Chronic administration of membrane sealant prevents severe cardiac injury and ventricular dilatation in dystrophic dogs (original) (raw)
GRMD: single-cardiac myocyte studies. Cardiomyopathy in the mdx mouse is relatively mild compared with that in either human DMD patients or GRMD dogs, suggesting that there may be significant differences in the pathophysiology of the disease between these species. In the mdx mouse, a reduction in the passive compliance of myocytes is an important feature of the disease (Figure 1; ref. 30). To determine the passive properties of GRMD myocytes, we isolated membrane-intact cardiac myocytes and subjected them to passive extension-tension studies using a unique micro-carbon fiber assay (30). Cardiac myocytes isolated from wild-type dogs well tolerated passive extensions throughout the physiological range of sarcomere lengths (1.8–2.2 μm; ref. 31). In marked contrast to wild-type myocytes, GRMD myocytes were highly sensitive to passive extension and showed blunted compliance (Figure 1, A–C). These effects in GRMD myocytes were more pronounced than in the mdx myocyte (Figure 1B; ref. 30). The marked sensitivity to passive extension could result from perturbations of the sarcomeric myofilaments. To directly assess this possibility, we chemically removed the surface membrane. This solubilization of the membrane allows direct investigation of the properties of the underlying myofilament-based tension-extension properties of the cell. Examination of GRMD dystrophic myofilaments revealed passive properties and calcium sensitivity similar to those of wild-type canine myocytes (Figure 1, D and E). These data indicate that the poor compliance of dystrophic myocytes stems from abnormalities requiring an intact sarcolemmal membrane.
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+].
Baseline in vivo hemodynamics in GRMD animals. In order to address the importance of poor myocyte compliance on global hemodynamic function of the heart, we subjected GRMD dogs to invasive catheter-based hemodynamic assessment. Eight adult GRMD dogs and 10 wild-type dogs were utilized in these studies (Table 1). Animals were anesthetized, and baseline blood samples were drawn. Serum levels of cardiac troponin I (cTnI) were significantly elevated in GRMD dogs immediately after induction of anesthesia compared with unaffected anesthetized wild-type dogs (Figure 2A). At rest, GRMD animals had normal serum cTnI levels (see below), indicating that general anesthesia alone provokes significant cardiac damage in GRMD but not in normal dogs. Anesthesia had no significant effect on elevated serum levels of creatine kinase (CK) (Figure 2B and below). In contrast to the mdx mouse model of DMD (30), dystrophic dog hearts had no significant reduction in end-diastolic volume (Figure 3), nor was end-diastolic pressure elevated (Table 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.
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.
Initial ages, weights, selected baseline hemodynamic parameters, and randomized treatment groups in GRMD and wild-type dogs
Summary of pretreatment hemodynamic and electrocardiographic parameters at baseline and during acute dobutamine infusion
GRMD dogs had significant hypertension, with increased ventricular and femoral artery systolic pressures, compared with wild-type dogs (Table 2). Arterial elastance and total peripheral resistance were increased (Table 2), suggesting differences within the systemic vasculature of the dystrophic dogs. Differences in arterial function may also explain the increase in maximal rate of relaxation (dP/dtmin) in the absence of other indicators of improved diastolic function (tau and 90% relaxation time; Table 2). Early relaxation of ventricular pressure is aided by the filling of the coronary vasculature, and poor arterial compliance increases the reflection of the systolic pressure pulse back toward the heart. This increase in the reflected pressure pulse increases the pressure in the coronary vasculature and in turn increases the rate of early ventricular relaxation (32).
After baseline measurements, dobutamine was infused (15 μg/kg/min) until a peak effect was observed, at which time dobutamine infusion was stopped. In response to dobutamine, dystrophic dogs showed evidence of significant cardiac reserve function, with increased systolic and diastolic parameters similar to those observed in wild-type dogs (Figure 3 and Table 2). After dobutamine infusion, a dynamic change in ventricular volume was observed in wild-type dogs; however, these changes were significantly attenuated in dystrophic dogs (Figure 3, E and F).
