Cardiomyocyte mitochondrial mono-ADP-ribosylation dictates cardiac tolerance to sepsis by configuring bioenergetic reserve

doi:10.21203/rs.3.rs-4644147/v1

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Cardiomyocyte mitochondrial mono-ADP-ribosylation dictates cardiac tolerance to sepsis by configuring bioenergetic reserve

https://doi.org/10.21203/rs.3.rs-4644147/v1

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The metabolic plasticity of tissues determines the degree and reversibility of organ damage under inflammatory challenges. Still, countermeasures for myocardial metabolic breakdown are absent while treating septic cardiomyopathy (SCM). Nicotinamide adenine dinucleotide signaling is fundamental to cellular metabolic homeostasis and inflammatory reactions. Here we revealed that genetic and pharmacological inactivation of mono-ADP-ribosyl hydrolase MacroD1 enriched in cardiomyocytes counteracted myocardial metabolic impairment, inflammation, dysfunction, and mortality risk induced by lipopolysaccharide and cecal ligation and puncture in mice. Mechanistically, MacroD1 selectively modulated the mitochondrialcomplex I (MCI) activity most vulnerable to early sepsis. Its inhibition enhanced mono-ADP-ribosylation of NDUFB9, an accessory assembly factor of MCI proton-pumping module ND5, and therefore binding to ND5 for preserving MCI activity in sepsis, restraining bioenergetic deficiency, oxidative stress-coupled NLRP3 inflammasome activation, and pyroptosis of cardiomyocytes. Thus, MacroD1 dictates cardiac tolerance to sepsis by configuring MCI-coupled bioenergetic reserve and pyroptosis of cardiomyocytes. Blockade of MacroD1 promises specific prevention of SCM.

Health sciences/Cardiology/Cardiovascular biology/Cardiovascular diseases/Heart failure

Biological sciences/Cell biology/Mechanisms of disease

Biological sciences/Molecular biology/Post-translational modifications

Septic cardiomyopathy (SCM) is a common manifestation of sepsis that affects the heart, resulting in myocardial depression. Approximately 40% of sepsis patients admitted to intensive care units are at risk of developing SCM, which complicates hemodynamic evaluation during intensive treatment. The mortality in patients with SCM is up to 70–90% despite adequate management of infection, and even if survived, approximately 5.5% of them are rehospitalized due to heart failure(1, 2). No consensus guidelines or specific therapies are available for the management of SCM.

The development of SCM involves a complex interplay among microbial toxins, inflammatory mediators, mitochondrial dysfunction, and other factors. Histopathological studies have reported inflammation infiltrates, interstitial and intracellular edema, and intra-cardiomyocyte lipid accumulation(3, 4) without significant cardiomyocyte death and sarcomeric structural damage(5) in the myocardium of septic patients and animal models. High diffusion of TNF-α expression across different types of cardiac cells and the breakdown of almost all bioenergetic pathways highlight the essence of myocardial inflammometabolic disorders(3, 6, 7). However, despite high levels of circulating inflammatory cytokines in septic patients, they are irrelevant to cardiac dysfunction(8). Treatments with TNF-neutralizing antibodies, soluble TNF receptors, Toll-like receptor-4 antibodies, or IL-1 receptor antagonists could not achieve the desired effects(8–10). In contrast, myocardial bioenergetic status links the intensity of inflammatory insult to contractile working(11). These scenarios support the idea of concurrent management of myocardial metabolism for SCM besides anti-inflammation.

A wide range of myocardial metabolic abnormalities exists in SCM(7), including impaired oxidative utilization of fatty acids and lipid accumulation in the myocardium. Glycolysis, glucose oxidation, glycogenolysis, ketone body degradation, and uptake of essential and branched-chain amino acids are also depressed. Mitochondrial dysfunction emerges as a critical mechanism for sepsis-induced multiple organ failure. Functional traits of mitochondria exhibit heterogeneous responses across different types of cells and organs in diseases, including sepsis(12). For example, the depressed activity of mitochondrial complex I (MCI), which is most vulnerable to early sepsis(13), favors hepatocytes against fulminant endotoxemic injury(14) but disfavors other multiple types of cells(15). Therefore, identifying cardiomyocyte-specific mediators programming myocardial metabolism against sepsis remains imperative to achieving safe therapy for SCM.

