Myosin Light Chain-2v Promoter Research Articles

The Cytomegalovirus Enhancer Induces an Immediate Response to the Myosin Light Chain 2v Promoter during P19CL6 Cell Differentiation

The P19CL6 mouse embryonic carcinoma cells efficiently differentiate into cardiac muscle cells in the presence of DMSO. A reporter plasmid for cardiac muscle differentiation was constructed by connecting the CMV enhancer and a 250 bp MLC-2v promoter in front of the GFP gene to further evaluate the role of the CMV enhancer. This plasmid (pCBVenh/MLC-2vpro/EGFP) was stably introduced into P19CL6 cells, and the transfectant differentiated into cardiomyocytes with DMSO. Upon DMSO addition, GFP was immediately transcribed (within 2 days) and the amount of the transcript increased with cultivation. Concomitantly, GFP fluorescence was detected in the cells under a microscope. However, native MLC-2v was transcribed later on day 4. This expression time course is different from that of GFP. Clearly the CMV enhancer responded immediately to DMSO. Since GATA DNA-binding proteins play crucial roles in the initiation of cardiomyocyte differentiation, such a response could be ascribed to the presence of multiple GATA motifs in the enhancer sequence but not in the native MLC-2v promoter. Thus the CMV enhancer may be not only useful for gene therapy and monitoring cell differentiation but also the study of the role of GATA transcription factors expressed in P19CL6 cells.

Construction of recombinant HIF-1α and NIS lentiviral expression plasmid and its functional identification

Objective To construct a recombinant lentivirus vector containing the human NIS gene and HIF-1α with the myosin light chain-2v(MLC-2v) as a promoter and to investigate the specific expression and feasibility of NIS as a reporter gene in cardiomyocytes.Methods The target gene HIF-1α and NIS were subcloned into the lentivirus (Lv)-elongation factor (EF)1-HIF-1α-internal ribosome entry site (IRES)-NIS and Lv-MLC-HIF-1α-IRES-NIS lentivirus vectors.The recombinated vectors were transfected into Hela cells by lipofectamine 2000.The expression of HIF-1α and NIS in the transfected Hela cells was detected by indirect immunofluorescence and Western blot.The H9C2 cells were exposed to different multiplicities of infection (MOI; 5,10,20,40) with packaged virus particles.The infection efficiency was detected by Western blot.MOI 20 was used for H9C2,NIH-3T3 and L6 cell lines and the specificity of the MLC-2v promoter was detected by the count of NIS protein in the 3 different cell lines with Western blot.The function and features of NIS protein were evaluated by dynamic iodine uptake and NaClO4 iodine uptake inhibition tests in vitro.Two-sample t test was used to analyze the data.Results The two recombinant lentivirus vectors were constructed successfully.The HIF-1α protein was expressed in the cytoplasm and the NIS protein was expressed on the cell membrane in Hela cells.The grey levels of NIS and HIF-1α proteins in the positive control were 69.8 and 71.9,respectively,which were 109.4 and 92.7 after being prompted by EF1,and 141.9 and 132.4 by MLC-2v.The expression of these proteins was much higher by EF1 promoter than that by MLC-2v promoter.The optimal MOI for the Lv-MLC-HIF-1α-IRES-NIS virus to infect H9C2 cells was 20.With the MOI of 20,the grey levels of NIS protein promoted by EF1 were 23.4,29.8 and 28.6 for H9C2,NIH-3T3 and L6 cells infected with Lv-EF1-HIF-1α-IRES-NIS virus,respectively.The expression of NIS protein promoted by MLC-2v was much higher in H9C2 cells than the other two cell lines.The grey level of NIS protein was 157.9 in H9C2 cells,178.8 in L6 cells and 217.3 in NIH-3T3 cells.The NIS protein expressed in infected H9C2 cells showed high radioiodine uptake.The peak of iodine uptake was 4 287.2 counts · min-1 at 40 min which was 16.85 times of the control group (254.4 counts · min-1) (t=5.34,P＜ 0.01).The inhibition rate of iodine uptake was up to 85.5％ (3 666.4/4 287.2,t=21.3,P＜0.01) by NaClO4.Conclusions MLC-2v promoter allows specific expression of the external gene HIF-1α and NIS in myocardium.The cardiomyocytes transfected with NIS gene acquires the function of iodine uptake.Therefore,NIS may have a potential to be the reporter gene to monitor the external gene therapy in ischemic cardiomyopathy. Key words: Hypoxia-inducible factor 1, alpha subunit; DNA, recombinant; Plasmids; Transfection ; Sodium/iodide symporter

