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The principal regulator of cellular response to low oxygen is hypoxia-inducible factor (HIF)-1, which is stabilized in several forms of heart failure. Our laboratory developed a mouse strain in which a stable form of HIF-1 can be inducibly expressed in cardiomyocytes. Strikingly, these mice show a rapid decrease in cardiac contractility and a rapid loss of SERCA2 protein, which is also seen in heart failure. Interestingly, while the SERCA2 transcript decreased, it did not fully account for the observed decrease in protein. We therefore investigated whether HIF-1-regulated microRNA could impair SERCA translation. Multiple screening analyses identified the microRNA miR-29c to be substantially upregulated upon HIF-1 induction and to have complementarity to SERCA, and therefore be a potential regulator of SERCA2 expression in hypoxia. Subsequent evaluation confirmed that miR-29c reduced SERCA2 expression and Ca2+ reuptake. Additionally, administration of an antagonist sequence (antimir) improved cardiac contractility and SERCA2 expression in HIF transgenic mice. To extend the significance of these findings, we examined miR-29c expression in physiological hypoxia. Surprisingly, miR-29c decreased in these settings. We also treated mice with antimir before infarction to see if further suppression of miR-29c could improve cardiac function. While no improvement in contractility or SERCA2 was observed, reduction of heart size after infarction indicated that the antimir could modulate cardiac physiology. These results demonstrate that while a HIF-1- regulated microRNA, miR-29c, can reduce SERCA2 expression and contractility, additional factors in the ischemic milieu may limit these effects. Efforts to develop miRNA-based therapies will need to explore and account for these additional countervailing effects. [ABSTRACT FROM AUTHOR]

Most cases of congenital obstructive nephropathy are the result of ureteropelvic junction obstructions, and despite their high prevalence, we have a poor understanding of their etiology and scarcity of genetic models. The eight-protein exocyst complex regulates polarized exocytosis of intracellular vesicles in a large variety of cell types. Here we report generation of a conditional knockout mouse for Sec10, a central component of the exocyst, which is the first conditional allele for any exocyst gene. Inactivation of Sec10 in ureteric bud-derived cells using Ksp1.3-Cre mice resulted in severe bilateral hydronephrosis and complete anuria in newborns, with death occurring 6–14 hours after birth. Sec10FL/FL;Ksp-Cre embryos developed ureteropelvic junction obstructions between E17.5 and E18.5 as a result of degeneration of the urothelium and subsequent overgrowth by surrounding mesenchymal cells. The urothelial cell layer that lines the urinary tract must maintain a hydrophobic luminal barrier again urine while remaining highly stretchable. This barrier is largely established by production of uroplakin proteins that are transported to the apical surface to establish large plaques. By E16.5, Sec10FL/FL;Ksp-Cre ureter and pelvic urothelium showed decreased uroplakin-3 protein at the luminal surface, and complete absence of uroplakin-3 by E17.5. Affected urothelium at the UPJ showed irregular barriers that exposed the smooth muscle layer to urine, suggesting this may trigger the surrounding mesenchymal cells to overgrow the lumen. Findings from this novel mouse model show Sec10 is critical for the development of the urothelium in ureters, and provides experimental evidence that failure of this urothelial barrier may contribute to human congenital urinary tract obstructions. [ABSTRACT FROM AUTHOR]

A close interconnection between nutrient metabolism and virulence factor expression contributes to the pathophysiology of Pseudomonas aeruginosa as a successful pathogen. P. aeruginosa fatty acid (FA) degradation is complicated with multiple acyl-CoA synthetase homologs (FadDs) expressed in vivo in lung tissue during cystic fibrosis infections. The promoters of two genetically linked P. aeruginosa fadD genes (fadD1 and fadD2) were mapped and northern blot analysis indicated they could exist on two different transcripts. These FadDs contain ATP/AMP signature and FA-binding motifs highly homologous to those of the Escherichia coli FadD. Upon introduction into an E. coli fadD-/fadR- double mutant, both P. aeruginosa fadDs functionally complemented the E. coli fadD-/fadR- mutant, allowing degradation of different chain-length FAs. Chromosomal mutagenesis, growth analysis, induction studies, and determination of kinetic parameters suggested that FadD1 has a substrate preference for long-chain FAs while FadD2 prefers shorter-chain FAs. When compared to the wild type strain, the fadD2 mutant exhibited decreased production of lipase, protease, rhamnolipid and phospholipase, and retardation of both swimming and swarming motilities. Interestingly, fadD1 mutant showed only increased swarming motility. Growth analysis of the fadD mutants showed noticeable deficiencies in utilizing FAs and phosphatidylcholine (major components of lung surfactant) as the sole carbon source. This defect translated into decreased in vivo fitness of P. aeruginosa in a BALB/c mouse lung infection model, supporting the role of lipids as a significant nutrient source for this bacterium in vivo. [ABSTRACT FROM AUTHOR]