The core roles of nicotinamide adenine dinucleotide (NAD⁺) in cell or organ homeostasis have been receiving particular attention, i.e., driving circadian physiology of mitochondrial oxidative metabolism(16), fueling inflammasome activation(17, 18), and linking mitochondrial homeostasis to signaling transduction(19). Intracellular NAD⁺ is highly enriched in mitochondria(19), where almost all NAD⁺-consuming signaling reactions occur(20). The latest report suggests mitochondria as the sentinel of infections(21) and aberrant NAD⁺ metabolism underlies a root cause of sepsis (22). The heart is a mitochondria-enriched organ, and further investigation of cardiac-specific NAD⁺ signaling pathways for SCM is warranted.

Herein, we profiled and characterized cardiomyocyte-enriched NAD⁺-signaling reaction mediators linked to myocardial inflammometabolic disorders in sepsis. Mono-ADP-ribosyl hydrolyzing MacroD1 was preferentially expressed in striated muscle cells, including cardiomyocytes, across organs among all NAD⁺-signaling enzymes known. Its inhibition potentiated myocardial tolerance to sepsis mainly via NDUFB9 mono-ADP-ribosylation (MARylation), which enhanced MCI activity and consequent bioenergetic reserve to withstand NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation-coupled cardiomyocyte pyroptosis. MacroD1 blockade protected the heart against sepsis, promising specific prevention of SCM.

All animal experiments were approved by the Animal Care Committee of Shanghai Jiaotong University School of Medicine and conducted under agency guidelines. A detailed methodology is available in the online data supplement. The source data supporting the results unshown can be available from the corresponding author upon reasonable request. Detailed information on primer pairs, siRNAs, and antibodies was listed in Supplementary Tables 1–5.

Statistical analysis. The results were expressed as mean ± SD. The Shapiro-Wilk test was used to determine if data were normally distributed, and the Brown-Forsythe test determined whether data variances were equal. The two-tailed unpaired Student's t-test was performed for the normally distributed data with equal variance, and the Mann-Whitney nonparametric test for the nonnormally distributed data or data with unequal variance for two groups. When more than two groups were compared, one-way ANOVA and subsequent Tukey's posthoc tests were employed for homogeneous variances, and Welch's ANOVA and subsequent Games-Howell's multiple comparison tests for heterogeneous variances. While for non-normal data, the nonparametric Kruskal-Wallis test was further conducted. The Kaplan-Meier method was used to calculate the survival rate and the log-rank (Mantel-Cox) test for evaluating the survival curve difference between groups. A p-value less than 0.05 was statistically significant.

Upregulation of cardiomyocyte-enriched MacroD1 links to SCM.

To identify cardiomyocyte-enriched NAD⁺-signaling effectors or regulators in response to sepsis, we first systematically analyzed their expression profiles in different types of cardiac cells using a single-cell RNA-sequencing dataset GSE109774(23). MacroD1 and deacetylase sirtuin-3 (Sirt3) demonstrated a selective expression in cardiomyocytes of mouse hearts (Figs. 1A, B; Supplementary Fig. 1), differing from other members in the family of NAD⁺-signaling enzymes (Supplementary Fig. 1). Despite well-documented mitochondrial localization(24, 25), the pan-organ effects of Sirt3 rendered it not to be considered here. MacroD1 protein was enriched in the striated muscles of mice and humans and mitochondria (Figs. 1C to F; Supplementary Fig. 2), raising our concern.

Myocardial MacroD1 protein levels but not mRNA were upregulated upon challenging with an exogenous toxin lipopolysaccharide (LPS, 10 mg/kg, i.p.) for 18 hours (Figs. 1G, H; Supplementary Fig. 3A). In the in vitro sepsis model of neonatal rat ventricular myocytes (NRCM), MacroD1 protein expression was enhanced by a high rather than low dose of LPS (Figs. 1I, J; Supplementary Fig. 3B). A similar scenario was recapitulated in AC16 human cardiomyocytes (Figs. 1K, L; Supplementary Fig. 3C). In this case, elevation of MacroD1 protein level in mitochondrial fractions was especially prominent (Supplementary Figs. 3D, E). The responsive upregulation of MacroD1 protein expression in cardiomyocytes induced by severe endotoxemia was not associated with its transcription (Supplementary Figs. 3F to I). These data implicate the distinct pathobiology of cardiomyocyte-enriched MacroD1 in cardiac response to sepsis.

Knockout of cardiomyocyte MacroD1 gene enables myocardial tolerance to sepsis.

To examine the functional consequence of MacroD1 in SCM, we generated the mouse line with the tamoxifen-induced cardiomyocyte-specific knockout of the MacroD1 gene [MacroD1^flox/flox/αMHC^MerCreMer (cKO)] (Figs. 2A, B; Supplementary Figs. 4A to D). Such conditional gene knockout did not exhibit effects on MacroD1 proteins in skeletal muscles compared to MacroD1^flox/flox controls (Fig. 2B), highlighting the specificity of genetic manipulation used.

