Aortic microcalcification is associated with elastin fragmentation in Marfan syndrome

Marfan syndrome (MFS) is a connective tissue disorder in which aortic rupture is the major cause of death. MFS patients with an aortic diameter below the advised limit for prophylactic surgery (<5 cm) may unexpectedly experience an aortic dissection or rupture, despite yearly monitoring. Hence, there is a clear need for improved prognostic markers to predict such aortic events. We hypothesize that elastin fragments play a causal role in aortic calcification in MFS, and that microcalcification serves as a marker for aortic disease severity. To address this hypothesis, we analysed MFS patient and mouse aortas. MFS patient aortic tissue showed enhanced microcalcification in areas with extensive elastic lamina fragmentation in the media. A causal relationship between medial injury and microcalcification was revealed by studies in vascular smooth muscle cells (SMCs); elastin peptides were shown to increase the activity of the calcification marker alkaline phosphatase (ALP) and reduce the expression of the calcification inhibitor matrix GLA protein in human SMCs. In murine Fbn1C1039G/+ MFS aortic SMCs, Alpl mRNA and activity were upregulated as compared with wild-type SMCs. The elastin peptide-induced ALP activity was prevented by incubation with lactose or a neuraminidase inhibitor, which inhibit the elastin receptor complex, and a mitogen-activated protein kinase kinase-1/2 inhibitor, indicating downstream involvement of extracellular signal-regulated kinase-1/2 (ERK1/2) phosphorylation. Histological analyses in MFS mice revealed macrocalcification in the aortic root, whereas the ascending aorta contained microcalcification, as identified with the near-infrared fluorescent bisphosphonate probe OsteoSense-800. Significantly, microcalcification correlated strongly with aortic diameter, distensibility, elastin breaks, and phosphorylated ERK1/2. In conclusion, microcalcification co-localizes with aortic elastin degradation in MFS aortas of humans and mice, where elastin-derived peptides induce a calcification process in SMCs via the elastin receptor complex and ERK1/2 activation. We propose microcalcification as a novel imaging marker to monitor local elastin degradation a

The MEF2 transcriptional target DMPK induces loss of sarcomere structure and cardiomyopathy

Aims The pathology of heart failure is characterized by poorly contracting and dilated ventricles. At the cellular level, this is associated with lengthening of individual cardiomyocytes and loss of sarcomeres. While it is known that the transcription factor myocyte enhancer factor-2 (MEF2) is involved in this cardiomyocyte remodelling, the underlying mechanism remains to be elucidated. Here, we aim to mechanistically link MEF2 target genes with loss of sarcomeres during cardiomyocyte remodelling. Methods Neonatal rat cardiomyocytes overexpressing MEF2 elongated and lost their sarcomeric structure. We identified and results myotonic dystrophy protein kinase (DMPK) as direct MEF2 target gene involved in this process. Adenoviral overexpression of DMPK E, the isoform upregulated in heart failure, resulted in severe loss of sarcomeres in vitro, and transgenic mice overexpressing DMPK E displayed disruption of sarcomere structure and cardiomyopathy in vivo. Moreover, we found a decreased expression of sarcomeric genes following DMPK E gain-of-function. These genes are targets of the transcription factor serum response factor (SRF) and we found that DMPK E acts as inhibitor of SRF transcriptional activity. Conclusion Our data indicate that MEF2-induced loss of sarcomeres is mediated by DMPK via a decrease in sarcomeric gene expression by interfering with SRF transcriptional activity. Together, these results demonstrate an unexpected role for DMPK as a direct mediator of adverse cardiomyocyte remodelling and heart failure

Orphan nuclear receptor Nur77 affects cardiomyocyte calcium homeostasis and adverse cardiac remodelling

