The cardioprotective role of NO in pre-clinical models of acute MI is well established (Gonzalez et al., 2008; Dezfulian et al., 2009; Duranski et al., 2005; Hendgen-Cotta et al., 2008). However, the ability of NO donors to reduce I/R damage has been disappointing in the clinical setting (Siddiqi et al., 2014; Jones et al., 2015). One explanation for these failures is the absence of drug targets or cardioprotective mechanisms by which to assess efficacy of NO donors. This challenge is circumvented through enzymatically regulated S-nitrosylation, where the target enzyme has been genetically validated. In particular, the denitrosylase SCoR2 (Anand et al., 2014) regulates multiple metabolic pathways, including ketolysis, glycolysis, PPP, and polyol metabolism, that have established roles in cardioprotection, but were not previously known to be coordinately regulated. Our studies thus identify a comprehensive role for SCoR2 in regulating cardiac metabolism and suggest a new approach to cardioprotection through global metabolic reprogramming.

A large body of evidence supports the benefits of ketones in acute myocardial injury. BDH1 catalyzes the initial step in the breakdown of the ketone BHB in extrahepatic tissues, and cardiac-specific knockout of BDH1 in mice leads to more severe ventricular dysfunction following ischemia (Horton et al., 2019). Conversely, cardiac-specific BDH1 overexpression improves contractile function in response to injury (Uchihashi et al., 2017). Ketone bodies secreted from the liver are a critical source of myocardial energy during fasting (Mitchell et al., 1995; Homilius et al., 2023; Cotter et al., 2013) and also in the failing (Aubert et al., 2016; Nielsen et al., 2019) and ischemic heart (Zuurbier et al., 2020), where they reduce infarct size and improve post-I/R contractile function (Marina Prendes et al., 2005; Snorek et al., 2012; Hron et al., 1978). Exogenous BHB reduces infarct size in rats (Zou et al., 2002) and mice (Yu et al., 2018) subjected to I/R injury by increasing ATP production and is beneficial in patients with cardiogenic shock (Berg-Hansen et al., 2023), providing evidence that ketolysis contributes to superior metabolism in the setting of acute I/R injury. Circulating ketone bodies are elevated in humans with congestive heart failure (Lommi et al., 1996) and are associated with improved cardiac function (Selvaraj et al., 2020) and a lower risk of adverse events in acute MI (Sato et al., 2023). Cardiac ketone body metabolism, beginning with BDH1-mediated breakdown of BHB, is thus a validated protective pathway in acute I/R injury and is beneficial in the context of human cardiovascular disease.

In this context, we have discovered that SCoR2 regulates S-nitrosylation of cardiac BDH1 at Cys115, thereby governing its stability. BDH1 expression is thus increased in SCoR2-/- hearts, in accord with its increased S-nitrosylation. Increased expression of BDH1 increases capacity for ketolysis, improving energy reserves (i.e., p-Cr) during ischemia (Homilius et al., 2023), as we confirm here. Many studies, including over 40 controlled trials in patients with CAD, HF, or cardiac surgery (Landoni et al., 2016), have demonstrated that p-Cr is cardioprotective (Landoni et al., 2016; Sharov et al., 1986; Prabhakar et al., 2003; Zhang et al., 2015; Qaed et al., 2019; Wang et al., 2021), an effect attributed to increased energy availability. Strategies to increase p-Cr in the post-ischemic myocardium are thus a promising avenue to reduce post-MI tissue damage. In this light, we show that inhibition of SCoR2 results in increased p-Cr.

PKM2 is a pyruvate kinase splice variant expressed in development, injury, and cancer (Cheon et al., 2016; Wang et al., 2012). PKM2 inhibition is strongly linked to tissue protection, regeneration, and repair (Zhou et al., 2019; Siragusa et al., 2019; Hauck et al., 2021; Cheng et al., 2017), including in the heart where inhibition (Cheng et al., 2017) or genetic deletion (Hauck et al., 2021) leads to reduced infarct size. S-nitrosylation of PKM2 inhibits activity and thus progression of intermediates through glycolysis (Zhou et al., 2019); instead, glucose 6-phosphate is shunted into the PPP, where it is used to synthesize the antioxidant NADPH and 5-carbon sugars for biosynthesis (TeSlaa et al., 2023). This is protective in the setting of MI (Hauck et al., 2021; Cheng et al., 2017). The PPP is responsible for producing a portion of the cardiac NADPH pool, and this pathway is upregulated in stressed or injured cardiac tissue (Gupte et al., 2006; Wang et al., 2022; Zoccarato et al., 2023). SNO-PKM2, a validated SCoR2 substrate, protects against ischemic kidney injury and endothelial damage (Siragusa et al., 2019) via increased flux through the PPP shunt (Zhou et al., 2019; Zhou et al., 2023b). We demonstrate that deletion of SCoR2 leads to an increase in SNO-PKM2 in the heart, accompanied by inhibition of glycolysis (decreased lactate) and stimulation of the PPP (increased NADPH, erythrose 4-phosphate, and ribose). We also observe an increase in other inputs into the PPP (primarily those converging on xylulose, such as galactaric acid, xylose, and lyxose, among others), supporting a comprehensive role for SCoR2 in regulation of the protective PPP shunt in the context of acute ischemic injury.

