Alexander C. Zambon

GenMAPP 2: new features and resources for pathway analysis

BackgroundMicroarray technologies have evolved rapidly, enabling biologists to quantify genome-wide levels of gene expression, alternative splicing, and sequence variations for a variety of species. Analyzing and displaying these data present a significant challenge. Pathway-based approaches for analyzing microarray data have proven useful for presenting data and for generating testable hypotheses.ResultsTo address the growing needs of the microarray community we have released version 2 of Gene Map Annotator and Pathway Profiler (GenMAPP), a new GenMAPP database schema, and integrated resources for pathway analysis. We have redesigned the GenMAPP database to support multiple gene annotations and species as well as custom species database creation for a potentially unlimited number of species. We have expanded our pathway resources by utilizing homology information to translate pathway content between species and extending existing pathways with data derived from conserved protein interactions and coexpression. We have implemented a new mode of data visualization to support analysis of complex data, including time-course, single nucleotide polymorphism (SNP), and splicing. GenMAPP version 2 also offers innovative ways to display and share data by incorporating HTML export of analyses for entire sets of pathways as organized web pages.ConclusionGenMAPP version 2 provides a means to rapidly interrogate complex experimental data for pathway-level changes in a diverse range of organisms.

Resident fibroblast lineages mediate pressure overload–induced cardiac fibrosis

Activation and accumulation of cardiac fibroblasts, which result in excessive extracellular matrix deposition and consequent mechanical stiffness, myocyte uncoupling, and ischemia, are key contributors to heart failure progression. Recently, endothelial-to-mesenchymal transition (EndoMT) and the recruitment of circulating hematopoietic progenitors to the heart have been reported to generate substantial numbers of cardiac fibroblasts in response to pressure overload-induced injury; therefore, these processes are widely considered to be promising therapeutic targets. Here, using multiple independent murine Cre lines and a collagen1a1-GFP fusion reporter, which specifically labels fibroblasts, we found that following pressure overload, fibroblasts were not derived from hematopoietic cells, EndoMT, or epicardial epithelial-to-mesenchymal transition. Instead, pressure overload promoted comparable proliferation and activation of two resident fibroblast lineages, including a previously described epicardial population and a population of endothelial origin. Together, these data present a paradigm for the origins of cardiac fibroblasts during development and in fibrosis. Furthermore, these data indicate that therapeutic strategies for reducing pathogenic cardiac fibroblasts should shift from targeting presumptive EndoMT or infiltrating hematopoietically derived fibroblasts, toward common pathways upregulated in two endogenous fibroblast populations.

Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation

Two major goals of regenerative medicine are to reproducibly transform adult somatic cells into a pluripotent state and to control their differentiation into specific cell fates. Progress toward these goals would be greatly helped by obtaining a complete picture of the RNA isoforms produced by these cells due to alternative splicing (AS) and alternative promoter selection (APS). To investigate the roles of AS and APS, reciprocal exon–exon junctions were interrogated on a genome-wide scale in differentiating mouse embryonic stem (ES) cells with a prototype Affymetrix microarray. Using a recently released open-source software package named AltAnalyze, we identified 144 genes for 170 putative isoform variants, the majority (67%) of which were predicted to alter protein sequence and domain composition. Verified alternative exons were largely associated with pathways of Wnt signaling and cell-cycle control, and most were conserved between mouse and human. To examine the functional impact of AS, we characterized isoforms for two genes. As predicted by AltAnalyze, we found that alternative isoforms of the gene Serca2 were targeted by distinct microRNAs (miRNA-200b, miRNA-214), suggesting a critical role for AS in cardiac development. Analysis of the Wnt transcription factor Tcf3, using selective knockdown of an ES cell-enriched and characterized isoform, revealed several distinct targets for transcriptional repression (Stmn2, Ccnd2, Atf3, Klf4, Nodal, and Jun) as well as distinct differentiation outcomes in ES cells. The findings herein illustrate a critical role for AS in the specification of ES cells with differentiation, and highlight the utility of global functional analyses of AS.

Time- and exercise-dependent gene regulation in human skeletal muscle.

BackgroundSkeletal muscle remodeling is a critical component of an organisms response to environmental changes. Exercise causes structural changes in muscle and can induce phase shifts in circadian rhythms, fluctuations in physiology and behavior with a period of around 24 hours that are maintained by a core clock mechanism. Both exercise-induced remodeling and circadian rhythms rely on the transcriptional regulation of key genes.ResultsWe used DNA microarrays to determine the effects of resistance exercise (RE) on gene regulation in biopsy samples of human quadriceps muscle obtained 6 and 18 hours after an acute bout of isotonic exercise with one leg. We also profiled diurnal gene regulation at the same time points (2000 and 0800 hours) in the non-exercised leg. Comparison of our results with published circadian gene profiles in mice identified 44 putative genes that were regulated in a circadian fashion. We then used quantitative PCR to validate the circadian expression of selected gene orthologs in mouse skeletal muscle.ConclusionsThe coordinated regulation of the circadian clock genes Cry1, Per2, and Bmal1 6 hours after RE and diurnal genes 18 hours after RE in the exercised leg suggest that RE may directly modulate circadian rhythms in human skeletal muscle.

