Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Dec 20;68(24):2652-2666.
doi: 10.1016/j.jacc.2016.09.946.

Gene Therapy With Angiotensin-(1-9) Preserves Left Ventricular Systolic Function After Myocardial Infarction

Caroline Fattah  1 , Katrin Nather  1 , Charlotte S McCarroll  1 , Maria P Hortigon-Vinagre  1 , Victor Zamora  1 , Monica Flores-Munoz  2 , Lisa McArthur  1 , Lorena Zentilin  3 , Mauro Giacca  3 , Rhian M Touyz  1 , Godfrey L Smith  1 , Christopher M Loughrey  1 , Stuart A Nicklin  4
Affiliations

Gene Therapy With Angiotensin-(1-9) Preserves Left Ventricular Systolic Function After Myocardial Infarction

Caroline Fattah et al. J Am Coll Cardiol. .

Abstract

Background: Angiotensin-(1-9) [Ang-(1-9)] is a novel peptide of the counter-regulatory axis of the renin-angiotensin-aldosterone system previously demonstrated to have therapeutic potential in hypertensive cardiomyopathy when administered via osmotic mini-pump. Here, we investigate whether gene transfer of Ang-(1-9) is cardioprotective in a murine model of myocardial infarction (MI).

Objectives: The authors evaluated effects of Ang-(1-9) gene therapy on myocardial structural and functional remodeling post-infarction.

Methods: C57BL/6 mice underwent permanent left anterior descending coronary artery ligation and cardiac function was assessed using echocardiography for 8 weeks followed by a terminal measurement of left ventricular pressure volume loops. Ang-(1-9) was delivered by adeno-associated viral vector via single tail vein injection immediately following induction of MI. Direct effects of Ang-(1-9) on cardiomyocyte excitation/contraction coupling and cardiac contraction were evaluated in isolated mouse and human cardiomyocytes and in an ex vivo Langendorff-perfused whole-heart model.

Results: Gene delivery of Ang-(1-9) reduced sudden cardiac death post-MI. Pressure volume measurements revealed complete restoration of end-systolic pressure, ejection fraction, end-systolic volume, and the end-diastolic pressure volume relationship by Ang-(1-9) treatment. Stroke volume and cardiac output were significantly increased versus sham. Histological analysis revealed only mild effects on cardiac hypertrophy and fibrosis, but a significant increase in scar thickness. Direct assessment of Ang-(1-9) on isolated cardiomyocytes demonstrated a positive inotropic effect via increasing calcium transient amplitude and contractility. Ang-(1-9) increased contraction in the Langendorff model through a protein kinase A-dependent mechanism.

Conclusions: Our novel findings showed that Ang-(1-9) gene therapy preserved left ventricular systolic function post-MI, restoring cardiac function. Furthermore, Ang-(1-9) directly affected cardiomyocyte calcium handling through a protein kinase A-dependent mechanism. These data emphasized Ang-(1-9) gene therapy as a potential new strategy in the context of MI.

Keywords: adeno-associated virus; calcium; inotropy; renin angiotensin system.

