reverse transcribed using random oligo-dT primers and a Verso cDNA Synthesis Kit (Thermo Fisher, AB1453) according to the manufacturer's instructions. Real-time PCR was performed using SsoAdvanced SYBR Green (Bio-Rad, 6090), and Rpl7 expression was used for normalization. The following primer sets were used to identify transcripts: collagen 1a1, 5′-AATGGCACGGCTGTGTGCGA and 5′-AACGGGTCCCCTTG-GGCCTT; collagen 3a1, 5′-TCCCCTGGAATCTGTGAATC and 5′-TGAGTCGAATTGGGGAGAAT; periostin, 5′-ACGGAGCTCAGG-GCTGAAGATG and 5′-GTTTGGGCCCTGATCCCGAC.Cell death analysis. At 90% confluence, primary skin fibroblasts were treated with 200 nM staurosporine for 36 hours or vehicle (DMSO). Cell death was determined by the Muse Count & Viability Assay (Millipore, MCH100102) as previously described (62). Briefly, the medium was collected with the trypsin-liberated cells, which were centrifuged and washed twice with PBS and then incubated with the Muse Count & Viability reagent. The cells were then quantified on a Muse cell analyzer (Millipore) at 5,000 counts per sample.Statistics. One-way ANOVA with post hoc Tukey's honest significant difference (HSD) or Student's t test was used to determine statistical significance, depending on the type of data analyzed and number of comparisons. P values of less than 0.05 were considered statistically significant. Averaged data are presented with SEM to indicate variability.Study approval. Mice were observed daily and cages changed weekly by certified veterinary technicians at Cincinnati Children's Hospital Medical Center. Mice were also closely assessed for their well-being, monitored by adequate physical activity and food intake on a daily basis. Housing conditions and husbandry conformed to AAALAC standards as well as the standard guidelines from the NIH Office of Laboratory Animal Welfare (http://grants.nih.gov/grants/olaw/animal_use. htm). The institution also retains ongoing certification by AAALAC.

Inhibiting FN polymerization or cardiac fibroblast gene expression attenuates pathological properties of MFs in vitro and ameliorates adverse cardiac remodeling and fibrosis in an in vivo model of heart failure. Interfering with FN polymerization may be a new therapeutic strategy for treating cardiac fibrosis and heart failure.

Inflammation is a major factor in heart disease. IκB kinase (IKK) and its downstream target NF-κB are regulators of inflammation and are activated in cardiac disorders, but their precise contributions and targets are unclear. We analyzed IKK/NF-κB function in the heart by a gain-of-function approach, generating an inducible transgenic mouse model with cardiomyocyte-specific expression of constitutively active IKK2. In adult animals, IKK2 activation led to inflammatory dilated cardiomyopathy and heart failure. Transgenic hearts showed infiltration with CD11b + cells, fibrosis, fetal reprogramming, and atrophy of myocytes with strong constitutively active IKK2 expression. Upon transgene inactivation, the disease was reversible even at an advanced stage. IKK-induced cardiomyopathy was dependent on NF-κB activation, as in vivo expression of IκBα superrepressor, an inhibitor of NF-κB, prevented the development of disease. Gene expression and proteomic analyses revealed enhanced expression of inflammatory cytokines, and an IFN type I signature with activation of the IFN-stimulated gene 15 (ISG15) pathway. In that respect, IKK-induced cardiomyopathy resembled Coxsackievirus-induced myocarditis, during which the NF-κB and ISG15 pathways were also activated. Vice versa, in cardiomyocytes lacking the regulatory subunit of IKK (IKKγ/NEMO), the induction of ISG15 was attenuated. We conclude that IKK/NF-κB activation in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure by inducing an excessive inflammatory response and myocyte atrophy.transcription factors | transgenic mice

Heart-specific expression of constitutively active FoxO3 leads to reversible heart atrophy. The reversibility of the phenotype suggests a remarkable ability of the adult myocardium to respond to different regulatory cues.