Cardiac troponin I directly binds and inhibits mitochondrial ATP synthase with a noncanonical role in the post-ischemic heart.

Cardiac troponin I (cTnI) is a key regulator of cardiomyocyte contraction. However, its role in mitochondria is unknown. Here we show that cTnI localized to mitochondria in the heart, inhibited mitochondrial functions when stably expressed in noncardiac cells and increased the opening of the mitochondrial permeability transition pore under oxidative stress. Direct, specific and saturable binding of cTnI to F1FO-ATP synthase was demonstrated in vitro using immune-captured ATP synthase and in cells using proximity ligation assay. cTnI binding doubled ATPase activity, whereas skeletal troponin I and several human pathogenic cTnI variants associated with familial hypertrophic cardiomyopathy did not. A rationally designed peptide, P888, inhibited cTnI binding to ATP synthase, inhibited cTnI-induced increase in ATPase activity in vitro and reduced cardiac injury following transient ischemia in vivo. We suggest that cTnI-bound ATP synthase results in lower ATP levels, and releasing this interaction during cardiac ischemia-reperfusion may increase the reservoir of functional mitochondria to reduce cardiac injury.

Uncovering newly identified aldehyde dehydrogenase 2 genetic variants that lead to acetaldehyde accumulation after an alcohol challenge.

BACKGROUND: Aldehyde dehydrogenase 2 (ALDH2) is critical for alcohol metabolism by converting acetaldehyde to acetic acid. In East Asian descendants, an inactive genetic variant in ALDH2, rs671, triggers an alcohol flushing response due to acetaldehyde accumulation. As alcohol flushing is not exclusive to those of East Asian descent, we questioned whether additional ALDH2 genetic variants can drive facial flushing and inefficient acetaldehyde metabolism using human testing and biochemical assays. METHODS: After IRB approval, human subjects were given an alcohol challenge (0.25 g/kg) while quantifying acetaldehyde levels and the physiological response (heart rate and skin temperature) to alcohol. Further, by employing biochemical techniques including human purified ALDH2 proteins and transiently transfected NIH 3T3 cells, we characterized two newly identified ALDH2 variants for ALDH2 enzymatic activity, ALDH2 dimer/tetramer formation, and reactive oxygen species production after alcohol treatment. RESULTS: Humans heterozygous for rs747096195 (R101G) or rs190764869 (R114W) had facial flushing and a 2-fold increase in acetaldehyde levels, while rs671 (E504K) had facial flushing and a 6-fold increase in acetaldehyde levels relative to wild type ALDH2 carriers. In vitro studies with recombinant R101G and R114W ALDH2 enzyme showed a reduced efficiency in acetaldehyde metabolism that is unique when compared to E504K or wild-type ALDH2. The effect is caused by a lack of functional dimer/tetramer formation for R101G and decreased Vmax for both R101G and R114W. Transiently transfected NIH-3T3 cells with R101G and R114W also had a reduced enzymatic activity by ~ 50% relative to transfected wild-type ALDH2 and when subjected to alcohol, the R101G and R114W variants had a 2-3-fold increase in reactive oxygen species formation with respect to wild type ALDH2. CONCLUSIONS: We identified two additional ALDH2 variants in humans causing facial flushing and acetaldehyde accumulation after alcohol consumption. As alcohol use is associated with a several-fold higher risk for esophageal cancer for the E504K variant, the methodology developed here to characterize ALDH2 genetic variant response to alcohol can lead the way precision medicine strategies to further understand the interplay of alcohol consumption, ALDH2 genetics, and cancer.

Cleavage of Hsp70.1 causes lysosomal cell death under stress conditions.

Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Heat shock protein 70.1 (Hsp70.1) exhibits dual functions as a chaperone protein and a lysosomal membrane stabilizer. Since chaperone-mediated autophagy participates in the recycling of ∼30% cytosolic proteins, its disorder causes cell susceptibility to stress conditions. Cargo proteins destined for degradation such as amyloid precursor protein and tau protein are trafficked by Hsp70.1 from the cytosol into lysosomes. Hsp70.1 is composed of an N-terminal nucleotide-binding domain (NBD) and a C-terminal domain that binds to cargo proteins, termed the substrate-binding domain (SBD). The NBD and SBD are connected by the interdomain linker LL1, which modulates the allosteric structure of Hsp70.1 in response to ADP/ATP binding. After the passage of the Hsp70.1-cargo complex through the lysosomal limiting membrane, high-affinity binding of the positive-charged SBD with negative-charged bis(monoacylglycero)phosphate (BMP) at the internal vesicular membranes activates acid sphingomyelinase to generate ceramide for stabilizing lysosomal membranes. As the integrity of the lysosomal limiting membrane is critical to ensure cargo protein degradation within the acidic lumen, the disintegration of the lysosomal limiting membrane is lethal to cells. After the intake of high-fat diets, however, ß-oxidation of fatty acids in the mitochondria generates reactive oxygen species, which enhance the oxidation of membrane linoleic acids to produce 4-hydroxy-2-nonenal (4-HNE). In addition, 4-HNE is produced during the heating of linoleic acid-rich vegetable oils and incorporated into the body via deep-fried foods. This endogenous and exogenous 4-HNE synergically causes an increase in its serum and organ levels to induce carbonylation of Hsp70.1 at Arg469, which facilitates its conformational change and access of activated µ-calpain to LL1. Therefore, the cleavage of Hsp70.1 occurs prior to its influx into the lysosomal lumen, which leads to lysosomal membrane permeabilization/rupture. The resultant leakage of cathepsins is responsible for lysosomal cell death, which would be one of the causative factors of lifestyle-related diseases.

Extracellular release of damaged mitochondria induced by prehematopoietic stem cell transplant conditioning exacerbates GVHD.

ABSTRACT: Despite therapeutic advancements, graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HSCT). In current models of GVHD, tissue injury induced by cytotoxic conditioning regimens, along with translocation of microbes expressing pathogen-associated molecular patterns, result in activation of host antigen-presenting cells (APCs) to stimulate alloreactive donor T lymphocytes. Recent studies have demonstrated that in many pathologic states, tissue injury results in the release of mitochondria from the cytoplasm to the extracellular space. We hypothesized that extracellular mitochondria, which are related to archaebacteria, could also trigger GVHD by stimulation of host APCs. We found that clinically relevant doses of radiation or busulfan induced extracellular release of mitochondria by various cell types, including cultured intestinal epithelial cells. Conditioning-mediated mitochondrial release was associated with mitochondrial damage and impaired quality control but did not affect the viability of the cells. Extracellular mitochondria directly stimulated host APCs to express higher levels of major histocompatibility complex II (MHC-II), costimulatory CD86, and proinflammatory cytokines, resulting in increased donor T-cell activation, and proliferation in mixed lymphocyte reactions. Analyses of plasma from both experimental mice and a cohort of children undergoing HSCT demonstrated that conditioning induced extracellular mitochondrial release in vivo. In mice undergoing MHC-mismatched HSCT, administration of purified syngeneic extracellular mitochondria increased host APC activation and exacerbated GVHD. Our data suggest that pre-HSCT conditioning results in extracellular release of damaged mitochondria, which increase alloreactivity and exacerbate GVHD. Therefore, decreasing the extracellular release of damaged mitochondria after conditioning could serve as a novel strategy for GVHD prevention.

ALDH2 dysfunction and alcohol cooperate in cancer stem cell enrichment.

The alcohol metabolite acetaldehyde is a potent human carcinogen linked to esophageal squamous cell carcinoma (ESCC) initiation and development. Aldehyde dehydrogenase 2 (ALDH2) is the primary enzyme that detoxifies acetaldehyde in the mitochondria. Acetaldehyde accumulation causes genotoxic stress in cells expressing the dysfunctional ALDH2E487K dominant negative mutant protein linked to ALDH2*2, the single nucleotide polymorphism highly prevalent among East Asians. Heterozygous ALDH2*2 increases the risk for the development of ESCC and other alcohol-related cancers. Despite its prevalence and link to malignant transformation, how ALDH2 dysfunction influences ESCC pathobiology is incompletely understood. Herein, we characterize how ESCC and preneoplastic cells respond to alcohol exposure using cell lines, three-dimensional organoids and xenograft models. We find that alcohol exposure and ALDH2*2 cooperate to increase putative ESCC cancer stem cells with high CD44 expression (CD44H cells) linked to tumor initiation, repopulation and therapy resistance. Concurrently, ALHD2*2 augmented alcohol-induced reactive oxygen species and DNA damage to promote apoptosis in the non-CD44H cell population. Pharmacological activation of ALDH2 by Alda-1 inhibits this phenotype, suggesting that acetaldehyde is the primary driver of these changes. Additionally, we find that Aldh2 dysfunction affects the response to cisplatin, a chemotherapeutic commonly used for the treatment of ESCC. Aldh2 dysfunction facilitated enrichment of CD44H cells following cisplatin-induced oxidative stress and cell death in murine organoids, highlighting a potential mechanism driving cisplatin resistance. Together, these data provide evidence that ALDH2 dysfunction accelerates ESCC pathogenesis through enrichment of CD44H cells in response to genotoxic stressors such as environmental carcinogens and chemotherapeutic agents.