Isoflurane Preconditioning Elicits Competent Endogenous Mechanisms of Protection from Oxidative Stress in Cardiomyocytes Derived from Human Embryonic Stem Cells

Human embryonic stem cell (hESC)-derived cardiomyocytes potentially represent a powerful experimental model complementary to myocardium obtained from patients that is relatively inaccessible for research purposes. We tested whether anesthetic-induced preconditioning (APC) with isoflurane elicits competent protective mechanisms in hESC-derived cardiomyocytes against oxidative stress to be used as a model of human cardiomyocytes for studying preconditioning. H1 hESC cell line was differentiated into cardiomyocytes using growth factors activin A and bone morphogenetic protein-4. Living ventricular hESC-derived cardiomyocytes were identified using a lentiviral vector expressing a reporter gene (enhanced green fluorescent protein) driven by a cardiac-specific human myosin light chain-2v promoter. Mitochondrial membrane potential, reactive oxygen species production, opening of mitochondrial permeability transition pore, and survival of hESC-derived cardiomyocytes were assessed using confocal microscopy. Oxygen consumption was measured in contracting cell clusters. Differentiation yielded a high percentage (∼85%) of cardiomyocytes in beating clusters that were positive for cardiac-specific markers and exhibited action potentials resembling those of mature cardiomyocytes. Isoflurane depolarized mitochondria, attenuated oxygen consumption, and stimulated generation of reactive oxygen species. APC protected these cells from oxidative stress-induced death and delayed mitochondrial permeability transition pore opening. APC elicits competent protective mechanisms against oxidative stress in hESC-derived cardiomyocytes, suggesting the feasibility to use these cells as a model of human cardiomyocytes for studying APC and potentially other treatments/diseases. Our differentiation protocol is very efficient and yields a high percentage of cardiomyocytes. These results also suggest a promising ability of APC to protect and improve engraftment of hESC-derived cardiomyocytes into the ischemic heart.

Promoter-specific lentivectors for long-term, cardiac-directed therapy of Fabry disease

In Fabry disease a deficiency of α-galactosidase A (α-gal A) activity leads to accumulation of globotriaosylceramide (Gb3) in various tissues including the heart. A specific cardiac variant of Fabry disease has also been described. Previously we have demonstrated the feasibility of gene therapy for Fabry disease. Here, to provide efficient transfer and increased specificity of transgene expression, we synthesized lentiviral vectors (LVs) with myocardial-specific promoters including: α-myosin heavy chain (α-MHC), myosin light chain (MLC2v), and cardiac troponin T (cTnT). Initially, neonatal Balb/c mice were injected with such LV constructs engineering expression of luciferase. One month post-injection, we found specific expression of luciferase in hearts of recipient animals when compared with transgene expression driven by the standard EF1-α promoter. To examine the feasibility of long-term therapy specifically targeting the heart, recombinant LV/α-gal A therapeutic vectors with analogous cardiac promoters were generated and injected into numerous neonatal Fabry mice. No immune response against the corrective α-gal A hydrolase was observed in the treated mice. Serum α-gal A activity of 10-week-old Fabry mice was increased in LV/α-gal A-injected animals compared to controls. In 28-week-old Fabry mice we observed significantly decreased Gb3 accumulation. Neonatal injections with LVs harboring cardiac-specific promoters may thus be an effective long-term treatment strategy for heart manifestations and cardiac variant Fabry disease. These results can be also extended to other progressive pathologies of the heart.

