Eric C. Svensson

Efficient and Stable Transduction of Cardiomyocytes After Intramyocardial Injection or Intracoronary Perfusion With Recombinant Adeno-Associated Virus Vectors

BACKGROUND The delivery of recombinant genes to cardiomyocytes holds promise for the treatment of a variety of cardiovascular diseases. Previous gene transfer approaches that used direct injection of plasmid DNA or replication-defective adenovirus vectors have been limited by low transduction frequencies and transient transgene expression due to immune responses, respectively. In this report, we have tested the feasibility of using intramyocardial injection or intracoronary infusions of recombinant adeno-associated virus (rAAV) vectors to program transgene expression in murine cardiomyocytes in vivo. METHODS AND RESULTS We constructed an rAAV containing the LacZ gene under the transcriptional control of the cytomegalovirus (CMV) promoter (AAVCMV-LacZ). We then injected 1x10(8) infectious units (IU) of this virus into the left ventricular myocardium of adult CD-1 mice. Control hearts were injected with the AdCMV-LacZ adenovirus vector. Hearts harvested 2, 4, and 8 weeks after AAVCMV-LacZ injection demonstrated stable beta-galactosidase (beta-gal) expression in large numbers of cardiomyocytes without evidence of myocardial inflammation or myocyte necrosis. In contrast, the AdCMV-LacZ-injected hearts displayed transient beta-gal expression, which was undetectable by 4 weeks after injection. Explanted C57BL/6 mouse hearts were also perfused via the coronary arteries with 1.5x10(9) IU of AAVCMV-LacZ and assayed 2, 4, and 8 weeks later for beta-gal expression. beta-Gal expression was detected in <1% of cardiomyocytes at 2 weeks after perfusion but was detected in up to 50% of cardiomyocytes 4 to 8 weeks after perfusion. CONCLUSIONS Direct intramyocardial injection or coronary artery perfusion with rAAV vectors can be used to program stable transgene expression in cardiomyocytes in vivo. rAAV appears to represent a useful vector for the delivery of therapeutic genes to the myocardium.

A syndrome of tricuspid atresia in mice with a targeted mutation of the gene encoding Fog-2.

Tricuspid atresia (TA) is a common form of congenital heart disease, accounting for 1–3% of congenital cardiac disorders. TA is characterized by the congenital agenesis of the tricuspid valve connecting the right atrium to the right ventricle and both an atrial septal defect (ASD) and a ventricular septal defect (VSD). Some patients also have pulmonic stenosis, persistence of a left-sided superior vena cava or transposition of the great arteries. Most cases of TA are sporadic, but familial occurrences with disease in multiple siblings have been reported. Gata4 is a zinc-finger transcription factor with a role in early cardiac development. Gata4-deficient mice fail to form a ventral heart tube and die of circulatory failure at embryonic day (E) 8.5 (refs 6,7). Zfpm2 (also known as Fog-2) is a multi-zinc-finger protein that is co-expressed with Gata4 in the developing heart beginning at E8.5 (refs 8–10). Zfpm2 interacts specifically with the N-terminal zinc finger of Gata4 and represses Gata4-dependent transcription. Here we use targeted mutagenesis to explore the role of Zfpm2 in normal cardiac development. Zfpm2-deficient mice died of congestive heart failure at E13 with a syndrome of tricuspid atresia that includes an absent tricuspid valve, a large ASD, a VSD, an elongated left ventricular outflow tract, rightward displacement of the aortic valve and pulmonic stenosis. These mice also display hypoplasia of the compact zone of the left ventricle. Our findings indicate the importance of Zfpm2 in the normal looping and septation of the heart and suggest a genetic basis for the syndrome of tricuspid atresia.

A Functionally Conserved N-terminal Domain of the Friend of GATA-2 (FOG-2) Protein Represses GATA4-Dependent Transcription

GATA4 is a transcriptional activator of cardiac-restricted promoters and is required for normal cardiac morphogenesis. Friend of GATA-2 (FOG-2) is a multizinc finger protein that associates with GATA4 and represses GATA4-dependent transcription. To better understand the transcriptional repressor activity of FOG-2 we performed a functional analysis of the FOG-2 protein. The results demonstrated that 1) zinc fingers 1 and 6 of FOG-2 are each capable of interacting with evolutionarily conserved motifs within the N-terminal zinc finger of mammalian GATA proteins, 2) a nuclear localization signal (RKRRK) (amino acids 736–740) is required to program nuclear targeting of FOG-2, and 3) FOG-2 can interact with the transcriptional co-repressor, C-terminal-binding protein-2 via a conserved sequence motif in FOG-2 (PIDLS). Surprisingly, however, this interaction with C-terminal-binding protein-2 is not required for FOG-2-mediated repression of GATA4-dependent transcription. Instead, we have identified a novel N-terminal domain of FOG-2 (amino acids 1–247) that is both necessary and sufficient to repress GATA4-dependent transcription. This N-terminal repressor domain is functionally conserved in the related protein, Friend of GATA1. Taken together, these results define a set of evolutionarily conserved mechanisms by which FOG proteins repress GATA-dependent transcription and thereby form the foundation for genetic studies designed to elucidate the role of FOG-2 in cardiac development.