Changes of neurofilament light chain in patients with alcohol dependence following withdrawal and the genetic effect from ALDH2 Polymorphism.

Neurofilament light chain (NFL), as a measure of neuroaxonal injury, has recently gained attention in alcohol dependence (AD). Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme which metabolizes the alcohol breakdown product acetaldehyde. An ALDH2 single nucleotide polymorphism (rs671) is associated with less ALDH2 enzyme activity and increased neurotoxicity. We examined the blood NFL levels in 147 patients with AD and 114 healthy controls using enzyme-linked immunosorbent assay and genotyped rs671. We also followed NFL level, alcohol craving and psychological symptoms in patients with AD after 1 and 2 weeks of detoxification. We found the baseline NFL level was significantly higher in patients with AD than in controls (mean ± SD: 264.2 ± 261.8 vs. 72.1 ± 35.6 pg/mL, p < 0.001). The receiver operating characteristic curve revealed that NFL concentration could discriminate patients with AD from controls (area under the curve: 0.85; p < 0.001). The NFL levels were significantly reduced following 1 and 2 weeks of detoxification, with the extent of reduction correlated with the improvement of craving, depression, and anxiety (p < 0.001). Carriers with the rs671 GA genotype, which is associated with less ALDH2 activity, had higher NLF levels either at baseline or after detoxification compared with GG carriers. In conclusion, plasma NFL level was increased in patients with AD and reduced after early abstinence. Reduction in NFL level corroborated well with the improvement of clinical symptoms. The ALDH2 rs671 polymorphism may play a role in modulating the extent of neuroaxonal injury and its recovery.

4-Hydroxynonenal impairs miRNA maturation in heart failure via Dicer post-translational modification.

BACKGROUND AND AIMS: Developing novel therapies to battle the global public health burden of heart failure remains challenging. This study investigates the underlying mechanisms and potential treatment for 4-hydroxynonenal (4-HNE) deleterious effects in heart failure. METHODS: Biochemical, functional, and histochemical measurements were applied to identify 4-HNE adducts in rat and human failing hearts. In vitro studies were performed to validate 4-HNE targets. RESULTS: 4-HNE, a reactive aldehyde by-product of mitochondrial dysfunction in heart failure, covalently inhibits Dicer, an RNase III endonuclease essential for microRNA (miRNA) biogenesis. 4-HNE inhibition of Dicer impairs miRNA processing. Mechanistically, 4-HNE binds to recombinant human Dicer through an intermolecular interaction that disrupts both activity and stability of Dicer in a concentration- and time-dependent manner. Dithiothreitol neutralization of 4-HNE or replacing 4-HNE-targeted residues in Dicer prevents 4-HNE inhibition of Dicer in vitro. Interestingly, end-stage human failing hearts from three different heart failure aetiologies display defective 4-HNE clearance, decreased Dicer activity, and miRNA biogenesis impairment. Notably, boosting 4-HNE clearance through pharmacological re-activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) using Alda-1 or its improved orally bioavailable derivative AD-9308 restores Dicer activity. ALDH2 is a major enzyme responsible for 4-HNE removal. Importantly, this response is accompanied by improved miRNA maturation and cardiac function/remodelling in a pre-clinical model of heart failure. CONCLUSIONS: 4-HNE inhibition of Dicer directly impairs miRNA biogenesis in heart failure. Strikingly, decreasing cardiac 4-HNE levels through pharmacological ALDH2 activation is sufficient to re-establish Dicer activity and miRNA biogenesis; thereby representing potential treatment for patients with heart failure.