GO-Elite

Summary: We introduce GO-Elite, a flexible and powerful pathway analysis tool for a wide array of species, identifiers (IDs), pathways, ontologies and gene sets. In addition to the Gene Ontology (GO), GO-Elite allows the user to perform over-representation analysis on any structured ontology annotations, pathway database or biological IDs (e.g. gene, protein or metabolite). GO-Elite exploits the structured nature of biological ontologies to report a minimal set of non-overlapping terms. The results can be visualized on WikiPathways or as networks. Built-in support is provided for over 60 species and 50 ID systems, covering gene, disease and phenotype ontologies, multiple pathway databases, biomarkers, and transcription factor and microRNA targets. GO-Elite is available as a web interface, GenMAPP-CS plugin and as a cross-platform application. Availability: http://www.genmapp.org/go_elite Contact: nsalomonis@gladstone.ucsf.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

Cyclic AMP is both a pro-apoptotic and anti-apoptotic second messenger.

The second messenger cyclic AMP (cAMP) can either stimulate or inhibit programmed cell death (apoptosis). Here, we review examples of cell types that show pro‐apoptotic or anti‐apoptotic responses to increases in cAMP. We also show that cells can have both such responses, although predominantly having one or the other. Protein kinase A (PKA)‐promoted changes in phosphorylation and gene expression can mediate pro‐apoptotic responses, such as in murine S49 lymphoma cells, based on evidence that mutants lacking PKA fail to undergo cAMP‐promoted, mitochondria‐dependent apoptosis. Mechanisms for the anti‐apoptotic response to cAMP likely involve Epac (Exchange protein activated by cAMP), a cAMP‐regulated effector that is a guanine nucleotide exchange factor (GEF) for the low molecular weight G‐protein, Rap1. Therapeutic approaches that activate PKA‐mediated pro‐apoptosis or block Epac‐mediated anti‐apoptotisis may provide a means to enhance cell killing, such as in certain cancers. In contrast, efforts to block PKA or stimulate Epac have the potential to be useful in diseases settings (such as heart failure) associated with cAMP‐promoted apoptosis.

Ca2+/Calmodulin-dependent protein kinase II δ mediates myocardial ischemia/reperfusion injury through nuclear factor-κB.

Rationale: Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated as a maladaptive mediator of cardiac ischemic injury. We hypothesized that the inflammatory response associated with in vivo ischemia/reperfusion (I/R) is initiated through CaMKII signaling. Objective: To assess the contribution of CaMKII&dgr; to the development of inflammation, infarct, and ventricular dysfunction after in vivo I/R and define early cardiomyocyte–autonomous events regulated by CaMKII&dgr; using cardiac-specific knockout mice. Methods and Results: Wild-type and CaMKII&dgr; knockout mice were subjected to in vivo I/R by occlusion of the left anterior descending artery for 1 hour followed by reperfusion for various times. CaMKII&dgr; deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis, and improved functional recovery. CaMKII&dgr; deletion also attenuated I/R-induced inflammation and upregulation of nuclear factor-&kgr;B (NF-&kgr;B) target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-&kgr;B activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKII&dgr; knockout mice indicate that NF-&kgr;B activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes leads to I&kgr;B kinase phosphorylation and concomitant increases in nuclear p65. Experiments using an I&kgr;B kinase inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-&kgr;B activation. Conclusions: This is the first study demonstrating that CaMKII&dgr; mediates NF-&kgr;B activation in cardiomyocytes after in vivo I/R and suggests that CaMKII&dgr; serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.Rationale: Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) has been implicated as a maladaptive mediator of cardiac ischemic injury. We hypothesized that the inflammatory response associated with in vivo ischemia/reperfusion (I/R) is initiated through CaMKII signaling. Objective: To assess the contribution of CaMKIIδ to the development of inflammation, infarct and ventricular dysfunction following in vivo I/R and define early cardiomyocyte-autonomous events regulated by CaMKIIδ using cardiac-specific knockout (KO) mice. Methods and Results: Wild-type (WT) and CaMKIIδ KO mice were subjected to in vivo I/R by occlusion of the left anterior descending (LAD) artery for 1-hr followed by reperfusion for various times. CaMKIIδ deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis and improved functional recovery. CaMKIIδ deletion also attenuated I/R induced inflammation and upregulation of NF-κB target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-κB activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKIIδ knockout mice indicate that NF-κB activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes lead to I kappa B kinase (IKK) phosphorylation and concomitant increases in nuclear p65. Experiments using an IKK inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-κB activation. Conclusions: This is the first study demonstrating that CaMKIIδ mediates NF-κB activation in cardiomyocytes following in vivo I/R and suggests that CaMKIIδ serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.