Distinct stressors may induce heart failure. As compensation, β-adrenergic stimulation enhances myocardial contractility by elevating cardiomyocyte intracellular Ca2+ ([Ca2+]i). However, chronic β-adrenergic stimulation promotes adverse cardiac remodelling. Cardiac expression of nuclear receptor Nur77 is enhanced by β-adrenergic stimulation, but its role in cardiac remodelling is still unclear. We show high and rapid Nur77 upregulation in cardiomyocytes stimulated with β-adrenergic agonist isoproterenol. Nur77 knockdown in culture resulted in hypertrophic cardiomyocytes. Ventricular cardiomyocytes from Nur77-deficient (Nur77-KO) mice exhibited elevated diastolic and systolic [Ca2+]i and prolonged action potentials compared to wild type (WT). In vivo, these differences resulted in larger cardiomyocytes, increased expression of hypertrophic genes

Macrophage-specific overexpression of group IIa sPLA2 increases atherosclerosis and enhances collagen deposition

Atherosclerosis is a chronic inflammatory disease of the vessel wall characterized by the accumulation of lipid-laden macrophages and fibrotic material. The initiation of the disease is accompanied by the accumulation of modified lipoproteins in the vessel wall. Group IIa secretory phospholipase A2 (sPLA2 IIa) is a key candidate player in the enzymatic modification of low density lipoproteins. To study the role of sPLA2 IIa in macrophages during atherogenesis, transgenic mice were generated using the human sPLA2 IIa gene and the CD11b promoter. Bone marrow transplantation to LDL receptor-deficient mice was performed to study sPLA2 IIa in atherosclerosis. After 10 weeks of high-fat diet, mice overexpressing sPLA2 IIa in macrophages showed 2.3-fold larger lesions compared with control mice. Pathological examination revealed that sPLA2 IIa-expressing mice had increased collagen in their lesions, independent of lesion size. However, smooth muscle cells or fibroblasts in the lesions were not affected. Other parameters studied, including T-cells and cell turnover, were not significantly affected by overexpression of sPLA2 IIa in macrophages. These data clearly show that macrophage sPLA2 IIa is a proatherogenic factor and suggest that the enzyme regulates collagen production in the plaque and thus fibrotic cap developmen

Macrophage secretory phospholipase A(2) group X enhances anti-inflammatory responses, promotes lipid accumulation, and contributes to aberrant lung pathology

Secreted phospholipase A2 group X (sPLA(2)-X) is one of the most potent enzymes of the phospholipase A(2) lipolytic enzyme superfamily. Its high catalytic activity toward phosphatidylcholine (PC), the major phospholipid of cell membranes and low-density lipoproteins (LDL), has implicated sPLA(2)-X in chronic inflammatory conditions such as atherogenesis. We studied the role of sPLA(2)-X enzyme activity in vitro and in vivo, by generating sPLA(2)-X-overexpressing macrophages and transgenic macrophage-specific sPLA(2)-X mice. Our results show that sPLA(2)-X expression inhibits macrophage activation and inflammatory responses upon stimulation, characterized by reduced cell adhesion and nitric oxide production, a decrease in tumor necrosis factor (TNF), and an increase in interleukin (IL)-10. These effects were mediated by an increase in IL-6, and enhanced production of prostaglandin E-2 (PGE(2)) and 15-deoxy-Delta 12,14-prostaglandin J(2) (PGJ(2)). Moreover, we found that overexpression of active sPLA(2)-X in macrophages strongly increases foam cell formation upon incubation with native LDL but also oxidized LDL (oxLDL), which is mediated by enhanced expression of scavenger receptor CD36. Transgenic sPLA(2)-X mice died neonatally because of severe lung pathology characterized by interstitial pneumonia with massive granulocyte and surfactant-laden macrophage infiltration. We conclude that overexpression of the active sPLA(2)-X enzyme results in enhanced foam cell formation but reduced activation and inflammatory responses in macrophages in vitro. Interestingly, enhanced sPLA(2)-X activity in macrophages in vivo leads to fatal pulmonary defects, suggesting a crucial role for sPLA(2)-X in inflammatory lung disease