Vascular Endothelial Growth Factor Gene Regulation by HEXIM1 in Heart

See related article, pages 415–422 Hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) was cloned as an upregulated gene in vascular smooth muscle cells after treatment with the differentiating agent HMBA.1 A recent report has shown that HEXIM1 is an inhibitor of positive transcription elongation factor (P-TEF)b, which plays an important role in regulation of RNA polymerase II elongation.2–4 HEXIM1 was previously cloned as a potential cardiac transcriptional regulatory factor suppressing the cardiac myosin light chain-2v promoter and termed cardiac lineage protein (CLP)-1.5 HEXIM1 was also cloned as the novel inhibitor of breast cell growth estrogen-downregulated gene (EDG)1.6 HEXIM1 protein level is downregulated by estrogens. HEXIM1 also suppresses estrogen receptor-α transcriptional activity.6,7 Gene and protein expression indicate the broad expression of HEXIM1 during postnatal development, with highest levels in heart, skeletal muscle, and brain. Protein localization of endogenous HEXIM1 in vascular smooth muscle cells and primary cardiomyocytes suggests that HEXIM1 is a nuclear protein. Furthermore, HEXIM1 has been shown to be involved in many biological processes, including cancers, AIDS, cardiac hypertrophy, and inflammation, through transcriptional repression.4 In vitro experiments demonstrate that HEXIM1 functions as a transcriptional repressor by inhibiting P-TEFb, a protein complex composed of cyclin-dependent kinase 9 and a cyclin partner that regulates RNA polymerase II elongation.2–4 HEXIM1 interacts with 7SK small nuclear RNA (snRNA), a component of P-TEFb complex containing cyclin-dependent kinase 9 and cyclin T1. This interaction occurs through the KHRR 7SK snRNA binding motif of …

Abstract 1271: IKKβ/NF-κB Pathway Protects Hearts from Hemodynamic Stress Mediated through Regulating MnSOD Expression

Objectives: We have previously reported that NF-κB contributes to GPCR agonist-induced hypertrophy in cultured cardiomyocytes. However, the in vivo role of this pathway in the pathogenesis of cardiac remodeling remains to be elucidated. Although IκB kinase β (IKKβ)/NF-κB pathway is a major negative regulator of cell death, it can sensitize cells to death-inducing stimuli in some instances, thus it can be either anti- or pro-apoptotic. In this study, we aimed to clarify the role of IKKβ/NF-κB signaling in cardiac remodeling using cardiac-specific IKKβ deficient mice. Methods and Results: We crossed mice bearing an IKK β flox allele with mice expressing the Cre recombinase under the control of the myosin light chain 2v promoter ( MLC2v-Cre +/− ) to generate IKK β flox/flox ; MLC2v-Cre +/− mice (conditional knockout:CKO). Then, CKO mice (n=14) and control littermates bearing IKK β flox/flox (CTRL, n=14) were subjected to pressure overload by means of transverse aortic constriction (TAC). EMSA analysis revealed NF-κB DNA binding activity after TAC had attenuated in CKO hearts. One week after TAC, echocardiography showed significantly lower left ventricular fractional shortening (26.9±2.7% vs. 41.4±0.9%, p<0.01), and higher left ventricular end-diastolic dimension (4.02±0.14 mm vs. 3.47±0.08 mm, p<0.01) and lung weight/body weight ratio (11.1±1.4 vs. 5.5±0.1, p<0.01) in CKO mice compared with CTRL mice, indicating the development of heart failure in CKO mice. Number of apoptotic cells had increased in CKO hearts after TAC, suggesting that the enhanced apoptosis is a cause for heart failure. The expression levels of MnSOD mRNA and protein after TAC, which is one of NF-κB target genes, were significantly lower in CKO than those in CTRL mice. As a consequence, oxidative stress and JNK activation in CKO hearts after TAC had significantly increased compared with those in CTRL heart, suggesting that increased oxidative stress and enhanced JNK activity resulted in cardiomyocyte apoptosis in CKO hearts. Conclusion: These results show that IKKβ/NF-κB pathway in cardiomyocyte plays a protective role mediated through attenuation of oxidative stress and JNK activation in response to pressure overload.

Functional arginine vasopressin system in early heart maturation

Since the neurohypophyseal hormone 8-arginine vasopressin (AVP) is involved in cardiovascular tissue hypertrophy and myocyte differentiation, it is possible that local AVP plays a role in heart maturation. AVP-specific RIA, RT-PCR, and immunoblot measurement of AVP receptors (VR) were used to investigate heart tissues from newborn and adult rats. To test AVP's role in differentiation and specialization into ventricle-like cardiomyocytes, we studied GFP-P19Cl6 stem cells, which express green fluorescence protein (GFP) reporter under transcriptional control of the myosin light chain-2v promoter. VR(1) transcripts and proteins were higher in adult than in newborn rat hearts. In contrast, VR(2) increased from postnatal day 1 to 5 and was barely detected in the adult rat heart. In cardiomyocytes expressing troponin C, immunofluorescence revealed VR(2) and VR(1). Intracellular cAMP increased 6.5- and 8.9-fold in response to the selective VR(2) agonist 1-desamino-8-D-AVP (DDAVP) after 1 and 24 h, respectively. Cardiac AVP was high in 1- and 5-day-old (330 +/- 26 and 276 +/- 53 pg/mg protein, respectively) but low in 66-day-old (98 +/- 15 pg/mg protein) rats. AVP immunostaining was detected in the tunica adventitia and endothelium of the coronary vessels. The possible role of AVP in cardiomyogenesis was indicated by DDAVP-AVP-dependent differentiation of GFP-P19Cl6 stem cells into contracting cells displaying GATA-4, a cardiac-specific marker, and ventricle-specific myosin light chain. Together, it is suggested that the AVP system is implicated in postnatal cardiac maturation.

