Figure 3

Early iron-overload cardiomyopathy is driven by downregulation of SERCA2a: rescue with adenoviral transfer of SERCA2a and RSV.

(A,B) Western blot analysis and quantification shows a marked decreased in myocardial SERCA2a level (A) which was prevented by in vivo adeno-viral gene therapy (AAV9) confirmed in isolated adult ventricular cardiomyocytes following in vivo AAV9 delivery of green fluorescent protein (GFP) showing a high yield of efficient gene delivery to the heart (B). Assessment of diastolic function using transthoracic echocardiography showing in vivo gene delivery of SERCA2 normalized the diastolic dysfunction associated with early iron-overload (C). (D,E) Western blot analysis revealed a dramatic corrective action of resveratrol (RSV) on the reduced SERCA2a levels (D) which correlated with the ability of RSV to prevent iron-induced downregulation of Serca2a mRNA expression in mouse (m) and human (h) LV cardiomyocytes (E,F) Western blot analysis of sodium-calcium exchanger-1 (NCX-1) showing increased levels in early iron-overload which was normalized in response to RSV. (G) Functional assessment of heart function showing diastolic dysfunction in early iron-overloaded wildtype mice was completely normalized by RSV therapy. (H) Ca²⁺ transients in ventricular cardiomyocytes showing elevated diastolic Ca²⁺ levels and prolongation of Ca²⁺ decay and correction by SERCA2a gene therapy and RSV. R.R. = relative ratio; R.E. = relative expression; E = early LV transmitral filling velocity; A = LV transmitral filling due to atrial contraction; DT = deceleration time; LA = left atrial; EF = ejection fraction. E’ = early tissue Doppler velocity; A’ = tissue Doppler due to atrial contraction; IVRT = isovolumetric relaxation time. n = 8–12 for functional studies; n = 8 for expression analysis and n = 3–4 for Western blot analysis. *p < 0.05 compared with all other groups; ^#p < 0.05 compared with the placebo group.