Four days after the LPS challenge, MacroD1^flox/flox mice suffered approximately a 25% death rate but all cKO animals survived (Fig. 2C). Despite comparable cardiac function at baseline, myocardial depression evident in MacroD1^flox/flox mice 18 hours post-injection of LPS was significantly counteracted by cKO, as demonstrated by echocardiographic evaluation of left ventricular ejection fraction, fractional shortening (Figs. 2D to F), and internal diameters (Supplementary Figs. 5A, B and Table 6). Also, the elevation of circulating LDH and cTnT in MacroD1^flox/flox mice under LPS insults was depressed in cKO animals (Figs. 2G, H), implicating restrained myocardial damage.

Consistent with the previous report(26), myocardial immunohistochemistry showed a marked increase of myeloperoxidase (MPO)-positive neutrophil cells in MacroD1^flox/flox hearts subjected to LPS insults (Figs. 2I, J). Masson’s trichrome staining revealed collagen deposition in line with myofibroblast activation detected by immunostaining of α-smooth muscle actin (Figs. 2I to K). These myocardial pathologies were much mitigated in LPS-stressed cKO mice, further suggesting protection against endotoxemia-induced myocardial damage.

Cecal ligation and puncture (CLP) surgery was also performed to mimic bacteremia-induced cardiac dysfunction. As expected, cardiac function (Figs. 2L to N; Supplementary Figs. 5C, D and Table 7) and survival rate (Fig. 2O) were improved in cKO than in MacroD1^flox/flox mice. Circulating levels of myocardial damage markers (Figs. 2P, Q) and intensity of myocardial inflammation infiltrate and myofibroblast activation (Figs. 2R, S) were lower in cKO mice, underscoring the importance of MacroD1 in SCM.

Preserved bioenergetic metabolism underpins cKO cardioprotection against sepsis.

To ascertain biological underpinnings for myocardial tolerance while MacroD1 inactivation, we continued to analyze transcriptomics of septic hearts. RNA sequencing of the ventricular myocardium revealed that cKO elicited minimal differential gene expression at baseline. LPS challenge led to the massive upregulation of gene sets associated with inflammatory signals in MacroD1^flox/flox and cKO hearts (Supplementary Figs. 6A to D). In this case, genes for the citrate cycle, one carbon, amino acid metabolism, and fatty acid degradation were widely downregulated in MacroD1^flox/flox but less changed in cKO hearts (Figs. 3A to C), which was validated by quantitative PCR assays (Fig. 3D). Strikingly, growth/differentiation factor GDF15, an inflammation-induced central mediator of tissue tolerance that promotes metabolic adaptation(27), was significantly upregulated in cKO hearts compared to MacroD1^flox/flox under LPS challenging (Supplementary Figs. 7A, B).

We also interrogated whether MacroD1 impacted mitochondrial bioenergetics and oxidative stress. LPS induced an intensity of redox-sensitive fluorescent probe dihydroethidine (DHE) in the myocardium of MacroD1^flox/flox stronger than in cKO mice, albeit with comparable levels under basal conditions (Figs. 3E, F). Accordingly, the content of myocardial ATP was preserved in cKO mice (Fig. 3G). Further ultrastructural analysis revealed less accumulation of lipid droplets in cKO hearts compared with MacroD1^flox/flox controls in response to LPS stress, with comparable mitochondrial number and size but a significantly higher mitochondrial cristae density in cKO cardiomyocytes (Figs. 3H to L). The sarcomeres were relatively intact in MacroD1^flox/flox and cKO hearts. Collectively, the preservation of metabolic pathways, especially mitochondrial oxidative phosphorylation, is tightly linked to cKO-mediated myocardial tolerance to sepsis.

MacroD1 downregulation maintains the bioenergetic process under endotoxemic stress.

To confirm the regulation of MacroD1 on mitochondrial functions, we assayed mitochondrial respiration capacity and superoxide production using NRCMs. As shown in Fig. 4A and B, mitochondrial ROS (mitoROS) level was comparable at baseline but depressed under LPS treatment in cardiomyocytes with MacroD1 knockdown (Supplementary Fig. 8). Basal and maximal oxygen consumption rates and ATP-linked respiration of NRCMs depressed by LPS were improved when MacroD1 was downregulated despite being similar at baseline (Figs. 4C to F). MacroD1 knockdown did not affect the spare respiration of NRCMs with and without LPS treatment (Fig. 4G). In sharp contrast to the scenarios with MacroD1 knockdown, an enhanced mitoROS generation was observed in NRCMs overexpressing MacroD1 in response to LPS insult (Supplementary Figs. 9A to C). Meanwhile, depression of basal and maximal oxygen consumption and ATP-linked and spare respiration deteriorated (Supplementary Figs. 9D to H).

