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

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.

Currently being explored therapeutically in cardiovascular diseases including HF and pulmonary hypertension, ACE2 shows promising therapeutic effects (3). Ang-(1-7) acts via the receptor Mas to block detrimental effects of Ang II and mediates direct therapeutic effects in cardiovascular disease (4,5).  is in clinical trials to treat diabetic foot ulcers and cancer (6,7), emphasizing translational approaches targeting the counter-regulatory RAAS axis.
Assessment of RAAS peptides as therapeutics is limited by short circulatory half-life, requiring osmotic mini-pumps for sustained release in vivo in experimental models. Accordingly, alternative delivery strategies are required for clinical translation. Viral gene therapy is being pursued for HF, including clinical trials using adeno-associated virus (AAV) vector-mediated delivery of sarcoplasmic endoreticulum calcium adenosine triphosphatase 2a (SERCA2a), emphasizing safety and clinical utility (12).
Angiotensin peptides are not produced from genes, but are generated extracellularly in the circulation.
Synthetic expression cassettes for Ang II, Ang-(1-7), and Ang-(1-9) have been utilized in transgenic models and in gene transfer approaches (13)(14)(15). Here, for the first time, in vivo AAV-mediated gene transfer of Ang-(1-9) via a synthetic expression cassette has been utilized to study cardiac effects in a murine model of MI.

Detailed methods are presented in the Online
Appendix. Briefly, an Ang-(1-9) expression cassette (13) was sub-cloned into plasmid adeno-associated virus-multiple cloning site (pAAV-MCS) and AAV9 vectors produced via standard protocols (16). Surgical procedures were performed in accordance with the Animals Scientific Procedures Act (1986) and approved by the University of Glasgow Animal Welfare and Ethical Review Panel and UK Home Office.
Fibrosis was assessed by Picrosirius red staining as described (8)  Ang-(1-9) Cardiac Gene Therapy following LAD ligation ( Figure 1A). High enhanced GFP expression was observed throughout the myocardium at all time points ( Figure 1B). Quantification of enhanced GFP expression in cardiac lysates revealed enhanced GFP expression was detectable at 1 week and increased at 4 and 8 weeks ( Figure 1C).
Next, animals were subjected to sham procedure, MI, MI/AAVGFP, or MI/AAVAng-(1-9) to assess effects on cardiac function and remodeling. MI in presence or absence of AAVGFP produced higher mortality than sham in the acute recovery phase due to cardiac rupture (sham: 100% survival; MI: 73% survival; MI/AAVGFP: 67% survival) ( Figures 1D  and 1E). Delivery of AAVAng-(1-9) increased survival to 91% in MI-induced animals. (p < 0.05). Importantly, EF was normalized to sham level, whereas CO was significantly increased compared to sham (p < 0.05). However, maximum derivative of change in systolic pressure over time (dP/dt max ) remained significantly reduced to 78.5% of sham ( Figure 3E) (p < 0.001). There were no significant differences in end-diastolic pressure, dP/dt min , and the rate constant of LV pressure decline (Tau) following AAVAng-(1-9) delivery ( Figures 3F to 3H).