Implications of S-glutathionylation of sarcomere proteins in cardiac disorders, therapies, and diagnosis

Abstract

The discovery that cardiac sarcomere proteins are substrates for S-glutathionylation and that this post-translational modification correlates strongly with diastolic dysfunction led to new concepts regarding how levels of oxidative stress affect the heartbeat. Major sarcomere proteins for which there is evidence of S-glutathionylation include cardiac myosin binding protein C (cMyBP-C), actin, cardiac troponin I (cTnI) and titin. Our hypothesis is that these S-glutathionylated proteins are significant factors in acquired and familial disorders of the heart; and, when released into the serum, provide novel biomarkers. We consider the molecular mechanisms for these effects in the context of recent revelations of how these proteins control cardiac dynamics in close collaboration with Ca²⁺ fluxes. These revelations were made using powerful approaches and technologies that were focused on thin filaments, thick filaments, and titin filaments. Here we integrate their regulatory processes in the sarcomere as modulated mainly by neuro-humoral control of phosphorylation inasmuch evidence indicates that S-glutathionylation and protein phosphorylation, promoting increased dynamics and modifying the Frank-Starling relation, may be mutually exclusive. Earlier studies demonstrated that in addition to cTnI as a well-established biomarker for cardiac disorders, serum levels of cMyBP-C are also a biomarker for cardiac disorders. We describe recent studies approaching the question of whether serum levels of S-glutathionylated-cMyBP-C could be employed as an important clinical tool in patient stratification, early diagnosis in at risk patients before HFpEF, determination of progression, effectiveness of therapeutic approaches, and as a guide in developing future therapies.

Keywords: cardiac troponin I; cross-bridge kinetics; length dependent activation; myosin binding protein C; protein phosphorylation; titin.

FIGURE 1

Functional units of C-zone sarcomeres in relaxed and active states. Shown are functional units of cardiac sarcomeres in the C-zone containing c(MyBP-C). The bottom panel shows a sarcomere the Ca-regulatory site on cTnC not occupied and with myosin in a super relaxed state (SRX) in which the heads are sequestered and not participating in the heartbeat. In the left middle panel, the cross-bridges are in a disordered relaxed state (DRX) poised to interact with the thin filament but blocked by the actions of the troponin complex proteins with an inhibitory peptide (Ip) and mobile domain of troponin I (cTnI) locking tropomyosin (Tm) a position sterically blocking the actin-cross-bridge interaction. This blocking position of Tm relies on an interaction of troponin T (cTnT) with the overlap region of Tm. In this state there is an interaction of the N-terminal domain of myosin binding protein C (cMyBP-C) with actin-Tm moving Tm away from it’s steric blocking position. As indicated, there are also interactions between the middle regions of cMyBP-C and myosin producing a drag on shortening capability. The right middle panel shows that with Ca-binding to the N-terminal domain of troponin C (cTnC) the thin filament is released from inhibition permitting activation of cross-bridge interactions with actin promoting force and shortening of sarcomere. The kinetics of the actin-cross-bridge reaction depend on interactions with the N-terminus of cMyBP-C (see text and Figure 2 for discussion).

FIGURE 2

Modulation of cMyBP-C by phosphorylation and oxidative stress. The top panel shows an active sarcomere emphasizing cMyBP-C interactions at the N-terminal domains with the thin filament and myosin and C-terminal domains interacting with tails of myosin and titin. These interactions are inhibited with site specific phosphorylations (yellow star) of an N-terminal domain (yellow star) and modulated by oxidative stress demonstrated by the action reactive oxygen species (ROS) to increase the ration of reduced glutathione to oxidized glutathione. As shown the oxidized glutathione reacts with available Cys residues generating S-glutathionylated (S-Glu) cMyBP-C, which, as discussed in the text, may block the effects of phosphorylation to increase cross-bridge kinetics. Cys residues identified in various studies (41, 42, 57) are shown to exist in N-terminal and C-terminal regions as well as domains in the middle panel.

FIGURE 3

Oxidative stress and length dependent stabilization of an unfolded state of titin distal Ig domains induced by exposure and S-glutathionylation of cryptic cysteine residues. (A) Illustration of stretch of titin in diastolic conditions with oxidant stress at a relatively short sarcomere length showing few exposed cysteines (yellow circles). (B) Illustration of stretch of titin with oxidant stress to a relatively long sarcomere length showing exposure of buried cysteine residues and S-glutathionylation. This figure is based on evidence reported by Loescher et al. (12) demonstrating that with mechanical and oxidant stress, stabilization of the state of distensibility of titin occurs with aggregation of distal Ig domains of parallel titin strands. Variations in this mechanism produce a range of values of passive tension and can affect length dependent activation (LDA). Evidence indicates that stretch of titin signals interactions with the C-terminal domains of cMyBP-C that are transmitted to the cMyBP-C N-terminal domains, which promote interactions with actin-Tm and activation of the thin filament. See text for further discussion.

FIGURE 4

Modulation of thin filament activation by cTnI N-terminal phosphorylation. Shown is a cartoon based on recent studies (7) of a region of a Ca-activated thin filament showing the effect of phosphorylation of Ser 23 and Ser 24 in the unique N-terminal peptide of cardiac troponin I (cTnI). In the dephosphorylated state the N-terminal peptide is mobile as indicated by the double yellow arrow. Also shown are the positions of the cardiac troponin I switch peptide (Sw), inhibitory peptide (Ip), and troponin I-troponin T interaction (I-T arm) described in Figure 1. Phosphorylation induces an interaction of the N-terminal peptide with actin-tropomyosin and as shown by the arrow in the figure moves tropomyosin (Tm) to a position favoring the off state of the thin filament, thereby decreasing myofilament response to Ca²⁺ and promoting relaxation. Cys residues reported to be S-glutathionylated (42) are located in the region of the I-T arm (Cys-80) and in a helix N-terminal to the Ip (Cys 97). See text for further discussion.