Implication of the cooking oil-peroxidation product "hydroxynonenal" for Alzheimer's disease.

Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that reduces cell injuries via detoxification of lipid-peroxidation product, 4-hydroxy-2-nonenal (hydroxynonenal). It is generated exogenously via deep-frying of linoleic acid-rich cooking oils and/or endogenously via oxidation of fatty acids involved in biomembranes. Although its toxicity for human health is widely accepted, the underlying mechanism long remained unknown. In 1998, Yamashima et al. have formulated the "calpain-cathepsin hypothesis" as a molecular mechanism of ischemic neuronal death. Subsequently, they found that calpain cleaves Hsp70.1 which became vulnerable after the hydroxynonenal-induced carbonylation at the key site Arg469. Since it is the pivotal aberration that induces lysosomal membrane rupture, they suggested that neuronal death in Alzheimer's disease similarly occurs by chronic ischemia via the calpain-cathepsin cascade triggered by hydroxynonenal. For nearly three decades, amyloid ß (Aß) peptide was thought to be a root substance of Alzheimer's disease. However, because of both the insignificant correlations between Aß depositions and occurrence of neuronal death or dementia, and the negative results of anti-Aß medicines tested so far in the patients with Alzheimer's disease, the strength of the "amyloid cascade hypothesis" has been weakened. Recent works have suggested that hydroxynonenal is a mediator of programmed cell death not only in the brain, but also in the liver, pancreas, heart, etc. Increment of hydroxynonenal was considered an early event in the development of Alzheimer's disease. This review aims at suggesting ways out of the tunnel, focusing on the implication of hydroxynonenal in this disease. Herein, the mechanism of Alzheimer neuronal death is discussed by focusing on Hsp70.1 with a dual function as chaperone protein and lysosomal stabilizer. We suggest that Aß is not a culprit of Alzheimer's disease, but merely a byproduct of autophagy/lysosomal failure resulting from hydroxynonenal-induced Hsp70.1 disorder. Enhancing ALDH2 activity to detoxify hydroxynonenal emerges as a promising strategy for preventing and treating Alzheimer's disease.

Impact of common ALDH2 inactivating mutation and alcohol consumption on Alzheimer's disease.

Aldehyde dehydrogenase 2 (ALDH2) is an enzyme found in the mitochondrial matrix that plays a central role in alcohol and aldehyde metabolism. A common ALDH2 polymorphism in East Asians descent (called ALDH2*2 or E504K missense variant, SNP ID: rs671), present in approximately 8% of the world's population, has been associated with a variety of diseases. Recent meta-analyses support the relationship between this ALDH2 polymorphism and Alzheimer's disease (AD). And AD-like pathology observed in ALDH2-/- null mice and ALDH2*2 overexpressing transgenic mice indicate that ALDH2 deficiency plays an important role in the pathogenesis of AD. Recently, the worldwide increase in alcohol consumption has drawn attention to the relationship between heavy alcohol consumption and AD. Of potential clinical significance, chronic administration of alcohol in ALDH2*2/*2 knock-in mice exacerbates the pathogenesis of AD-like symptoms. Therefore, ALDH2 polymorphism and alcohol consumption likely play an important role in the onset and progression of AD. Here, we review the data on the relationship between ALDH2 polymorphism, alcohol, and AD, and summarize what is currently known about the role of the common ALDH2 inactivating mutation, ALDH2*2, and alcohol in the onset and progression of AD.

A common East-Asian ALDH2 mutation causes metabolic disorders and the therapeutic effect of ALDH2 activators.

