Therapeutic potential of AAV9-S15D-RLC gene delivery in humanized MYL2 mouse model of HCM

Abstract

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder characterized by ventricular hypertrophy, myofibrillar disarray and fibrosis, and is primarily caused by mutations in sarcomeric genes. With no definitive cure for HCM, there is an urgent need for the development of novel preventive and reparative therapies. This study is focused on Aspartic acid-to-Valine (D166V) mutation in the myosin regulatory light chain, RLC (MYL2 gene), associated with a malignant form of HCM. Since myosin RLC phosphorylation is critical for normal cardiac function, we aimed to exploit this post-translational modification via phosphomimetic-RLC gene therapy. We hypothesized that mimicking/modulating cardiac RLC phosphorylation in nonphosphorylatable D166V myocardium would improve heart function of HCM-D166V mice. Adeno-Associated Virus, serotype-9 (AAV9) was used to deliver phosphomimetic human RLC variant with Serine-to-Aspartic acid substitution at Ser15-RLC phosphorylation site (S15D-RLC) into the hearts of humanized HCM-D166V mice. Improvement of heart function was monitored by echocardiography, invasive hemodynamics (PV-loops) and muscle contractile mechanics. A significant increase in cardiac output and stroke work and a decrease in relaxation constant, Tau, shown to be prolonged in HCM mice, were observed in AAV-vs. PBS-injected HCM mice. Strain analysis showed enhanced myocardial longitudinal shortening in AAV-treated vs. control mice. In addition, increased maximal contractile force was observed in skinned papillary muscles from AAV-injected HCM hearts. Our data suggest that myosin RLC phosphorylation may have important translational implications for the treatment of RLC-induced HCM and possibly play a role in other disease settings accompanied by depressed Ser15-RLC phosphorylation.

Ablation of the N terminus of cardiac essential light chain promotes the super‐relaxed state of myosin and counteracts hypercontractility in hypertrophic cardiomyopathy mutant mice

“…To gain insight into A57G‐ versus Δ43‐related heart remodeling in vivo , cardiac morphology and function were assessed in all experimental groups of mice using B‐ and M‐mode echocardiography, and pulse‐wave and tissue Doppler analysis, as described in Yadav et al . . Heart rate (HR) and body temperature (37.0 ± 1 °C) were carefully monitored and controlled during data acquisition to avoid any effects on cardiac function and subsequent data analysis.…”

“…A Vevo‐2100 imaging system, equipped with MS 400 transducer, was used to assess the LV diastolic function of mice that was analyzed using the values derived from the mitral inflow velocity curves obtained by pulse‐wave Doppler method, as described previously . Doppler indexes included the ratio of peak velocity of early to late diastolic filling of mitral inflow (E/A), isovolumetric contraction time (IVCT), isovolumetric relaxation time (IVRT), and ejection time (ET).…”

“…The mouse LV PM were isolated from the hearts of F and M mice from all groups, dissected into small bundles, and chemically skinned for 24 h at 4 °C. The skinning solution contained 50% glycerol and 50% pCa 8 solution [10 −8 m (Ca 2+ ), 1 m m free (Mg 2+ ), total MgPr (propionate) =3.88 m m , 7 m m EGTA, 2.5 m m (Mg‐ATP 2− ), 20 m m MOPS pH 7.0, 15 m m creatine phosphate, 15 U·mL −1 of phosphocreatine kinase, ionic strength = 150 m m (adjusted with KPr), and 1% Triton X‐100] . Muscles were then transferred to the fresh solution without Triton X‐100 and stored at −20 °C until used (5–10 days).…”

“…As recently described , high‐resolution long‐axis B‐mode images were used to perform STE analyses using the vevo strain software ( vevo lab 2.1.0). Changes in length (in the radial axis from the center of the ventricular cavity outward and in the longitudinal axis from the apex to the base of the heart) relative to initial length (final length/initial length) and strain rate, describing the rate of change of deformation over time, were measured and analyzed.…”

“…Global LV longitudinal and radial strain and strain rates were evaluated in both epicardial and endocardial regions. In addition, B‐mode images were recorded, with vector diagrams depicting magnitude and direction of endocardial deformation . The LV global longitudinal strain (GLS) assessments, depicting the maximal shortening of myocardial longitudinal length during systole, were compared to those in diastole and the resting myocardial length.…”