Brian Bostick

Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy

Sarcolemma-associated neuronal NOS (nNOS) plays a critical role in normal muscle physiology. In Duchenne muscular dystrophy (DMD), the loss of sarcolemmal nNOS leads to functional ischemia and muscle damage; however, the mechanism of nNOS subcellular localization remains incompletely understood. According to the prevailing model, nNOS is recruited to the sarcolemma by syntrophin, and in DMD this localization is altered. Intriguingly, the presence of syntrophin on the membrane does not always restore sarcolemmal nNOS. Thus, we wished to determine whether dystrophin functions in subcellular localization of nNOS and which regions may be necessary. Using in vivo transfection of dystrophin deletion constructs, we show that sarcolemmal targeting of nNOS was dependent on the spectrin-like repeats 16 and 17 (R16/17) within the rod domain. Treatment of mdx mice (a DMD model) with R16/17-containing synthetic dystrophin genes effectively ameliorated histological muscle pathology and improved muscle strength as well as exercise performance. Furthermore, sarcolemma-targeted nNOS attenuated alpha-adrenergic vasoconstriction in contracting muscle and improved muscle perfusion during exercise as measured by Doppler and microsphere circulation. In summary, we have identified the dystrophin spectrin-like repeats 16 and 17 as a novel scaffold for nNOS sarcolemmal targeting. These data suggest that muscular dystrophy gene therapies based on R16/17-containing dystrophins may yield better clinical outcomes than the current therapies.

A Single Intravenous Injection of Adeno-associated Virus Serotype-9 Leads to Whole Body Skeletal Muscle Transduction in Dogs

The success of many gene therapy applications hinges on efficient whole body transduction. In the case of muscular dystrophies, a therapeutic vector has to reach every muscle in the body. Recent studies suggest that vectors based on adeno-associated virus (AAV) are capable of body-wide transduction in rodents. However, translating this finding to large animals remains a challenge. Here we explored systemic gene delivery with AAV serotype-9 (AAV-9) in neonatal dogs. Previous attempts to directly deliver AAV to adult canine muscle have yielded minimal transduction due to a strong cellular immune response. However, in neonatal dogs we observed robust skeletal muscle transduction throughout the body after a single intravenous injection. Importantly, systemic transduction was achieved in the absence of pharmacological intervention or immune suppression and it lasted for at least 6 months (the duration of study). We also observed several unique features not predicted by murine studies. In particular, cardiac muscle was barely transduced in dogs. Many muscular dystrophy patients can be identified by neonatal screening. The technology described here may lead to an effective early intervention in these patients.

Prevention of Dystrophin-Deficient Cardiomyopathy in Twenty-One-Month-Old Carrier Mice by Mosaic Dystrophin Expression or Complementary Dystrophin/Utrophin Expression

A cure for dystrophin-deficient muscular dystrophy requires treating both skeletal muscle and the heart. Whereas mosaic dystrophin expression has been shown to protect skeletal muscle, controversy exists over whether mosaic expression is protective in the heart. We have shown recently that mosaic dystrophin expression prevents stress-induced heart damage in young carrier mice. Although an interesting finding, the clinical relevance remains to be established because young dystrophin-null mdx mice do not have heart disease. On the other hand, heart failure has been reported in human carriers. To resolve this mouse/human discrepancy, we evaluated the cardiac phenotype in 21-month-old mdx, carrier, and normal mice. We found dilated cardiomyopathy in old mdx mice but not in age-matched carrier mice. All anatomical parameters and physiological assay results (ECG and closed-chest Millar catheter) were within the normal range in old carrier mice. Focal myocardial inflammation was found in a small fraction of old carrier mice, but it had no major impact on heart function. Dobutamine stress revealed a near normal hemodynamic profile except for a marginal reduction in systolic pressure in old carrier mice. Immunostaining and Western blot showed dystrophin expression in 50% cardiomyocytes in old carrier mice. Interestingly, utrophin was upregulated in dystrophin-negative heart cells in carrier mice. In summary, we have provided the first clear-cut evidence that dilated cardiomyopathy in old mdx mice was prevented by mosaic dystrophin expression or complementary dystrophin/utrophin expression. Our results raise the hope for ameliorating dystrophic cardiomyopathy through partial gene and/or cell therapy.

Adeno-Associated Virus Serotype-9 Microdystrophin Gene Therapy Ameliorates Electrocardiographic Abnormalities in mdx Mice

Adeno-associated virus (AAV)-mediated microdystrophin gene therapy holds great promise for treating Duchenne muscular dystrophy (DMD). Previous studies have revealed excellent skeletal muscle protection. Cardiac muscle is also compromised in DMD patients. Here we show that a single intravenous injection of AAV serotype-9 (AAV-9) microdystrophin vector efficiently transduced the entire heart in neonatal mdx mice, a dystrophin-deficient mouse DMD model. Furthermore, microdystrophin therapy normalized the heart rate, PR interval, and QT interval. The cardiomyopathy index was also significantly improved in treated mdx mice. Our study demonstrates for the first time that AAV microdystrophin gene therapy can ameliorate the electrocardiographic abnormalities in a mouse model for DMD.