Identification and selection of cardiomyocytes during human embryonic stem cell differentiation

Human embryonic stem cells (hESC) are pluripotent lines that can differentiate in vitro into cell derivatives of all three germ layers, including cardiomyocytes. Successful application of these unique cells in the areas of cardiovascular research and regenerative medicine has been hampered by difficulties in identifying and selecting specific cardiac progenitor cells from the mixed population of differentiating cells. We report the generation of stable transgenic hESC lines, using lentiviral vectors, and single-cell clones that express a reporter gene (eGFP) under the transcriptional control of a cardiac-specific promoter (the human myosin light chain-2V promoter). Our results demonstrate the appearance of eGFP-expressing cells during the differentiation of the hESC as embryoid bodies (EBs) that can be identified and sorted using FACS (purity>95%, viability>85%). The eGFP-expressing cells were stained positively for cardiac-specific proteins (>93%), expressed cardiac-specific genes, displayed cardiac-specific action-potentials, and could form stable myocardial cell grafts following in vivo cell transplantation. The generation of these transgenic hESC lines may be used to identify and study early cardiac precursors for developmental studies, to robustly quantify the extent of cardiomyocyte differentiation, to label the cells for in vivo grafting, and to allow derivation of purified cell populations of cardiomyocytes for future myocardial cell therapy strategies.

Genetically selected stem cells from human adipose tissue express cardiac markers

In the present study, the potential of human adipose-derived stem cells to differentiate into cells with characteristics of cardiomyocytes was investigated. Adipose tissue-derived stem cells (ADSCs) were transduced with two different lentiviral vectors simultaneously: (1) a lentiviral vector expressing eGFP controlled by the Nkx2.5 promoter and (2) a lentiviral vector expressing DsRed2 controlled by the myosin light chain-2v promoter (MLC-2v). Nkx2.5-eGFP and MLC-2v-DsRed2 dual positive cells were isolated by FACS. Immunostaining and RT-PCR analysis of the dual positive cells revealed that these cells are positive for Nkx2.5, cardiac troponin I, and L-type calcium channel α-1c subunit. Electrophysiology studies demonstrated the presence of functional voltage-dependent calcium and potassium channels. These observations confirm that cardiac progenitor cells can be isolated and enriched from human adipose-derived stem cells using lentiviral selection, and they might represent a new source for cell therapy for myocardial infarction and heart failure.

Nitric Oxide Signaling in Oxytocin-Mediated Cardiomyogenesis

Oxytocin (OT), a hormone recently identified in the heart, induces embryonic and cardiac somatic stem cells to differentiate into cardiomyocytes (CM), possibly through nitric oxide (NO). We verified this hypothesis using P19 cells and P19 Clone 6 derivatives expressing a green fluorescent protein (GFP) reporter linked to cardiac myosin light chain-2v promoter. OT treatment of these cells induced beating cell colonies that were fully inhibited by N,G-nitro-L-arginine-methyl-ester (L-NAME), an inhibitor of NO synthases (NOS), partially reduced by 1400W, an inhibitor of inducible NOS, and ODQ, an inhibitor of NO-sensitive guanylyl cyclases. The NO generator S-nitroso-N-acetylpenicillamine (SNAP) reversed the L-NAME inhibition of cell beating and GFP expression. In OT-induced cells, L-NAME significantly decreased transcripts of the cardiac markers Nkx2.5, MEF2c, alpha-myosin heavy chain, and less, GATA4, endothelial NOS, and atrial natriuretic peptide, as well as the skeletal myocyte (SM) marker myogenin. Image analysis of OT-induced P19Cl6-GFP cells revealed ventricular CM coexpressing sarcomeric alpha-actinin and GFP, with some cells exclusively expressing alpha-actinin, most likely of the SM phenotype. The OT-mediated production of CM, but not SM, was diminished by L-NAME. In P19 cells, exogenously added OT stimulated the expression of its own transcript, which was reduced in the presence of L-NAME. Surprisingly, L-NAME alone decreased the expression of anti-stage specific embryonic antigen-1 marker of the undifferentiated state and induced some beating colonies as well as GFP in P19Cl6-GFP cells. Collectively, our data suggest that the pleiotropic action of NO is involved in the initiation of CM differentiation of P19 cells and maintenance of their undifferentiated state.

