Preclinical Proof-of-Concept of a Minimally Invasive Direct Cardiac Compression Device for Pediatric Heart Support.

PURPOSE: For pediatric patients, extracorporeal membrane oxygenation (ECMO) remains the predominant mechanical circulatory support (MCS) modality for heart failure (HF) although survival to discharge rates remain between 50 and 60% for these patients. The device-blood interface and disruption of physiologic hemodynamics are significant contributors to poor outcomes. METHODS: In this study, we evaluate the preclinical feasibility of a minimally invasive, non-blood-contacting pediatric DCC prototype for temporary MCS. Proof-of-concept is demonstrated in vivo in an animal model of HF. Hemodynamic pressures and flows were examined. RESULTS: Minimally invasive deployment on the beating heart was successful without cardiopulmonary bypass or anticoagulation. During HF, device operation resulted in an immediate 43% increase in cardiac output while maintaining pulsatile hemodynamics. Compared to the pre-HF baseline, the device recovered up to 95% of ventricular stroke volume. At the conclusion of the study, the device was easily removed from the beating heart. CONCLUSIONS: This preclinical proof-of-concept study demonstrated the feasibility of a DCC device on a pediatric scale that is minimally invasive and non-blood contacting, with promising hemodynamic support and durability for the initial intended duration of use. The ability of DCC to maintain pulsatile MCS without blood contact represents an opportunity to mitigate the mortality and morbidity observed in non-pulsatile, blood-contacting MCS.

Assessment of systemic AAV-microdystrophin gene therapy in the GRMD model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the DMD gene. Although mouse models of DMD provide insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)-microdystrophin (µDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups [control, 1 × 1013 vector genomes per kilogram (vg/kg), 1 × 1014 vg/kg, and 2 × 1014 vg/kg; n = 3 each], treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-µDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day -7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; µDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.

Natural History of Histopathologic Changes in Cardiomyopathy of Golden Retriever Muscular Dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked inherited myopathy that causes progressive skeletal and cardiac muscle disease. Heart lesions were described in the earliest DMD reports, and cardiomyopathy is now the leading cause of death. However, diagnostics and treatment for cardiomyopathy have lagged behind those for appendicular and respiratory skeletal muscle disease. Most animal model studies have been done in the mdx mouse, which has a relatively mild form of cardiomyopathy. Dogs with the genetically homologous condition, Golden Retriever muscular dystrophy (GRMD), develop progressive cardiomyopathy analogous to that seen in DMD. Previous descriptive studies of GRMD cardiomyopathy have mostly been limited to selective sampling of the hearts from young dogs. METHODS AND RESULTS: We systematically assessed cardiac lesions in 31 GRMD and carrier dogs aged 3 to 76 months and a separate cohort of 2-10-year-old normal hounds. Both semi-quantitative lesion scoring and quantitation of the cross-sectional area of fibrosis distinguished dogs with GRMD disease from normal dogs. The carriers generally had intermediate involvement but had even greater fibrosis than GRMD dogs. Fatty infiltration was the most prominent feature in some older GRMD dogs. Vascular hypertrophy was increased in GRMD dogs and correlated positively with lesion severity. Purkinje fiber vacuolation was also increased but did not correlate with lesion severity. Histopathologic changes correlated with late gadolinium enhancement on cardiac MRI. CONCLUSION: These features are generally compatible with those of DMD and further validate GRMD as a useful model to study cardiomyopathy pathogenesis and treatment. Additionally, the nature of some degenerative lesions suggests that functional hypoxia or non-thrombotic ischemia may contribute to disease progression.

Short-term treatment of golden retriever muscular dystrophy (GRMD) dogs with rAAVrh74.MHCK7.GALGT2 induces muscle glycosylation and utrophin expression but has no significant effect on muscle strength.

We have examined the effects of intravenous (IV) delivery of rAAVrh74.MHCK7.GALGT2 in the golden retriever muscular dystrophy (GRMD) model of Duchenne Muscular Dystrophy (DMD). After baseline testing, GRMD dogs were treated at 3 months of age and reassessed at 6 months. This 3-6 month age range is a period of rapid disease progression, thus offering a relatively short window to establish treatment efficacy. Measures analyzed included muscle AAV transduction, GALGT2 transgene expression, GALGT2-induced glycosylation, muscle pathology, and muscle function. A total of five dogs were treated, 4 at 2x1014vg/kg and one at 6x1014vgkg. The 2x1014vg/kg dose led to transduction of regions of the heart with 1-3 vector genomes (vg) per nucleus, while most skeletal muscles were transduced with 0.25-0.5vg/nucleus. GALGT2-induced glycosylation paralleled levels of myofiber vg transduction, with about 90% of cardiomyocytes having increased glycosylation versus 20-35% of all myofibers across the skeletal muscles tested. Conclusions from phenotypic testing were limited by the small number of dogs. Treated dogs had less pronounced fibrosis and overall lesion severity when compared to control groups, but surprisingly no significant changes in limb muscle function measures. GALGT2-treated skeletal muscle and heart had elevated levels of utrophin protein expression and GALGT2-induced expression of glycosylated α dystroglycan, providing further evidence of a treatment effect. Serum chemistry, hematology, and cardiac function measures were largely unchanged by treatment. Cumulatively, these data show that short-term intravenous treatment of GRMD dogs with rAAVrh74.MHCK7.GALGT2 at high doses can induce muscle glycosylation and utrophin expression and may be safe over a short 3-month interval, but that such treatments had only modest effects on muscle pathology and did not significantly improve muscle strength.