PubMed Disclaimer

Figures

None
Graphical abstract
Figure 1
Figure 1
AAV Delivery (A) Study design. (B) Immunohistochemistry for enhanced GFP at 1, 2, and 8 weeks following intravenous delivery of AAVGFP. (Original magnification ×4 for upper panel and ×40 for lower panel; scale = 100 μm.) (C) Quantification of GFP in transduced heart lysates using GFP assay. Fluorescence normalized to negative control heart tissue basal fluorescence and total protein concentration. (D) Mortality for each animal group. Group sizes are n = 10, n = 15, n = 15, and n = 11 for sham, MI, MI/AAVGFP, and MI/AAV Ang-(1-9), respectively. (E) Percent survival and cause of mortality. AAV = adeno-associated virus; Ang-(1-9) = angiotensin-(1-9); GFP = green fluorescence protein; MI = myocardial infarction.
Figure 2
Figure 2
Cardiac Function (A) Eight-week M-mode images (scale = 5 mm and 1 s). Effect of AAVAng-(1-9) varied by parameter: (B) Serial FS; (C) LVESD; (D) LVEDD; (E) posterior LV thickness; and (F) EF. *p <0.05 versus sham; #p <0.05 versus MI and MI/AAVGFP; ∼p <0.05 MI/AAVGFP versus MI/AAVAng-(1-9). (G) Average E/A ratio measurements (n = 6 per group). Data presented as mean ± SEM. A = after wave; E = early wave; EF = ejection fraction; FS = fractional shortening; LV = left ventricular; LVEDD = left ventricular end diastolic dimension; LVESD = left ventricular end systolic dimension; other abbreviations as in Figure 1.
Figure 3
Figure 3
Hemodynamic Indexes LV hemodynamic measurements at 8 weeks were determined using a PV-loop system with true blood volume calculated using Wei’s equation. Shown are (A) PV-loop relationship example; the systolic functional indexes of (B) ESP, (C) EF, (D) CO; and (E) dP/dtmax, the diastolic functional indexes of (F) end-diastolic pressure (EDP), (G) dP/dtmin, (H) Tau, and (I) EDPVR; and the volume indexes of (J) EDV, (K) ESV, (L) SV, and (M) ESPVR. *p <0.05, **p <0.01, ***p <0.001 versus sham; #p <0.05, ##p <0.01, ###p <0.001 versus MI and MI/AAVGFP; ∼p <0.05 versus MI/AAVGFP only. n = 9, 9, 9, and 8 for, sham, MI, MI/AAVGFP, and MI/AAVAng-(1-9), respectively. Data presented as mean ± SEM. CO = cardiac output; EDP = end diastolic pressure; EDPVR = EDP volume relationship; EDV = end diastolic volume; ESP = end systolic pressure; ESPVR = ESP volume relationship; ESV = end systolic volume; PV = pressure volume; SV = stroke volume; other abbreviations as in Figures 1 and 2.
Figure 4
Figure 4
Cardiomyocyte Hypertrophy (A) Heart images at 8 weeks (scale bar = 5 mm). (B) Ratio of HW to TL. *p <0.05, **p <0.01 versus sham. n = 10, 10, 9, and 8 for, sham, MI, MI/AAVGFP, and MI/AAVAng-(1-9), respectively. Data presented as mean ± SEM. (C) Cardiac cross sections in transverse axis (original main image magnification ×25; scale = 50 μm; inset zoom image scale = 12.5 μm). (D) LV cardiomyocyte diameter in hearts. ***p <0.001 versus sham. n = 10, 10, 9, and 8 for sham, MI, MI/AAVGFP, and MI/AAVAng-(1-9), respectively. (E) Cardiac cross sections in longitudinal axis (original main image magnification = ×25; scale = 50 μm; inset zoom image scale = 50 μm). (F) LV cardiomyocyte length. n = 10, 10, 9, and 8 for sham, MI, MI/AAVGFP, and MI/AAVAng-(1-9), respectively. Data presented as mean ± SEM with average cell size taken as average of a group of cells evenly distributed across LV. HW = heart weight; TL = tibia length; other abbreviations as in Figures 1 and 2.
Figure 5
Figure 5
Cardiac Fibrosis (A) Picrosirius red staining of heart sections (original magnification ×1.25; scale = 1 mm; zoom insert image scale = 0.5 mm). (B) Quantification of total cardiac fibrosis of the scar, LV, right ventricular, and septum regions. (C) Scar thickness for each MI group. n = 10, 10, 9, and 8 for sham, MI, MI/AAVGFP and MI/AAVAng-(1-9), respectively. Data presented as mean ± SEM. *p <0.05, **p <0.01, ***p <0.001 versus sham region; #p <0.05, ##p <0.01 versus MI and MI/AAVGFP region. Abbreviations as in Figures 1 and 2.
