(A) Schematic representation of wild-type and mutant apoptosis signal-regulating kinase 1 (ASK1) proteins. The kinase domain is shown as red boxes. ASK(KR) and ASKN(KR) represent a dominant-negative mutant (Lys709>Arg) and the N-terminal truncated form of ASK(KR), respectively. (B) Effect of ASKN(KR) on phenylephrine (PE)-induced mitogen-activated protein kinase kinase (MKK6) activation. (Upper panel) rat neonatal cardiomyocytes were infected with hemagglutinin (HA)-tagged ASKN(KR) at multiplicity of infection (MOI) of 1 or 100, and stimulated with 100 µmol/l PE for 10 min. Cell lysates were immunoprecipitated (IP) with anti-MKK6 antibody, and MKK6 activity was measured with an immune complex kinase assay using His-p38 as a substrate. (Lower panel) Western blot (WB) using anti-MKK6 antibody. (C) LacZ expression in heart and liver 14 weeks after recombinant adeno-associated virus (rAAV)/LacZ gene transfer. (D) HA-tagged ASKN(KR) (95-kDa) or endogenous ASK1 (160-kDa) protein level in hamster hearts. (E) ASK1 activity in hamster hearts. Immune complex kinase assay of ASK1 was performed as described in the Methods section. IB = immunoblot.

Normal F1B (orange bars, n = 10), rAAV/LacZ (blue bars, n = 12), and rAAV/ASKN(KR) (red bars, n = 19) hamsters were analyzed with echocardiography before, 6 weeks, and 14 weeks after gene transfer (GT). (A) Representative M-mode echocardiographic tracings 14 weeks after GT. (B) Left ventricular fractional shortening (LVFS), (C) LV ejection fraction (LVEF), (D) LV end-diastolic dimension (LVEDD), (E) LV end-systolic dimension (LVESD), (F) diastolic wall thickness of interventricular septum (IVSd), (G) diastolic wall thickness of LV posterior wall (LVPWT), and (H) LVEDD/LVPWT (index of LV diastolic wall stress). Pre-, 6 weeks, and 14 weeks represent before, 6, and 14 weeks after gene transfer, respectively. *p < 0.05 versus rAAV/LacZ at the corresponding time point. p < 0.05 versus normal hamsters. Abbreviations as in Figure 1.

Hemodynamic parameters of rAAV/LacZ (blue bars, n = 7) and rAAV/ASKN(KR) (red bars, n = 6) hamsters were examined 14 weeks after gene transfer. (A) Left ventricular systolic pressure (LVSP), (B) maximum first derivative of LV pressure (max LVdp/dt), (C) minimum first derivative of LV pressure (min LVdp/dt), and (D) heart rate (HR). *p < 0.05 versus rAAV/LacZ. Abbreviations as in Figure 1.

Lower graphs show results of densitometric analysis. The ratio of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), or ß-myosin heavy chain (ß-MHC) to glyceraldehyde 3 phosphate dehydrogenase (GAPDH) in a normal hamster was expressed as 1. Orange, blue, and red bars represent normal, rAAV/LacZ, and rAAV/ASKN(KR) hamsters, respectively (n = 3 for each group). *p < 0.05 versus rAAV/LacZ, p < 0.05 versus normal hamsters. Abbreviations as in Figure 1.

Hematoxylin and eosin-stained (A to C) and Masson-trichrome-stained (D to F) sections of normal hamsters (A and D), rAAV/LacZ-treated (B and E), and rAAV/ASKN(KR)-treated hamsters (C and F). Each bar represents 100 µm. (G) mRNA levels of 1 type I and III collagen were evaluated by dot blot analysis (type I collagen: n = 3 for each group; type III collagen: n = 5 for normal hamsters, n = 4 for rAAV/LacZ or rAAV/ASKN(KR)). The ratio of 1 type I or III collagen to GAPDH in a normal hamster was expressed as 1. *p < 0.05 versus rAAV/LacZ, p < 0.05 versus normal hamsters. Abbreviations as in Figures 1 and 4.

(A) Images of apoptotic cardiomyocytes. Triple staining with propidium iodide, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), and anti--sarcomeric actin antibody. (B) Number of TUNEL-positive cardiomyocytes. *p < 0.05 versus rAAV/LacZ (n = 5). (C) Immunohistochemical staining of activated caspase 3 (left panels). Normal rabbit immunoglobulin (Ig)G was used as a negative control (right panels). Arrows indicate activated caspase 3-positive cells. (D) Number of activated caspase 3-positive cardiomyocytes. *p < 0.05 versus rAAV/LacZ (n = 5). Abbreviations as in Figure 1.

(A) Western blot analysis for phospho- and total-c-Jun NH₂-terminal kinase (JNK), p38 MAP kinase, or extracellular signal-regulated kinase (ERK) 4 weeks after gene transfer. (B to D) Densitometric analysis of blots. Ratio of phospho- and total-JNK, p38 MAPK, or ERK in a rAAV/LacZ-treated heart was expressed as 1. Blue and red bars represent rAAV/LacZ (n = 6) and rAAV/ASKN(KR) (n = 5) groups, respectively. Abbreviations as in Figure 1.

(A) Western blot analysis for -, ß-, and -sarcoglycan (SG) in hamster hearts 14 weeks after gene transfer. (B) Western blot analysis for dystrophin in hamster hearts 14 weeks after gene transfer. Full-length (arrow) and degraded (arrowheads) dystrophin were detected. (C) Western blot analysis for µ- and m-calpain. Arrows indicate autolyzed form of µ-calpain. (D) [Ca²⁺]_i after change to Na⁺-free solution. (Left panel) Changes in the ratio of fluorescent intensity of fura-2 at 340 nm to that at 380 nm after change to Na⁺-free solution. A representative averaged recording from at least 3 cardiomyocytes infected with AdV/LacZ or AdV/ASKN(KR) is shown. The right panel shows maximum fold changes in the fluorescent intensity ratio of fura-2 (n = 3). (E) Activation of ASK1 induced by Ca²⁺ overload. ASK1 activity was assessed by immune complex kinase assay with MKK6 as a substrate. (F) Calpain activity during Ca²⁺ overload. Left panel, changes in the fluorescent intensity of Boc-Leu-Met-CMAC (CMAC) during Ca²⁺ overload. A representative averaged recording from at least 3 cardiomyocytes is shown. To assess calpain-dependent protease activity, the cells were treated with or without calpeptin, a calpain inhibitor. Right panel shows slopes of CMAC fluorescent intensity curve between 10 and 30 min after change to Na⁺-free solution (n = 3). *p < 0.05 versus AdV/LacZ-infected cardiomyocytes. Abbreviations as in Figure 1.