Whether MacroD1 modulated fatty acid oxidation utilization in mitochondria was also evaluated by substrate-dependent oxygen consumption in NRCMs. In the presence of etomoxir but without palmitoylcarnitine, MacroD1 knockdown did not affect basal, maximal, and acute responsive respiration of NRCMs with LPS treatment compared to negative controls (NC) (Figs. 4H to K). When NRCMs were co-treated by palmitoylcarnitine, MacroD1 knockdown protected against basal and maximal oxygen consumption reduction compared to the NC under LPS treatment (Figs. 4L to O). Thus, MacroD1 downregulation decreases mitochondrial superoxide generation and preserves bioenergetic process and fatty acid utilization in endotoxemic challenges.

MCI subunit NDUFB9 underpins bioenergetic preservation induced by cKO.

We proceeded to figure out the critical mediators for MacroD1 inactivation-dependent bioenergetic metabolism. Using blue native polyacrylamide gel electrophoresis, we resolved that MacroD1^flox/flox hearts had mitochondrial membrane protein complexes with comparable levels to cKO ones under basal conditions and an 18-hour LPS challenge (Fig. 5A; Supplementary Figs. 10A, B). Strikingly, cKO selectively elevated MCI activity at baseline and counteracted the depression of MCI and mitochondrial complex V (MCV) induced by LPS (Figs. 5B to F). Whereas overexpression of MacroD1 increased mitoROS and further depressed MCI activity and ATP production in AC16 human cardiomyocytes treated with LPS. Deletion of the Macro domain in MacroD1, opposite to MacroD1 overexpression, preserved the mitochondrial function of AC16 cells treated with or without LPS, implicating the importance of the Macro domain in MacroD1-dependent mitochondrial function (Figs. 5G to K).

Selective effects of MacroD1 on basal MCI activity prompted us to focus on identifying mitochondrial targets that are MARylated, especially for MCI-associated entities. MacroD1 antibody immunoprecipitated 572 proteins compared to IgG-based captures. A mono-ADP ribose antibody-based proteomics identified 261 proteins differentially enriched in cKO compared to MacroD1^flox/flox hearts. By cross-referencing with the mouse MitoCarta3.0 inventory(28), three proteins related to MCI function were identified (Fig. 5L), and only NDUFB9 showed MARylation. Physical interaction between MacroD1 and NDUFB9 was verified using respective antibodies- and epitopes-based immunoprecipitation in mouse hearts and HEK293 cells expressing the two recombinants (Figs. 5M, N). Experiments with truncated forms revealed that the Macro domain mediates the binding of MacroD1 to NDUFB9 (Fig. 5O).

NDUFB9 is an accessory assembly factor of MCI proton-pumping module MT-ND5, a rate-limiting step for overall electron transfer in MCI and supercomplex MCI/III/IV assembly(29). Its interaction with MT-ND5 is required for this module's stability. An increased binding of NDUFB9 to MT-ND5 was detected in cKO cardiomyocytes. This physical interaction between NDUFB9 and MT-ND5 was depressed in MacroD^flox/flox hearts by the LPS challenge but maintained while cKO (Supplementary Fig. 11A), which may underpin MCI activity compromise during sepsis and its preservation by MacroD1 blocking.

NDUFB9 MARylation at R173 favors myocardial bioenergetics in sepsis.

We further examined whether and how MacroD1 modulates NDUFB9 MARylation and function. At baseline, cKO did not change NDUFB9 protein expression but enhanced its MARylation. Upon LPS challenge, NDUFB9 MARylation was reduced in MacroD1^flox/flox whereas maintained in cKO hearts (Fig. 6A). NDUFB9 MARylation reduced by LPS and preserved by MacroD1 knockdown was reproduced in AC16 cells (Fig. 6B). Overexpression of MacroD1 de-MARylated NDUFB9 compared to MacroD1 knockdown and NC groups, whereas truncated forms of MacroD1 did not (Fig. 6C).