Extracellular ATP and cAMP as Paracrine and Interorgan Regulators of Renal Function P2Y Receptors of MDCK Cells: Epithelial Cell Regulation by Extracellular Nucleotides

1. Madin–Darby canine kidney (MDCK) cells, a well‐ differentiated renal epithelial cell line derived from distal tubule/collecting duct, respond to extracellular nucleotides by altering ion flux and the production of arachidonic acid‐derived products, in particular prostaglandin E2 (PGE2). Our work has defined the receptors and signalling events involved in such responses.

Ca2+/Calmodulin-Dependent Protein Kinase II δ Mediates Myocardial Ischemia/Reperfusion Injury Through Nuclear Factor-κBNovelty and Significance

Rationale: Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated as a maladaptive mediator of cardiac ischemic injury. We hypothesized that the inflammatory response associated with in vivo ischemia/reperfusion (I/R) is initiated through CaMKII signaling. Objective: To assess the contribution of CaMKII&dgr; to the development of inflammation, infarct, and ventricular dysfunction after in vivo I/R and define early cardiomyocyte–autonomous events regulated by CaMKII&dgr; using cardiac-specific knockout mice. Methods and Results: Wild-type and CaMKII&dgr; knockout mice were subjected to in vivo I/R by occlusion of the left anterior descending artery for 1 hour followed by reperfusion for various times. CaMKII&dgr; deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis, and improved functional recovery. CaMKII&dgr; deletion also attenuated I/R-induced inflammation and upregulation of nuclear factor-&kgr;B (NF-&kgr;B) target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-&kgr;B activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKII&dgr; knockout mice indicate that NF-&kgr;B activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes leads to I&kgr;B kinase phosphorylation and concomitant increases in nuclear p65. Experiments using an I&kgr;B kinase inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-&kgr;B activation. Conclusions: This is the first study demonstrating that CaMKII&dgr; mediates NF-&kgr;B activation in cardiomyocytes after in vivo I/R and suggests that CaMKII&dgr; serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.Rationale: Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) has been implicated as a maladaptive mediator of cardiac ischemic injury. We hypothesized that the inflammatory response associated with in vivo ischemia/reperfusion (I/R) is initiated through CaMKII signaling. Objective: To assess the contribution of CaMKIIδ to the development of inflammation, infarct and ventricular dysfunction following in vivo I/R and define early cardiomyocyte-autonomous events regulated by CaMKIIδ using cardiac-specific knockout (KO) mice. Methods and Results: Wild-type (WT) and CaMKIIδ KO mice were subjected to in vivo I/R by occlusion of the left anterior descending (LAD) artery for 1-hr followed by reperfusion for various times. CaMKIIδ deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis and improved functional recovery. CaMKIIδ deletion also attenuated I/R induced inflammation and upregulation of NF-κB target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-κB activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKIIδ knockout mice indicate that NF-κB activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes lead to I kappa B kinase (IKK) phosphorylation and concomitant increases in nuclear p65. Experiments using an IKK inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-κB activation. Conclusions: This is the first study demonstrating that CaMKIIδ mediates NF-κB activation in cardiomyocytes following in vivo I/R and suggests that CaMKIIδ serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.

Effects of (-)-epicatechin on myocardial infarct size and left ventricular remodeling after permanent coronary occlusion.

OBJECTIVES We examined the effects of the flavanol (-)-epicatechin on short- and long-term infarct size and left ventricular (LV) structure and function after permanent coronary occlusion (PCO) and the potential involvement of the protective protein kinase B (AKT)/extracellular signal-related kinase (ERK) signaling pathways. BACKGROUND (-)-epicatechin reduces blood pressure in hypertensive patients and limits infarct size in animal models of myocardial ischemia-reperfusion injury. However, nothing is known about its effects on infarction after PCO. METHODS (-)-epicatechin (1 mg/kg daily) treatment was administered via oral gavage to 250 g male rats for 10 days before PCO and was continued afterward. The PCO controls received water. Sham animals underwent thoracotomy and treatment in the absence of PCO. Immunoblots assessed AKT/ERK involvement 2 h after PCO. The LV morphometric features and function were measured 48 h and 3 weeks after PCO. RESULTS In the 48-h group, treatment reduced infarct size by 52%. There were no differences in hemodynamics among the different groups (heart rate and aortic and LV pressures). Western blots revealed no differences in AKT or ERK phosphorylation levels. At 3 weeks, PCO control animals demonstrated significant increases in LV end-diastolic pressure, heart and body weight, and LV chamber diameter versus sham. The PCO plus (-)-epicatechin group values were comparable with those of the sham plus (-)-epicatechin group. Treatment resulted in a 33% decrease in myocardial infarction size. The LV pressure-volume curves demonstrated a right shift in control PCO animals, whereas the (-)-epicatechin curves were comparable with those of the sham group. The LV scar area strains were significantly improved with (-)-epicatechin. CONCLUSIONS These results demonstrate the unique capacity of (-)-epicatechin to confer cardioprotection in the setting of a severe form of myocardial ischemic injury. Protection is sustained over time and preserves LV structure and function. The cardioprotective mechanism(s) of (-)-epicatechin seem to be unrelated to AKT or ERK activation. (-)-epicatechin warrants further investigation as a cardioprotectant.