Neonatal gene transfer of Serca2a delays onset of hypertrophic remodeling and improves function in familial hypertrophic cardiomyopathy

Abstract

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant genetic disorder linked to numerous mutations in the sarcomeric proteins. The clinical presentation of FHC is highly variable, but it is a major cause of sudden cardiac death in young adults with no specific treatments. We tested the hypothesis that early intervention in Ca(2+) regulation may prevent pathological hypertrophy and improve cardiac function in a FHC displaying increased myofilament sensitivity to Ca(2+) and diastolic dysfunction. A transgenic (TG) mouse model of FHC with a mutation in tropomyosin at position 180 was employed. Adenoviral-Serca2a (Ad.Ser) was injected into the left ventricle of 1-day-old non-transgenic (NTG) and TG mice. Ad.LacZ was injected as a control. Serca2a protein expression was significantly increased in NTG and TG hearts injected with Ad.Ser for up to 6 weeks. Compared to TG-Ad.LacZ hearts, the TG-Ad.Ser hearts showed improved whole heart morphology. Moreover, there was a significant decline in ANF and β-MHC expression. Developed force in isolated papillary muscle from 2- to 3-week-old TG-Ad.Ser hearts was higher and the response to isoproterenol (ISO) improved compared to TG-Ad.LacZ muscles. In situ hemodynamic measurements showed that by 3 months the TG-Ad.Ser hearts also had a significantly improved response to ISO compared to TG-Ad.LacZ hearts. The present study strongly suggests that Serca2a expression should be considered as a potential target for gene therapy in FHC. Moreover, our data imply that development of FHC can be successfully delayed if therapies are started shortly after birth.

Figure 1

A. Representative hearts from a mouse injected with saline (left heart) or Ad.LacZ one day after birth and stained for lacZ. B. Expression of exogenous Serca2a detected by PCR in samples from 1 week, 3 week and 6 week old mice. C. Representative hearts from (non transgenic) NTG and transgenic Tm180 (TG) mice injected with Ad.LacZ or Ad.Ser at 6 week of age. D. Heart weight to body weight ratio in NTG and TG mice injected with Ad.LacZ or Ad.Ser at 3 weeks and 3–4 months of age. Data are presented as mean ± SEM, n=8–9 per group E. Hyroxyproline content in ventricles from NTG and TG mice injected with Ad.LacZ or Ad.Ser at 3–4 months of age. Data are presented as mean ± SEM, n=3–4 per group.

Figure 2

Message levels of atrial natriuretic factor (ANF), β-myosin heavy chain (βMHC), phospholamban (PLB) and endogenous Serca2a at different time points in NTG and TG hearts injected with Ad.LacZ or Ad.Ser. A. Expression of ANF. B. Expression of βMHC. C. Expression of PLB. D. Expression of endogenous Serca2a. Expression was normalized to GAPDH and presented as mean±SEM. n=5–10 per group

Figure 3

Expression of total Serca2a, PLB and phosphorylated PLB (phos-PLB) at Ser16 in NTG and TG mice injected with Ad.LacZ or Ad.Ser at different time points. A. A representative Western blot for total Serca2a expression at 1 week of age. B. The average expression of total Serca2a at 1 week of age. C. The average expression of total Serca 2a at 3 week of age. D. The average expression of total Serca 2a at 3–4 month of age. Actin was used as internal control for loading. E. A representative Western blot for PLB and phospho-PLB (Ser 16) at 3 weeks of age. F. A representative Western blot for PLB and phospho-PLB (Ser 16) at 3–4 months of age. Data are presented as mean ± SEM. n=4–8 per group.

Figure 4

The effect of isoproterenol (ISO) on developed force and relaxation time in papillary muscles isolated from NTG and TG mice injected with Ad.LacZ or Ad.Ser. A. Developed force at baseline and during ISO stimulation. B. Time to 90% of relaxation normalized to developed force at baseline and during ISO stimulation. Data are presented as mean ± SEM, n=5–11 per group.

Figure 5

Hemodynamic parameters during basal state and isoproterenol (ISO) stimulation in NTG and TG mice injected with Ad.LacZ or Ad.Ser. A. Developed pressure (DP). B. Heart rate (HR). C. Rate of contraction (dP/dt_max) and D. Rate of relaxation (dP/dt_min) at baseline. E. Percentage change of DP, HR, dP/dt_max and dP/dt_min during low dose of ISO stimulation (0.08 ng/g b.w./min). F. Percentage change of DP, HR, dP/dt_max and dP/dt_min during max dose of ISO stimulation (0.32 ng/g b.w./min). Data are presented as mean ± SEM. n=5–10 per group.

Figure 6

Quantification of myofilament protein phosphorylation of NTG and TG mice injected with Ad.LacZ and Ad.Ser. TnT_{3 or 4}, troponin T (isoform 3 or 4); p, phosphorylated; MLC₂, regulatory myosin light-chain; U, un-phosphorylated; Tm, tropomyosin; Note the TG animals express both the TG (Tm180) and wild-type (WT) form of Tm. A. Representative 2D-DIGE gel showing region of interest from 3 week old mice injected with Ad.LacZ. Myofibrillar fractions were labeled separately and equally mixed. The red signal corresponds to NTG-Ad.LacZ and the green signal corresponds to TG-Ad.LacZ. The legend to the right identifies the labels on the x-axis of the bar graphs (B–E). B. Representative 2D-DIGE gel region of Tm from 3 week old TG-Ad.LacZ and TG-Ad.Ser mice. The bar graph shows the 3 week old mice Tm phosphorylation levels. C. Representative 2D-DIGE gel region of Tm from 3–4 month old TG-Ad.LacZ and TGAd. Ser mice. The bar graph shows the 3–4 month old mice Tm phosphorylation levels. D. Representative 2D-DIGE gel images showing region of MLC₂ from 3 week old transgenic and wild type mice treated with Ad.LacZ. The bar graph to the right shows the 3 week old mice MLC₂ phosphorylation levels. E. Representative 2D-DIGE gel images showing region of MLC₂ from 3–4 month old transgenic and wild type mice injected with Serca2a. The bar graph to the right shows the 3–4 month old mice MLC₂ phosphorylation levels. Values are means±SEM. n=4 per group.