Background: The response to hypoxia in tissues is regulated by the heterodimeric transcription factor Hypoxia Inducible Factor-1 (HIF-1). Methodology/Principal Findings: We have created a strain of mice with inducible cardiomyocyte-specific expression of a mutated, oxygen-stable, form of HIF-1α. Cardiac function steadily decreased with transgene expression, but recovered after the transgene was turned off. Using long-oligo microarrays, we identified 162 transcripts more than 3-fold dysregulated in these hearts after transgene expression. Among the down-regulated genes the transcript for SERCA was reduced 46% and the protein 92%. This led us to an evaluation of calcium flux that showed diminished reuptake of cytoplasmic calcium in myocytes from these hearts, suggesting a mechanism for cardiac dysfunction. Conclusions/Significance: These results provide a deeper understanding of transcriptional activity of HIF in the heart, and show that enhanced HIF-1 activity is sufficient to cause contractile dysfunction in the adult heart. HIF is stabilized in the myocardium of patients with ischemic cardiomyopathy, and our results suggest that HIF could be contributing directly to the contractile dysfunction in this disease. [ABSTRACT FROM AUTHOR]

The article discusses various reports published within the issue, including one about the process of low energy permeabilization, and a study that examines the role of endocytic pathways when low acoustic pressure ultrasound is applied with microbubbles.