Mono-ADP ribose antibody-based affinity proteomics identified MARylation on the arginine residues at position 173 of NDUFB9 (Fig. 6D), which possesses evolutionary conservation among humans, mice, and rats (Fig. 6E). Using a site-directed mutagenesis strategy with alanine in the place of arginine (Fig. 6F), in combination with immunoprecipitation, we identified no significant MARylation on mutated NDUFB9 (NDUFB9^R173A) while LPS stress deMARylated wild-type NDUFB9 (Fig. 6G). Knockdown and overexpression of MacroD1 enhanced and depressed MARylation of wild-type NDUFB9, respectively, without any effects on NDUFB9^R173A (Fig. 6H).

We then validated the function of MARylation at R173 of NDUFB9. In AC16 cells subjected to MacroD1 knockdown (Supplementary Fig. 11B), MCI activity and ATP production were not significantly affected by LPS treatment but reduced while concurrently downregulating NDUFB9 (Figs. 6I, J; Supplementary Fig. 11C). Overexpression of synonymous mutant of NDUFB9, which bypassed the affection of interference sequences targeting wild-type NDUFB9 (Supplementary Figs. 12A, B), rescued the above effects of co-treatments with MacroD1 and NDUFB9 silencing and LPS. In such cases, further introduction of synonymous NDUFB9 mutant with the substitution of arginine at position 173 by alanine (SM-NDUFB9^R173A) acted similarly to NDUFB9 knockdown (Figs. 6I, J). Moreover, given the essential role of MCI dysfunction in oxidative stress, we also evaluated the contribution of NDUFB9 MARylation to mitoROS generation. Resembling effects on MCI and ATP, NDUFB9 knockdown abolished the resistance of MacroD1 downregulation to mitoROS production induced by LPS, whereas SM-NDUFB9 overexpression, instead of SM-NDUFB9^R173A overexpression, rescued effects of NDUFB9 knockdown (Figs. 6K, L). Thus, NDUFB9 MARylation at R173 mediates the preservation of mitochondrial metabolic homeostasis afforded by MacroD1 inhibition during septic challenging.

cKO intercepts MCI dysfunction-coupled inflammatory damage of cardiomyocytes.

Cellular bioenergetic status programs inflammatory response(30), in which inflammasome is a core player, and a key component NLRP3 mediates caspase-1 activation, gasdermin D (GSDMD) cleavage, the production and release of pro-inflammatory cytokines IL-1β/IL-18, and pyroptotic cell death(31). We thus analyzed whether MacroD1-mediated mitochondrial metabolism affected NLRP3 inflammasome activation and pyroptosis of cardiomyocytes in sepsis.

The 18-hour LPS or 24-hour CLP challenges induced a comparable increase of serum IL-1β and TNFα in MacroD1^flox/flox and cKO mice (Supplementary Figs. 13A to D). However, depressed expression of the apoptosis-associated speck-like protein (ASC), NLRP3, IL1β, IL18, and TNFα mRNAs was observed and validated in cKO hearts exposed to LPS stress or CLP surgery (Figs. 7A to D; Supplementary Figs. 13E to G). Western blotting examinations revealed downregulation of pyroptotic mediators cleaved caspase-1 and GSDMD, inflammasome components NLRP3 and ASC, and general pro-inflammatory cytokine TNFα proteins (Figs. 7E to H). Levels of ASC and NLRP3 proteins translocated to mitochondria, a critical step for NLRP3 inflammasome activation, were significantly lower in cKO mice (Figs. 7I to K). Immunoprecipitation in the myocardium and proximity ligation assay in isolated adult mice ventricular cardiomyocytes further showed reduced binding of ASC to NLRP3 proteins (Figs. 7L to N). Similar phenomena were reproduced in NRCMs subjected to LPS insult (Figs. 7O to S; Supplementary Figs. 14A to C), highlighting that MacroD1 regulates the priming and activation of NLRP3 inflammasome in cardiomyocytes during septic challenging.

Moreover, we interrogated whether MCI mediated MacroD1-dependent NLRP3 inflammasome activation. As shown in Supplementary Figs. 15A to E, MCI inhibition with rotenone abolished the protection of MacroD1 knockdown against mitoROS generation, ATP compromise, and increased expression of pro-inflammatory cytokines IL1β and TNFα induced by LPS treatment in NRCMs. Conversely, scavenging of mitoROS produced effects similar to MacroD1 inactivation. Adenovirus-mediated MacroD1 overexpression facilitated mitoROS generation, ATP breakdown, and expression of IL1β and TNFα under LPS stimulation, which was well counteracted by a mitochondria-targeted SOD mimetic mito-TEMPO (Supplementary Figs. 16A to E).

Taken together, MacroD1 modulates pyroptotic damage of cardiomyocytes via the MCI-mitoROS-NLRP3 inflammasome pathway during sepsis.

Therapeutic potential of MacroD1 blockade for SCM.