Cardiomyocyte-specific miRNA-30c over-expression causes dilated cardiomyopathy

MicroRNAs (miRNAs) regulate many aspects of cellular function and their deregulation has been implicated in heart disease. MiRNA-30c is differentially expressed in the heart during the progression towards heart failure and in vitro studies hint to its importance in cellular physiology. As little is known about the in vivo function of miRNA-30c in the heart, we generated transgenic mice that specifically overexpress miRNA-30c in cardiomyocytes. We show that these mice display no abnormalities until about 6 weeks of age, but subsequently develop a severely dilated cardiomyopathy. Gene expression analysis of the miRNA-30c transgenic hearts before onset of the phenotype indicated disturbed mitochondrial function. This was further evident by the downregulation of mitochondrial oxidative phosphorylation (OXPHOS) complexes III and IV at the protein level. Taken together these data indicate impaired mitochondrial function due to OXPHOS protein depletion as a potential cause for the observed dilated cardiomyopathic phenotype in miRNA-30c transgenic mice. We thus establish an in vivo role for miRNA-30c in cardiac physiology, particularly in mitochondrial functio

Differentially regulated pathways at 4 weeks of age.

<p>Significantly down- and upregulated pathways based on the microarray expression data of left ventricular tissue in line A. Count represents the number of differentially expressed genes and Genes is the total number of genes in the given pathway.</p

MiRNA-30c transgenic mice have a mitochondrial phenotype.

<p>(a) Western blot of mitochondrial OXPHOS proteins from wildtype and transgenic littermates, before the onset of the phenotype, at 4 weeks of age in line B. (b) Quantification of western blots of mitochondrial OXPHOS proteins of panel A (N = 6). (c) Western blot of mitochondrial OXPHOS proteins in line B at 10 weeks of age, after DCM starts to develop. (d) Overview images of electron microscopy from wildtype (left) and miRNA-30c TG mice (right) of line B. Mitochondria are indicated with a black arrowhead. (e) Quantification of mitochondrial area (N = 3, 18 images per heart) at 4 weeks of age. (f) Quantification of mitochondrial to genomic DNA ratio by qPCR (N = 4) at 4 weeks of age in line B. (g) Mitochondrial citrate synthase activity at 4 weeks of age (N = 4) in line B. Error bars represent s.e.m. and * denotes a p-value ≤0.05.</p

MiRNA-30c expression in the heart and the generation of αMHC-miRNA-30c transgenic mice (miRNA-30c TG).

<p>(a) MiRNA-30c in situ hybridization on adult wildtype hearts shows expression in the nuclei of both cardiomyocytes (black arrowheads) and interstitial cells (grey arrowheads). The cytoplasm is also miRNA-30c positive. (b) Schematic overview of the miRNA-30c overexpression construct used for the generation of transgenic mice. (c) Northern blot for miRNA-30c in wildtype and transgenic littermates in line B at 8 weeks of age. U6 was used as a loading control and shows similar loading. (d) Quantification of miRNA-30c overexpression by qPCR in both transgenic lines at 4 weeks of age (N≥6). (e) MiRNA-30c expression in left en right ventricular tissue of line B at 4 weeks of age (N = 6). Error bars represent s.e.m. and * denotes a p-value ≤0.05.</p

MiRNA-30c transgenic mice develop severely dilated cardiomyopathy.

<p>(a) Kaplan-Meier survival curve for both transgenic lines. We observed no mortality in wildtype littermates. 50% mortality was reached at 37 and 21 weeks for line A and B, respectively. (b) Transgenic mice of both lines develop dilated hearts with enlarged right atria during the end-stage of disease progression. In line A this occurs after an age of 12 weeks, while in line B the first signs of dilation are noted at 6 weeks of age. .(c and d) Cardiac function as determined by echocardiography in line B at 6 weeks of age (N = 3). LVID;d/TL denotes LV internal diameter during diastole, corrected for tibia length. LVID;s/TL denotes LV internal diameter during systole, corrected for tibia length. Differences between LVID;d/TL and LVID;s/TL were analysed by 2-way ANOVA for repeated measures (diagonal line indicates significant interaction effect). (e) Heart weight corrected for tibia length shows no significant difference between wildtype and miRNA-30c TG at 6 weeks of age in line B (N = 3). (f) ANF mRNA expression as evaluated by qPCR (N≥3) shows a significant increase in transgenic mice in line B. Error bars represent s.e.m. and * denotes a p-value ≤0.05.</p