Calreticulin Induces Dilated Cardiomyopathy

Background

Calreticulin, a Ca²⁺-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.

Methodology/Principal Findings

We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca²⁺ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca²⁺-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca²⁺-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.

Conclusions/Significance

We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca²⁺ handling genes, gap junction components and left ventricle remodeling.     

Increased Cardiac Myocyte PDE5 Levels in Human and Murine Pressure Overload Hypertrophy Contribute to Adverse LV Remodeling

Background

The intracellular second messenger cGMP protects the heart under pathological conditions. We examined expression of phosphodiesterase 5 (PDE5), an enzyme that hydrolyzes cGMP, in human and mouse hearts subjected to sustained left ventricular (LV) pressure overload. We also determined the role of cardiac myocyte-specific PDE5 expression in adverse LV remodeling in mice after transverse aortic constriction (TAC).

Methodology/Principal Findings

In patients with severe aortic stenosis (AS) undergoing valve replacement, we detected greater myocardial PDE5 expression than in control hearts. We observed robust expression in scattered cardiac myocytes of those AS patients with higher LV filling pressures and BNP serum levels. Following TAC, we detected similar, focal PDE5 expression in cardiac myocytes of C57BL/6NTac mice exhibiting the most pronounced LV remodeling. To examine the effect of cell-specific PDE5 expression, we subjected transgenic mice with cardiac myocyte-specific PDE5 overexpression (PDE5-TG) to TAC. LV hypertrophy and fibrosis were similar as in WT, but PDE5-TG had increased cardiac dimensions, and decreased dP/dt_max and dP/dt_min with prolonged tau (P<0.05 for all). Greater cardiac dysfunction in PDE5-TG was associated with reduced myocardial cGMP and SERCA2 levels, and higher passive force in cardiac myocytes in vitro.

Conclusions/Significance

Myocardial PDE5 expression is increased in the hearts of humans and mice with chronic pressure overload. Increased cardiac myocyte-specific PDE5 expression is a molecular hallmark in hypertrophic hearts with contractile failure, and represents an important therapeutic target.     

Calreticulin affects fibronectin-based cell-substratum adhesion via the regulation of c-Src activity

Calreticulin is an endoplasmic reticulum Ca2+-storage protein, which influences gene expression and cell adhesion. In this study, we show that calreticulin induces fibronectin gene expression and matrix deposition, leading to differences in cell spreading and focal adhesion formation in cells differentially expressing calreticulin. We further show that these effects of calreticulin occur via a c-Src-regulated pathway and that c-Src activity is inversely related to calreticulin abundance. Since c-Src is an important regulator of focal contact turnover, we investigated the effect of c-Src inhibition on cells differentially expressing calreticulin. Inhibition of c-Src rescued the poorly adhesive phenotype of the calreticulin-underexpressing cells in that they became well spread, commenced formation of numerous focal contacts, and deposited a rich fibronectin matrix. Importantly, we show that c-Src activity is dependent on releasable Ca2+ from the endoplasmic reticulum, thus implicating Ca2+-sensitive pathways that are affected by calreticulin in cell-substratum adhesion. We propose that calreticulin affects fibronectin synthesis and matrix assembly via the regulation of fibronectin gene expression. In parallel, calcium-dependent effects of calreticulin on c-Src activity influence the formation and/or stability of focal contacts, which are instrumental in matrix assembly and remodeling.     

A rapid and sensitive method for the quantitation of montelukast in sheep plasma using liquid chromatography/tandem mass spectrometry

A rapid LC-MS/MS method was developed and partially validated for the quantitation of montelukast in spiked sheep plasma. A total run time of 1.5 min was achieved using a short monolithic column and employing a rapid gradient. Sample preparation involved protein precipitation with twofold acetonitrile by volume during which a deuterated internal standard (montelukast D-6) was incorporated. The MRM transitions for montelukast and the deuterated internal standard were 586/422 and 592/427, respectively. A linear dynamic range of 0.25-500 ng/mL with a correlation coefficient of 0.9999 was achieved. Precision was below 5% at all levels except at the LOQ (0.36 ng/mL) which demonstrated an overall of R.S.D. of 8%. Post-column infusion experiments were performed with precipitated plasma matrix and showed minimal interference with the peaks of interest.     