Cardiac Expression of a Mini-dystrophin That Normalizes Skeletal Muscle Force Only Partially Restores Heart Function in Aged Mdx Mice

Duchenne muscular dystrophy (DMD) affects both skeletal and cardiac muscle. It is currently unclear whether the strategies developed for skeletal muscle can ameliorate cardiomyopathy. Synthetic mini-/micro-dystrophin genes have yielded impressive skeletal muscle protection in animal models. The 6-kb DeltaH2-R19 minigene is particularly promising because it completely restores skeletal muscle force to wild-type levels. Here, we examined whether expressing this minigene in the heart, but not skeletal muscle, could normalize cardiac function in the mdx model of DMD cardiomyopathy. Transgenic mdx mice were generated to express the DeltaH2-R19 minigene under the control of the alpha-myosin heavy-chain promoter. Heart structure and function were examined in adult and very old mice. The DeltaH2-R19 minigene enhanced cardiomyocyte sarcolemmal strength and prevented myocardial fibrosis. It also restored the dobutamine response and enhanced treadmill performance. Surprisingly, heart-restricted DeltaH2-R19 minigene expression did not completely normalize electrocardiogram and hemodynamic abnormalities. Overall, systolic function and ejection fraction were restored to normal levels but stroke volume and cardiac output remained suboptimal. Our results demonstrate that the skeletal muscle-proven DeltaH2-R19 minigene can correct cardiac histopathology but cannot fully normalize heart function. Novel strategies must be developed to completely restore heart function in DMD.

Dipeptidyl peptidase inhibition prevents diastolic dysfunction and reduces myocardial fibrosis in a Mouse model of Western diet induced obesity

OBJECTIVE Consumption of a high-fat/high-fructose Western diet (WD) is linked to rising obesity and heart disease, particularly diastolic dysfunction which characterizes early obesity/metabolic cardiomyopathy. Mounting evidence supports a role for inflammation, oxidative stress and fibrosis in the pathophysiology of metabolic cardiomyopathy. Dipeptidyl peptidase-4 (DPP-4) is a circulating exopeptidase recently reported to be elevated in the plasma of patients with insulin resistance (IR), obesity and heart failure. We hypothesized that a model of WD induced obesity/metabolic cardiomyopathy would exhibit increased DPP-4 activity and cardiac fibrosis with DPP-4 inhibition preventing cardiac fibrosis and the associated diastolic dysfunction. MATERIALS/METHODS Four-week-old C57BL6/J mice were fed a high-fat/high-fructose WD with the DPP-4 inhibitor MK0626 for 16 weeks. Cardiac function was examined by high-resolution cine-cardiac magnetic resonance imaging (MRI). Phenotypic analysis included measurements of body and heart weight, systemic IR and DPP-4 activity. Immunohistochemistry and transmission electron microscopy (TEM) were utilized to identify underlying pathologic mechanisms. RESULTS We found that chronic WD consumption caused obesity, IR, elevated plasma DPP-4 activity, heart enlargement and diastolic dysfunction. DPP-4 inhibition with MK0626 in WD fed mice resulted in >75% reduction in plasma DPP-4 activity, improved IR and normalized diastolic relaxation. WD consumption induced myocardial oxidant stress and fibrosis with amelioration by MK0626. TEM of hearts from WD fed mice revealed abnormal mitochondrial and perivascular ultrastructure partially corrected by MK0626. CONCLUSIONS This study provides evidence of a role for increased DPP-4 activity in metabolic cardiomyopathy and a potential role for DPP-4 inhibition in prevention and/or correction of oxidant stress/fibrosis and associated diastolic dysfunction.

Uric Acid Promotes Left Ventricular Diastolic Dysfunction in Mice Fed a Western Diet