To explore the potential of targeting MacroD1 to prevent SCM, we adopted the cell-permeable MRS2578 that inhibits MacroD1 activity(32, 33). In in vitro cultured cardiomyocytes of neonatal mice (NMCMs), NDUFB9 MARylation was significantly increased at baseline and preserved under co-treatment with MRS2578 and LPS (Supplementary Fig. 17A), confirming the effectiveness of the blocking compound. LPS-induced MCI activity compromise and mitoROS generation were depressed by MRS2578 treatment (Supplementary Figs. 17B to D). MRS2578 treatment, similar to MacroD1 knockdown, elevated oxygen consumption response to LPS (Supplementary Figs. 17E to I). Also, it depressed the priming and activation of NLRP3 inflammasome, as evaluated by quantitative PCR assay of IL-1β and TNFα mRNA expression (Supplementary Figs. 17J, K) and Western blotting test of NLRP3, ASC, and TNFα proteins and the cleavage of caspase-1 and GSDMD (Supplementary Figs. 17L to Q).

We then conducted intraperitoneal injection of MRS2578 in mice, once 8 hours before and after the LPS challenge (Fig. 8A). No death event was observed 96 hours post-injection of LPS in mice receiving the compound, in contrast to an approximately 25% mortality rate in controls (Fig. 8B). Echocardiographic analysis demonstrated that MRS2578 did not affect left ventricular ejection fraction and fraction shortening at baseline and counteracted their depression induced by 18-hour LPS stress (Figs. 8C to E; Supplementary Table 8). The restrain of elevated circulating cTnT and LDH and myocardial IL1β and TNFα mRNA expression induced by LPS indicated the prevention of MRS2578 against inflammatory myocardial damage (Figs. 8F, G; Supplementary Figs. 18A, B).

Histopathological analysis with MPO, DHE, and Masson’s stains further revealed alleviation of inflammatory infiltration, oxidative stress, and collagen deposition in the hearts of mice subjected to MRS2578 injection (Figs. 8H to K). Myocardial ATP was preserved (Fig. 8L) when MRS2578 maintained MCI and MCV activity under LPS treatment without affecting other mitochondrial complexes (Supplementary Figs. 19A to E). Overall, the chemical blockade of MacroD1 holds therapeutic potential for preventing SCM (Fig. 8M).

Identifying cardiac-specific mediators programming metabolic plasticity is a prerequisite for safely preventing SCM. The current study reveals an essential role of cardiomyocyte-enriched MacroD1 in modulating myocardial tolerance to sepsis. Conditional inactivation of the MacroD1 gene in cardiomyocytes enhances NDUFB9 MARylation whereby MCI activity and bioenergetics reserve, restraining superoxide reactions, inflammasome activation-coupled myocardial damage, dysfunction, and mortality in sepsis. The pharmacological blockade of MacroD1 recapitulates the cardioprotective benefits of its genetic inactivation and offers a new approach to preventing SCM.

Metabolic reprogramming of tissues is a crucial response to systemic inflammation and determines the degree and reversibility of organ damage(34). Bioenergetic derangement directly links to septic multi-organ dysfunction(11, 35). Despite NAD⁺ metabolism being vital to sepsis, anti-inflammatory effects by blocking NAD⁺ biosynthesis or supplying NAD⁺ precursors(36, 37) complicate its roles in SCM. High doses of NAD(H)-loaded nanoparticles(38) decreased septic mortality, being a lack of evaluating myocardial damage and function. Herein, MacroD1 inhibition significantly resisted LPS-induced reduction in NAD⁺ contents and NAD⁺/NADH ratio (Supplementary Figs. 20A, B), consistent with increased activity of MCI in which NADH was oxidized to NAD⁺ and for fueling bioenergy. MCI subunit NDUFB9 MARylation downregulated by LPS and enhanced by MacroD1 inhibition were NAD⁺ dependent because intracellular NAD⁺ depletion abolished such phenomena (Supplementary Fig. 20C). Similar scenarios occurred in regulating MCI activity and inflammasome activation (Supplementary Figs. 20D-F). Overall, the NAD⁺ underpins the MacroD1/NDUFB9/MCI/inflammasome pathway in cardiomyocytes. In sepsis with myocardial NAD⁺ starvation, MacroD1 blocking preserves the NAD⁺/NADH ratio critical for cellular oxidative states and bioenergy, restraining inflammatory damage to cardiomyocytes.

