Prednisolone attenuates improvement of cardiac and skeletal contractile function and histopathology by lisinopril and spironolactone in the mdx mouse model of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is an inherited disease that causes striated muscle weakness. Recently, we showed therapeutic effects of the combination of lisinopril (L), an angiotensin converting enzyme (ACE) inhibitor, and spironolactone (S), an aldosterone antagonist, in mice lacking dystrophin and haploinsufficient for utrophin (utrn(+/-);mdx, het mice); both cardiac and skeletal muscle function and histology were improved when these mice were treated early with LS. It was unknown to what extent LS treatment is effective in the most commonly used DMD murine model, the mdx mouse. In addition, current standard-of-care treatment for DMD is limited to corticosteroids. Therefore, potentially useful alternative or additive drugs need to be both compared directly to corticosteroids and tested in presence of corticosteroids. We evaluated the effectiveness of this LS combination in the mdx mouse model both compared with corticosteroid treatment (prednisolone, P) or in combination (LSP). We tested the additional combinatorial treatment containing the angiotensin II receptor blocker losartan (T), which is widely used to halt and treat the developing cardiac dysfunction in DMD patients as an alternative to an ACE inhibitor. Peak myocardial strain rate, assessed by magnetic resonance imaging, showed a negative impact of P, whereas in both diaphragm and extensor digitorum longus (EDL) muscle contractile function was not significantly impaired by P. Histologically, P generally increased cardiac damage, estimated by percentage area infiltrated by IgG as well as by collagen staining. In general, groups that only differed in the presence or absence of P (i.e. mdx vs. P, LS vs. LSP, and TS vs. TSP) demonstrated a significant detrimental impact of P on many assessed parameters, with the most profound impact on cardiac pathology.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Cine Cardiac Magnetic Resonance Imaging was performed on a subset of mice.

Peak circumferential strain rate, assessed at the base of the left ventricle, was negatively impacted when Prednisolone was added to the treatment regimen. C: C57BL/10, n = 4; U: untreated mdx mice, n = 5, P: Prednisolone treated mdx mice, n = 6, LS: Lisinopril-Spironolactone treated mdx mice, n = 5; LSP, Lisinopril-Spironolactone-Prednisolone treated mdx mice n = 5; TS: Losartan-Spironolactone treated mdx mice, n = 5; TSP: Losartan-Spironolactone-Prednisolone treated mdx mice, n = 7. * indicates a significantly lower strain rate compared to the equivalent primary treatment without Prednisolone, P<0.05.

Figure 2. Diaphragm contractile function was assessed in isolated strips, at optimal length, at 37°Celsius.

C: C57BL/10, n = 9; U: untreated mdx mice, n = 7, P: Prednisolone treated mdx mice, n = 10, LS: Lisinopril-Spironolactone treated mdx mice, n = 9; LSP, Lisinopril-Spironolactone-Prednisolone treated mdx mice n = 6; TS: Losartan-Spironolactone treated mdx mice, n = 11; TSP: Losartan-Spironolactone-Prednisolone treated mdx mice, n = 8. * ANOVA indicated the only significant difference was between wild-type mice and Prednisolone-treated mdx mice (P<0.05). Difference between wild-type and untreated mdx mice was very similar but not quite significant (P = 0.08).

Figure 3. EDL contractile function was assessed in isolated whole EDL muscle, at optimal length, at 30°Celsius.

A: Twitch force: Twitch force at optimal length was not significantly different between groups. B: Total force: tetanic force at optimal length was not significantly different between groups. C: Specific force: force normalized to the muscle’s cross-sectional area revealed C57BL/10 mice have significantly higher forces than untreated mdx mice, TS, and TSP groups, *P<0.05. C: C57BL/10, n = 8; U: untreated mdx mice, n = 6, P: Prednisolone treated mdx mice, n = 9, LS: Lisinopril-Spironolactone treated mdx mice, n = 10; LSP, Lisinopril-Spironolactone-Prednisolone treated mdx mice n = 6; TS: Losartan-Spironolactone treated mdx mice, n = 11; TSP: Losartan-Spironolactone-Prednisolone treated mdx mice, n = 8.

Figure 4. Body weight and EDL weight analysis.

