Akt2 knockout mitigates chronic iNOS inhibition-induced cardiomyocyte atrophy and contractile dysfunction despite persistent insulin resistance

Abstract

Increased levels of inducible nitric oxide synthase (iNOS) during cardiac stress such as ischemia-reperfusion, sepsis and hypertension may display both beneficial and detrimental roles in cardiac contractile performance. However, the precise role of iNOS in the maintenance of cardiac contractile function remains elusive. This study was designed to determine the impact of chronic iNOS inhibition on cardiac contractile function and the underlying mechanism involved with a special focus on the NO downstream signaling molecule Akt. Male C57 or Akt2 knockout [Akt2(-/-)] mice were injected with the specific iNOS inhibitor 1400W (2 mg/kg/d) or saline for 7 days. Both 1400W and Akt2 knockout dampened glucose and insulin tolerance without additive effects. Treatment of 1400W decreased heart and liver weights as well as cardiomyocyte cross-sectional area in C57 but not Akt2 knockout mice. 1400W but not Akt2 knockout compromised cardiomyocyte mechanical properties including decreased peak shortening and maximal velocity of shortening/relengthening, prolonged relengthening duration, reduced intracellular Ca(2+) release and decay rate, the effects of which were ablated or attenuated by Akt2 knockout. Akt2 knockout but not 1400W increased the levels of intracellular Ca(2+) regulatory proteins including SERCA2a and phospholamban phosphorylation. 1400W reduced the level of anti-apoptotic protein Bcl-2, the effect of which was unaffected by Akt2 knockout. Neither 1400W nor Akt2 knockout significantly affected ER stress, autophagy, the post-insulin receptor signaling Akt, GSK3β and AMPK, as well as the stress signaling IκB, JNK, ERK and p38 with the exception of elevated IκB phosphorylation with jointed effect of 1400W and Akt2 knockout. Taken together, these data indicated that an essential role of iNOS in the maintenance of cardiac morphology and function possibly through an Akt2-dependent mechanism.

Fig. 1

Effect of chronic iNOS inhibition using 1400W (2 mg/kg/d for 7 days, s.c.) on glucose homeostasis in control and Akt2 deficient mice. A: IPGTT following glucose challenge (2 g/kg, b.w.); B: AUC for IPGTT C: IPITT following insulin challenge (1.5 U/kg, b.w.); and D: AUC for IPITT. Mean ± SEM, n = 6–7 mice per group, * p < 0.05 vs. Control group.

Fig. 2

Effect of chronic iNOS inhibition using 1400W (2 mg/kg/d for 7 days, s.c.) on biometric parameters in control and Akt2 deficient mice. A: Body weight; B: Fasting blood glucose; C: Heart weight; D: Heart-to- body weight ratio; E: Liver weight; F: Liver-to-body weight ratio; G: Kidney weight; and H: Kidney-to-body weight ratio. Mean ± SEM, n = 5–6 mice per group, * p < 0.05 vs. Control group, # p < 0.05 vs. 1400W group.

Fig. 3

Effect chronic iNOS inhibition using 1400W (2 mg/kg/d for 7 days, s.c.) on cardiac morphology in control and Akt2 deficient mice. A: Representative images of transverse cardiac sections stained with a Lectin-FitC conjugate; and B: Quantitative analysis of cardiomyocyte cross-sectional area, Mean ± SEM, n = 150 images from 3 – 4 mice per group, * p < 0.05 vs. Control group, # p < 0.05 vs. 1400W group.

Fig. 4

Contractile properties of cardiomyocytes in control and Akt2 deficient mice treated with or without the iNOS inhibitor 1400W (2 mg/kg/d for 7 days, s.c.). A: Resting cell length; B: Peak shortening (PS, normalized to resting cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (−dL/dt); E: Time-to-PS (TPS); and F: Time-to-90% relengthening (TR₉₀). Mean ± SEM, n = 88–110 cells per group, * p < 0.05 vs. Control group, # p < 0.05 vs. 1400W group.

Fig. 5

Intracellular Ca²⁺ properties in cardiomyocytes from control and Akt2 deficient mice treated with or without the iNOS inhibitor 1400W (2 mg/kg/d for 7 days, s.c.). A: Resting fura-2 fluorescence intensity (FFI); B: Peak FFI (360/380 Ratio); C: Electrically-stimulated rise in FFI (ΔFFI); and D: Intracellular Ca²⁺ decay. Mean ± SEM, n = 53–55 cells per group, * p < 0.05 vs. Control group, # p < 0.05 vs. 1400W group.

Fig. 6

Western blot analysis of SERCA2a, phospholamban (pan and phosphorylated), BCL-xL, Bcl-2, Bip, Beclin-1, and LC3 in myocardium from control and Akt2 deficient mice treated with or without the iNOS inhibitor 1400W (2 mg/kg/d for 7 days, s.c.). A: Representative gel blots of SERCA2a, pan and phosphorylated phospholamban, BCL-xL, Bcl-2, Bip, Beclin-1, LC3 and GAPDH (loading control) using specific antibodies; B: SERCA2a; C: SERCA2a-to-PLB ratio; D: phosphorylated PLB (pPLB)-to-PLB ratio; E: BCL-xL; F: Bcl-2; G: Bip; H: Beclin-1; and I: LC3-II-to-LC3-I ratio. Mean ± SEM, n = 3–4 mice per group, * p < 0.05 vs. Control group, # p < 0.05 vs. 1400W group.

Fig. 7

Western blot analysis of pan and phosphorylated Akt, GSK3β and AMPK in myocardium from control and Akt2 deficient mice treated with or without the iNOS inhibitor 1400W (2 mg/kg/d for 7 days). A: Representative gel blots of Akt, pAkt, GSK3β, pGSK3β, AMPK, pAMPK, and GAPDH (loading control) using specific antibodies; B: pAkt-to-Akt ratio; C: pGSK3β-to-GSK3β ratio; and D: pAMPK-to-AMPK ratio. Mean ± SEM, n = 3 – 4 mice per group.

Fig. 8

Western blot analysis of pan and phosphorylated IΚB, JNK, ERK, and p38 in myocardium from control and Akt2 deficient mice treated with or without the iNOS inhibitor 1400W (2 mg/kg/d for 7 days). A: pIΚB-to-IΚB ratio; B: pJNK-to-JNK ratio; C: pERK-to-ERK ratio; and D: pp38-to-p38 ratio. Mean ± SEM, n = 3–4 mice per group, * p < 0.05 vs. Control group, # p < 0.05 vs. 1400W group.