Obesity and type 2 diabetes have reached pandemic proportion. ALDH2 (acetaldehyde dehydrogenase 2, mitochondrial) is the key metabolizing enzyme of acetaldehyde and other toxic aldehydes, such as 4-hydroxynonenal. A missense Glu504Lys mutation of the ALDH2 gene is prevalent in 560 million East Asians, resulting in reduced ALDH2 enzymatic activity. We find that male Aldh2 knock-in mice mimicking human Glu504Lys mutation were prone to develop diet-induced obesity, glucose intolerance, insulin resistance, and fatty liver due to reduced adaptive thermogenesis and energy expenditure. We find reduced activity of ALDH2 of the brown adipose tissue from the male Aldh2 homozygous knock-in mice. Proteomic analyses of the brown adipose tissue from the male Aldh2 knock-in mice identifies increased 4-hydroxynonenal-adducted proteins involved in mitochondrial fatty acid oxidation and electron transport chain, leading to markedly decreased fatty acid oxidation rate and mitochondrial respiration of brown adipose tissue, which is essential for adaptive thermogenesis and energy expenditure. AD-9308 is a water-soluble, potent, and highly selective ALDH2 activator. AD-9308 treatment ameliorates diet-induced obesity and fatty liver, and improves glucose homeostasis in both male Aldh2 wild-type and knock-in mice. Our data highlight the therapeutic potential of reducing toxic aldehyde levels by activating ALDH2 for metabolic diseases.

Targeting an allosteric site in dynamin-related protein 1 to inhibit Fis1-mediated mitochondrial dysfunction.

The large cytosolic GTPase, dynamin-related protein 1 (Drp1), mediates both physiological and pathological mitochondrial fission. Cell stress triggers Drp1 binding to mitochondrial Fis1 and subsequently, mitochondrial fragmentation, ROS production, metabolic collapse, and cell death. Because Drp1 also mediates physiological fission by binding to mitochondrial Mff, therapeutics that inhibit pathological fission should spare physiological mitochondrial fission. P110, a peptide inhibitor of Drp1-Fis1 interaction, reduces pathology in numerous models of neurodegeneration, ischemia, and sepsis without blocking the physiological functions of Drp1. Since peptides have pharmacokinetic limitations, we set out to identify small molecules that mimic P110's benefit. We map the P110-binding site to a switch I-adjacent grove (SWAG) on Drp1. Screening for SWAG-binding small molecules identifies SC9, which mimics P110's benefits in cells and a mouse model of endotoxemia. We suggest that the SWAG-binding small molecules discovered in this study may reduce the burden of Drp1-mediated pathologies and potentially pathologies associated with other members of the GTPase family.

A Common East Asian aldehyde dehydrogenase 2*2 variant promotes ventricular arrhythmia with chronic light-to-moderate alcohol use in mice.

Chronic heavy alcohol use is associated with lethal arrhythmias. Whether common East Asian-specific aldehyde dehydrogenase deficiency (ALDH2*2) contributes to arrhythmogenesis caused by low level alcohol use remains unclear. Here we show 59 habitual alcohol users carrying ALDH2 rs671 have longer QT interval (corrected) and higher ventricular tachyarrhythmia events compared with 137 ALDH2 wild-type (Wt) habitual alcohol users and 57 alcohol non-users. Notably, we observe QT prolongation and a higher risk of premature ventricular contractions among human ALDH2 variants showing habitual light-to-moderate alcohol consumption. We recapitulate a human electrophysiological QT prolongation phenotype using a mouse ALDH2*2 knock-in (KI) model treated with 4% ethanol, which shows markedly reduced total amount of connexin43 albeit increased lateralization accompanied by markedly downregulated sarcolemmal Nav1.5, Kv1.4 and Kv4.2 expressions compared to EtOH-treated Wt mice. Whole-cell patch-clamps reveal a more pronounced action potential prolongation in EtOH-treated ALDH2*2 KI mice. By programmed electrical stimulation, rotors are only provokable in EtOH-treated ALDH2*2 KI mice along with higher number and duration of ventricular arrhythmia episodes. The present research helps formulate safe alcohol drinking guideline for ALDH2 deficient population and develop novel protective agents for these subjects.

Analysis of well-annotated next-generation sequencing data reveals increasing cases of SARS-CoV-2 reinfection with Omicron.

SARS-CoV-2 has extensively mutated creating variants of concern (VOC) resulting in global infection surges. The Omicron VOC reinfects individuals exposed to earlier variants of SARS-CoV-2 at a higher frequency than previously seen for non-Omicron VOC. An analysis of the sub-lineages associated with an Omicron primary infection and Omicron reinfection reveals that the incidence of Omicron-Omicron reinfections is occurring over a shorter time interval than seen after a primary infection with a non-Omicron VOC. Our analysis suggests that a single infection from SARS-CoV-2 may not generate the protective immunity required to defend against reinfections from emerging Omicron lineages. This analysis was made possible by Next-generation sequencing (NGS) of a Danish cohort with clinical metadata on both infections occurring in the same individual. We suggest that the continuation of COVID-19 NGS and inclusion of clinical metadata is necessary to ensure effective surveillance of SARS-CoV-2 genomics, assist in treatment and vaccine development, and guide public health recommendations.

