Mitochondrial production of reactive oxygen species contributes to the β-adrenergic stimulation of mouse cardiomycytes

Abstract

The sympathetic adrenergic system plays a central role in stress signalling and stress is often associated with increased production of reactive oxygen species (ROS). Furthermore, the sympathetic adrenergic system is intimately involved in the regulation of cardiomyocyte Ca2+ handling and contractility. In this study we hypothesize that endogenously produced ROS contribute to the inotropic mechanism of β-adrenergic stimulation in mouse cardiomyocytes. Cytoplasmic Ca2+ transients, cell shortening and ROS production were measured in freshly isolated cardiomyocytes using confocal microscopy and fluorescent indicators. As a marker of oxidative stress, malondialdehyde (MDA) modification of proteins was detected with Western blotting. Isoproterenol (ISO), a β-adrenergic agonist, increased mitochondrial ROS production in cardiomyocytes in a concentration- and cAMP–protein kinase A-dependent but Ca2+-independent manner. Hearts perfused with ISO showed a twofold increase in MDA protein adducts relative to control. ISO increased Ca2+ transient amplitude, contraction and L-type Ca2+ current densities (measured with whole-cell patch-clamp) in cardiomyocytes and these increases were diminished by application of the general antioxidant N-acetylcysteine (NAC) or the mitochondria-targeted antioxidant SS31. In conclusion, increased mitochondrial ROS production plays an integral role in the acute inotropic response of cardiomyocytes to β-adrenergic stimulation. On the other hand, chronically sustained adrenergic stress is associated with the development of heart failure and cardiac arrhythmias and prolonged increases in ROS may contribute to these defects.

Figure 1. β-Adrenergic stimulation of mouse cardiomyocytes results in increased mitochondrial ROS production

A, mean data (±SEM) of the relative increase in mitochondrial superoxide production measured with MitoSOX Red following 10 min of 1 Hz stimulation under control conditions (white bar) or exposure to: ISO (1–100 nm as indicated); forskolin (1 μm); ISO (100 nm) in cells pre-exposed to H89 (5 μm), PKI (5 μm) or BAPTA AM (50 μm); (–)-Bay K 8644 (1 μm). Data in each group were obtained from ≥8 cells from at least 2 mice. *P < 0.05 and **P < 0.001 vs. the control group. B, MitoSOX Red fluorescence in cardiomyocytes exposed to H₂O₂ (1 mm) in the presence or absence (Control) of the respiratory chain inhibitor rotenone (2.4 μm) (n≥ 13 cells).

Figure 2. β-Adrenergic stimulation increases mitochondrial ROS, whereas it does not affect mitochondrial membrane potential (ΔΨ_m) or mitochondrial [Ca²⁺]

A, representative confocal images showing two MitoSOX Red-loaded cardiomyocytes under control conditions and after exposure to 1 nm ISO. Measurements show ∼10% increase in fluorescence in both cells after ISO exposure (black bars). B, confocal images showing three cardiomyocytes loaded with TMRE to measure ΔΨ_m. Images were obtained under control conditions, after exposure to ISO (100 nm), and after dissipating ΔΨ_m with FCCP (10 μm). There was no noticeable effect of ISO exposure, whereas the fluorescence decreased after application of FCCP. C, confocal images of three cardiomyocytes loaded with rhod-2 to measure mitochondrial [Ca²⁺] under control conditions and after 10 min exposure to ISO (100 nm), which had no noticeable effect on the fluorescence. Common scale bar for B and C.

Figure 3. β-Adrenergic stimulation of mouse hearts increases MDA protein adducts

Western blots and mean (±SEM) densitometric quantifications of total MDA protein adducts in hearts perfused under control conditions (Ctrl) or in the presence of ISO (100 nm; A and B) or H₂O₂ (0.2 mm; C and D). Data normalized to the mean value in control, which was set to 1.0; n = 3–4 hearts in each group. **P < 0.01, ***P < 0.001.

Figure 4. The β-adrenergic stimulatory effect on cardiomyocyte Ca²⁺ transient amplitude and cell shortening is blunted by the antioxidant NAC

A, representative Ca²⁺ transients from cardiomyocytes in the presence of ISO (100 nm) and/or NAC (5 mm) as indicated. Average (±SEM; n≥ 19 cells from at least three mice) amplitude of Ca²⁺ transients expressed as ΔF/F₀ (B) or translated into [Ca²⁺]_i (C), Ca²⁺ transient decay time constant (D), and fractional cell shortening (E) without and with ISO as indicated and in the absence (white bars) or presence (grey bars) of NAC. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. The β-adrenergic stimulatory effect on cardiomyocyte Ca²⁺ transient amplitude is blunted by the mitochondria-targeted antioxidant SS31

A, representative Ca²⁺ transients from cardiomyocytes in the presence of ISO (100 nm) and/or SS31 (200 nm) as indicated. Average (±SEM; n≥ 10 cells from at least two mice) amplitude of Ca²⁺ transients expressed as ΔF/F₀ (B) or translated into [Ca²⁺]_i (C), and Ca²⁺ transient decay time constant (D) without and with ISO as indicated and in the absence (white bars) or presence (grey bars) of SS31. *P < 0.05.

Figure 6. The β-adrenergic stimulation of cardiomyocyte L-type Ca²⁺ currents is inhibited by the antioxidant NAC

A, representative records of the L-type Ca²⁺ current (I_CaL) in cardiomyocytes ±100 nm ISO (upper panel, voltage step from −80 mV holding potential to −10 mV) and mean data (±SEM) of the peak current density (lower panel) (n = 6 cells in each group). B, same as A but in the presence of 5 mm NAC (n = 7 cells in each group).