228. AAV Mediated Hypoxia-Inducible VEGF Expression and Neovascular Formation in Ischemic Pig Myocardium

Rapid, efficient overexpression of angiogenic growth factors in the peri-infarct zone is a promising approach for gene therapy of acute myocardium infarction. Patients suffering from an acute myocardial infarction often require invasive treatment, and gene transfer could be performed in combination with intravascular revascularization strategies or with bypass surgery. Many angiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietins, fibroblast growth factor-4 (FGF-4) and placental growth factor (PlGF) have been shown to be potent inducers of arteriogenesis. VEGF is the most potent among these factors as it is capable of increasing proliferation and migration of endothelial and vascular smooth muscle cells, and attracting stem cell homing. In our previous studies, we have induced neovascular formation in ischemic mouse hearts with adeno-associated viral vector (AAV) mediated VEGF gene transfer. To avoid the harmful effect of excessive angiogenesis, we have constructed an AAV vector (MLCVEGF) with 9 copies of the hypoxia-response element (HRE) of the human erythropoietin (EPO) gene and the promoter of the rat myosin light chain 2v (MLC-2v) controlling VEGF gene expression. Since information derived from mouse model may not be directly applicable to human and the pig heart closely resembles the human heart closely, we tested the hypotheses that MLCVEGF can also mediate hypoxia- inducible VEGF expression and induce neovascular formation in the pig heart. We injected 1|[times]|1011 genomes of MLCVEGF or CMVVEGF (AAV vector with CMV promoter driving VEGF expression) into normal and ischemic pig myocardium after 90 minutes of occlusion and reperfusion of the left anterior descending coronary artery (LAD). CMVLacZ vector (AAV vector with CMV promoter driving LacZ gene expression) was used as control. All vectors were packaged in AAV serotype 1 capsid. Myocardiums were collected 2 weeks after the vector injection. Gene expression was analyzed by real-time RT-PCR and immunohistochemical staining. Capillaries and a-actin positive vessels were quantitated on anti PECAM-1 and smooth muscle a-actin antibody stained sections. We observed robust LacZ and VEGF gene expression at CMVLacZ and CMVVEGF injection sites and found more capillaries and a-actin positive vessels at VEGF vector injected sites than CMVLacZ injection sites. VEGF expression mediated by MLCVEGF vector was low in normal myocardium and was induced about 40-fold in ischemic myocardium 2 weeks after myocardial infarction and vector injection. Conclusions: 1) AAV serotype 1 can infect pig myocardium efficiently. 2) AAV vector containing VEGF controlled by MLC-2v promoter and 9 copies of EPO HRE can mediate hypoxia-inducible gene expression and induce neovascular formation in the pig myocardium.

