*2.1.3. Generation of ROS*

Mitochondria are also a large cellular source of ROS. ROS includes the superoxide anion radical (O2 ·−) and hydroxyl radical (· OH), as well as nonradical oxidants, such as hydrogen peroxide (H2 O2 ) and singlet oxygen (1 O2 ) [21]. They can be converted from one to the other by enzymatic and nonenzymatic mechanisms. The most abundant form of ROS in the body is O2 ·−, which is enzymatically or spontaneously dismutated to H2 O2 . In the human body, there are three superoxide dismutase (SOD) isoforms with precise subcellular compartmentalization: the Cu,Zn-dependent isoform (Cu,Zn SOD, SOD1) is found in the cytosol; the Mn-dependent isoform (Mn SOD, SOD2) is located in the mitochondrial matrix; and Cu,Zn SOD is located in the extracellular space (ecSOD, SOD3) [22]. Mitochondrial ROS have emerged as an important mechanism of disease and redox signaling in the cardiovascular system.

O2 ·− is the proximal mitochondrial ROS and is produced by the one-electron reduction of oxygen [23]. Mitochondrial O2 ·− production takes place at redox-active prosthetic groups within proteins where the kinetic factors are key to the production of O2 ·− formation [23]. Under physiological conditions, the balance between ROS generation and ROS scavenging is highly controlled. ROS generation can initiate diverse cellular responses, which include triggering signaling pathways involved in cell protection, initiating coordinated activation of mitochondrial fission and autophagy to optimize removal of abnormal mitochondria and cells, and ensuring that the damage does not spread to neighboring mitochondria and cells [21]. Both high levels of ROS (oxidative stress) and excessively low levels of ROS (reductive stress) are harmful and may play causative roles in the pathologies related to the dramatic change of redox environment [21]. Excess ROS production in the heart under pathophysiological conditions leads to mitochondrial dysfunction and bioenergetic decline and contributes to a number of cell pathologies in the heart. For example, ROS is favored by high membrane potential, low ATP formation, and hampering the flow of electrons through the complexes in cardiomyocytes. In addition, ROS formation is the result of the uncoupling of respiration as seen during the opening of the mPTP [21]. Although many studies have detected O2 ·− produced in isolated mitochondria, there are few reliable methods that can be used to measure the mitochondrial ROS production *in vivo* [24].

The molecular mechanisms of ROS generation in the cardiac mitochondrion remain unclear. It has been showed that complex I (NADH-ubiquinone oxidoreductase) is the main source of ROS in the mitochondrion. However, the ROS production at complex I is high under pathological conditions, not physiological condition [21]. Further mechanistic studies suggest that the major site of ROS production in complex I is either upstream of a rotenone-binding site or tightly coupled to the increased level of NAD(P)H after rotenone supplementation [21]. ROS production at complex II is low at physiological concentrations of succinate, suggesting that complex II is not a key contributor to the mitochondrial ROS. ROS production at complex III only occurs after the binding of antimycin A, suggesting that conformational changes that occur on antimycin A binding may be responsible for the production of ROS [21].