Epicardial–Myocardial Signaling Directing Coronary Vasculogenesis

The establishment of the coronary circulation is critical for the development of the embryonic heart. Over the last several years, there has been tremendous progress in elucidating the pathways that control coronary development. Interestingly, many of the pathways that regulate the development of the coronary vasculature are distinct from those governing vasculogenesis in the rest of the embryo. It is becoming increasingly clear that coronary development depends on a complex communication between the epicardium, the subepicardial mesenchyme, and the myocardium mediated in part by secreted growth factors. This communication coordinates the growth of the myocardium with the formation of the coronary vasculature. This review summarizes our present understanding of the role of these growth factors in the regulation of coronary development. Continued progress in this field holds the potential to lead to novel therapeutics for the treatment of patients with coronary artery disease.

The N Termini of Friend of GATA (FOG) Proteins Define a Novel Transcriptional Repression Motif and a Superfamily of Transcriptional Repressors

Members of the Friend of GATA (FOG) family of transcriptional co-factors are required for the development of both the cardiovascular and hematopoietic systems. FOG proteins physically interact with members of the GATA family of transcriptional activators and modulate their activity. We have previously shown that FOG-2 can bind to the N-terminal zinc finger of GATA4 and, via this interaction, repress GATA4-mediated transcriptional activation of various cardiac promoters. In this report we further characterize the domain of FOG-2 necessary for repression of GATA4 transcriptional activity. We show that FOG-2-mediated repression is not blocked by the histone deacetylase inhibitor tricostatin A, suggesting that FOG-2 repression of GATA4 occurs via a histone deacetylase independent mechanism. N-terminal deletion mutants of FOG-2 revealed that the first 12 amino acids of FOG-2 are necessary for FOG-2-mediated repression. Fusion of these 12 amino acids to the DNA binding domain of GAL4 demonstrated that this region is sufficient to mediate transcriptional repression even when recruited to a heterologous promoter. Single amino acid substitutions within this N-terminal domain of FOG-2 defined the critical amino acid sequence as RRKQxxPxxI. Interestingly, a search of the NCBI protein data base identified several other partially characterized zinc finger transcriptional repressors from various vertebrate species that contained this motif at their N terminus. Taken together, these observations define a novel transcriptional repression motif and a superfamily of zinc finger transcriptional repressors.

Muscle-based gene therapy: realistic possibilities for the future

The past five years have witnessed tremendous growth in the field of gene therapy, with pre-clinical and clinical gene therapy trials for diseases as diverse as cancer, AIDS and atherosclerosis. These studies have utilized many different vectors and target organs in order to achieve therapeutic effects. In this review, we examine the rationale for using skeletal muscle as a target tissue for gene therapy, discuss the wide array of vectors that have been used for muscle-based gene therapy, summarize the disease-targets that have been approached using these techniques, and discuss some of the obstacles that remain to be overcome en route to successful muscle-based human gene therapy.

Ets1 is required for proper migration and differentiation of the cardiac neural crest

Defects in cardiac neural crest lead to congenital heart disease through failure of cardiac outflow tract and ventricular septation. In this report, we demonstrate a previously unappreciated role for the transcription factor Ets1 in the regulation of cardiac neural crest development. When bred onto a C57BL/6 genetic background, Ets1−/− mice have a nearly complete perinatal lethality. Histologic examination of Ets1−/− embryos revealed a membranous ventricular septal defect and an abnormal nodule of cartilage within the heart. Lineage-tracing experiments in Ets1−/− mice demonstrated that cells of the neural crest lineage form this cartilage nodule and do not complete their migration to the proximal aspects of the outflow tract endocardial cushions, resulting in the failure of membranous interventricular septum formation. Given previous studies demonstrating that the MEK/ERK pathway directly regulates Ets1 activity, we cultured embryonic hearts in the presence of the MEK inhibitor U0126 and found that U0126 induced intra-cardiac cartilage formation, suggesting the involvement of a MEK/ERK/Ets1 pathway in blocking chondrocyte differentiation of cardiac neural crest. Taken together, these results demonstrate that Ets1 is required to direct the proper migration and differentiation of cardiac neural crest in the formation of the interventricular septum, and therefore could play a role in the etiology of human congenital heart disease.