Besides effects on mitochondrial bioenergetics, MacroD1 inhibition counteracted sepsis-induced depression of fatty oxidation utilization that provides 60–90% of bioenergetics for normal cardiac function. This accounted for the reduced lipid accumulation in cKO hearts during sepsis. The restrain of dysregulated expression of genes related to lipid, citric cycle, amino acid, and one-carbon metabolism may be partly attributed to the retrograde regulation of MacroD1-inactivated mitochondria on nuclear gene transcription. For example, GDF15 upregulation induced by cKO could program triglyceride metabolism to protect the heart against sepsis(27, 39) and implicates the roles of MacroD1 in cardio-metabolic disorders.

MCI, a proximal mitochondrial electron transport unit most vulnerable to early sepsis(13), dominates ATP production by controlling the formation of mitochondrial supercomplexes containing MCIII and MCIV(40) and is identified as a core metabolic effector of MacroD1 inhibition. The enhanced interaction between NDUFB9 and MT-ND5 (Supplementary Fig. 11A) favors MCI stability(41, 42) and ensures adequate bioenergetics reserve capacity to support cardiac function. Stable MCI activity may also promote autophagy(43) to protect against SCM(26). However, direct intervention of MCI is unsuitable for SCM treatment due to its differential effects in various organs(14, 15). Instead, selective modulation of MacroD1 enriched in cardiomyocytes promises specific management of myocardial bioenergetics in sepsis.

MCI is also the initiation site of ROS in cardiac mitochondrial dysfunction(44). Excessive mitoROS links NLRP3 inflammasome activation to cell pyroptosis, which involves nuclear remodeling of pro-inflammatory cytokine expression and post-translational modifications for inflammasome assembly(17, 45). MacroD1 modulated mitoROS generation and subsequent activation of the NLRP3 inflammasome via MCI. Its inhibition preserved MCI activity and bioenergetics to limit mitoROS output, NLRP3 inflammasome activation, and cardiomyocyte pyroptosis that drives SCM(46, 47). By scavenging mitoROS, the MacroD1-MCI-mitoROS-NLRP3 cascade was disrupted. GDF15 upregulated by MacroD1 inhibition should also have anti-inflammatory effects(48), although the mechanisms are not yet understood. Of note, the blockade of MacroD1 did not affect serum levels of inflammatory cytokines, including IL1β and TNFα. This case supports the point of no correlation between pro-inflammatory cytokines and SCM(8) and that cardiomyocyte inflammatory response may only affect cardiac recruitment of systemic inflammatory cytokines.

Myocardial inflammation is usually characterized by dynamic spatiotemporal crosstalk between cardiac resident cells and the recruited immune cells. Cardiac fibroblasts(8, 49), endothelial cells(50), and immune cells(51) participate in myocardial inflammation and affect the contractile activity of cardiomyocytes via paracrine or juxtacrine effects under sepsis. The cardiomyocytes have recently been accepted as an initiating site for myocardial inflammation under nonischemic stresses(52). They can produce cytokines that depress the ventricle(53) and synchronize immune cell recruitment. Cardiomyocyte-specific regulatory property of MacroD1 on inflammatory response should render a direct target for breaking the vicious cycle of myocardial inflammation and damage in SCM.

Extraordinary mortality of SCM at the onset of sepsis in both patients and animal models underscores early prevention. Despite the importance of oxidative stress in septic organ failure, there have not yet been conclusive benefits for mitochondria-targeted antioxidants. The elusive efficacy may be attributable to indiscriminately blocking ROS across all cell types because oxidative stress initiates survival programming in a given cell type but induces an inflammatory response in other cells(54). Targeting MacroD1 may circumvent such a dilemma to enable cardiomyocyte ROS control. Cardioprotection of early administration of MacroD1 blockade against myocardial inflammation and fibrosis implicates the potential to resist late cardiac remodeling despite survivors with normal cardiac function and comparable myocardial oxidative states and inflammation response on day seven post-LPS injection in mice with and without MacroD1 blocking (Supplementary Fig. 21; Supplementary Table 9).

Because MCI inhibition-induced acetyl-CoA accumulation could facilitate mitochondrial protein acetylation and burden Sirt3 most studied in organ metabolic diseases including SCM(55, 56), MacroD1-MCI signaling axis may involve Sirt3. However, expression of Sirt3 protein was not susceptible to MacroD1 at baseline (Supplement Fig. 22A). Sirt3 knockdown did not affect NDUFB9 MARylation and MCI activity preserved by MacroD1 blocking (Supplement Figs. 22B, C) while enhanced mitochondrial ROS production and partially weakened anti-oxidative effects mediated by MacroD1 knockdown (Supplementary Figs. 22D, E), suggesting their differential modulation on oxidative stress in cardiomyocytes during sepsis. Sirt3 knockout did not significantly affect myocardial inflammasome activation during sepsis(25), further supporting the roles of Sirt3 different from MacroD1 in SCM.

