Pyroptosis in cardiovascular diseases: Pumping gasdermin on the fire.

Pyroptosis is a form of programmed cell death associated with activation of inflammasomes and inflammatory caspases, proteolytic cleavage of gasdermin proteins (forming pores in the plasma membrane), and selective release of proinflammatory mediators. Induction of pyroptosis results in amplification of inflammation, contributing to the pathogenesis of chronic cardiovascular diseases such as atherosclerosis and diabetic cardiomyopathy, and acute cardiovascular events, such as thrombosis and myocardial infarction. While engagement of pyroptosis during sepsis-induced cardiomyopathy and septic shock is expected and well documented, we are just beginning to understand pyroptosis involvement in the pathogenesis of cardiovascular diseases with less defined inflammatory components, such as atrial fibrillation. Due to the danger that pyroptosis represents to cells within the cardiovascular system and the whole organism, multiple levels of pyroptosis regulation have evolved. Those include regulation of inflammasome priming, post-translational modifications of gasdermins, and cellular mechanisms for pore removal. While pyroptosis in macrophages is well characterized as a dramatic pro-inflammatory process, pyroptosis in other cell types within the cardiovascular system displays variable pathways and consequences. Furthermore, different cells and organs engage in local and distant crosstalk and exchange of pyroptosis triggers (oxidized mitochondrial DNA), mediators (IL-1ß, S100A8/A9) and antagonists (IL-9). Development of genetic tools, such as Gasdermin D knockout animals, and small molecule inhibitors of pyroptosis will not only help us fully understand the role of pyroptosis in cardiovascular diseases but may result in novel therapeutic approaches inhibiting inflammation and progression of chronic cardiovascular diseases to reduce morbidity and mortality from acute cardiovascular events.

Passing Behavior of Oligonucleotides through a Stacked DNA Nanochannel with Featured Path Design.

DNA nanotechnology has emerged as a useful tool for constructing artificial channels penetrating the lipid bilayer. In this work, we introduce a stacked DNA origami nanochannel device characterized by a width-variable pathway, consisting of narrow entrance and exit channels coupled with a wide, modifiable lumen. This design modulates the translocation behavior of oligonucleotides, revealing distinct stages of signal patterns in the recorded current traces. The observed prolonged dwell times indicate oligonucleotide retention, specifically due to the transition from the wide lumen to the narrower exit channel, while variations in current recovery between events suggested intermediate channel states between conducting and blocking. Further, by incorporating sequence-specific overhangs within the channel lumen, we achieved unique asymmetric current profiles during ATP aptamer translocation events. Featured stages also highlighted the aptamer binding dynamics and ATP-induced release. The distinguished oligonucleotide passing behaviors afforded by the stacked DNA origami channel with interior decoration demonstrated the strategic and profitable attempts at DNA nanochannel engineering for nanodevice development and applications.

Novel strategies for targeting neutrophil against myocardial infarction.

Inflammation is a crucial factor in cardiac remodeling after acute myocardial infarction (MI). Neutrophils, as the first wave of leukocytes to infiltrate the injured myocardium, exacerbate inflammation and cardiac injury. However, therapies that deplete neutrophils to manage cardiac remodeling after MI have not consistently produced promising outcomes. Recent studies have revealed that neutrophils at different time points and locations may have distinct functions. Thus, transferring neutrophil phenotypes, rather than simply blocking their activities, potentially meet the needs of cardiac repair. In this review, we focus on discussing the fate, heterogeneity, functions of neutrophils, and attempt to provide a more comprehensive understanding of their roles and targeting strategies in MI. We highlight the strategies and translational potential of targeting neutrophils to limit cardiac injury to reduce morbidity and mortality from MI.

Bone marrow-derived naïve B lymphocytes improve heart function after myocardial infarction: a novel cardioprotective mechanism for empagliflozin.

The role of adaptive immunity in myocardial recovery post myocardial infarction (MI), particularly the immune response by B lymphocytes, remains elusive. Bone marrow immune microenvironment in response to MI is remotely regulated by the hypothalamic pituitary adrenal (HPA) axis. We utilized the cardioprotective actions of SGLT2 inhibitor to identify and characterize bone marrow B cell subsets that respond to myocardial injury. Initially, we preformed ligation of left anterior descendant (LAD) coronary artery in male C57BL/6J mice to monitor the dynamic changes of immune cells across tissues. Mechanistic insights from mouse models demonstrated arrest of bone marrow B cell maturation and function 24 h post MI. A secondary MI model (twice MIs) in mice was established for the first time to evaluate the dosage-dependent cardioprotection of empagliflozin (EMPA). Single-cell RNA-Seq further demonstrated that EMPA restored bone marrow naïve B cell (B220+CD19+CD43-IgM+IgD+) counts and function. Additionally, we recruited 14 acute MI patients with single LAD disease, and profiled B cells post percutaneous coronary intervention (PCI) (compared to 18 matched no-MI controls). We revealed a positive correlation of increased B cell counts with enhanced ejection fraction in MI patients with PCI while lymphopenia was associated with patients with heart failure. Mechanistically, MI triggers the release of glucocorticoids from neuroendocrine system, inducing NHE1-mediated autophagic death of bone marrow B cells while repressing B cell progenitor proliferation and differentiation. Infusion of B cells derived from bone marrow significantly improved cardiac function and diminished infarct size post MI. These findings provide new mechanistic insights into regulation of adaptive immune response post MI, and support targeting bone marrow B cell development for improved ventricular remodeling and reduced heart failure after MI.