Introduction: PRKCBP1 is known to cooperatively bind to acetylated and methylated nucleosomes through its bromo- and PHD domains. It can modulate transcription and cancer progression by histone methylation modifications affecting enhancer and promoter regions of genes. Mutations and dysregulation of PRKCBP1 have been found in multiple cancer types, however its role in the cardiac myocardium is unexplored. Hypoxia-inducible factor 1α (HIF-1α) upregulation and stabilization is a common feature of both cancer and myocardial ischemia, promoting cellular functions such as proliferation, glucose metabolism and angiogenesis. Here we investigated the role of PRKCBP1 in a model of cardiac HIF-1 activation. Hypothesis: PRKCBP1 inhibits the HIF-1 response in hypoxic heart by decreasing enhancer activity of HIF-1 target genes. Methods & Results: We expressed an inducible, cardiac-specific, oxygen-stable form of HIF-1α in mice that express increased PRKCBP1 (P+). Compared to wild-type (WT) mice, P+ mice did not exhibit the expected HIF-1 phenotype of increased heart weight to body weight (6.4±0.3 vs. 4.8±0.2 vs. 5.1±0.3 mg/g, WT vs. P+ vs. uninduced; p<0.05) with reduced ventricular function and associated chamber dilation. Semi-quantitative real-time PCR analysis of HL-1 and H9C2 cardiac cell lines transfected with CMV-driven expression plasmids for PRKCBP1 and oxygen-stable HIF-1α showed striking reduction of multiple HIF-1 target genes such as PDK1 (45% reduction) compared to the HIF-1α plasmid alone. RNAi mediated knockdown of PRKCBP1 removed this negative regulation (65% increase). Bioinformatic analysis of human PRKCBP1 and HIF-1α ChIP-seq data indicates that PRKCBP1 binds to the enhancer of 78% of HIF-1 regulated genes. Furthermore, global DNA 5-mC is substantially reduced in P+ heart cells compared to WT, suggesting an additional mechanism of gene regulation. Conclusion: We have discovered a new regulator of HIF-1 action that modifies the hypoxic response, likely through chromatin remodeling. This new form of regulation may modify the pathophysiology of ischemia and provide new targets for therapy. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates gene response during hypoxia, including regulating angiogenesis, the formation of new blood vessels, and glycolytic metabolism. This heterodimeric protein is composed of HIF-1α and HIF-1α subunits and consists of a basic helix-loop-helix-PAS domain. HIF-1α is expressed constitutively, whereas HIF-1α is regulated by oxygen concentration and inducible by hypoxia. HIF- 1α possesses an oxygen-dependent degradation domain, which causes it to be targeted for rapid proteosomal degradation during normoxia; hypoxia slows the destruction of HIF-1α. Since HIF-1 plays an important role in angiogenesis, it is a subject of interest in the heart where hypoxia occurs due to coronary artery disease, the leading cause of morbidity and mortality on the Western world. This research project involved the over expression of an oxygenstable form of HIF-1α in the murine heart, to identify the genes regulated by HIF-1. In doing so we are able to determine how HIF-1 directly regulates angiogenesis and glucose metabolism. The study hypothesizes that HIF-1 promotes the expression of genes to stimulate angiogenesis and shifts the citric acid cycle from aerobic to anaerobic glycolysis. To identify the genes, mouse models, chromatin immunoprecipitation (ChIP) assays and other validation techniques, such as electrophoresis mobility shift assay (EMSA) gels and computational analyses were used. Bi-transgenic mice were used as animal models, with a cardiac specific promoter and a mutated form of human HIF-1α, under Tet-off control. The Tet-off system controls gene expression with the use of Doxycycline as an inhibitor to promoter complex binding, preventing the transcription of the transgene. A ChIP assay was used to identify DNA regions that are bound to transcription factors, thereby determining which genes are directly regulated. Since HIF-1α is being over expressed, the DNA sequence identified is composed of the promoter regions regulated by HIF-1. One gene of particular interest that was identified via ChIP analysis was pkc binding protein 1 isoform 2, which may play a role in the regulation of HIF-1. This gene has not been fully characterized, but has been implicated in HIF regulation previously. Genes will be further verified by using additional EMSA and computational analyses. Identifying the genes that HIF-1 directly binds to will give insight to the role that HIF-1 plays in ischemia and coronary artery disease. This will provide important information to assist in the development of HIF-1 gene therapy. [ABSTRACT FROM AUTHOR]

The chromatin immunoprecipitation (ChIP) assay is a versatile technique used to evaluate the association of proteins with specific DNA regions both in vivo and in vitro. This assay can be used to identify proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome associated with a particular protein. The ChIP assay can also be used to analyze binding of transcription factors, transcription cofactors, DNA replication factors, and DNA repair proteins. Here we describe a useful ChIP-qPCR protocol to examine the interaction of NFkB with the VEGF promoter in adult rat primary cardiomyocytes that have been mechanically stretched after attaching to the extracellular matrix protein laminin.

Background: The heterodimeric, oxygen-sensitive transcription factor Hypoxia Inducible Factor-1 (HIF-1) orchestrates angiogenesis and plays a key role in the response to ischemia and the growth of cancers., Methods: We developed a transgenic mouse line in which expression of an oxygen-stable HIF-1α construct was controlled by a tetracycline-responsive promoter. HIF-1α expression was induced for up to 28 days in adult mouse heart, resulting in angiogenesis and progressive ventricular dysfunction., Results: Gross inspection demonstrated enlarged hearts with large epicardial vessels with prominent side branches. Perfusion curves obtained by ultrasound contrast analysis demonstrated a significant increase in the myocardial red cell volume after 28 days of HIF-1α expression. Corrosion casts of cardiac vessels were made with a new low-viscosity resin that can fill the vasculature down to the level of the capillaries. Scanning electron microscopy of these casts reveal "lakes" of capillaries forming off of larger vessels after HIF expression, and support the rapid formation of mature neovascularization. Pro-angiogenic factors DLL-4, Notch-1, and PDGF-β, were evaluated by immunohistochemistry and Western blots, and support a pattern of progressive functional neoangiogenesis., Conclusions: This study demonstrates the structural characteristics of HIF-directed angiogenesis and supports the utility of manipulation of HIF signaling to enhance perfusion and treat ischemia.

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.