Acute infusion of membrane sealants in GRMD animals. During the initial hemodynamic protocol, an acute dose of P188 (460 mg/kg) was administered i.v. over a period of 15 minutes in a total volume of 3 ml/kg. This dose was sufficient to normalize the end-diastolic volume of the mdx mouse (30). In the GRMD heart, this dose of P188 had no significant acute effect on the end-diastolic volume (Figure 4A). No significant changes in end-diastolic volume were observed in wild-type animals infused with equivalent doses of P188. Histological examination of heart sections from 1-year-old GRMD dogs not involved in the chronic study revealed the presence of extensive fibrosis when stained with Sirius red. This fibrosis takes 2 forms: large lesions associated with diseased myocardium and increased levels of fibrosis surrounding relatively normal myocardium (Figure 4B). Analysis of wild-type sections revealed only small amounts of collagen between layers of myocardium (Figure 4C).
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.
We addressed the potential acute effects of P188 on the vasculature by determining several measures of vascular function. The elastic property of the large arteries was not altered by the acute administration of P188 (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI41329DS1). Similarly, total peripheral resistance and peripheral blood pressure were unaffected by P188 (Supplemental Figure 1). These data are consistent with the selective action of P188 on the membranes of dystrophic cardiac myocytes.
During the hemodynamic assay, arrhythmic events were infrequent and not different between wild-type and GRMD dogs. Dystrophic dogs had significantly increased baseline heart rate and shortened P-R interval compared with wild-type dogs, neither of which were different after administration of dobutamine (Table 2). The ratio of Q-wave to R-wave amplitude was significantly elevated in dystrophic dogs, but this effect was not altered by acute dobutamine (Table 2). Acute infusion of P188 had no significant effects on ECG measurements in wild-type or dystrophic dogs (Supplemental Figure 2).
Chronic infusion of poloxamer in GRMD animals. Continuous intravascular infusion of P188 or saline vehicle control was initiated in adult GRMD animals. Animals were randomized to treatment group. Baseline hemodynamic function was similar in the groups prior to chronic infusion of P188 or saline (Table 1). After the initial hemodynamic assessment, a vascular access port was implanted on the flank and connected to an indwelling jugular catheter. The carotid artery was repaired, all incisions were closed, and the animal was allowed to recover from anesthesia. The next day, an infusion of 0.9% saline (0.4 ml/kg/h) was initiated in both groups, lasting 1 week. The serum half-life of P188 is 18 hours in dogs (33); thus, 1 week is sufficient to wash out any P188 remaining from the acute administration. After this washout and a surgery recovery period, the saline group continued receiving 0.9% NaCl at a rate of 0.4 ml/kg/h, and the P188 treatment group began receiving 60 mg/kg/h P188 in 0.9% NaCl at the same rate (0.4 ml/kg/h) for 8 weeks total treatment (Figure 5A).
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.
During the 8-week chronic infusion period, blood samples were taken periodically to monitor cardiac injury biomarkers. Saline-treated GRMD animals had significantly elevated serum concentrations of cTnI, evidence of cardiac injury. In contrast, serum cTnI levels were completely normal in the P188-treated GRMD animals (Figure 5B; P < 0.05 vs. saline). Serum CK levels were unchanged by P188 infusion (Figure 5C). The safety of P188 for both the renal and hepatic systems was assessed. Figure 5, D and E, shows that during the course of the 8-week infusion, the dogs receiving P188 had no change in liver enzymes or evidence of azotemia. These results indicate that administration of P188 at 60 mg/kg/h for 8 weeks does not result in kidney or liver damage in GRMD animals.