926. Recombinant AAV Vectors Pseudotyped with Serotype-1 Capsid Mediates Early Onset of Gene Expression in Mouse Hearts

Top of pageAbstract Adeno-associated virus serotype 2 (AAV2) has been used most frequently as vector in experiments on cardiac gene therapy. We have used this vector to deliver vascular endothelial growth factor (VEGF) gene controlled by the hypoxia response element (HRE) and a cardiac myosin light chain 2v promoter. We achieved cardiac-specific and hypoxia-responsive gene expression that induced neovascular formation in ischemic murine hearts and improved cardiac function. However, gene expression mediated by AAV2 vector in hearts was delayed and did not reach the maximum level until about 4 weeks after intramyocardial injection. Irreversible myocardial injury could have occurred during this period of delay. Because hypoxia inducible factor 1 (HIF-1) is induced immediately at the onset of hypoxia, earlier onset of the HRE regulated gene expression should be more effective in inducing therapeutic angiogenesis. Recently, studies on AAV with different serotypes have shown that they differ in their transduction efficiency with different tissues. To identify a serotype that can efficiently mediate early expression of transgene in the myocardium, we packaged into serotypes 1 to 5 capsids, AAV vector carrying CMV promoter driving the mouse erythropoietin (Epo) gene. The packaged vectors were injected into murine hearts intramyocardially. Gene expression was studied by real-time RT-PCR. The kinetics of Epo expression was studied by measuring the hematocrit after AAVEpo gene transduction at different time points. We found that AAV1, 4 and 5 mediated earlier and higher gene expression than AAV2. Gene expression mediated by AAV1 increased faster than AAV4 and 5, and became the highest after day 4. To compare the early gene expression mediated by AAV serotype 1 and 2, we packaged into these capsids AAV-LacZ vectors that carried a CMV promoter driving LacZ gene expression and injected them to normal and ischemic hearts. Gene expression was detected by LacZ staining. The LacZ expression was detected in the normal and ischemic myocardium one day after AAV1-LacZ injection, while no LacZ expression could be detected in the hearts injected with AAV2-LacZ at this time. At day 14, 100% of the cardiomyocytes around needle injection sites were infected by AAV1-LacZ vector, whereas only sporadic cardiomyocytes were infected by AAV2-LacZ. These results indicate that AAV1 can mediate gene expression within one day. HRE regulated VEGF gene packaged in this capsid will induce very early gene expression and therefore enhance the therapeutic effect.

Adeno-associated viral vector delivers cardiac-specific and hypoxia-inducible VEGF expression in ischemic mouse hearts

It has been shown that the adeno-associated virus (AAV) vector can deliver the VEGF gene efficiently into the ischemic mouse myocardium. However, the AAV genomes can be found in extracardiac organs after intramyocardial injection. To limit unwanted VEGF expression in organs other than the heart, we tested the use of the cardiac myosin light chain 2v (MLC-2v) promoter and the hypoxia-response element to mediate cardiac-specific and hypoxia-inducible VEGF expression. An AAV vector, MLCVEGF, with 250 bp of the MLC-2v promoter and nine copies of the hypoxia-response element driving VEGF expression, was constructed. Gene expression was studied in vitro by infection of rat cardiomyocytes, rat skeletal myocytes, and mouse fibroblasts with the vector and in vivo by direct injection of the vector into normal and ischemic mouse hearts. With MLCVEGF infection, VEGF expression was higher in cardiomyocytes than the other two cell lines and was hypoxiainducible. VEGF expression was also higher in ischemic hearts than in normal hearts. No VEGF expression was detectable in organs with detectable MLCVEGF vectors other than the heart. MLCVEGF-injected ischemic hearts had more capillaries and small vessels around the injection site, smaller infarct size, and better cardiac function than the negative controls. Hence, MLCVEGF can mediate cardiac-specific and hypoxia-inducible VEGF expression, neoangiogenesis, infarct-size reduction, and cardiac functional improvement.

Chronic ventricular myocyte-specific overexpression of angiotensin II type 2 receptor results in intrinsic myocyte contractile dysfunction

ANG II type 2 receptor (AT(2)) is upregulated in failing hearts, but its effect on myocyte contractile function is not known. We measured fractional cell shortening and intracellular Ca(2+) concentration transients in left ventricular myocytes derived from transgenic mice in which ventricle-specific expression of AT(2) was driven by the myosin light chain 2v promoter. Confocal microscopy studies confirmed upregulation of AT(2) in the ventricular myocytes and partial colocalization of AT(2) with AT(1). Three components of contractile performance were studied. First, baseline measurements (0.5 Hz, 1.5 mmol/l extracellular Ca(2+) concentration, 25 degrees C) and study of contractile reserve at faster pacing rates (1-5 Hz) revealed Ca(2+)-dependent contractile dysfunction in myocytes from AT(2) transgenic mice. Comparison of two transgenic lines suggested a dose-dependent relationship between magnitude of contractile dysfunction and level of AT(2) expression. Second, activity of the Na(+)/H(+) exchanger, a dominant transporter that regulates beat-to-beat intracellular pH, was impaired in the transgenic myocytes. Third, the inotropic response to beta-adrenergic versus ANG II stimulation differed. Both lines showed impaired contractile response to beta-adrenergic stimulation. ANG II elicited an increase in contractility and intracellular Ca(2+) in wild-type myocytes but caused a negative inotropic effect in myocytes from AT(2) transgenic mice. In contrast with beta-adrenergic response, the depressed response to ANG II was related to level of AT(2) overexpression. The depressed response to ANG II was also present in myocytes from young transgenic mice before development of heart failure. Thus chronic overexpression of AT(2) has the potential to cause Ca(2+)- and pH-dependent contractile dysfunction in ventricular myocytes, as well as loss of the inotropic response to ANG II.

