Early treatment with lisinopril and spironolactone preserves cardiac and skeletal muscle in Duchenne muscular dystrophy mice

Abstract

Background: Nearly universal cardiomyopathy in Duchenne muscular dystrophy (DMD) contributes to heart failure and death. Because DMD patients show myocardial fibrosis well before functional impairment, we postulated that earlier treatment using drugs with antifibrotic effect may be beneficial.

Methods and results: Three groups of 10 utrn(+/-);mdx, or "het" mice, deficient for dystrophin and haploinsufficient for utrophin with skeletal myopathy and cardiomyopathy that closely mimics clinical DMD were studied. One het group received spironolactone and lisinopril starting at 8 weeks of life (het-treated-8); a second received the same starting at 4 weeks of life (het-treated-4), and the third het group was untreated. At 20 weeks, all mice had normal ejection fractions though circumferential strain rate was abnormal (-0.21±0.08) in untreated hets. This improved to -0.40±0.07 in het-treated-8 mice (P=0.003) and further improved to -0.56±0.10 in het-treated-4 mice (P=0.014 for het-treated-4 versus het-treated-8). Treated mice showed less cardiomyocyte damage, with a 44% reduction in intracardiomyocyte serum immunoglobulin G localization in het-treated-8 mice (P<0.0001) and a further 53% reduction in het-treated-4 mice (P=0.0003 versus het-treated-8); matrix metalloproteinases were similarly reduced. Cardiac, limb, and diaphragm function by ex vivo muscle testing remained at 80% of normal with early treatment compared to a decline to 40% of normal skeletal muscle function without treatment.

Conclusions: These findings offer clinically available medications with proven antifibrotic effect as a new therapeutic strategy in DMD. Early initiation greatly attenuated myocardial disease and, for the first time with these drugs, improved skeletal myopathy. Thus, early initiation of such agents warrants further clinical evaluation to maintain ambulatory, respiratory, and cardiac function for patients with DMD and related myopathies.

Figure 1

In vivo cine CMR in the het mouse showed normal global systolic function in all mice, treated and untreated (data not shown). A–B) Comparison of an end-diastolic frame (a) to an end-systolic frame (b) indicates systolic thickening in all regions, resulting in a normal LV ejection fraction. C) Measurement of myocardial deformation using vector velocity image segmentation allows measurement of LV circumferential strain rate, a sensitive measure of subtle abnormalities in both systolic and diastolic function. D) Despite normal LVEF, untreated het mice had abnormal circumferential strain rate that was significantly improved with treatment initiation at 8 weeks with lisinopril and spironolactone (p=0.003 compared to het-untreated); this improvement was even more marked if treatment was started at 4 weeks of life (p=0.014 comparing het-treated-8 to het-treated-4; p<0.0001 het-untreated vs. het-treated-4).

Figure 2

In vitro muscle physiology showed that compared to untreated het mice, trabeculae isolated from het-treated-4 mice trended to improve active developed force at 4 Hz, 37 °C (A), improve maximal rates of contraction and relaxation (B), and faster timing kinetics (C). Cardiac regulatory systems regarding length-dependency (D), frequency-dependency (E), and β-adrenergic responsiveness were assessed similarly as previously reported and trended to be normalized to levels in control mice. In EDL muscles, specific force (150 Hz tetanus for 500 ms at 30 °C) was significantly improved upon treatment (G), and resistance to eccentric contractions was also improved (H). In isolated diaphragm muscle (180 Hz tetanus for 500 ms at 37 °C), specific force development was also significantly improved. n=6–9 group, * P<0.05 vs. C57BL/10 control, ^$ P<0.05 vs. het-untreated animals.

Figure 3

Drug treatment improves histological parameters of heart and skeletal muscles. A) Hematoxylin and Eosin (H&E)-stained left ventricular sections show the cardiac damage prevalent throughout het-untreated hearts that is almost completely prevented in both treatment groups. Intracellular localization of mouse IgG (green) indicates damaged myocardium that is significantly attenuated in het-treated-8 and even further improved in het-treated-4 hearts. Gelatinase in situ zymography (ISZ) shows the combined activity of matrix metalloproteinases 2 and 9 (bright green), indicative of ventricular remodeling, that is also attenuated in the het-treated-8 hearts, and almost entirely prevented in the het-treated-4 hearts. B) IgG localization (green) in quadriceps skeletal muscle sections indicates a profound and significant reduction of ongoing myofiber damage in the het-treated-4 group, with intermediate effects in the het-treated-8 group compared to untreated hets. Localization of Collagen I (red) in the matrix surrounding individual muscle fibers is shown to demonstrate the intracellular localization of the IgG staining. Bar = 50 μm.

Figure 4

A) The average percentage (± SE) of section area stained for mouse IgG for heart (left) and quadriceps skeletal muscles from het-untreated and treated groups shows significant reductions in ongoing muscle damage. B) Blinded visual scoring of gelatinase activity from in situ zymography supports reductions of MMP remodeling in treated groups. * P<0.03 vs. untreated, ** P<0.05 vs. 8-week. Panel A, n=7–10/group. Panel B, n=5/group.

Figure 5

ERTR7 immunolocalization (red) in heart sections shows that the mononuclear cell infiltrate (nuclei stained blue) in damaged regions of het-untreated cardiac muscle is mostly composed of fibroblasts. The less frequent areas of damage in the het-treated-8 group still contain fibroblasts, suggesting that the drug treatment is not acting directly on fibroblast proliferation or migration. Since very few areas of cardiac damage are present in het-treated-4 hearts, clusters of activiated fibroblasts are not typically present.