Gene expression dynamics in deer antler: mesenchymal differentiation toward chondrogenesis

Annual re-growth of deer antler represents a unique example of complete organ regeneration. Because antler mesenchymal cells retain their embryonic capacity to develop into cartilage or bone, studying antler development provides a natural system to follow gene expression changes during mesenchymal differentiation toward chondrogenic/osteogenic lineage. To identify novel genes involved either in early events of mesenchymal cell specialization or in robust bone development, we have introduced a 3 K heterologous microarray set-up (deer cDNA versus mouse template). Fifteen genes were differentially expressed; genes for housekeeping, regulatory functions (components of different signaling pathways, including FGF, TGFβ, Wnt), and genes encoding members of the Polycomb group were represented. Expression dynamics for genes are visualized by an expression logo. The expression profile of the gene C21orf70 of unknown function is described along with the effects when over-expressed; furthermore the nuclear localization of the cognate protein is shown. In this report, we demonstrate the particular advantage of the velvet antler model in bone research for: (1) identification of mesenchymal and precartilaginous genes and (2) targeting genes upregulated in robust cartilage development. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Characterisation of the spectrum and genetic dependence of collateral mutations induced by translesion DNA synthesis

Translesion DNA synthesis (TLS) is a fundamental damage bypass pathway that utilises specialised polymerases with relaxed template specificity to achieve replication through damaged DNA. Misinsertions by low fidelity TLS polymerases may introduce additional mutations on undamaged DNA near the original lesion site, which we termed collateral mutations. In this study, we used whole genome sequencing datasets of chicken DT40 and several human cell lines to obtain evidence for collateral mutagenesis in higher eukaryotes. We found that cisplatin and UVC radiation frequently induce close mutation pairs within 25 base pairs that consist of an adduct-associated primary and a downstream collateral mutation, and genetically linked their formation to TLS activity involving PCNA ubiquitylation and polymerase κ. PCNA ubiquitylation was also indispensable for close mutation pairs observed amongst spontaneously arising base substitutions in cell lines with disrupted homologous recombination. Collateral mutation pairs were also found in melanoma genomes with evidence of UV exposure. We showed that collateral mutations frequently copy the upstream base, and extracted a base substitution signature that describes collateral mutagenesis in the presented dataset regardless of the primary mutagenic process. Using this mutation signature, we showed that collateral mutagenesis creates approximately 10–20% of non-paired substitutions as well, underscoring the importance of the process.     

Ovarian carcinoma of low malignant potential treated at the 1(st) Department of Obstetrics and Gynecology Semmelweis University Faculty of Medicine,between 1990 and 2000

The authors analyzed the epidemiologic and histological characteristics and the management of ovarian carcinoma of low malignant potential (LMP) at a university hospital between 1990 and 2000. The authors carried out a retrospective study reviewing hospital charts. Based on the records experience with 29 such tumors is peresented. Of these 20 (74%) were of the serous variety, 7 (26%) were mucinous. LMP tumors accounted for 16% of proliferating epithelial ovarian tumors. They occured at a mean age of 45 years. The LMP tumors were bilateral in 12% of the cases. The majority of patients (87%) with LMP tumors presented with early stage disease. Tumor markers such as CA-125 were not always elevated as in invasive ovarian carcinoma. Laboratory investigations have not demonstrated that these tumors represent an intermediate step between benign ovarian tumors and carcinoma. The recommended therapy is surgical, consisting of total abdominal hysterectomy, bilateral salpingo-oophorectomy, peritoneal washings, and tumor debulking. Conservative surgery consisting of unilateral salpingo-oophorectomy is considered to be an appropriate treatment for young women with early stage LMP ovarian tumors who wish to retain their fertility potential. 50 percent of women who underwent conservative surgery subsequently conceived in this study. There were no recurrences in the study group, so the authors conclude that the long term outcome of LMP tumors is extremely favorable.