Polyols are relatively understudied organic metabolites containing more than 1 hydroxyl group. The ‘polyol pathway’ historically refers specifically to sorbitol Garg and Gupta, 2022; however, there are many other polyols that are metabolically relevant in mammals, as first described in the mid-20th century (Hutcheson et al., 1956; Touster and Harwell, 1958). Endogenous production of polyols, well characterized in microorganisms (Lactobacillus, E. avium, L. casei, and others) (Rice et al., 2020; Monedero et al., 2010), has remained underappreciated and relatively uncharacterized in humans. Recent evidence of endogenous erythritol synthesis in human blood (Hootman et al., 2017), and in A549 lung cancer cells by the reductases ADH and SORD (Schlicker et al., 2019), casts doubt on the view that polyols are not endogenously produced in humans (Hiele et al., 1993). Additionally, heritable genetic mutations (i.e., in transketolase Boyle et al., 2016, transaldolase Verhoeven et al., 2001, and ribose-5-phosphate isomerase Huck et al., 2004) may lead to aberrant accumulation of certain polyols (ribitol, arabitol, and erythritol), suggesting close connections to the PPP. Many polyols are structurally related to PPP intermediates, such as arabitol, xylitol, and ribitol, and are likely derived at least in part from these compounds (Wamelink and Williams, 2022; Huck et al., 2004). Most studies appear to presume that any polyols produced are simply excreted from the body (Boyle et al., 2016).

However, there is tantalizing new evidence that polyols impact cardiovascular health (Witkowski et al., 2023; Witkowski et al., 2024). Erythritol, xylitol, threitol, arabitol, and myo-inositol are highly associated with incident risk for major adverse cardiovascular events, and erythritol, in particular, is reported to enhance in vitro platelet reactivity and in vivo thrombosis rate (Witkowski et al., 2023). Our untargeted metabolomic analysis revealed that SCoR2 deletion causes a marked reduction in these polyol ‘off-shoots’ from the PPP, particularly arabitol/ribitol (indistinguishable in our analysis) and sorbitol, a difference that widens after 4 hr of reperfusion relative to sham injury. We present strong evidence that this effect is not mediated through direct reduction of carbohydrates by SCoR2, in contrast with a recent report (Hoshino et al., 2024) (where the actual data are negative, supporting our findings) and literature viewpoint. Rather, the many observed changes in carbohydrates and corresponding polyols are seemingly well rationalized by the widespread alterations in S-nitrosylation of metabolic enzymes (Supplementary file 1) accompanying SCoR2 deletion. Indeed, multiple reductase enzymes were identified in the SCoR2-dependent nitrosoproteome (e.g., ALDH1B1 and DHRS7B, Supplementary file 1), some of which may catalyze direct reduction of carbohydrates to polyols in an S-nitrosylation- and SCoR2-dependent manner. Multiple reductase and aldolase enzymes were recently identified as interacting partners with polyols (such as arabitol) and related carbohydrates (such as arabinose, ribulose, xylose, ribose, and xylulose) (Hicks et al., 2023), including aldolase B, MDH2, PDH, and IDH3 that all have been identified in SNO-proteomic screens (Mnatsakanyan et al., 2019) and may be SCoR2-regulated. Reduction in polyol levels thus represents a fourth SCoR2-regulated metabolic pathway contributing to comprehensive metabolic reprogramming during I/R injury.

The emerging concept that protein S-nitrosylation can be regulated independently of NO synthesis (i.e., by SNO metabolism) changes the therapeutic paradigm in myocardial ischemia. Protein denitrosylases, including both GSNOR (Lima et al., 2009; Tang et al., 2023; Castillo et al., 2021; Grimmett et al., 2021; Hatzistergos et al., 2015) and thioredoxin (Perone and Lembo, 2020; Tao et al., 2004), have been shown to play important roles in cardiac injury and repair. However, a general schema for how these enzymes confer protection has been missing. Here we establish a cardioprotective framework for SCoR2 through metabolic regulation, whereby SCoR2 inhibition coordinately orchestrates multiple metabolic pathways for therapeutic gain: stimulation of ketolysis, inhibition of glycolysis, increased PPP shunting, and reduction in polyol compounds converge to protect the heart (Figure 5L). These findings help to cement SCoR2 as a major regulator of metabolism in the injured myocardium and new class of pharmaceutical target.

Limitations

The results presented here are purposefully limited to the acute response to cardiac tissue injury and do not explore whether deletion or inhibition of SCoR2 may be beneficial in long-term recovery from MI. Metabolic regulation in chronic injury may involve alternative pathways. The protection offered by SCoR2-mediated metabolic reprogramming is also specific to MI injury, although suggestive findings are shown in human cardiomyopathy. We identified the critical enzymes PKM2 and BDH1 via unbiased multi-omic screens, and we surmise they coordinately regulate increased flux through the PPP and ketolysis pathways upon function-regulating S-nitrosylation, thereby explaining improved energy balance, but isotope tracing experiments would be necessary to confirm this.