Frameshift mutations in the TTN gene encoding titin are a major cause for inherited forms of dilated cardiomyopathy (DCM), a heart disease characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. To date, there are no specific treatment options for DCM patients but heart transplantation. Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326. Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression. AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals. These results demonstrate that disruption of the titin reading frame due to a truncating DCM mutation can be restored by exon skipping in both patient cardiomyocytes in vitro and mouse heart in vivo, indicating RNA-based strategies as a potential treatment option for DCM.

Background: Mesenchymal stem cells (MSC) contribute to cardiac repair after myocardial injury. Underlying molecular mechanisms remain unexplored. Results: Activated platelets inhibit recruitment of MSC to apoptotic cardiac myocytes and fibroblasts via HMGB1/TLR-4-mediated down-regulation of HGF receptor MET. Conclusion:We identify a novel mechanism by which platelets impair MSC migration to damaged cardiac cells. Significance: The cross-talk between platelets and MSC might be critical for myocardial repair.

Objective— Atherosclerosis, an inflammatory disease of arterial vessel walls, requires migration and matrix metalloproteinase (MMP)-9–dependent invasion of monocytes/macrophages into the vascular wall. MMP-9 expression is stimulated by transcription factor nuclear factor-κB, which is regulated by inhibitor κB (IκB) and thus IκB kinase. Regulators of nuclear factor-κB include serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored involvement of SGK1 in vascular inflammation and atherogenesis. Approach and Results— Gene-targeted apolipoprotein E (ApoE)–deficient mice without ( apoe −/− sgk1 +/+ ) or with ( apoe −/− sgk1 −/− ) additional SGK1 knockout received 16-week cholesterol-rich diet. According to immunohistochemistry atherosclerotic lesions in aorta and carotid artery, vascular CD45 + leukocyte infiltration, Mac-3 + macrophage infiltration, vascular smooth muscle cell content, MMP-2, and MMP-9 positive areas in atherosclerotic tissue were significantly less in apoe −/− sgk1 −/− mice than in apoe −/− sgk1 +/+ mice. As determined by Boyden chamber, thioglycollate-induced peritonitis and air pouch model, migration of SGK1-deficient CD11b + F4/80 + macrophages was significantly diminished in vitro and in vivo. Zymographic MMP-2 and MMP-9 production, MMP-9 activity and invasion through matrigel in vitro were significantly less in sgk1 −/− than in sgk1 +/+ macrophages and in control plasmid–transfected or inactive K127N SGK1-transfected than in constitutively active S422D SGK1-transfected THP-1 cells. Confocal microscopy revealed reduced macrophage number and macrophage MMP-9 content in plaques of apoe −/− sgk1 −/− mice. In THP-1 cells, MMP-inhibitor GM6001 (25 μmol/L) abrogated S422D SGK1-induced MMP-9 production and invasion. According to reverse transcription polymerase chain reaction, MMP-9 transcript levels were significantly reduced in sgk1 −/− macrophages and strongly upregulated in S422D SGK1-transfected THP-1 cells compared with control plasmid–transfected or K127N SGK1-transfected THP-1 cells. According to immunoblotting and confocal microscopy, phosphorylation of IκB kinase and inhibitor κB and nuclear translocation of p50 were significantly lower in sgk1 −/− macrophages than in sgk1 +/+ macrophages and significantly higher in S422D SGK1-transfected THP-1 cells than in control plasmid–transfected or K127N SGK1-transfected THP-1 cells. Treatment of S422D SGK1-transfected THP-1 cells with IκB kinase-inhibitor BMS-345541 (10 μmol/L) abolished S422D SGK1-induced increase of MMP-9 transcription and gelatinase activity. Conclusions— SGK1 plays a pivotal role in vascular inflammation during atherogenesis. SGK1 participates in the regulation of monocyte/macrophage migration and MMP-9 transcription via regulation of nuclear factor-κB.

Tissue damage due to apoptotic or necrotic cell death typically initiates distinct cellular responses, leading either directly to tissue repair and regeneration or to immunological processes first, to clear the site, for example, of potentially damage-inducing agents. Mesenchymal stem cells (MSC) as well as immature dendritic cells (iDC) and monocytes migrate to injured tissues. MSC have regenerative capacity, whereas monocytes and iDC have a critical role in inflammation and induction of immune responses, including autoimmunity after tissue damage. Here, we investigated the influence of apoptotic and necrotic cell death on recruitment of MSC, monocytes and iDC, and identified hepatocyte growth factor (HGF) and the alarmin high mobility group box 1 (HMGB1) as key factors differentially regulating these migratory responses. MSC, but not monocytes or iDC, were attracted by apoptotic cardiomyocytic and neuronal cells, whereas necrosis induced migration of monocytes and iDC, but not of MSC. Only apoptotic cell death resulted in HGF production and HGF-mediated migration of MSC towards the apoptotic targets. In contrast, HMGB1 was predominantly released by the necrotic cells and mediated recruitment of monocytes and iDC via the receptor of advanced glycation end products. Moreover, necrotic cardiomyocytic and neuronal cells caused an HMGB1/toll-like receptor-4-dependent inhibition of MSC migration towards apoptosis or HGF, while recruitment of monocytes and iDC by necrosis or HMGB1 was not affected by apoptotic cells or HGF. Thus, the type of cell death differentially regulates recruitment of either MSC or monocytes and iDC through HGF and HMGB1, respectively, with a dominant, HMGB1-mediated role of necrosis in determining tropism after tissue injury.

Sphingosine 1-phosphate (S1P) is a powerful regulator of platelet formation. Enzymes generating S1P include sphingosine kinase 1. The present study thus explored the role of sphingosine kinase 1 in platelet formation and function. Activation-dependent platelet integrin αIIbβ3 activation and secretion of platelets lacking functional sphingosine kinase 1 (sphk1(-/-)) and of wild-type platelets (sphk1(+/+)) were determined utilizing flow cytometry and chronolume luciferin assay. Cytosolic Ca(2+) activity ([Ca(2+)]i) and aggregation were measured using fura-2 fluorescence and aggregometry, respectively. In vitro platelet adhesion and thrombus formation were evaluated using a flow chamber with shear rates of 1,700 s(-1). Activation-dependent increase of [Ca(2+)]i, degranulation (release of alpha and dense granules), integrin αIIbβ3 activation, and aggregation were all significantly increased in sphk1(-/-) platelets compared with sphk1(+/+) platelets. Moreover, while platelet adhesion and thrombus formation under arterial shear rates were significantly augmented in Sphk1-deficient platelets, bleeding time and blood count were unaffected in sphk1(-/-) mice. In conclusion, sphingosine kinase 1 is a powerful negative regulator of platelet function counteracting degranulation, aggregation, and thrombus formation.