Natural History of Cardiomyopathy in Adult Dogs With Golden Retriever Muscular Dystrophy.

Background Duchenne muscular dystrophy (DMD) is an X-linked disease that causes progressive muscle weakness. Affected boys typically die from respiratory or cardiac failure. Golden retriever muscular dystrophy (GRMD) is genetically homologous with DMD and causes analogous skeletal and cardiac muscle disease. Previous studies have detailed features of GRMD cardiomyopathy in mostly young dogs. Cardiac disease is not well characterized in adult GRMD dogs, and cardiac magnetic resonance (CMR) imaging studies have not been completed. Methods and Results We evaluated echocardiography and CMR in 24 adult GRMD dogs at different ages. Left ventricular systolic and diastolic functions, wall thickness, and myocardial strain were assessed with echocardiography. Features evaluated with CMR included left ventricular function, chamber size, myocardial mass, and late gadolinium enhancement. Our results largely paralleled those of DMD cardiomyopathy. Ejection fraction and fractional shortening correlated well with age, with systolic dysfunction occurring at ≈30 to 45 months. Circumferential strain was more sensitive than ejection fraction in early disease detection. Evidence of left ventricular chamber dilatation provided proof of dilated cardiomyopathy. Late gadolinium enhancement imaging showed DMD-like left ventricular lateral wall lesions and earlier involvement of the anterior septum. Multiple functional indexes were graded objectively and added, with and without late gadolinium enhancement, to give cardiac and cardiomyopathy scores of disease severity. Consistent with DMD, there was parallel skeletal muscle involvement, as tibiotarsal joint flexion torque declined in tandem with cardiac function. Conclusions This study established parallels of progressive cardiomyopathy between dystrophic dogs and boys, further validating GRMD as a model of DMD cardiac disease.

Glucose Metabolism as a Pre-clinical Biomarker for the Golden Retriever Model of Duchenne Muscular Dystrophy.

PURPOSE: Metabolic dysfunction in Duchenne muscular dystrophy (DMD) is characterized by reduced glycolytic and oxidative enzymes, decreased and abnormal mitochondria, decreased ATP, and increased oxidative stress. We analyzed glucose metabolism as a potential disease biomarker in the genetically homologous golden retriever muscular dystrophy (GRMD) dog with molecular, biochemical, and in vivo imaging. PROCEDURES: Pelvic limb skeletal muscle and left ventricle tissue from the heart were analyzed by mRNA profiling, qPCR, western blotting, and immunofluorescence microscopy for the primary glucose transporter (GLUT4). Physiologic glucose handling was measured by fasting glucose tolerance test (GTT), insulin levels, and skeletal and cardiac positron emission tomography/X-ray computed tomography (PET/CT) using the glucose analog 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). RESULTS: MRNA profiles showed decreased GLUT4 in the cranial sartorius (CS), vastus lateralis (VL), and long digital extensor (LDE) of GRMD vs. normal dogs. QPCR confirmed GLUT4 downregulation but increased hexokinase-1. GLUT4 protein levels were not different in the CS, VL, or left ventricle but increased in the LDE of GRMD vs. normal. Microscopy revealed diffuse membrane expression of GLUT4 in GRMD skeletal but not cardiac muscle. GTT showed higher basal glucose and insulin in GRMD but rapid tissue glucose uptake at 5 min post-dextrose injection in GRMD vs. normal/carrier dogs. PET/ CT with [18F]FDG and simultaneous insulin stimulation showed a significant increase (p = 0.03) in mean standard uptake values (SUV) in GRMD skeletal muscle but not pelvic fat at 5 min post-[18F]FDG /insulin injection. Conversely, mean cardiac SUV was lower in GRMD than carrier/normal (p < 0.01). CONCLUSIONS: Altered glucose metabolism in skeletal and cardiac muscle of GRMD dogs can be monitored with molecular, biochemical, and in vivo imaging studies and potentially utilized as a biomarker for disease progression and therapeutic response.

Suspected acute myocardial infarction in a dystrophin-deficient dog.

Golden retriever muscular dystrophy (GRMD) is a model for the genetically homologous human disease, Duchenne muscular dystrophy (DMD). Unlike the mildly affected mdx mouse, GRMD recapitulates the severe DMD phenotype. In addition to skeletal muscle involvement, DMD boys develop cardiomyopathy. While the cardiomyopathy of DMD is typically slowly progressive, rare early episodes of acute cardiac decompensation, compatible with myocardial infarction, have been described. We report here a 7-month-old GRMD dog with an apparent analogous episode of myocardial infarction. The dog presented with acute signs of cardiac disease, including tachyarrhythmia, supraventricular premature complexes, and femoral pulse deficits. Serum cardiac biomarkers, cardiac-specific troponin I (cTnI) and N-terminal prohormone of B-type natriuretic peptide (NT-proBNP), were markedly increased. Echocardiography showed areas of hyperechoic myocardial enhancement, typical of GRMD cardiomyopathy. Left ventricular dyskinesis and elevated cTnI were suggestive of acute myocardial damage/infarction. Over a 3-year period, progression to a severe dilated phenotype was observed.