FOG-1-mediated recruitment of NuRD is required for cell lineage re-enforcement during haematopoiesis

The transcriptional co‐factor Friend of GATA1 (FOG‐1) has been shown to interact with subunits of the nucleosome remodelling and histone deacetylase (NuRD) complex through a specific motif located at its N‐terminus. To test the importance of FOG‐1/NuRD interaction for haematopoiesis in vivo, we generated mice with a mutation that specifically disrupts FOG‐1/NuRD interaction (FOG‐1R3K5A). Homozygous FOG‐1R3K5A mice were found to have splenomegaly, extramedullary erythropoiesis, granulocytosis and thrombocytopaenia secondary to a block in megakaryocyte maturation. FOG‐1R3K5A/R3K5A megakaryocytes and erythroid progenitors expressed increased levels of GATA2, showing that FOG‐1/NuRD interaction is required for the earlier described ‘GATA Switch’. In addition, ablation of FOG‐1/NuRD interaction led to inappropriate expression of mast cell and eosinophil‐specific genes in the megakaryocyte and erythroid lineages. Chromatin immunoprecipitation experiments revealed that the NuRD complex was not properly recruited to a mast cell gene promoter in FOG‐1R3K5A/R3K5A megakaryocytes, suggesting that FOG‐1/NuRD interaction is required for the direct suppression of mast cell gene expression. Taken together, these results underscore the importance of the FOG‐1/NuRD interaction for the re‐enforcement of lineage commitment during erythropoiesis and megakaryopoiesis in vivo.

Adenoviral-mediated gene transfer of a constitutively active form of the retinoblastoma gene product attenuates neointimal thickening in experimental vein grafts ☆ ☆☆

PURPOSE Inappropriate or excessive vascular smooth muscle cell proliferation leads to the development of occlusive lesions in up to 50% of vein grafts. The purpose of this study was to test the hypothesis that induced overexpression of a cytostatic nonphosphorylatable form of the retinoblastoma protein (DeltaRb) would attenuate neointimal thickening in experimental vein grafts. METHODS A replication-deficient adenovirus vector that encoded a nonphosphorylatable, constitutively active form of DeltaRb was constructed (AdDeltaRb) and contained an NH2-terminal epitope tag from the influenza hemagglutinin molecule (HA). Forty-eight male New Zealand white rabbits underwent surgical exposure of the external jugular vein for transfection with either 3 x 10(10) plaque-forming units/mL AdDeltaRb (n = 16), 3 x 10(10) plaque-forming units/mL control adenovirus (AdBglII, n = 15), or vehicle (n = 17) for 10 minutes at 120 mm Hg. After vector exposure, the vein was excised and interposed end-to-end into the carotid circulation. After 5 days, 12 grafts (four from each group) were excised and assayed for genomic DeltaRb DNA with the polymerase chain reaction or for hemagglutinin molecule expression and localization with immunohistochemistry. The remainder of the grafts (n = 36) were perfusion-fixed after 4 weeks, and 5 microm sections prepared for digital planimetric analysis. RESULTS Polymerase chain reaction results identified the DeltaRb gene only in the grafts that were transfected with AdDeltaRb. Immunohistochemical analysis results revealed transgene expression in most of the endothelial cells and in many of the smooth muscle cells. After 4 weeks, the grafts that were exposed to AdDeltaRb exhibited a 22% reduction in neointimal thickness (vehicle, 77 +/- 7 microm; AdBglII, 75 +/- 5 microm; AdDeltaRb, 60 +/- 5 microm; P =.05), and medial thickness, luminal diameter, and other parameters were unchanged (medial thickness: vehicle, 72 +/- 10 microm; AdBglII, 85 +/- 7 microm; AdDeltaRb, 69 +/- 9 microm; P = NS; luminal diameter: vehicle, 4.5 +/- 0.2 mm; AdBglII, 4.4 +/- 0.2 mm; AdDeltaRb, 4.7 +/- 0.1 mm; P = NS). CONCLUSION With this delivery system, adenoviral-mediated gene transfer is highly efficient and induced overexpression of DeltaRb leads to a reduction in vein graft neointimal thickening.

Translational control of FOG-2 expression in cardiomyocytes by microRNA-130a.

MicroRNAs are increasingly being recognized as regulators of embryonic development; however, relatively few microRNAs have been identified to regulate cardiac development. FOG-2 (also known as zfpm2) is a transcriptional co-factor that we have previously shown is critical for cardiac development. In this report, we demonstrate that FOG-2 expression is controlled at the translational level by microRNA-130a. We identified a conserved region in the FOG-2 3′ untranslated region predicted to be a target for miR-130a. To test the functional significance of this site, we generated an expression construct containing the luciferase coding region fused with the 3′ untranslated region of FOG-2 or a mutant version lacking this microRNA binding site. When these constructs were transfected into NIH 3T3 fibroblasts (which are known to express miR-130a), we observed a 3.3-fold increase in translational efficiency when the microRNA target site was disrupted. Moreover, knockdown of miR-130a in fibroblasts resulted in a 3.6-fold increase in translational efficiency. We also demonstrate that cardiomyocytes express miR-130a and can attenuate translation of mRNAs with a FOG-2 3′ untranslated region. Finally, we generated transgenic mice with cardiomyocyte over-expression of miR-130a. In the hearts of these mice, FOG-2 protein levels were reduced by as much as 80%. Histological analysis of transgenic embryos revealed ventricular wall hypoplasia and ventricular septal defects, similar to that seen in FOG-2 deficient hearts. These results demonstrate the importance of miR-130a for the regulation of FOG-2 protein expression and suggest that miR-130a may also play a role in the regulation of cardiac development.