A: Body weight was significantly reduced in presence of Prednisolone. B: EDL weight showed a similar pattern to body weight. C: Body weight and EDL weight show a close correlation. D: Total EDL force/BW shows a similar ratio in all groups, with a non-significant (P = 0.09) trend to be lower in untreated mdx mice. C57BL/10, n = 4–8; U: untreated mdx mice, n = 6, P: Prednisolone treated mdx mice, n = 10, LS: Lisinopril-Spironolactone treated mdx mice, n = 10; LSP, Lisinopril-Spironolactone-Prednisolone treated mdx mice n = 6; TS: Losartan-Spironolactone treated mdx mice, n = 11; TSP: Losartan-Spironolactone-Prednisolone treated mdx mice, n = 8. * indicates a significantly lower value compared to the equivalent primary treatment without Prednisolone, P<0.05.

Figure 5. Hematoxylin and eosin (H&E) and IgG stained representative heart sections.

Cardiac damage in 20 week-old mdx mice is very mild and slightly attenuated by LS treatment. However, P treatment augments the damage present in mdx hearts as observed by both IgG stained myocytes as well as regions where cardiac muscle has been replaced by fibrotic scars as observed in H&E stained sections. C: C57BL/10; U: untreated mdx mice, P: Prednisolone treated mdx mice, LS: Lisinopril-Spironolactone treated mdx mice; LSP, Lisinopril-Spironolactone-Prednisolone treated mdx mice; TS: Losartan-Spironolactone treated mdx mice; TSP: Losartan-Spironolactone-Prednisolone treated mdx mice. Bar = 100 µm.

Figure 6. Hematoxylin & eosin (H&E) and IgG stained representative quadriceps sections from each treatment and control group of mice.

Small patches of damaged myofibers, representing ongoing skeletal muscle damage at 20 weeks-of-age are present in mdx quadriceps muscles (bright green IgG staining), but are completely absent from 20 week-old wild-type mice (C57BL/10). LS and TS treatment reduces the amount of ongoing damage, but P treatment results in larger and more prevalent patches of ongoing skeletal muscle damage in mdx quadriceps muscles. The same pattern of increased damage can be observed in P treated H&E stained sections. C: C57BL/10; U: untreated mdx mice, P: Prednisolone treated mdx mice, LS: Lisinopril-Spironolactone treated mdx mice; LSP, Lisinopril-Spironolactone-Prednisolone treated mdx mice; TS: Losartan-Spironolactone treated mdx mice; TSP: Losartan-Spironolactone-Prednisolone treated mdx mice. Bar = 100 µm.

Figure 7. Quantification of histological analysis.

A: Cardiac damage, quantified by the percentage of IgG staining, was significantly greater with Prednisolone inclusion in treatment (ANOVA). B: Quadriceps damage, quantified by % IgG staining, was significantly greater with Prednisolone in mdx mice, but had no significant impact in other groups. C57BL/10, n = 8; U: untreated mdx mice, n = 10, P: Prednisolone treated mdx mice, n = 9–10, LS: Lisinopril-Spironolactone treated mdx mice, n = 10; LSP, Lisinopril-Spironolactone-Prednisolone treated mdx mice n = 6; TS: Losartan-Spironolactone treated mdx mice, n = 10–11; TSP: Losartan-Spironolactone-Prednisolone treated mdx mice, n = 7–8. * indicates a significantly different value (P<0.05) compared to all other groups. $ indicates a significantly higher value compared to the equivalent primary treatment without Prednisolone, P<0.05.

Figure 8. Collagen I immunostaining of hearts from Prednisolone treated mice confirms the presence of Collagen scarring replacing cardiac muscle.

The damage present in hearts from P treated mice has progressed to collagen-containing fibrotic scarring in 20 week-old mice. LS shows no negative effects on mdx hearts. C: C57BL/10; U: untreated mdx mice, P: Prednisolone treated mdx mice, LS: Lisinopril-Spironolactone treated mdx mice; LSP, Lisinopril-Spironolactone-Prednisolone treated mdx mice; TS: Losartan-Spironolactone treated mdx mice; TSP: Losartan-Spironolactone-Prednisolone treated mdx mice. Bar = 100 µm.