192. Cardiac-Specific and Hypoxia-Responsive Vascular Endothelial Growth Factor Expression in Ischemic Hearts

A problem associated with angiogenic gene therapy is that uncontrolled gene expression of angiogenic factors may cause several side effects, including: hemangioma formation, retinopathy, occult tumor growth and atherosclerotic plaque progression. Previously, we used the hypoxia response element (HRE) isolated from the erythropoietin (Epo) gene enhancer to render vascular endothelial growth factor (VEGF) expression responsive to hypoxia induction in vitro and in ischemic myocardium in vivo. We found in our experiment that adeno-associate viral vector (AAV) can be detected in other organs in addition to the heart after intra-myocardial injection. Thus, unwanted effects may still occur even with HRE controlled VEGF expression, since hypoxia in other tissues in patients with heart failure may results in extra-cardiac VEGF gene expression. To ensure the safety of angiogenic therapy for ischemic heart disease, it is necessary that the gene expression is cardiac specific and hypoxia responsive. In this study, we explored the feasibility of using the cardiac myosin light-chain-2v (MLC-2v) promoter and Epo HRE to mediate cardiac-specific and hypoxia-responsive VEGF gene expression. We constructed an AAV vector with MLC-2v promoter and 9 copies of Epo HRE driving VEGF (MLCVEGF) expression. Gene expression mediated by this vector was studied in vitro by transfection of this vector into a rat cardiac muscle cell line (H9c2), a rat skeletal muscle cell line (L6) and a mouse fibroblast cell line (NIH3T3), and in vivo by direct injection of the vector to normal and ischemic mouse hearts. AAV vectors with CMV driving VEGF (CMVVEGF), and 9HRE plus minimum SV40 promoter driving VEGF (H9VEGF) were used as controls. RESULTS: With MLCVEGF infection, VEGF expression is higher in H9c2 than the other two non-cardiac cell lines, and the expression is hypoxia inducible. In vivo studies indicated that MLCVEGF increased VEGF expression by 74% in ischemic heart compared to normal heart. VEGF expression was detected in liver, kidney, spleen and gonad of CMVVEGF inoculated mice by RT-PCR, but no VEGF expression was detected in organs other than heart and liver of MLCVEGF inoculated mice. CONCLUSION: Our result indicates that MLC-2v promoter and 9 copies of HRE can mediate cardiac-specific and hypoxia-inducible gene expression in vitro and in vivo. Induction of angiogenesis and improvement of cardiac function by this vector, studies currently in progress, may provide a safe approach for the treatment of ischemic heart disease.

927. Cell Therapy To Restore Perfusion and Cardiac Function in Infarcted Heart Is Improved by EPAS1 Gene Transfer

Background: Although constitutive expression of human heme oxygenase-1(hHO-1) could provide a useful approach for cellular protection in the ischemic heart. However, uncontrollable over-expression of hHO-1 can lead to unwanted side effects. We developed a novel vigilant vector system, which is composed of myosin light chain-2v promoter (MLC-2v) and a gene switch that is based on an oxygen dependent degradation domain (ODD) from the hypoxia inducible factor-1-alpha(HIF-1alpha). We hypothesis that vigilant system could control hHO-1 expression and protect ischemic myocardium from ischemic damage.

Protection from ischemic heart injury by a vigilant heme oxygenase-1 plasmid system.

Although human heme oxygenase-1 (hHO-1) could provide a useful approach for cellular protection in the ischemic heart, constitutive overexpression of hHO-1 may lead to unwanted side effects. To avoid this, we designed a hypoxia-regulated hHO-1 gene therapy system that can be switched on and off. This vigilant plasmid system is composed of myosin light chain-2v promoter and a gene switch that is based on an oxygen-dependent degradation domain from the hypoxia inducible factor-1-alpha. The vector can sense ischemia and switch on the hHO-1 gene system, specifically in the heart. In an in vivo experiment, the vigilant hHO-1 plasmid or saline was injected intramyocardially into myocardial infarction mice or sham operation mice. After gene transfer, expression of hHO-1 was only detected in the ischemic heart treated with vigilant hHO-1 plasmids. Masson trichrome staining showed significantly fewer fibrotic areas in vigilant hHO-1 plasmids-treated mice compared with saline control (43.0%+/-4.8% versus 62.5%+/-3.3%, P<0.01). The reduction of interstitial fibrosis is accompanied by an increase in myocardial hHO-1 expression in peri-infarct border areas, concomitant with higher Bcl-2 levels and lower Bax, Bak, and caspase 3 levels in the ischemic myocardium compared with saline control. By use of a cardiac catheter, heart from vigilant hHO-1 plasmids-treated mice showed improved recovery of contractile and diastolic performance after myocardial infarction compared with saline control. This study documents the beneficial regulation and therapeutic potential of vigilant plasmid-mediated hHO-1 gene transfer. This novel gene transfer strategy can provide cardiac-specific protection from future repeated bouts of ischemic injury.

