Acute AT1R blockade prevents isoproterenol-induced injury in mdx hearts

Abstract

Background: Duchenne muscular dystrophy (DMD) is an X-linked disease characterized by skeletal muscle degeneration and a significant cardiomyopathy secondary to cardiomyocyte damage and myocardial loss. The molecular basis of DMD lies in the absence of the protein dystrophin, which plays critical roles in mechanical membrane integrity and protein localization at the sarcolemma. A popular mouse model of DMD is the mdx mouse, which lacks dystrophin and displays mild cardiac and skeletal pathology that can be exacerbated to advance the disease state. In clinical and pre-clinical studies of DMD, angiotensin signaling pathways have emerged as therapeutic targets due to their adverse influence on muscle remodeling and oxidative stress. Here we aim to establish a physiologically relevant cardiac injury model in the mdx mouse, and determine whether acute blockade of the angiotensin II type 1 receptor (AT₁R) may be utilized for prevention of dystrophic injury.

Methods and results: A single IP injection of isoproterenol (Iso, 10 mg/kg) was used to induce cardiac stress and injury in mdx and wild type (C57Bl/10) mice. Mice were euthanized 8 h, 30 h, 1 week, or 1 month following the injection, and hearts were harvested for injury evaluation. At 8 and 30 h post-injury, mdx hearts showed 2.2-fold greater serum cTnI content and 3-fold more extensive injury than wild type hearts. Analysis of hearts 1 week and 1 month after injury revealed significantly higher fibrosis in mdx hearts, with a more robust and longer-lasting immune response compared to wild type hearts. In the 30-hour group, losartan treatment initiated 1 h before Iso injection protected dystrophic hearts from cardiac damage, reducing mdx acute injury area by 2.8-fold, without any significant effect on injury in wild type hearts. However, both wild type and dystrophic hearts showed a 2-fold reduction in the magnitude of the macrophage response to injury 30 h after Iso with losartan.

Conclusions: This work demonstrates that acute blockade of AT₁R has the potential for robust injury prevention in a model of Iso-induced dystrophic heart injury. In addition to selectively limiting dystrophic cardiac damage, blocking AT₁R may serve to limit the inflammatory nature of the immune response to injury in all hearts. Our findings strongly suggest that earlier adoption of angiotensin receptor blockers in DMD patients could limit myocardial damage and subsequent cardiomyopathy.

Keywords: Angiotensin; Duchenne muscular dystrophy; Dystrophic cardiomyopathy; Fibrosis; Immune infiltration.

Figure 1:. Effects of a single dose of isoproterenol on healthy and dystrophic hearts.

(A) After one bolus injection of 10 mg/kg isoproterenol (Iso), mice were sacrificed at timepoints of 8 hours, 30 hours, 1 week, or 1 month. (B) Left: Acute Iso-induced injury, measured by IgG incorporation, was prevalent as early as 8 hours, with a peak at 30 hours. Evidence of acute injury was totally removed by 1 week. (*** indicates p<0.001 vs. baseline; interaction p=0.01; n = 6-17 mice per group). Right: Wild type (C10) hearts displayed a peak acute injury area of only 6±2%, while mdx peak injury was 3-fold higher at 19±3%. (*** indicates p<0.001). (C) Representative images of data shown in panel B. Whole hearts are shown with WGA (green) marking the total tissue area and IgG (red) indicating areas of acute myocyte injury. Scale bar = 1mm.

Figure 2:. Sarcolemmal injury triggers cardiomyocyte destruction as early as 8 hours after Iso.

(A) In both wild type (C10) and dystrophic hearts, uptake of IgG into injured myocytes corresponds with profound disruption of sarcomere structure as early as 8 hours following a bolus of Iso, compared to uninjured cardiomyocyte sarcomeres (right). Sarcomeres were visualized by α-actinin staining (green), myocyte injury was marked by endogenous IgG uptake (red), and extracellular matrix was visualized by WGA staining (magenta). Scale bar = 20 μm. (B) In wild type mouse hearts, myocytes that had taken up IgG also lost dystrophin at the sarcolemma, evidenced by absence of dystrophin staining (green). As expected, mdx hearts did not exhibit dystrophin staining. Scale bar = 50 μm. (C) Cardiac troponin I (cTnI) concentration was measured in serum collected from wild type and mdx mice as an index of myocardial damage. Dystrophic mice showed significantly higher serum levels of cTnI 8 hours after Iso. (*** indicates p<0.001; n ≥ 8 mice per group).

Figure 3:. Fibrotic replacement of Iso-induced cardiac injury is dynamic.

