTL

Heat shock or transfection with heat shock protein 70 (Hsp70) genes has been shown to protect tumor cell lines against immune mechanisms of cytotoxicity. We have reported previously that heat shock confers resistance to CTL in the rat myeloma cell line Y3 that is Hsp70 defective. Evidence is now presented that Hsp70 is able to prevent the induction of the resistant phenotype. In Con A-stimulated lymphocytes and in lymphocyte × Y3 somatic cell hybrid clones a severe, non-Hsp70-inducing heat shock elicits resistance to CTL in contrast to a heat shock that results in Hsp70 expression. Thus, Hsp70 expression appears to be negatively associated with the development of resistance. Furthermore, loading of Y3 cells with recombinant Hsp70 protein before heat shock is able to prevent resistance. Because apoptosis induced in Y3 cells by heat shock is not affected, Hsp70 appears to interfere selectively with the CTL-induced lethal pathway that is found to be calcium but not caspase dependent. It is suggested that after heat shock Hsp70 enhances the CTL-induced apoptotic pathway by chaperoning certain proteins in the target cell that are involved in the execution of cell death. Thus, although shown to confer protection against many cytotoxic mechanisms, Hsp70 does not appear to be generally cytoprotective. This observation could also be of relevance when interpreting the effectiveness of tumor immunity.

Immunohistochemical Characterization of Neotissues and Tissue Reactions to Septal Defect–Occlusion Devices

Background—We sought to evaluate tissue reactions within and at the surface of devices for interventional therapy of septal defects and to identify antigen characteristics of neotissues. Methods and Results—Atrial or ventricular septal defect–occlusion devices (Amplatzer, n=7; Cardioseal/Starflex, n=3) were processed using a uniform protocol after surgical removal from humans (implantation time, 5 days to 4 years). Devices were fixed in formalin and embedded in methylmethacrylate. Serial sections were obtained by sectioning with a diamond cutter and grinding, thus saving the metal/tissue interface for histologic evaluation. Immunohistochemical staining was performed using conventional protocols. Superficial endothelial cells stained positive for von Willebrand factor. Within the newly formed tissues, fibroblast-like cells were identified with a time-dependent expression of smooth muscle cell maturation markers (smooth muscle actin, smooth muscle myosin, h-caldesmon, and desmin) beside extracellular matrix components. Neovascularization of the newly formed tissues was demonstrated with the typical immunohistochemical pattern of capillaries and small vessels. Inflammatory cells could be identified as macrophages (CD68+) and both T-type and B-type lymphocytes (CD3+, CD79+). Conclusions—This is the first presentation of results from serial immunohistochemical staining of a collection of explanted human septal-occlusion devices. A time-dependent maturation pattern of the fibroblast-like cells in the neotissues around the implants could be described. Neoendothelialization was seen in all specimens with implantation times of 10 weeks or more. The time course of neoendothelialization, as seen in our study, further supports the clinical practice of anticoagulant or antiplatelet therapy for 6 months after implantation. This time interval should be sufficient to prevent thromboembolic events due to thrombus formation at the foreign surface of cardiovascular implants.

Metformin differentially activates ER stress signaling pathways without inducing apoptosis.

SUMMARY Endoplasmic reticulum stress signaling (ERSS) plays an important role in the pathogenesis of diabetes and heart disease. The latter is a common comorbidity of diabetes and worsens patient outcome. Results from clinical studies suggest beneficial effects of metformin – a widely used oral drug for the treatment of type 2 diabetes – on the heart of diabetic patients with heart failure. We therefore analyzed the effect of metformin on ERSS in primary rat cardiomyocytes. We found that metformin activates the PERK-ATF4 but not the ATF6 or IRE1-XBP1 branch in ERSS and leads to a strong upregulation of CHOP mRNA and protein. Surprisingly, long-term induction of CHOP by metformin is not accompanied by apoptosis even though CHOP is regarded to be a mediator of ER-stress-induced apoptosis. In conclusion, metformin induces distinct ER stress pathways in cardiomyocytes and our results indicate that CHOP is not necessarily a mediator of apoptosis. Metformin might exert its cardioprotective effect through selective activation of ERSS pathways in the cardiomyocyte.

A novel method for processing resin-embedded specimens with metal implants for immunohistochemical labelling

A major technical problem in the processing of resin-embedded tissues is the adhesion of the tissue sample on glass slides for immunohistochemical labelling. We therefore established a novel protocol for processing such specimens with improved attachment of the tissue sample during resin removal (deplastification). In order to demonstrate the feasibility of the procedure we employed a panel of smooth muscle cell maturation markers. The technique makes use of a silicone glue (Elastosil E41; Wacker Chemie, München, Germany) to attach the tissue samples to the glass slides. This allows resin dissolution in xylene/2-methoxyethylacetate without detachment of the sample from the slide. Our results demonstrate successful immunohistochemical labelling with primary antibodies directed against: smooth muscle actin, smooth muscle myosin, h-caldesmon, desmin, vimentin and von Willebrand factor. In conclusion, we have established a new and successful method for resin-embedded sample adhesion on glass slides. The developed protocol is feasible for investigation of cells which are involved in intimal proliferation following stent implantation.

Down-regulation of Ku 70 and Ku 80 mRNA expression in transitional cell carcinomas of the urinary bladder related to tumor progression.

