Ischemic heart injury leads to HIF1-dependent differential splicing of CaMK2γ.

Ischemic heart disease is a leading cause of heart failure and hypoxia inducible factor 1 (HIF1) is a key transcription factor in the response to hypoxic injury. Our lab has developed a mouse model in which a mutated, oxygen-stable form of HIF1α (HIF-PPN) can be inducibly expressed in cardiomyocytes. We observed rapid cardiac dilation and loss of contractility in these mice due to lower expression of excitation-contraction coupling genes and reduced calcium flux. As alternative splicing plays an underappreciated role in transcriptional regulation, we used RNA sequencing to search for splicing changes in calcium-handling genes of HIF-PPN hearts and compared them to previous sequencing data from a model of myocardial infarction (MI) to select for transcripts that are modified in a pathological setting. We found overlap between genes differentially expressed in HIF-PPN and post-MI mice (54/131 genes upregulated in HIF-PPN hearts at 1 day and/or 3 days post-MI, and 45/78 downregulated), as well as changes in alternative splicing. Interestingly, calcium/calmodulin dependent protein kinase II, gamma (CAMK2G) was alternatively spliced in both settings, with variant 1 (v1) substantially decreased compared to variants 2 (v2) and 3 (v3). These findings were also replicated in vitro when cells were transfected with HIF-PPN or exposed to hypoxia. Further analysis of CAMK2γ protein abundance revealed only v1 was detectable and substantially decreased up to 7 days post-MI. Rbfox1, a splicing factor of CAMK2G, was also decreased in HIF-PPN and post-MI hearts. Subcellular fractionation showed CAMK2γ v1 was found in the nuclear and cytoplasmic fractions, and abundance decreased in both fractions post-MI. Chromatin immunoprecipitation analysis of HIF1 in post-MI hearts also demonstrated direct HIF1 binding to CAMK2G. CaMK2 is a key transducer of calcium signals in both physiological and pathological settings. The predominantly expressed isoform in the heart, CaMK2δ, has been extensively studied in cardiac injury, but the specific role of CaMK2γ is not well defined. Our data suggest that loss of CaMK2γ after MI is HIF1-dependent and may play an important role in the heart's calcium signaling and transcriptional response to hypoxia.

A Comparison of Focused and Unfocused Ultrasound for Microbubble-Mediated Gene Delivery.

We compared focused and unfocused ultrasound-targeted microbubble destruction (UTMD) for delivery of reporter plasmids to the liver and heart in mice. Optimal hepatic expression was seen with double-depth targeting at 5 and 13 mm in vivo, incorporating a low pulse repetition frequency and short pulse duration. Reporter expression was similar, but the transfection patterns were distinct, with intense foci of transfection using focused UTMD (F-UTMD). We then compared both approaches for cardiac delivery and found 10-fold stronger levels of reporter expression for F-UTMD and observed small areas of intense luciferase expression in the left ventricle. Non-linear contrast imaging of the liver before and after insonation also showed a substantially greater change in signal intensity for F-UTMD, suggesting distinct cavitation mechanisms for both approaches. Overall, similar levels of hepatic transgene expression were observed, but cardiac-directed F-UTMD was substantially more effective. Focused ultrasound presents a new frontier in UTMD-directed gene therapy.

Protein kinase C binding protein 1 inhibits hypoxia-inducible factor-1 in the heart.

AIMS: Hypoxia-inducible factor-1 alpha (HIF-1α) is a key transcription factor responsible for the induction of genes that facilitate adaptation to hypoxia. To study HIF-1 signalling in the heart, we developed a mouse model in which an oxygen-stable form of HIF-1α can be inducibly expressed in cardiac myocytes, under the regulation of tetracycline. METHODS AND RESULTS: Remarkably, expression of the transgene in mice generated two distinct phenotypes. One was the expected expression of HIF-regulated transcripts and associated changes in cardiac angiogenesis and contractility. The other was an unresponsive phenotype with much less expression of typical HIF-response genes and substantial expression of a zinc-finger protein, Protein Kinase C Binding Protein 1 (PRKCBP1). We have demonstrated that this second phenotype is due to an insertion of a fragment of DNA upstream of the PRKCBP1 gene that contains two additional canonical HIF binding sites and leads to substantial HIF binding, assessed by chromatin immunoprecipitation, and transcriptional activation. This insertion is found only in the FVB strain of mice that contributed the αMHC-tet binding protein transgene to these biallelic mice. In HEK293 cells transfected with oxygen-stable HIF-1α and PRKCBP1, we demonstrated inhibition of HIF-1 activity by a luciferase reporter assay. Using mouse primary cells and cell lines, we show that transfection with oxygen-stable HIF-1α and PRKCBP1 reduced expression of direct HIF-1 gene targets and that knockdown of PRKCBP1 removes that negative inhibition. Consistent with previous reports suggesting that PRKCBP1 modulates the chromatin landscape, we found that HL-1 cells transfected with oxygen-stable HIF-1α and PRKCBP1 have reduced global 5-methyl cytosine compared to HIF-1 alone. CONCLUSION: We show genetic, transcriptional, biochemical, and physiological evidence that PRKCBP1 inhibits HIF activity. Identification of a new oxygen-dependent and previously unsuspected regulator of HIF may provide a target for new therapeutic approaches to ischaemic heart disease.

HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility.

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. NEW & NOTEWORTHY Our study demonstrated hypoxia-inducible factor-1-dependent upregulation of miR-29c, which, in turn, inhibited SERCA2 expression and reduced cardiac contractility in a transgenic overexpression system. Interestingly, these results were not recapitulated in a murine myocardial infarction model. These results underscore the complexity of the pathological environment and highlight the need for therapeutic target validation in physiologically relevant models.

Chromatin Immunoprecipitation Assay: Examining the Interaction of NFkB with the VEGF Promoter.

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.

Urothelial Defects from Targeted Inactivation of Exocyst Sec10 in Mice Cause Ureteropelvic Junction Obstructions.

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. Sec10 FL/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, Sec10 FL/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.

Ultrasound directs a transposase system for durable hepatic gene delivery in mice.

Our aim was to evaluate the delivery of transposase-based vectors by ultrasound targeted microbubble destruction (UTMD) in mice. DNA vectors were attached to cationic lipid microbubbles (1-3 µm in diameter), injected intravenously and delivered to the liver by destruction of the carrier bubbles with ultrasound in burst mode at 1.0 MHz, 20-µs pulse duration, 10-Hz pulse repetition frequency and ∼1.3-MPa acoustic peak negative pressure. We evaluated the expression and genomic integration of conventional (pcDNA3) and piggyBac transposase-based (pmGENIE) reporter vectors. In vivo, we observed UTMD-mediated liver-specific expression of pmGENIE for an average of 24 d, compared with 4 d with pcDNA3. Reporter expression was located predominately near blood vessels initially, whereas expression after 3 d was more evenly distributed through the parenchyma of the liver. We confirmed random genomic integration for pmGENIE in vitro; however, integration events for pmGENIE in vivo were targeted to specific areas of chromosome 14. Our results suggest that a combination of UTMD and non-viral DNA transposase vectors can mediate weeks of hepatic-specific gene transfer in vivo, and analyses performed by non-restrictive linear amplification-mediated (nrLAM) polymerase chain reaction, cloning and sequencing identify an unexpected tropism for integration within a specific sequence on chromosome 14 in mice. UTMD delivery of transgenes may be useful for the treatment of hepatic gene deficiency disorders.

Cardiac angiogenesis directed by stable Hypoxia Inducible Factor-1.

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-ß, 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.

Introduction to the ultrasound targeted microbubble destruction technique.

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.

Multiple FadD acyl-CoA synthetases contribute to differential fatty acid degradation and virulence in Pseudomonas aeruginosa.

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.

Conditional HIF-1alpha expression produces a reversible cardiomyopathy.

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-1alpha. 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.

Immunization with hybrid recombinant Mycobacterium tuberculosis H37Rv proteins increases the TH1 cytokine response in mice following a pulmonary instillation of irradiated mycobacteria.

The aim of this research was to identify subunit immunogens that can generate enhanced CD8 T cell and TH1 responses against Mycobacterium tuberculosis. A genomic comparison of the M. tuberculosis H(37)R(V) and M. bovis BCG identified 61 proteins that are unique to H(37)R(V). Further screening of these 61 proteins using in silico analyses mimicking proteasomal digestion, transporter-associated antigen processing and H-2 antigen presentation identified 13 proteins with high densities of predicted MHC class I epitopes. Two native proteins, Rv1986c and Rv3875, were selected on the basis of their secreted or transmembrane characteristics and relatively lower frequencies of predicted MHC class II epitopes. To further enhance the CD8 T cell and TH1 responses, a hybrid protein, H32, was constructed by combining the nucleotide sequences encoding the MHC class I antigen-rich segment of Rv1986c and the entire Rv3875 sequence. The two native proteins and the hybrid were used to immunize C57BL/6 and Balb/c mice, which was followed by pulmonary instillation with irradiated M. tuberculosis H(37)R(V). All three proteins elicited elevated IFN-gamma responses, with the hybrid showing significant increases over the native proteins in both mice. This strategy of immunogen selection might be used to improve the current subunit vaccines against M. tuberculosis as well as other intra-cellular pathogens.