(A) Left: In wild type and dystrophic hearts, the area of fibrosis was the highest 1 week after Iso-induced injury, and declined by 1 month (** indicates p=0.009 vs. baseline, *** indicates p<0.001 vs. baseline, ## indicates p=0.003 vs. 1 month; interaction p=0.02; n = 8-15 mice per group). Right: One month after injury, mdx hearts displayed significantly larger fibrotic area (13±1%) than control hearts (6±1%) (*** indicates p<0.001). (B) Representative montages of data displayed in panel A. Whole hearts are shown with Fast Green-stained myocardium and Sirius Red-stained fibrosis. Scale bar = 1mm. (C) One month after Iso-induced injury, fibrotic lesions in mdx hearts displayed dramatically increased birefringent area relative to 1-week lesions, suggesting that replacement fibrosis contracts during maturation. (*** indicates p<0.001, n = 76-86 lesions from 14 mice per group). (D) Representative images of data displayed in panel C. Matched brightfield, cross-polarized, and overlay images of mdx hearts show the change in the birefringence of 1-week and 1-month-old lesions; areas of birefringence are denoted in yellow on overlay images. Scale bar = 100 μm.

Figure 4:. Iso-induced injury triggered a significantly greater immune cell response in dystrophic hearts.

(A) CD45⁺ cell area was dramatically increased in mdx hearts 30 hours and 1 week after Iso administration, with a return toward baseline at 1 month after injury. Wild type (C10) hearts followed a similar pattern, with lower overall levels of infiltration and a faster return toward baseline levels (*** indicates p<0.001, ** indicates p=0.008 vs. baseline; ### indicates p<0.001, ## indicates p=0.004 difference between strains at the same timepoint; interaction p=0.02; n = 5-11 mice per group). (B) The expansion of the CD45⁺ cell population occurred primarily in lesioned areas of the heart, where the CD45⁺ area significantly increased compared to baseline in dystrophic hearts (*** indicates p<0.001, ** indicates p=0.008, * indicates p<0.05 vs. baseline). (C) Representative images for data shown in panels A and B. Cardiac lesions are represented by endogenous IgG uptake (red, baseline and 30 hours) and WGA accumulation (red, 1 week and 1 month). CD45⁺ cells are shown in green. Each panel is 1 mm². (D) CD68⁺ immune infiltrates were significantly increased in dystrophic hearts 30 hours and 1 week after Iso administration, with a return toward baseline by 1 month post-injury. Wild type hearts displayed a smaller and shorter-lived surge in cardiac CD68⁺ cells, with a significant increase observed only at 30 hours after Iso injection (*** indicates p<0.001, * indicates p=0.02 vs. baseline; ### indicates p<0.001, # indicates p=0.01 difference between strains at the same timepoint; interaction p=0.02; n = 4-12 mice per group). (E) CD68⁺ cell expansion occurs primarily in lesioned areas of the heart, where their numbers are significantly greater compared to baseline. Only wild type hearts showed any expansion of non-lesion CD68⁺ cell numbers at 30 hours after Iso injection (*** indicates p<0.001 vs. baseline, * indicates p=0.02 vs. baseline). (F) Representative images for data shown in panels D and E, with CD68⁺ cells shown in cyan. Each panel is 1 mm².

Figure 5:. Acute losartan administration dramatically reduces Iso-induced injury in dystrophic hearts.

(A) Angiotensin II type 1 receptors (AT₁R) are G protein-coupled receptors that can be activated by angiotensin II (Ang II) and blocked by losartan. Top: Mice were treated with bolus injections of losartan (green) 1 hour prior to Iso administration (red), and at regular intervals over 30 hours following Iso injection. (B) 30 hours after Iso, losartan treatment caused no significant change in wild type (C10) cardiac injury, resulting in similarly low levels of acute Iso-induced injury in wild type and dystrophic hearts with losartan treatment. Untreated C10 and mdx data originally presented in Fig. 1B, shown here for comparison. (*** indicates p<0.001; interaction p=0.02; n = 10-17 mice per group). (C) Representative images of data shown in panel B. Whole hearts are shown with WGA (green) marking the total tissue area and IgG (red) indicating areas of acute myocyte injury. Scale bar = 1mm. (D and E) CD45⁺ and CD68⁺ areas with and without losartan in wild type and mdx hearts, in both lesion and non-lesion areas. (*** indicates p<0.001, ** indicates p<0.01, * indicates p=0.03 vs. same domain without losartan; ### indicates p<0.001, ## indicates p<0.01 difference in total immune area; n = 9-13 mice per group). (F) Representative images for data shown in panels D and E, with CD45⁺ cells shown in green and CD68⁺ cells shown in cyan. Cardiac lesions are indicated by endogenous IgG uptake (red). Each panel is 0.25 mm².