The rising obesity rates parallel increased consumption of a Western diet, high in fat and fructose, which is associated with increased uric acid. Population-based data support that elevated serum uric acids are associated with left ventricular hypertrophy and diastolic dysfunction. However, the mechanism by which excess uric acid promotes these maladaptive cardiac effects has not been explored. In assessing the role of Western diet–induced increases in uric acid, we hypothesized that reductions in uric acid would prevent Western diet–induced development of cardiomyocyte hypertrophy, cardiac stiffness, and impaired diastolic relaxation by reducing growth and profibrotic signaling pathways. Four-weeks-old C57BL6/J male mice were fed excess fat (46%) and fructose (17.5%) with or without allopurinol (125 mg/L), a xanthine oxidase inhibitor, for 16 weeks. The Western diet–induced increases in serum uric acid along with increases in cardiac tissue xanthine oxidase activity temporally related to increases in body weight, fat mass, and insulin resistance without changes in blood pressure. The Western diet induced cardiomyocte hypertrophy, myocardial oxidative stress, interstitial fibrosis, and impaired diastolic relaxation. Further, the Western diet enhanced activation of the S6 kinase-1 growth pathway and the profibrotic transforming growth factor-&bgr;1/Smad2/3 signaling pathway and macrophage proinflammatory polarization. All results improved with allopurinol treatment, which lowered cardiac xanthine oxidase as well as serum uric acid levels. These findings support the notion that increased production of uric acid with intake of a Western diet promotes cardiomyocyte hypertrophy, inflammation, and oxidative stress that lead to myocardial fibrosis and associated impaired diastolic relaxation.

AAV micro-dystrophin gene therapy alleviates stress-induced cardiac death but not myocardial fibrosis in > 21-m-old mdx mice, an end-stage model of Duchenne muscular dystrophy cardiomyopathy

Duchenne muscular dystrophy (DMD) is a fatal genetic disease caused by the absence of the sarcolemmal protein dystrophin. Dilated cardiomyopathy leading to heart failure is a significant source of morbidity and mortality in DMD. We recently demonstrated amelioration of DMD heart disease in 16 to 20-m-old dystrophin-null mdx mice using adeno-associated virus (AAV) mediated micro-dystrophin gene therapy. DMD patients show severe heart disease near the end of their life expectancy. Similarly, mdx mice exhibit profoundly worsening heart disease when they reach beyond 21 months of age. To more rigorously test micro-dystrophin therapy, we treated mdx mice that were between 21.2 and 22.7-m-old (average, 22.1 ± 0.2 months; N=8). The ∆R4-23/∆C micro-dystrophin gene was packaged in the cardiotropic AAV-9 virus. 5×10(12) viral genome particles/mouse were delivered to mdx mice via the tail vein. AAV transduction, myocardial fibrosis and heart function were examined 1.7 ± 0.2 months after gene therapy. Efficient micro-dystrophin expression was observed in the myocardium of treated mice. Despite the robust dystrophin expression, myocardial fibrosis was not mitigated. Most hemodynamic parameters were not improved either. However, ECG abnormalities were partially corrected. Importantly, treated mice became more resistant to dobutamine-induced cardiac death. In summary, we have revealed for the first time the potential benefits and limitations of AAV micro-dystrophin therapy in end-stage Duchenne dilated cardiomyopathy. Our findings have important implications for the use of AAV gene therapy in dilated cardiomyopathy and heart failure.

AAV-microdystrophin Therapy Improves Cardiac Performance in Aged Female mdx Mice

Dystrophin deficiency leads to lethal dilated Duchenne cardiomyopathy. A promising therapy is to deliver a highly abbreviated microdystrophin gene to the heart using adeno-associated virus (AAV). Microdystrophin has been shown to mitigate dystrophin-deficient skeletal muscle disease. However, it is not clear whether microdystrophin is equally effective in treating Duchenne cardiomyopathy. To evaluate microdystrophin therapy in the heart, we injected 5 × 10(12) viral genome particles/mouse of AAV-9 ΔR4-23/ΔC microdystrophin vector via tail vein to ~16-20-month-old (average 18.7-month-old) female mdx mice, a manifesting model of Duchenne cardiomyopathy. Cardiac transduction and heart function were examined at 2-8 months after gene transfer. We observed robust myocardial microdystrophin expression. Electrocardiography (ECG) and left ventricular catheter hemodynamic assays also revealed significant improvement. Furthermore, AAV-microdystrophin therapy prevented dobutamine-stress induced acute cardiac death. We demonstrate for the first time that AAV microdystrophin therapy significantly ameliorates functional deficiency in a phenotypic model of Duchenne cardiomyopathy. Our results support further exploration of microdystrophin therapy to treat Duchenne cardiomyopathy.

Gender influences cardiac function in the mdx model of duchenne cardiomyopathy

Aged mdx mice represent an important model for studying Duchenne cardiomyopathy. Herein we compared the cardiac phenotypes of 22‐month‐old male and female mdx mice. Surprisingly, only females displayed the characteristic cardiac dilation on pressure–volume loop analysis. Female mdx mice also exhibited lower contractility, larger Q waves, and higher ratios of heart weight to body weight. Our results reveal significant gender disparity in mdx cardiac function. Gender should be considered when using the mdx model for the study of Duchenne cardiomyopathy. Muscle Nerve, 2010