Cardiac Gene Therapy With Relaxin Receptor 1 Overexpression Protects Against Acute Myocardial Infarction

Abstract

Relaxin is a pleiotropic hormone shown to confer cardioprotection in several preclinical models of cardiac ischemia-reperfusion injury. In the present study, the effects of up-regulating relaxin family peptide receptor 1 (RXFP1) via adeno-associated virus serotype 9 (AAV9) vectors were investigated in a mouse model of myocardial infarction. AAV9-RXFP1 vectors were generated and injected in adult male CD1 mice. Up-regulation of Rxfp1 was confirmed via quantitative polymerase chain reaction, and overexpressing animals showed increased sensitivity to relaxin-induced ventricular inotropic response. Overexpressing animals also demonstrated reduced infarct size and preserved cardiac function 24 hours after ischemia-reperfusion. Up-regulation of RXFP1 via AAV9 vectors has potential therapeutic utility in preventing adverse remodeling after myocardial infarction.

Keywords: AAV, adeno-associated virus; CMV, cytomegalovirus; GLS, global longitudinal strain; IR, ischemia-reperfusion; LV function; LV, left ventricular; MAPK, mitogen-activated protein kinase; MI, myocardial infarction; PV, pressure-volume; RXFP1; RXFP1, relaxin family peptide receptor 1; SIRO, simulated ischemia and reoxygenation; VEC, empty vector; eNOS, endothelial nitric oxide synthase; gene therapy; ischemia-reperfusion injury; mRNA, messenger ribonucleic acid; relaxin.

Graphical abstract

Figure 1

Feasibility Data for Using AAV-RXFP1 Vectors for Cardiotropic Expression (A) Schematic outlining the plasmid design with the transgene incorporated downstream of the cytomegalovirus (CMV) promoter. (B) Dose-response relationship measuring the messenger ribonucleic acid (mRNA) levels of relaxin family peptide receptor 1 (RXFP1) 4 weeks after injection of adeno-associated virus (AAV)–RXFP1; quantitative polymerase chain reaction analysis shows increased expression of Rxfp1 mRNA at all doses. For subsequent experimentation, 1 × 10¹¹ viral genomes per mouse was chosen. (C) Significantly higher mRNA expression of Rxfp1 in cardiac tissue lysates when treated with AAV-RXFP1 compared with empty vector (VEC) (1 × 10¹¹ genomes/mouse, n = 4 for all experimental groups). (D) Significantly higher expression of Rxfp1 mRNA was observed in cardiomyocytes isolated from AAV-RXFP1 mice when primary cells were isolated 4 weeks after injection with AAV-RXFP1 (n = 5 for all experimental groups). (E) Increased phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK) in cardiac tissue lysates extracted from AAV-RXFP1 mice (n = 6) compared with VEC mice (n = 3). For C, 1-way analysis of variance with Tukey post hoc analysis was used. For D, the Mann-Whitney U test was performed to compare the skewed distributions. For E, unpaired Student’s t-test was used for comparisons between the groups. ∗P < 0.05. ∗∗P < 0.01. ADU = arbitrary densitometric units.

Figure 2

Representative PV Loop Tracings Before and After Intervention in Control Mice (A) Representative pressure-volume (PV) loops showing cardiac function showing baseline function (pre) and function after injection (within 15 minutes; post) of either saline or serelaxin (SRLX) (10 μg/kg and 1 mg/kg) acute doses delivered intraperitoneally. End-systolic pressure and peak systolic pressure (P_max) are visibly elevated in animals injected with 1 mg/kg serelaxin (Supplemental Table 1). (B) Representative tracings of acute changes in PV loops upon stoppage of mechanical ventilation (leading to increased left ventricular [LV] filling). The slope of the end-systolic PV relationship (ESPVR) was lower when preload increase was facilitated prior to the injection of 1 mg/kg serelaxin (pre). After the intervention, and upon confirming the stabilization of any drug-induced effects on the PV loops, mechanical ventilation was turned off to obtain the ESPVR slope (post). The higher slope after intervention suggests that the observed increases in contractile pressures are preload independent. EDPVR = end-diastolic pressure-volume relationship; Ped = end-diastolic pressure; Pes = end-systolic pressure; RVU = relative volume unit; Ved = end-diastolic volume; Ves = end-systolic volume.

