Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy.

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.

Pivotal role of serum- and glucocorticoid-inducible kinase 1 in vascular inflammation and atherogenesis.

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.

High-frequency ultrasound-guided disruption of glycoprotein VI-targeted microbubbles targets atheroprogressison in mice.

Targeted contrast-enhanced ultrasound (CEU) using microbubble agents is a promising non-invasive imaging technique to evaluate atherosclerotic lesions. In this study, we decipher the diagnostic and therapeutic potential of targeted-CEU with soluble glycoprotein (GP)-VI in vivo. Microbubbles were conjugated with the recombinant fusion protein GPVI-Fc (MBGPVI) that binds with high affinity to atherosclerotic lesions. MBGPVI or control microbubbles (MBC) were intravenously administered into ApoE(-/-) or wild type mice and binding of the microbubbles to the vessel wall was visualized by high-resolution CEU. CEU molecular imaging signals of MBGPVI were substantially enhanced in the aortic arch and in the truncus brachiocephalicus in ApoE(-/-) as compared to wild type mice. High-frequency ultrasound (HFU)-guided disruption of MBGPVI enhanced accumulation of GPVI in the atherosclerotic lesions, which may interfere with atheroprogression. Thus, we establish targeted-CEU with soluble GPVI as a novel non-invasive molecular imaging method for atherosclerosis. Further, HFU-guided disruption of GPVI-targeted microbubbles is an innovate therapeutic approach that potentially prevents progression of atherosclerotic disease.

Sphingosine kinase 1 (Sphk1) negatively regulates platelet activation and thrombus formation.

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 αIIbß3 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 αIIbß3 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.

Activated platelets interfere with recruitment of mesenchymal stem cells to apoptotic cardiac cells via high mobility group box 1/Toll-like receptor 4-mediated down-regulation of hepatocyte growth factor receptor MET.

Recruitment of mesenchymal stem cells (MSC) following cardiac injury, such as myocardial infarction, plays a critical role in tissue repair and may contribute to myocardial recovery. However, the mechanisms that regulate migration of MSC to the site of tissue damage remain elusive. Here, we demonstrate in vitro that activated platelets substantially inhibit recruitment of MSC toward apoptotic cardiac myocytes and fibroblasts. The alarmin high mobility group box 1 (HMGB1) was released by platelets upon activation and mediated inhibition of the cell death-dependent migratory response through Toll-like receptor (TLR)-4 expressed on the MSC. Migration of MSC to apoptotic cardiac myocytes and fibroblasts was driven by hepatocyte growth factor (HGF), and platelet activation was followed by HMGB1/TLR-4-dependent down-regulation of HGF receptor MET on MSC, thereby impairing HGF-driven MSC recruitment. We identify a novel mechanism by which platelets, upon activation, interfere with MSC recruitment to apoptotic cardiac cells, a process that may be of particular relevance for myocardial repair and regeneration.