Highly Reactive Isolevuglandins Promote Atrial Fibrillation Caused by Hypertension

Visual Abstract


HIGHLIGHTS
IsoLGs are highly reactive lipid dicarbonyl metabolites that constitute a major component of oxidative stress-related injury, and they promote the formation of amyloid.
In a hypertensive murine model, IsoLG adducts and PAOs developed in the atria, along with inducible AF.
IsoLG and PAO accumulation and AF were prevented by the dicarbonyl scavenger 2-HOBA, but not by an inactive analog 4-hydroxybenzylamine.
Mechanically stretched atrial cells generated cytosolic IsoLG adducts and PAOs that were prevented by 2-HOBA.
Natriuretic peptides generated cytotoxic oligomers, a process accelerated by IsoLGs, contributing to atrial PAO formation.
These findings identify a novel pathway during oxidative stress to increase AF susceptibility, and they support the concept of preemptively scavenging reactive downstream mediators as a potential therapeutic approach to prevent AF.
These findings support the concept of pre-emptively scavenging reactive downstream oxidative stress mediators as a potential therapeutic approach to prevent AF. There is abundant evidence linking oxidative stress and reactive oxygen species (ROS) directly to the pathogenesis and progression of AF (2). Inflammatory cells generate ROS, and inflammationmediated AF is the most common and costly complication of cardiac surgery, as well as the mechanism of early recurrence following catheter ablation (3)(4)(5). In addition, multiple risk factors for AF, including hypertension, obesity, and aging, are mechanistically linked to oxidative stress (6,7). Boutaud, the National Institute of General Medical Sciences grant T32 GM007569 to Dr. Prinsen at the National Institutes of Health, the American Heart Association, Southeast Affiliate grant 2160035 to Dr. Murray, and National Center grant Unfortunately, "upstream therapy" targeting ROS levels directly with dietary antioxidants has been ineffective in clinical trials (8), in part because they fail to actually reduce oxidative injury in humans.
Nonspecific ROS scavenging may also interfere with physiological ROS signaling.
Polyunsaturated fatty acid oxidation leads to the formation of highly reactive aldehydes. The most reactive of these products are dicarbonyl compounds known as isolevuglandins (IsoLGs) (also called g-ketoaldehydes or isoketals [9,10]) ( Figure 1). They adduct proteins almost instantaneously, causing misfolding and crosslinks (9). Tissue IsoLG adducts are elevated early in multiple diseases linked to inflammation and oxidative stress, including hypertension, obesity, atherosclerosis, and Alzheimer's disease (11)(12)(13)(14)(15). Moreover, IsoLGs induce multiple effects that drive disease, including cytotoxicity, activation of inflammation and cytokine secretion, and acceleration of amyloidosis. In Alzheimer's, misfolded protein amyloid b 1-42 monomers coassemble initially to form soluble preamyloid oligomers (PAOs), now recognized to be the primary cytotoxic species correlating with disease progression rather than downstream amyloid fibril deposition (16,17). Importantly, IsoLGs markedly accelerate the oligomerization of amyloid b 1-42 (18,19), providing a pathophysiological link between oxidative stress and proteotoxicity. As in the brain, amyloidosis develops in the human atrium with aging (20)(21)(22), and we recently identified PAOs in human atrial tissue (23). In a cellular model simulating AF, we previously found that rapid stimulation of atrial cells caused the formation of IsoLG adducts and protein oligomers within hours (24). We hypothesized that IsoLGs are molecular drivers of the AF substrate, constituting a novel mechanism to increase arrhythmia susceptibility. We chose a model of hypertension to test this hypothesis for several reasons. First, we found that the presence of protein oligomers in the human atrium was linked to hypertension (23). Second, considerable evidence implicates oxidative damage and inflammation in the development of hypertension (11,25). Third, it was recently demonstrated that The 1,4-dicarbonyl (red box) IsoLGs interact rapidly with lysines to form lactam adducts and crosslinking of proteins. The phenolic amine pyridoxamine and its structural analog 2-HOBA (blue box) react with IsoLGs at a rate several orders of magnitude more rapidly than they react with lysines, thus serving as scavengers to prevent adduct formation. 2-HOBA ¼ 2-hydroxylbenzylamine; Prinsen et al. IsoLG adducts are indeed formed during experimental hypertension, serving as neoantigens to promote dendritic and T-cell activation (11). In the present studies, we report that IsoLGs and PAOs (1 g/l), or hydralazine þ hydrochlorothiazide (320 mg/l and 60 mg/l, respectively) was delivered via drinking water (11). International, Burlington, North Carolina) were exposed to 10% cyclical stretch at a rate of 1 Hz for 24 h using the Flexcell FX-5000 Tension System (Flexcell International) (27).
IsoLG ADDUCTS. Immunohistochemistry. Formalin fixed hearts were subjected to immunohistochemistry using an anti-IsoLG-lysyl adduct single-chain antibody (D11 ScFv) characterized previously (28). Images were captured using a high-throughput Leica SCN400 slide scanner automated digital image system from Leica Microsystems (Wetzlar, Germany). Whole slides were imaged at 20Â magnification to a resolution of 0.5 mm/pixel. Tissue cores were mapped using Ariol added, and the IsoLG-lysyl adducts were purified by solid-phase extraction and high-performance liquid chromatography before being quantified by liquid chromatography-tandem mass spectrometry assay using isotopic dilution as described previously (29).    2E). As for IsoLG adducts, this effect was abrogated by 2-HOBA ( Figures 2E and 2F), whereas the inactive structural analog 4-HOBA failed to prevent PAO formation ( Figure 2E).  with control mice, the total amount or burden of inducible AF was significantly increased in hypertensive mice ( Figure 4A). The AF substrate was reversible, with a 95% reduction in total AF burden within 2 weeks after stopping ang II ( Figure 4A) (associated with a 70% reduction in BP (Supplemental burden was comparable to that seen with animals receiving ang II alone. There were no effects of 2-HOBA on any ECG or electrophysiological parameters ( Table 1). Taken together with the results shown in Figure 2, these findings demonstrate that ang IImediated hypertension promotes the formation of atrial IsoLGs, PAOs, and AF susceptibility, with IsoLGs playing a critical role in the pathophysiological process.  (Figures 5A and 5B). However, when incubated in the presence of IsoLGs, PAO formation was markedly accelerated. We then examined whether natriuretic peptide oligomers were detrimental to atrial cells. Both ANP and BNP oligomers reduced ATP production in atrial HL-1 cells, indicating cytotoxicity (Figures 5C and 5D). This effect was most pronounced for oligomers formed during a 1-day incubation, whereas cytotoxicity progressively declined with longer incubation times, most prominently for ANP ( Figure 5C). This time course is analogous to that observed for amyloid b 1-42 neuronal injury: as monomers coalesce to oligomers and subsequently to less toxic fibrils, PAO formation and associated cytotoxicity develops and then declines in a time-and concentration-dependent manner (17).
Finally, adjacent sections of hypertensive mouse atria were immunostained for PAOs and either ANP or BNP, with results demonstrating evidence of partial colocalization of natriuretic peptides with atrial oligomers ( Figure 5E). Taken together, these findings support a role for cytotoxic ANP and BNP oligomers as potential mediators of atrial pathophysiology during hypertension.

