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Intravenous AAV8 Encoding Urocortin-2 Increases Function of the Failing Heart in Mice.

Lai NC, Gao MH, Giamouridis D, Suarez J, Miyanohara A, Parikh J, Hightower S, Guo T, Dillmann W, Kim YC, Diaz-Juarez J, Hammond HK - Hum. Gene Ther. (2015)

Bottom Line: UCn2 gene transfer was associated with increased peak systolic Ca(2+) transient amplitude and rate of Ca(2+) decline and increased SERCA2a expression.We conclude that a single IV injection of AAV8.UCn2 increases function of the failing heart.The simplicity of IV injection of a vector encoding a gene with beneficial paracrine effects to increase cardiac function is an attractive potential clinical strategy.

View Article: PubMed Central - PubMed

Affiliation: 1 VA San Diego Healthcare System , San Diego, CA 92161.

ABSTRACT
Urocortin-2 (UCn2) peptide infusion increases cardiac function in patients with heart failure, but chronic peptide infusion is cumbersome, is costly, and provides only short-term benefits. Gene transfer would circumvent these shortcomings. We previously showed that a single intravenous (IV) injection of AAV8.UCn2 increases plasma UCn2 and left ventricular (LV) systolic and diastolic function for at least 7 months in normal mice. Here we test the hypothesis that IV delivery of AAV8.UCn2 increases function of the failing heart. Myocardial infarction (MI, by coronary ligation) was used to induce heart failure, which was assessed by echocardiography 3 weeks after MI. Mice with LV ejection fraction (EF) <25% received IV delivery of AAV8.UCn2 (5×10(11) gc) or saline, and 5 weeks later echocardiography showed increased LV EF in mice that received UCn2 gene transfer (p=0.01). In vivo physiological studies showed a 2-fold increase in peak rate of LV pressure development (LV +dP/dt; p<0.0001) and a 1.6-fold increase in peak rate of LV pressure decay (LV -dP/dt; p=0.0007), indicating increased LV systolic and diastolic function in treated mice. UCn2 gene transfer was associated with increased peak systolic Ca(2+) transient amplitude and rate of Ca(2+) decline and increased SERCA2a expression. In addition, UCn2 gene transfer reduced Thr286 phosphorylation of Cam kinase II, and increased expression of cardiac myosin light chain kinase, findings that would be anticipated to increase function of the failing heart. We conclude that a single IV injection of AAV8.UCn2 increases function of the failing heart. The simplicity of IV injection of a vector encoding a gene with beneficial paracrine effects to increase cardiac function is an attractive potential clinical strategy.

No MeSH data available.


Related in: MedlinePlus

AAV8.CBA.UCn2 map and experimental protocol. (A) AAV8.CBA.UCn2 vector map. CBA, chicken β-actin promoter; CMV.en, human cytomegalovirus enhancer; ITR, inverted terminal repeat; SVpA, polyA from SV40 viral genome; UCn2, urocortin-2. (B) Experimental protocol. Normal mice underwent myocardial infarction (MI, by proximal left coronary ligation) to induce HF, which was assessed by echocardiography 3 weeks after MI. Mice with EF <25% were then randomized to receive AAV8.UCn2 (5×1011 gc, IV) or IV saline. Five weeks later, echocardiography was used to assess LV size and function. In vivo physiological studies were conducted to evaluate rates of LV pressure development (LV +dP/dt) and decay (LV −dP/dt), to assess LV systolic and diastolic function. Cross sections of LV (midpapillary level) show that the infarction is extensive (lower section), comprising the majority of the LV free wall, with only the interventricular septum spared. Data acquisition and analysis were blinded to group treatment. Color images available online at www.liebertpub.com/hum
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f1: AAV8.CBA.UCn2 map and experimental protocol. (A) AAV8.CBA.UCn2 vector map. CBA, chicken β-actin promoter; CMV.en, human cytomegalovirus enhancer; ITR, inverted terminal repeat; SVpA, polyA from SV40 viral genome; UCn2, urocortin-2. (B) Experimental protocol. Normal mice underwent myocardial infarction (MI, by proximal left coronary ligation) to induce HF, which was assessed by echocardiography 3 weeks after MI. Mice with EF <25% were then randomized to receive AAV8.UCn2 (5×1011 gc, IV) or IV saline. Five weeks later, echocardiography was used to assess LV size and function. In vivo physiological studies were conducted to evaluate rates of LV pressure development (LV +dP/dt) and decay (LV −dP/dt), to assess LV systolic and diastolic function. Cross sections of LV (midpapillary level) show that the infarction is extensive (lower section), comprising the majority of the LV free wall, with only the interventricular septum spared. Data acquisition and analysis were blinded to group treatment. Color images available online at www.liebertpub.com/hum

Mentions: A helper virus-free AAV8 vector encoding murine UCn2 driven by a chicken β-actin (CBA) promoter (AAV8.CBA.UCn2; Fig. 1) was produced by transient transfection of HEK293T cells with the vector plasmid pRep2/Cap8 and pAd-Helper plasmid.28 Plasmid pRep2/Cap8 was obtained from the University of Pennsylvania Vector Core. Cell lysates prepared after 72 hr of transfection were treated with benzonase and viruses were consolidated through 25% sucrose-cushion ultracentrifugation. The pellets were resuspended for further purification of the virus through anion-exchange column chromatography (Q-Sepharose; GE Health Science) and concentrated by 25% sucrose-cushion ultracentrifugation.29,30 Subsequently, the pellets were resuspended in 10 mM Tris-HCl (pH 7.9, 1 mM MgCl2, 3% sucrose). Virus titers were determined by real-time qPCR with virus genome DNA prepared from purified virus.