DNA-dependent protein kinase (DNA-PK) containing the regulatory subunits Ku 70 and Ku 80 plays a prominent role in the repair of double-stranded DNA breaks by a nonhomologous end-joining pathway maintaining genomic stability. In an attempt to elucidate the significance of the DNA-PK complex for human urothelial carcinogenesis, the expression of Ku 70 and Ku 80 was studied in 71 transitional cell carcinomas (TCC) of the urinary bladder of various grades and stages, and in relation to lifestyle and occupational bladder cancer risk factors. To analyse the mRNA expression of Ku 70 and Ku 80, real-time quantitative reverse transcription-polymerase chain reaction was used and the protein expression assessed by immunohistochemistry. Advanced high-grade, high-stage TCC expressed the mRNA of Ku 70 and Ku 80 at a lower level than superficial low-grade, low-stage carcinomas, suggesting down-regulation of the Ku system to be associated with progression of bladder cancer from a low to a high malignant potential. The protein expression of Ku 70 and Ku 80 was closely related and decreased consistently with increasing grades and stages, paralleling the expression of the mRNA. Among hazardous environmental bladder cancer risk factors, heavy consumption of coffee was associated with a twofold decreased Ku 70 and Ku 80 mRNA expression, whereas tobacco smoke did not substantially affect the activity of the Ku system, except for a trend towards a dose-response relationship in the expression of Ku 70 mRNA. There is some evidence that exposure to polycyclic hydrocarbons, paints and lacquer, and stone dust may modify the expression of Ku 70 mRNA. Although the underlying molecular genetic pathways are not yet clearly understood, our data indicate that down-regulation of the Ku system promotes progression of urothelial carcinogenesis to a more malignant and aggressive clinical behavior, presumably as a result of an impaired capacity for DNA repair.

Gene expression of the Na-Ca2+ exchanger, SERCA2a and calsequestrin after myocardial ischemia in the neonatal rabbit heart.

Background: Neonatal hearts are less susceptible to developing myocardial dysfunction after hypoxia and/or ischemia than adult hearts. Differences in intracellular calcium homeostasis may be responsible for reduced calcium overload of the immature myocardium leading to the observed protection against ischemia. Objective: To assess differences in baseline and post-ischemic gene expression of calcium handling proteins after ischemia in neonatal and adult rabbit hearts. Methods: We used isolated antegrade perfused rabbit hearts (age 2 days, 28 days, n = 32), which were exposed to ischemia and hypothermia simulating myocardial stunning comparable to neonatal asphyxia. Gene and protein expression of the sodium–calcium exchanger (NCX), the sarco-endoplasmatic reticulum Ca2+-ATPase 2a (SERCA) and calsequestrin (CSQ) were measured using quantitative real-time PCR and Western blotting. Results: After ischemia and reperfusion in neonatal and adult hearts, a significant decrease in myocardial performance was recorded. At the mRNA level, significant differences in the baseline expression of NCX, SERCA and CSQ between neonatal and adult hearts were observed. In neonatal post-ischemic hearts, NCX and CSQ expression were significantly higher at the mRNA level than in controls. In contrast, SERCA expression remained unchanged in neonatal hearts and decreased in adult hearts compared to the non-ischemic controls. Conclusion: These findings suggest that changes in gene expression of calcium handling proteins may be involved in the different susceptibility of neonatal compared to adult hearts to developing myocardial dysfunction after ischemia.

Characterization of maturation of neuronal voltage-gated sodium channels SCN1A and SCN8A in rat myocardium.

BackgroundSodium channels predominantly expressed in brain are expressed in myocardial tissue and play an important role in cardiac physiology. Alterations of sodium channels are known to result in neurological disease in infancy and childhood. It will be of interest to study the expression of brain-type sodium channels in the developing myocardium.MethodsThe expression of neuronal sodium channels (SCN1A, SCN8A) and the cardiac isoform SCN5A in the developing rat myocardium was studied by rtPCR, Western blot, and immunohistochemistry at different stages of antenatal and postnatal development.ResultsSignificant changes of sodium channel expression during development were detected. Whereas SCN5A RNA increased to maximum levels on day 21 after birth, the highest SCN1A RNA levels were detected on day 1 to 7 after birth. SCN8A RNA was maximally expressed during embryonic development. At the protein level, the amount of SCN5A protein increased along with the RNA level. SCN1A protein level decreased after birth in contrast to RNA expression. Western blot could not detect SCN8A protein in the myocardium at any stage of development. Immunohistochemistry however proved the presence of SCN8A protein in the developing rat myocardium.ConclusionsHeart- and brain-type sodium channels are differentially expressed during ontogenesis. The high expression level of SCN1A in the perinatal period and early infancy indicates its importance in preserving a regular cardiac rhythm in this early phase of life. Altered regulation of sodium channels might result in severe cardiac rhythm disturbances.

Abundant Expression of Spliced HDM2 in Hodgkin Lymphoma Cells does not Interfere with p14ARF and p53 Binding

Abstract Recently, comparative genomic hybridization (CGH)- and fluorescence in situ hybridization (FISH)-analyses of native Hodgkin and Reed-Sternberg (H&RS) cells extracted from Hodgkin lymphoma (HL) revealed a recurrent amplification of the HDM2 locus on chromosome 12. HDM2 is known to target, inactivate and to degrade p53. Wild type (wt) p53 protein is detected in high levels in HL. Simultaneously, stabilized wt p53 and spliced hdm2 transcripts have been observed in different tumors. Therefore, we examined the expression and structure of HDM2 in HL cell lines and possible effects on components of the p53 pathway. DNA integrity and induction potential of p53 was verified by DNA sequencing and detection of potential effector proteins (p21WAF/CIP, HDM2) using immunofluorescence, respectively. All HL cell lines show an overexpression of HDM2 protein. Furthermore, several different spliced hdm2 transcripts (mdm-sv) including five new variants lacking a functional p53 binding site were characterized. If expressed, corresponding proteins were shown to be not restricted to the nucleus. Co-localization of the potential binding partners HDM2/p14ARF and HDM2/p53 was found in HL cell lines. We suggest that HDM2-sv have no significant disturbing influence on the interaction of these proteins.