Chronic administration of membrane sealant prevents severe cardiac injury and ventricular dilatation in dystrophic dogs

Abstract

Duchenne muscular dystrophy (DMD) is a fatal disease of striated muscle deterioration caused by lack of the cytoskeletal protein dystrophin. Dystrophin deficiency causes muscle membrane instability, skeletal muscle wasting, cardiomyopathy, and heart failure. Advances in palliative respiratory care have increased the incidence of heart disease in DMD patients, for which there is no cure or effective therapy. Here we have shown that chronic infusion of membrane-sealing poloxamer to severely affected dystrophic dogs reduced myocardial fibrosis, blocked increased serum cardiac troponin I (cTnI) and brain type natriuretic peptide (BNP), and fully prevented left-ventricular remodeling. Mechanistically, we observed a markedly greater primary defect of reduced cell compliance in dystrophic canine myocytes than in the mildly affected mdx mouse myocytes, and this was associated with a lack of utrophin upregulation in the dystrophic canine cardiac myocytes. Interestingly, after chronic poloxamer treatment, the poor compliance of isolated canine myocytes remained evident, but this could be restored to normal upon direct application of poloxamer. Collectively, these findings indicate that dystrophin and utrophin are critical to membrane stability-dependent cardiac myocyte mechanical compliance and that poloxamer confers a highly effective membrane-stabilizing chemical surrogate in dystrophin/utrophin deficiency. We propose that membrane sealant therapy is a potential treatment modality for DMD heart disease and possibly other disorders with membrane defect etiologies.

Figures

Figure 1. Mechanical properties of adult cardiac myocytes isolated from GRMD dogs.

Membrane-intact adult cardiac myocytes were isolated by enzymatic digestion from the hearts of 2 untreated 1-year-old GRMD dogs. (A) Microcarbon fibers were attached to the isolated myocytes, allowing mechanical manipulations and tension measurements (photo). Passive tension tracings from membrane-intact cardiac myocytes extended to sarcomere lengths indicated below the tracing. mN, millinewton. (B) Summary passive extension-tension curves are shown for membrane-intact cardiac myocytes isolated from wild-type and GRMD dogs and mdx mice. (C) Summary of the maximal sarcomere length tolerated by the myocytes. *P < 0.05. (D) Solubilization of membranes by detergents permits direct assessment of myofilaments. In a solution of nominal calcium, passive extension of myofilaments from wild-type and GRMD myocytes revealed no differences (7–8 cells from 2 dogs for each genotype). (E) Assessment of myofilament calcium sensitivity in membrane-solubilized myocytes revealed no differences between GRMD and wild-type myocytes. Data are from 5–7 myocytes isolated from 2–3 animals. pCa, –log[Ca2+].

Figure 2. Elevations in serum cTnI after induction of anesthesia in GRMD animals.

Serum samples collected within minutes of induction of general anesthesia reveal significant elevations in a biomarker of cardiac injury (A). (B) Significant elevations in serum CK in GRMD dogs. Values are shown as mean ± SEM; n = 8–10. ‡P < 0.05 by t test.

Figure 3. Baseline hemodynamic function in GRMD animals.

(A and B) Representative LV pressure tracings during inferior vena cava occlusion in wild-type (A) and GRMD (B) hearts. (C and D) Summary of end-diastolic volume (C) and stroke volume (D) at rest (black bars) and in response to dobutamine (white bars). Summaries of the change in mean end-diastolic volume (EDV) (E) and end-systolic volume (ESV) (F) observed in response to dobutamine. Values are shown as mean ± SEM. *P < 0.05 versus wild-type.

Figure 4. Preexisting fibrotic lesions and ventricular volume in GRMD dogs.

(A) Assessment of end-diastolic volume before and after acute infusion of P188 in GRMD and wild-type canines. Values are shown as mean ± SEM. (B) Representative Sirius red–stained histopathological sections from untreated adult GRMD animals. In brightfield images (left), collagen appears red; in polarized light, Sirius red–stained collagen exhibits birefringence, allowing it to be visualized as green-yellow light (right). Upper images show a large lesion with extensive fibrosis and degenerating myocytes. Lower images show strands of collagen that extend through areas of relatively normal myocardium (arrows). (C) Identical stains of wild-type myocardium. Scale bars: 100 μm.

Figure 5. Summary of disease biomarkers during chronic administration of P188 in GRMD animals.