Intravenous AAV8 Encoding Urocortin-2 Increases Function of the Failing Heart in Mice.

Lai NC, Gao MH, Giamouridis D, Suarez J, Miyanohara A, Parikh J, Hightower S, Guo T, Dillmann W, Kim YC, Diaz-Juarez J, Hammond HK - Hum. Gene Ther. (2015)

AAV8.CBA.UCn2 map and experimental protocol. (A) AAV8.CBA.UCn2 vector map. CBA, chicken β-actin promoter; CMV.en, human cytomegalovirus enhancer; ITR, inverted terminal repeat; SVpA, polyA from SV40 viral genome; UCn2, urocortin-2. (B) Experimental protocol. Normal mice underwent myocardial infarction (MI, by proximal left coronary ligation) to induce HF, which was assessed by echocardiography 3 weeks after MI. Mice with EF <25% were then randomized to receive AAV8.UCn2 (5×1011 gc, IV) or IV saline. Five weeks later, echocardiography was used to assess LV size and function. In vivo physiological studies were conducted to evaluate rates of LV pressure development (LV +dP/dt) and decay (LV −dP/dt), to assess LV systolic and diastolic function. Cross sections of LV (midpapillary level) show that the infarction is extensive (lower section), comprising the majority of the LV free wall, with only the interventricular septum spared. Data acquisition and analysis were blinded to group treatment. Color images available online at www.liebertpub.com/hum
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4492611&req=5

f1: AAV8.CBA.UCn2 map and experimental protocol. (A) AAV8.CBA.UCn2 vector map. CBA, chicken β-actin promoter; CMV.en, human cytomegalovirus enhancer; ITR, inverted terminal repeat; SVpA, polyA from SV40 viral genome; UCn2, urocortin-2. (B) Experimental protocol. Normal mice underwent myocardial infarction (MI, by proximal left coronary ligation) to induce HF, which was assessed by echocardiography 3 weeks after MI. Mice with EF <25% were then randomized to receive AAV8.UCn2 (5×1011 gc, IV) or IV saline. Five weeks later, echocardiography was used to assess LV size and function. In vivo physiological studies were conducted to evaluate rates of LV pressure development (LV +dP/dt) and decay (LV −dP/dt), to assess LV systolic and diastolic function. Cross sections of LV (midpapillary level) show that the infarction is extensive (lower section), comprising the majority of the LV free wall, with only the interventricular septum spared. Data acquisition and analysis were blinded to group treatment. Color images available online at www.liebertpub.com/hum
Mentions: A helper virus-free AAV8 vector encoding murine UCn2 driven by a chicken β-actin (CBA) promoter (AAV8.CBA.UCn2; Fig. 1) was produced by transient transfection of HEK293T cells with the vector plasmid pRep2/Cap8 and pAd-Helper plasmid.28 Plasmid pRep2/Cap8 was obtained from the University of Pennsylvania Vector Core. Cell lysates prepared after 72 hr of transfection were treated with benzonase and viruses were consolidated through 25% sucrose-cushion ultracentrifugation. The pellets were resuspended for further purification of the virus through anion-exchange column chromatography (Q-Sepharose; GE Health Science) and concentrated by 25% sucrose-cushion ultracentrifugation.29,30 Subsequently, the pellets were resuspended in 10 mM Tris-HCl (pH 7.9, 1 mM MgCl2, 3% sucrose). Virus titers were determined by real-time qPCR with virus genome DNA prepared from purified virus.

Bottom Line: UCn2 gene transfer was associated with increased peak systolic Ca(2+) transient amplitude and rate of Ca(2+) decline and increased SERCA2a expression.We conclude that a single IV injection of AAV8.UCn2 increases function of the failing heart.The simplicity of IV injection of a vector encoding a gene with beneficial paracrine effects to increase cardiac function is an attractive potential clinical strategy.

View Article: PubMed Central - PubMed

Affiliation: 1 VA San Diego Healthcare System , San Diego, CA 92161.

ABSTRACT
Urocortin-2 (UCn2) peptide infusion increases cardiac function in patients with heart failure, but chronic peptide infusion is cumbersome, is costly, and provides only short-term benefits. Gene transfer would circumvent these shortcomings. We previously showed that a single intravenous (IV) injection of AAV8.UCn2 increases plasma UCn2 and left ventricular (LV) systolic and diastolic function for at least 7 months in normal mice. Here we test the hypothesis that IV delivery of AAV8.UCn2 increases function of the failing heart. Myocardial infarction (MI, by coronary ligation) was used to induce heart failure, which was assessed by echocardiography 3 weeks after MI. Mice with LV ejection fraction (EF) <25% received IV delivery of AAV8.UCn2 (5×10(11) gc) or saline, and 5 weeks later echocardiography showed increased LV EF in mice that received UCn2 gene transfer (p=0.01). In vivo physiological studies showed a 2-fold increase in peak rate of LV pressure development (LV +dP/dt; p<0.0001) and a 1.6-fold increase in peak rate of LV pressure decay (LV -dP/dt; p=0.0007), indicating increased LV systolic and diastolic function in treated mice. UCn2 gene transfer was associated with increased peak systolic Ca(2+) transient amplitude and rate of Ca(2+) decline and increased SERCA2a expression. In addition, UCn2 gene transfer reduced Thr286 phosphorylation of Cam kinase II, and increased expression of cardiac myosin light chain kinase, findings that would be anticipated to increase function of the failing heart. We conclude that a single IV injection of AAV8.UCn2 increases function of the failing heart. The simplicity of IV injection of a vector encoding a gene with beneficial paracrine effects to increase cardiac function is an attractive potential clinical strategy.

No MeSH data available.


Related in: MedlinePlus