Myocardial fibrosis is a consistent finding in both DMD patients and GRMD dogs (Figure 4 and refs. 5, 25, 34, 35). Fibrotic lesions are thought to result from myocyte necrosis and subsequent inflammatory response. These fibrotic lesions likely increase the mechanical strain on neighboring myocytes, causing further myocyte necrosis and subsequent fibrosis over time. To assess the ability of chronically administered P188 to limit fibrosis, we stained cardiac tissue with Sirius red to identify mature fibrotic lesions. Upon quantification of the fibrotic area, GRMD animals receiving P188 had significantly less (P < 0.05) fibrosis than the GRMD group receiving saline (Figure 6, A and B). While P188 blunted lesion formation in GRMD animals, it did not restore myocardial lesion content to wild-type levels (Figure 6B). Chronic P188 administration to adult GRMD animals did fully block increases in the heart failure biomarker BNP (Figure 6C; P < 0.05).
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.
Prior to randomization for the chronic study, there was no significant difference in LV geometry among GRMD saline- versus P188-treated animals (Table 1 and Figure 7, A and C). After the 8-week infusion period, a second hemodynamic protocol was performed on the dystrophic dogs. During the course of the 8-week infusion period, GRMD dogs receiving saline developed significant LV dilation (P < 0.05; Figure 7). Dramatically, GRMD animals receiving P188 had no alteration in LV geometry, with no progression toward a dilated cardiac phenotype. LV geometry differences between saline and P188-treated GRMD animals were evident throughout the hemodynamic testing protocol (Figure 7C) and were particularly pronounced after the administration of dobutamine (Figure 7, C–F). The dilation of ventricular geometry in the saline GRMD group is consistent with ongoing cardiac injury and necrosis/fibrosis. Hemodynamic data also showed a significant enhancement in the isovolumic relaxation function in P188-treated GRMD compared with saline-treated GRMD animals (Figure 7D). The mechanism by which chronic P188 mediates improvements in diastolic function is not clear, but its determination would likely be of further benefit in DMD patients, where diastolic dysfunction is an early marker of cardiac disease (36).
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.
Myocytes isolated from dystrophic dogs after infusion with P188 or saline showed some significant differences in calcium handling. The amplitude of calcium release and kinetics of relaxation were both improved in myocytes isolated from GRMD dogs receiving chronic P188 infusion (Supplemental Figure 3). These improvements in calcium handling resulted from the improved myocyte health in the group treated with P188, as opposed to a direct action of P188, which is washed out during the myocyte isolation procedure.
To determine the effects of chronic P188 administration on the primary cellular defect of dystrophin-deficient cardiac myocytes, we examined the passive tension-extension properties of isolated myocytes at the end of the infusion protocol (Figure 8A). As seen with myocytes from untreated GRMD dogs (Figure 1), single myocytes isolated from GRMD animals receiving saline were highly sensitive to even small extensions in length. In addition to showing poor myocyte compliance, saline-infused GRMD myocytes were highly susceptible to terminal contracture after mild sarcomeric extension (Figure 8, A and C). Myocytes isolated from GRMD dogs receiving chronic P188, but with P188 washed out during the isolation procedure, had passive extension properties similar to those of dystrophic myocytes from untreated animals (Figure 8A), demonstrating that the P188 activity is readily reversed. However, acute administration of P188 to isolated GRMD myocytes, regardless of previous treatment, corrected the passive tension-extension relationship to levels similar to those of dystrophin-replete canine myocytes (Figure 8, B and C). These data demonstrate that the severely attenuated passive extension-tension relationship of GRMD myocytes occurs secondary to the formation of membrane tears that are corrected in the presence of P188.
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.
Utrophin expression in GRMD myocardium. The mechanism underlying blunted cellular compliance of GRMD cardiac myocytes is unknown. The mdx heart shows a marked upregulation of the dystrophin homolog utrophin, which may explain a mechanism by which mdx mice are relatively spared from the severe pathology observed in dystrophin-deficient dogs or humans (37, 38). To determine whether similar upregulation occurs in GRMD hearts, we assessed utrophin levels in crude microsomal membranes isolated from the hearts of 2 untreated GRMD and 3 wild-type dogs. These studies revealed that, in contrast to the mdx mouse, the GRMD dogs lack upregulation of utrophin in their hearts (Figure 9). Furthermore, the expression of utrophin was unaltered by the chronic infusion of P188 (Supplemental Figure 4).
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.