Figure 6
Figure 6
Excitation Contraction Coupling in Isolated Cardiomyocytes (A) Representative Ca2+-transient traces from Ang-(1-9)–treated cells. (B) Average Ca2+-transient amplitude for cardiomyocytes untreated (control; n = 21) or pre-treated with 1 μmol/l Ang-(1-9) (n = 21). (C) Cell shortening traces. (D) Cell shortening. (E) Average sarcoplasmic reticulum Ca2+ content. *p < 0.05 versus control. (F) Average first Ca2+ transient traces after 10 mmol/l caffeine. (G) Average L-type Ca2+-transient amplitude [control, n = 10; Ang-(1-9), n = 11]. *p < 0.05 versus control. Data presented as mean ± SEM. Red trace = 1 Hz stimulation. Abbreviations as in Figure 1.
Figure 7
Figure 7
Inotropic Effects of Ang-(1-9) Upon achieving maximum developed pressure, hearts were paced at 320 beats/min and allowed to reach steady state for 10 min before addition of 1 μmol/l Ang-(1-9). The protein kinase A inhibitor H89 (1 μmol/l) was perfused 10 min prior to adding Ang-(1-9) and was present throughout perfusion. (A) There were significant differences in (A) LV developed pressure compared to control hearts, but not in (B) the first derivative of LV developed pressured (dP/dtmax). Control n = 5, Ang-(1-9) n = 6, and H89 + Ang-(1-9) n = 5. *p <0.05 versus control hearts; †p <0.05 versus Ang-(1-9). Abbreviations as in Figure 1.
Figure 8
Figure 8
Excitation Contraction Coupling in hiPSC-CMs (A) Representative Ca2+-transient traces from Ang-(1-9)-treated cells. (B) Average Ca2+-transient amplitude for iCell2 hiPSC-CM (Cellular Dynamics International, Madison, Wisconsin). (C) Contractility traces. (D) Average contraction amplitude for iCell2 hiPSC-CMs. *p <0.05 versus control. Data presented as mean ± SEM (n = 5). Red trace = 1 Hz stimulation. hiPSC-CM = human-induced pluripotent stem cell-derived cardiomyocytes; other abbreviation as in Figure 1.
Central Illustration
Central Illustration
Post-MI Gene Therapy With Ang-(1-9) Adeno-associated virus serotype 9–mediated delivery of Ang-(1-9) via tail vein in a murine model of MI following coronary artery ligation produced significantly improved CO, SV, EF, and FS. Incubating freshly isolated adult murine cardiomyocytes or human induced pluripotent stem cell-derived cardiomyocytes with Ang-(1-9) leads to elevated SR Ca2+ content through stimulation of the L type calcium channel and enhanced contraction. Ang-(1-9) = angiotensin-(1-9); CO = cardiac output; EF = ejection fraction; FS = fractional shortening; MI = myocardial infarction; SR = sarcoplasmic reticulum; SV = stroke volume.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Donoghue M., Hsieh F., Baronas E. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res. 2000;87:E1–E9. - PubMed
    1. Tipnis S.R., Hooper N.M., Hyde R., Karran E., Christie G., Turner A.J. A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. J Biol Chem. 2000;275:33238–33243. - PubMed
    1. Der Sarkissian S., Huentelman M.J., Stewart J., Katovich M.J., Raizada M.K. ACE2: a novel therapeutic target for cardiovascular diseases. Prog Biophys Mol Biol. 2006;91:163–198. - PubMed
    1. Ferreira A.J., Santos R.A., Almeida A.P. Angiotensin-(1-7): cardioprotective effect in myocardial ischemia/reperfusion. Hypertension. 2001;38:665–668. - PubMed
    1. Grobe J.L., Mecca A.P., Mao H., Katovich M.J. Chronic angiotensin-(1-7) prevents cardiac fibrosis in DOCA-salt model of hypertension. Am J Physiol Heart Circ Physiol. 2006;290:H2417–H2423. - PubMed