Heart-specific targeting of beta-galactosidase by the ventricle-specific cardiac myosin light chain 2 promoter using adenovirus vectors.

Adenoviruses are attractive vectors for gene transfer into cardiac muscle. However, their promiscuous tissue tropism, which leads to an ectopic expression of the transgene, is a considerable limitation. To restrict expression to cardiomyocytes, we have constructed two recombinant adenoviruses (Ad-MLC2-250betagal and Ad-MLC2-2100betagal) containing the beta-galactosidase reporter gene under the control of the 250- or 2100-bp rat ventricle-specific cardiac myosin light chain-2v promoter (MLC-2v). Our in vitro and in vivo data have evidenced that the 2100-bp promoter allows stronger beta-galactosidase activity than the 250-bp promoter and that the deleted promoter allows a weak beta-galactosidase expression in skeletal muscle-derived cells in vitro. In contrast to the in vitro results, the highly deleted MLC-2v promoter of 250 pb conserved its heart specificity in in ovo and in vivo when introduced into the adenovirus genome, indicating that the specificity of this promoter is neither altered by the inverted terminal repeat nor by the enhancer of the Ela promoter, both of which located in the 5' flanking region of the promoter. Systemic injections of both recombinant adenoviruses into chicken embryos showed beta-galactosidase expression mainly in the right ventricle of the heart. We have confirmed the cardiac specificity of both promoters in mammalian species after injection of both recombinant adenoviruses into the heart of adult rats in vivo. The comparison of both promoters in vitro and in vivo has shown that the 250-bp MLC-2v promoter is 80% less active than the 2100-bp MLC-2v promoter and has enabled us to conclude that the MLC-2v promoter of 2100 bp is the most appropriate for efficient expression of a reporter gene or a therapeutic cardiac gene (e.g., SERCA2a or minidystrophin gene).

EFIA/YB-1 Is a Component of Cardiac HF-1A Binding Activity and Positively Regulates Transcription of the Myosin Light-Chain 2v Gene

Transient assays in cultured ventricular muscle cells and studies in transgenic mice have identified two adjacent regulatory elements (HF-1a and HF-1b/MEF-2) as required to maintain ventricular chamber-specific expression of the myosin light-chain 2v (MLC-2v) gene. A rat neonatal heart cDNA library was screened with an HF-1a binding site, resulting in the isolation of EFIA, the rat homolog of human YB-1. Purified recombinant EFIA/YB-1 protein binds to the HF-1a site in a sequence-specific manner and contacts a subset of the HF-1a contact points made by the cardiac nuclear factor(s). The HF-1a sequence contains AGTGG, which is highly homologous to the inverted CCAAT core of the EFIA/YB-1 binding sites and is found to be essential for binding of the recombinant EFIA/YB-1. Antiserum against Xenopus YB-3 (100% identical in the DNA binding domain and 89% identical in overall amino acid sequence to rat EFIA) can specifically abolish a component of the endogenous HF-1a complex in the rat cardiac myocyte nuclear extracts. In cotransfection assays, EFIA/YB-1 increased 250-bp MLC-2v promoter activity by 3.4-fold specifically in the cardiac cell context and in an HF-1a site-dependent manner. EFIA/YB-1 complexes with an unknown protein in cardiac myocyte nuclear extracts to form the endogenous HF-1a binding activity. Immunocoprecipitation revealed that EFIA/YB-1 has a major associated protein of approximately 30 kDa (p30) in cardiac muscle cells. This study suggests that EFIA/YB-1, together with the partner p30, binds to the HF-1a site and, in conjunction with HF-1b/MEF-2, mediates ventricular chamber-specific expression of the MLC-2v gene.