(A) Outline of study design for the chronic administration of P188 (n = 4) or saline (n = 4) in GRMD animals. (B–E) Serum chemistry data from samples take biweekly throughout the chronic infusion period; shown are (B) cTnI (dashed line indicates wild-type canine values), (C) CK, (D) aspartate transferase (AST) and alkaline phosphatase (ALP), and (E) blood urea nitrogen (BUN) and creatinine. Values are shown as mean ± SEM; 4–8 animals per group. *P < 0.05, significant treatment effect by 2-way ANOVA analysis.

Figure 6. Chronic P188 treatment limits myocardial fibrosis and blocks elevation in BNP in GRMD animals.

(A) Sirius red staining of myocardial sections reveals extensive fibrosis in GRMD hearts. Collagen appears red in brightfield images and yellow-green in polarized images. Scale bars: 400 μm. (B) Quantification of collagen content from 16–24 sections from 4 dogs in each group. (C) Serum BNP levels taken before (GRMD-Pre) and after the chronic infusion protocol. Values are shown as mean ± SEM; 4–8 animals per group. *P < 0.05 versus wild-type; ‡P < 0.05 versus GRMD (saline) group. All data were analyzed with 1-way ANOVA and a Bonferroni’s multiple comparisons post-hoc test.

Figure 7. Eight weeks of P188 administration prevents LV remodeling in GRMD animals.

Catheter-based intraventricular recordings demonstrate significant ventricular remodeling in GRMD dogs receiving saline infusion. (A and B) Representative tracings of pressure-volume loops from before the treatment (A) and after the chronic infusion protocol (B). (C) Monitoring of LV end-diastolic volume during the terminal hemodynamic protocol. Offset at the far left are baseline data from the initial hemodynamic protocol prior to chronic infusion. *P < 0.05 (see Methods). (D–F) Comparisons of hemodynamic data taken during the peak of the dobutamine response (≈10 minutes after infusion); LV pressure tau (D), end-diastolic volume (E), and end-systolic volume (F). Groups consist of 3–4 dogs. ‡P < 0.05 by t test. For C–F, values are shown as mean ± SEM.

Figure 8. In vitro passive tension-extension relationships in intact GRMD cardiac myocytes after chronic infusion.

After the 8-week infusion, myocytes were isolated from P188- (red) and saline-infused (blue) GRMD and wild-type dogs. Passive tension-extension relationships of dystrophic cardiomyocytes in the absence (A) and presence (B) of acute application of 150 μM P188 are shown. The black curve in B is derived from wild-type myocyte data in A and is included as a reference. SL, sarcomere length. (C) Comparisons of maximum stable sarcomere length (Max SL) between the groups. *P < 0.05 versus wild-type; †P < 0.05 versus chronic saline GRMD group; ‡P < 0.05 versus chronic P188 GRMD group. Values are shown as mean ± SEM. All points were derived from 5–7 myocytes isolated from 4–5 dogs. All data were analyzed with 1-way ANOVA and Bonferroni’s multiple comparisons post-hoc test. MD, GRMD.

Figure 9. Utrophin expression in GRMD hearts.

(A) Western blot analysis of utrophin and dystrophin KCl-washed crude microsomal membranes isolated from wild-type and GRMD dog hearts. (B) Quantification of the levels of utrophin expression observed in A.

Figure 10. Model of the cellular basis for severe cardiomyopathy in GRMD animals.

(A) A schematic of membane-intact cardiac myocyte passive tension-extension curves. In the presence of dystrophin, myocytes show compliance throughout the working sarcomere length range (black line). In the absence of dystrophin, the cardiac myocytes become very noncompliant and resistant to passive extensions (gray line), as seen in GRMD myocytes. Upregulation of utrophin partially restores the passive extension-tension properties of the dystrophin-deficient myocytes (dotted line), as seen in the mdx myocytes. (B) Cellular model depicting the normal mechanical stabilizing function of dystrophin (blue) in wild-type animals. In wild-type cardiac myocytes, upon passive cell extension (lower scheme), dystrophin protects the membrane from damage. In the mdx myocytes, dystrophin is absent, but utrophin (red) is upregulated and partially replaces the mechanical buffering properties of dystrophin. This provides some, but not full protection of the membrane from mechanical damage. Hence, with cell extension, there is some membrane damage. In the dystrophin-deficient dog myocytes (GRMD), utrophin is not upregulated. Thus, upon cell extension, there is more damage to the membrane.