DISCUSSION
As the most common sustained cardiac arrhythmia, AF constitutes a significant public health problem for which optimal medical therapies are lacking. Elucidating early mechanisms that increase AF susceptibility are critical to develop effective preventative and therapeutic strategies. In this study, we identi-  (42), histones (43), and proteasomes (44). IsoLGs can also adduct to DNA and phosphatidylethanolamines (45,46). Tissue IsoLG adducts are elevated early in animal models of cardiovascular risk factors, including hypertension, obesity, and hyperlipidemia (41), as well as atherosclerosis (13,14). They are also increased in other diseases linked to oxidative injury/inflammation, such as chronic ethanol exposure (47), pulmonary fibrosis (48), Alzheimer's disease (15), and cancer (49). To date, IsoLG adducts identified in experimental models have emerged as critical mediators of oxidative injury in the brain during Alzheimer's disease, and in the vasculature during hypertension and atherosclerosis (11,13,14,50).
Multiple risk factors for developing AF are associated with increased atrial pressure that promotes atrial tension/enlargement, and our results support a critical role for atrial cell stretch in the pathophysiological process. Atrial myocyte stretch triggers a generalized stress response, with activation of immediate early genes, dedifferentiation, activation of hypertrophic signaling cascades, and increased release/production of natriuretic peptides (51,52).
Importantly, stretch of ventricular myocytes causes rapid production of superoxide (53). Similarly, in the present study, we found that atrial myocyte stretch causes IsoLG adduct formation, indicative of atrial ROS production. Prevention of AF susceptibility using a dicarbonyl scavenger is consistent with the concept that stretch-mediated oxidative stress is an early event in generating the AF substrate.
Diseases related to oxidative stress are increasingly linked to proteotoxicity as a contributing mechanism (16,31,54), in particular for neurological and cardiac dysfunction (16,17,31).  aging human atrium (20)(21)(22). Second, we showed that IsoLGs markedly accelerate the oligomerization of ANP and BNP in vitro and in cells, yielding cytotoxic oligomers, as occurs with amyloid b 1-42 (18,19).    Figure 1) is key to scavenger potency (34,35). For the related analog 4-HOBA, this structural proximity is lost-hence, this compound is a very poor scavenger of dicarbonyls, enabling it to serve as a negative control. Importantly, 2-HOBA and its analogs are not antioxidants in that they do not react with O₂_ˉ, OONO À , or H 2 O 2 (11), and the reduction in IsoLG adduct levels has been attributed directly to the dicarbonyl scavenging effect, and not to inhibition of ROS production and/or lipid peroxidation. Although 2-HOBA reacts with IsoLG (a 1,4-dicarbonyl) much more rapidly than with MDA (a 1,3-dicarbonyl) or methylglyoxal (a 1,2- Finally, although our data demonstrated a reduction in cytotoxicity with longer peptide incubation times for ANP supporting PAOs as the cytotoxic moiety, this was not observed for BNP. Nonetheless, these experiments were performed solely to assess oligomer cytotoxicity, rather than the kinetics of PAO/amyloid formation for the natriuretic peptides. whereby oxidative stress increases AF susceptibility is not known. This paper identifies a novel molecular pathway by which highly reactive lipid dicarbonyl metabolites constituting a major component of oxidative stress-related injury are mechanistically linked to AF susceptibility during hypertension, a disease also linked to oxidative stress. Our findings also define a novel potential mechanism whereby oxidative stress promotes amyloid formation in the atria.
TRANSLATIONAL OUTLOOK: These findings identify a novel pathway during oxidative stress to increase AF susceptibility, and they support the concept of pre-emptively scavenging reactive downstream mediators, rather than targeting generation of reactive oxidative species per se, as a potential therapeutic approach to prevent AF. The scavenger 2-HOBA has been well-tolerated in initial Phase 1 clinical trials, and a Phase 2 trial will start within the next few months to examine its efficacy to prevent AF.