Figure 3

Millar Tracings in Transfected Mice After Administration of Low-Dose Serelaxin (A) Representative PV loops tracings in VEC and AAV-RXFP1 mice before (pre) and after (post) administration of low-dose serelaxin (10 μg/kg) intraperitoneally. PV-associated parameters are subsequently quantified. (B) The change in peak systolic pressure (P_Max) was significantly higher in the AAV-RXFP1 group (13.75 ± 3.13 mm Hg/s vs −0.94 ± 4.46 mm Hg/s in VEC group). (C) The change in (dP/dt)_Max was also significantly higher with serelaxin treatment in AAV-RXFP1 group (1,191 ± 365.5 mm Hg/s vs −319.8 ± 125.3 mm Hg/s in VEC group). (D) No net changes in end-diastolic pressure (EDP) were detected between the groups (0.97 ± 0.70 mm Hg/s in AAV-RXFP1 group vs −0.11 ± 2.39 mm Hg/s in VEC group; P = NS). (E) The increase in end-systolic pressure (ESP) was significantly higher in the AAV-RXFP1 group with serelaxin (14.58 ± 3.428 mm Hg/s vs −1.33 ± 4.92 mm Hg/s in VEC group). (F) Increased magnitude of change in (dP/dt)_Min was observed in AAV-RXFP1 animals when treated with serelaxin (−682 ± 318.8 mm Hg/s vs 384 ± 133.3 mm Hg/s in VEC group). (G) The load-independent relaxation constant τ is lowered after treatment with relaxin in the AAV-RXFP1 group (net change −1.15 ± 1.03 in AAV-RXFP1 group and 5.83 ± 3.109 in VEC group). For B to G, n = 4 for VEC and n = 5 for AAV-RXFP1; unpaired student’s t-test was used to compare datasets. ∗P < 0.05. ∗∗P < 0.01. Abbreviations as in Figures 1 and 2.

Figure 4

Infarct Size and Functional Parameters Post-MI in Transfected Mice (A) Infarct size was significantly lower in AAV-RXFP1 mice (24.42% ± 5.95%) compared with VEC mice (54.22% ± 15.70%) when expressed as a percentage of area at risk. Bar represents 3.5 mm. Area at risk did not differ between the 2 groups (53.40% ± 2.16% in AAV-RXFP1 vs 51.77% ± 1.80% in VEC). (B) Fractional shortening (FS) was significantly higher in the AAV-RXFP1 group at 24 hours (27.81% ± 2.993% vs 14.94% ± 2.68% in VEC) and 7 days (31.68% ± 1.77% vs 12.64% ± 1.69% in VEC). (C) Left ventricular internal diameter at systole (LVID - s) was significantly smaller in AAV-RXFP1 mice at 24 hours (3.01 ± 0.14 mm vs 3.67 ± 0.15 mm in VEC) and 7 days (2.87 ± 0.15 mm vs 4.51 ± 0.28 mm in VEC). (D) Left ventricular internal diameter at diastole (LVID - d) was not significantly different at 24 hours between the groups but was significantly smaller in AAV-RXFP1 mice at 7 days (4.20 ± 0.12 mm vs 5.15 ± 0.26 mm in VEC). (E) Radial dyssynchrony among the left ventricular (LV) long-axis segments was not significantly different between the AAV-RXFP1 and VEC groups at 24 hours (19.39% ± 2.73% vs 31.14% ± 5.84%, respectively) but was significantly higher in the VEC group at 7 days (15.53% ± 5.87% vs 33.79% ± 5.039%). (F) Global longitudinal strain was significantly higher in magnitude in AAV-RXFP1 mice at 24 h (−13.84% ± 1.61% vs −6.86% ± 1.56% in VEC) and 7 days (−14.08% ± 2.07% vs −6.42% ± 1.17% in VEC). (G) LV scar size was significantly smaller at 7 days post–myocardial infarction (MI) in AAV-RXFP1 mice (3.86% ± 0.94%) than in VEC mice (10.71% ± 1.23%), expressed as a percentage of LV tissue (bar represents 3.5 mm). (H) The proportion of trypan blue–positive cells (compared with baseline in the corresponding non–simulated ischemia and reoxygenation [SIRO] controls) was significantly lower in myocytes isolated from AAV-RXFP1 animals compared with VEC (1.58 ± 0.22 vs 2.89 ± 0.46, respectively). For A and H, unpaired Student’s t-test was used (n = 5 for VEC and AAV-RXFP1). For B to F (n = 5-10), 2-way analysis of variance with post hoc Bonferroni test was used. For G, unpaired Student’s t-test was used to compare scar sizes between VEC (n = 7 for VEC, n = 5 for AAV-RXFP1). Bar represents 100 μm. ∗P < 0.05. ∗∗P < 0.01. ∗∗∗P < 0.001. PreIR = pre-ischemia/reperfusion; other abbreviations as in Figures 1 and 2.