Cardiac Gene Transfer of Short Hairpin RNA Directed Against Phospholamban Effectively Knocks Down Gene Expression but Causes Cellular Toxicity in Canines

Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways

Chronic Losartan Administration Reduces Mortality and Preserves Cardiac but Not Skeletal Muscle Function in Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a degenerative disorder affecting skeletal and cardiac muscle for which there is no effective therapy. Angiotension receptor blockade (ARB) has excellent therapeutic potential in DMD based on recent data demonstrating attenuation of skeletal muscle disease progression during 6–9 months of therapy in the mdx mouse model of DMD. Since cardiac-related death is major cause of mortality in DMD, it is important to evaluate the effect of any novel treatment on the heart. Therefore, we evaluated the long-term impact of ARB on both the skeletal muscle and cardiac phenotype of the mdx mouse. Mdx mice received either losartan (0.6 g/L) (n = 8) or standard drinking water (n = 9) for two years, after which echocardiography was performed to assess cardiac function. Skeletal muscle weight, morphology, and function were assessed. Fibrosis was evaluated in the diaphragm and heart by Trichrome stain and by determination of tissue hydroxyproline content. By the study endpoint, 88% of treated mice were alive compared to only 44% of untreated (p = 0.05). No difference in skeletal muscle morphology, function, or fibrosis was noted in losartan-treated animals. Cardiac function was significantly preserved with losartan treatment, with a trend towards reduction in cardiac fibrosis. We saw no impact on the skeletal muscle disease progression, suggesting that other pathways that trigger fibrosis dominate over angiotensin II in skeletal muscle long term, unlike the situation in the heart. Our study suggests that ARB may be an important prophylactic treatment for DMD-associated cardiomyopathy, but will not impact skeletal muscle disease

Skeletal muscle function in <i>mdx</i> mice at two years.

<p>(a) Specific force and (b) maximum tetanic force of the extensor digitorum longus (EDL), soleus, and diaphragm were quantified at two years in treated and untreated <i>mdx</i> mice. No significant differences were noted. Data represent mean±SD.</p

Cardiac Function As Assessed By 2-D Echocardiography At 2 Years.

a<p>HR: heart rate;^bIVS: interventricular septum;^cLVIDd: left ventricular inner diameter in diastole;^dLVFW: left ventricular free wall;^eLVIDs: left ventricular inner diameter in systole;^fFS: fractional shortening; ^gEDV: end diastolic volume; ^hESV: end systolic volume; ⁱEF: ejection fraction; ^jLV: left ventricular; ^kCO: cardiac output; ^lSV: stroke volume;^mCon: control treatment; ⁿValues reported as mean ± standard deviation;^oLos: losartan treatment; ^pp: p value.</p

Survival in treated and untreated <i>mdx</i> mice at two years.

<p>The survival curves for treated and untreated <i>mdx</i> mice are significantly different(p = 0.05). 88% of treated mice were alive at two years compared to only 44% of untreated mice. Data represent mean±SEM.</p