Drp1/p53 interaction mediates p53 mitochondrial localization and dysfunction in septic cardiomyopathy.

BACKGROUND: Previous studies have implicated p53-dependent mitochondrial dysfunction in sepsis induced end organ injury, including sepsis-induced myocardial dysfunction (SIMD). However, the mechanisms behind p53 localization to the mitochondria have not been well established. Dynamin-related protein 1 (Drp1), a mediator of mitochondrial fission, may play a role in p53 mitochondrial localization. Here we examined the role of Drp1/p53 interaction in SIMD using in vitro and murine models of sepsis. METHODS: H9c2 cardiomyoblasts and BALB/c mice were exposed to lipopolysaccharide (LPS) to model sepsis phenotype. Pharmacologic inhibitors of Drp1 activation (ψDrp1) and of p53 mitochondrial binding (pifithrin µ, PFTµ) were utilized to assess interaction between Drp1 and p53, and the subsequent downstream impact on mitochondrial morphology and function, cardiomyocyte function, and sepsis phenotype. RESULTS: Both in vitro and murine models demonstrated an increase in physical Drp1/p53 interaction following LPS treatment, which was associated with increased p53 mitochondrial localization, and mitochondrial dysfunction. This Drp1/p53 interaction was inhibited by ΨDrp1, suggesting that this interaction is dependent on Drp1 activation. Treatment of H9c2 cells with either ΨDrp1 or PFTµ inhibited the LPS mediated localization of Drp1/p53 to the mitochondria, decreased oxidative stress, improved cellular respiration and ATP production. Similarly, treatment of BALB/c mice with either ΨDrp1 or PFTµ decreased LPS-mediated mitochondrial localization of p53, mitochondrial ROS in cardiac tissue, and subsequently improved cardiomyocyte contractile function and survival. CONCLUSION: Drp1/p53 interaction and mitochondrial localization is a key prodrome to mitochondrial damage in SIMD and inhibiting this interaction may serve as a therapeutic target.

SGLT2 inhibitor ameliorates endothelial dysfunction associated with the common ALDH2 alcohol flushing variant.

The common aldehyde dehydrogenase 2 (ALDH2) alcohol flushing variant known as ALDH2*2 affects ∼8% of the world's population. Even in heterozygous carriers, this missense variant leads to a severe loss of ALDH2 enzymatic activity and has been linked to an increased risk of coronary artery disease (CAD). Endothelial cell (EC) dysfunction plays a determining role in all stages of CAD pathogenesis, including early-onset CAD. However, the contribution of ALDH2*2 to EC dysfunction and its relation to CAD are not fully understood. In a large genome-wide association study (GWAS) from Biobank Japan, ALDH2*2 was found to be one of the strongest single-nucleotide polymorphisms associated with CAD. Clinical assessment of endothelial function showed that human participants carrying ALDH2*2 exhibited impaired vasodilation after light alcohol drinking. Using human induced pluripotent stem cell-derived ECs (iPSC-ECs) and CRISPR-Cas9-corrected ALDH2*2 iPSC-ECs, we modeled ALDH2*2-induced EC dysfunction in vitro, demonstrating an increase in oxidative stress and inflammatory markers and a decrease in nitric oxide (NO) production and tube formation capacity, which was further exacerbated by ethanol exposure. We subsequently found that sodium-glucose cotransporter 2 inhibitors (SGLT2i) such as empagliflozin mitigated ALDH2*2-associated EC dysfunction. Studies in ALDH2*2 knock-in mice further demonstrated that empagliflozin attenuated ALDH2*2-mediated vascular dysfunction in vivo. Mechanistically, empagliflozin inhibited Na+/H+-exchanger 1 (NHE-1) and activated AKT kinase and endothelial NO synthase (eNOS) pathways to ameliorate ALDH2*2-induced EC dysfunction. Together, our results suggest that ALDH2*2 induces EC dysfunction and that SGLT2i may potentially be used as a preventative measure against CAD for ALDH2*2 carriers.