A humanin analog decreases oxidative stress and preserves mitochondrial integrity in cardiac myoblasts

Abstract

A potent analog (HNG) of the endogenous peptide humanin protects against myocardial ischemia-reperfusion (MI-R) injury in vivo, decreasing infarct size and improving cardiac function. Since oxidative stress contributes to the damage from MI-R we tested the hypotheses that: (1) HNG offers cardioprotection through activation of antioxidant defense mechanisms leading to preservation of mitochondrial structure and that, (2) the activity of either of a pair of non-receptor tyrosine kinases, c-Abl and Arg is required for this protection. Rat cardiac myoblasts (H9C2 cells) were exposed to nanomolar concentrations of HNG and to hydrogen peroxide (H2O2). Cells treated with HNG in the presence of H2O2 demonstrated reduced intracellular reactive oxygen species (ROS), preserved mitochondrial membrane potential, ATP levels and mitochondrial structure. HNG induced activation of catalase and glutathione peroxidase (GPx) within 5 min and decreased the ratio of oxidized to reduced glutathione within 30 min. siRNA knockdown of both Abl and Arg, but neither alone, abolished the HNG-mediated reduction of ROS in myoblasts exposed to H2O2. These findings demonstrate an HNG-mediated, Abl- and Arg-dependent, rapid and sustained activation of critical cellular defense systems and attenuation of oxidative stress, providing mechanistic insights into the observed HNG-mediated cardioprotection in vivo.

Keywords: Abelson murine leukemia viral mammalian homolog; Abl-related gene product; Arg; GPx; HN; HN in which the serine 14 is replaced by glycine; HNG; Humanin; MI–R; MMP; Mitochondria; Myocardial ischemia–reperfusion; Oxidative stress; ROS; SOD; c-Abl; glutathione peroxidase; humanin, an endogenous 24-amino acid peptide; mitochondrial membrane potential; myocardial ischemia–reperfusion; reactive oxygen species; superoxide dismutase.

Fig. 1. HNG prevents oxidative stress, maintains inner mitochondrial membrane potential (Δψm) and mitochondrial function

A Relative DCF fluorescence in saline controls, controls exposed to 100 µM H₂O₂and cells incubated in HNG (10 nM) with 100 µM H₂O₂ (* compared with H₂O₂-treated control, p = 0.003, ** p =0.001; 8 N per experiment, 7 experiments). B The ratio of red/green fluorescence (ψm ) from JC-10 loaded cells that had been pretreated with 40 µM H₂O₂ with or without HNG (10 nM) (compared with control treated cells, no H₂O₂*p = 0.0142, *** p = 5.0×10⁻⁶15 N per experiment, 3 experiments). C Intracellular ATP levels after 30 and 120 minutes of HNG exposure (compared with matched control, ** p = 0.0024, *** p = 0.0001, 3 N per experiment, 4 experiments).

Fig. 2. Immunofluorescent overlays support HNG-mediated preservation of mitochondrial structure

A saline controls, B saline for 10 minutes followed by addition of H₂O₂ (100 µM, 3 hours), C HNG (10 nM) for 10 minutes, then as in B. blue: Dapi stained nuclei, green: cytochrome c, red: MitotrackerCMXROS, 63× magnification.

Fig. 3. HNG decreases a glutathione-based measure of oxidative stress while rapidly and persistently enhancing the activity of critical antioxidant enzymes

A The time course of change in the ratio of oxidized to reduced glutathione in lysates of cells treated with HNG (10 nM) (**p = 0.0007, 10 N per experiment, 4 experiments). Both B GPx (**p = 4.0×10⁻⁶7 N per experiment, 4 experiments) and C catalase (*p = 0.0089, 18 N per experiment, 6 experiments) activities, normalized to total protein, following cell exposure to HNG (10 nM) and lysate prep. D Total cellular SOD activity (normalized to total protein) in lysates from cells exposed to HNG (10 nM) (*p = 0.004, 12 N per experiment, 4 experiments).

Fig. 4. Abl and Arg mRNA knockdowns demonstrate that one must be functional for HNG-mediated ROS reduction

A qRT-PCR results for siRNA knockdowns of Abl and Arg, each alone and in combination with the other (*** p = 0.0009, n=3). B Immunoblots prepared with lysates from cells treated as in A. C Following siRNA transfections, relative intercellular ROS levels were determined as described for Figure 1. (*p = 0.0139, **p = 0.003 for neg kd, and 0.005 for arg kd, 18 N per experiment, 3 experiments).