Deciphering mitochondrial MARylation remains challenging, unlike nuclear ADP-ribosylation for genotoxic stress response. On the one hand, MacroD1 is the only ADP-ribose-metabolizing enzyme localized to mitochondria(24), while mitochondrial ADP-ribosyltransferases remain unknown(57). On the other hand, there is no wide-acknowledged method, although multiple approaches are emerging to characterize MARylation(58, 59). Using antibodies to immunoprecipitate proteins, combined with mono-ADP ribose antibody-based validations and site-directed mutagenesis, NDUFB9 was identified as a functional effector of MacroD1-mediated inflammometabolic regulation. Even so, the potential contributions of other proteins binding to MacroD1 could not be precluded. In addition, differing from ADP-ribosylation of histidine and lysine residues in muscle mitochondrial NDUFB9 reported(60), its arginine residues were MARylated by MacroD1 inhibition. The underlying explanation may be either histidine and lysine residues are inclined to be poly-ADP ribosylated by poly-(ADP-ribose)-polymerases or MARylated by other NAD⁺-signaling enzymes than MacroD1. Given the effects of mitochondrial ADP-ribosylation on nuclear ADP-ribosylation(60), MacroD1 may mediate cardiac mitochondrial-nuclear crosstalk.

Mitochondrial MacroD1 holds potential pleiotropic effects on proteins and nucleic acids through its mono-ADP-ribose hydrolyzing activities(61). There is currently limited evidence for ADP-ribosylation within mitochondria. Whether MacroD1 performs enzymatic ADPr hydrolyzing of mitochondrial nucleic acids remains a pending issue, despite documented removal of ADPr from phosphorylated double-stranded DNAs by recombinant MacroD1 proteins in vitro experiment(61). We cannot preclude the potential modulation of MacroD1 on mitochondrial DNAs and RNAs besides the NDUFB9 protein identified. Nonetheless, comparable expression levels of mitochondrial complexes in cKO and MacroD1^flox/flox mice at baseline and septic challenge (Fig. 5A) suggest that MacroD1 does not affect mitochondrial DNAs encoding constitutive proteins of respiratory complexes.

The existence of MacroD1 in the nuclear and cytoplasm of cardiomyocytes (Fig. 1F), albeit in small amounts, implicates potential biological function. To examine whether non-mitochondrial MacroD1 involves MCI-coupled NLRP3 inflammasome activation, we constructed mutants truncating the mitochondrial targeting sequence in MacroD1 (MacroD1^ΔMTS). Overexpression of MacroD1^ΔMTS with a predominant cytoplasmic expression and the scarce in the nuclear in AC16 cardiomyocytes had almost no affection on MCI activity, mitoROS generation, and inflammatory response during LPS treatment, different from MacroD1 overexpression and knockdown (Supplementary Figs. 23A-G). Thus, cardioprotection of MacroD1 blocking against sepsis should most result from its regulatory effects on mitochondria.

In sum, the potentiation of mitochondrial bioenergetics metabolism plasticity in nonimmune cardiomyocytes is critical for restraining myocardial inflammometabolic disorders and dysfunction in sepsis. Targeting cardiomyocyte MacroD1-MCI-NLRP3 pathway holds promise for preventing SCM.

Acknowledgments: We thank Prof. Jiang Hong and Liqun Zhao at the Department of intensive medicine, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine for their constructive comments, MS. Shanshan Wang and Lianyan Jing at The Core Facility Center of CAS Center for Excellence in Molecular Plant Sciences for their assistance in proteomic assays.

Funding: This work was supported by the National Original Exploration Program of China (82150001, to Jun Li), the National Key Research & Development Program of China (2018YFC1312504, to Jun Li), the National Natural Science Foundation of China (81670360, 81870293, to Jun Li; 82000376, to Fangfang Li; 82000375, to Qitong Zhang), and the Popularization & Management Optimization Program of Diagnosis and Treatment Technique of Shanghai (SHDC22023241, to Hao Xu).

Competing interests: The authors declare no competing interests.

Data and materials availability：All data associated with this study are present in the paper or the Supplementary Materials. The raw and analyzed RNA-sequencing data produced in this study can be found in the Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/) under the GEO series accession number: GSE253829.

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Cardiomyocyte mitochondrial mono-ADP-ribosylation dictates cardiac tolerance to sepsis by configuring bioenergetic reserve

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