An NLRP3 inflammasome-triggered cytokine storm contributes to Streptococcal toxic shock-like syndrome (STSLS).

Infection with the Streptococcus suis (S. suis) epidemic strain can cause Streptococcal toxic shock-like syndrome (STSLS), which is characterized by a cytokine storm, dysfunction of multiple organs and a high incidence of mortality despite adequate treatment. Despite some progress concerning the contribution of the inflammatory response to STSLS, the precise mechanism underlying STSLS development remains elusive. Here, we use a murine model to demonstrate that caspase-1 activity is critical for STSLS development. Furthermore, we show that inflammasome activation by S. suis is mainly dependent on NLRP3 but not on NLRP1, AIM2 or NLRC4. The important role of NLRP3 activation in STSLS is further confirmed in vivo with the NLRP3 inhibitor MCC950 and nlrp3-knockout mice. By comparison of WT strain with isogenic strains with mutation of various virulence genes for inflammasome activation, Suilysin is essential for inflammasome activation, which is dependent on the membrane perforation activity to cause cytosolic K+ efflux. Moreover, the mutant strain msly (P353L) expressing mutagenic SLY without hemolytic activity was unable to activate the inflammasome and does not cause STSLS. In summary, we demonstrate that the high membrane perforation activity of the epidemic strain induces a high level of NLRP3 inflammasome activation, which is essential for the development of the cytokine storm and multi-organ dysfunction in STSLS and suggests NLRP3 inflammasome as an attractive target for the treatment of STSLS.

The prokaryotic Argonaute proteins enhance homology sequence-directed recombination in bacteria.

Argonaute proteins are present and conserved in all domains of life. Recently characterized prokaryotic Argonaute proteins (pAgos) participates in host defense by DNA interference. Here, we report that the Natronobacterium gregoryi Argonaute (NgAgo) enhances gene insertions or deletions in Pasteurella multocida and Escherichia coli at efficiencies of 80-100%. Additionally, the effects are in a homologous arms-dependent but guide DNA- and potential enzyme activity-independent manner. Interestingly, such effects were also observed in other pAgos fragments including Thermus thermophilus Argonaute (TtAgo), Aquifex aeolicus Argonaute (AaAgo) and Pyrococcus furiosus Argonaute (PfAgo). The underlying mechanism of the NgAgo system is a positive selection process mainly through its PIWI-like domain interacting with recombinase A (recA) to enhance recA-mediated DNA strand exchange. Our study reveals a novel system for enhancing homologous sequence-guided gene editing in bacteria.

Characteristics and clinical significance of myocardial injury in patients with severe coronavirus disease 2019.

AIMS: To investigate the characteristics and clinical significance of myocardial injury in patients with severe coronavirus disease 2019 (COVID-19). METHODS AND RESULTS: We enrolled 671 eligible hospitalized patients with severe COVID-19 from 1 January to 23 February 2020, with a median age of 63 years. Clinical, laboratory, and treatment data were collected and compared between patients who died and survivors. Risk factors of death and myocardial injury were analysed using multivariable regression models. A total of 62 patients (9.2%) died, who more often had myocardial injury (75.8% vs. 9.7%; P < 0.001) than survivors. The area under the receiver operating characteristic curve of initial cardiac troponin I (cTnI) for predicting in-hospital mortality was 0.92 [95% confidence interval (CI), 0.87-0.96; sensitivity, 0.86; specificity, 0.86; P < 0.001]. The single cut-off point and high level of cTnI predicted risk of in-hospital death, hazard ratio (HR) was 4.56 (95% CI, 1.28-16.28; P = 0.019) and 1.25 (95% CI, 1.07-1.46; P = 0.004), respectively. In multivariable logistic regression, senior age, comorbidities (e.g. hypertension, coronary heart disease, chronic renal failure, and chronic obstructive pulmonary disease), and high level of C-reactive protein were predictors of myocardial injury. CONCLUSION: The risk of in-hospital death among patients with severe COVID-19 can be predicted by markers of myocardial injury, and was significantly associated with senior age, inflammatory response, and cardiovascular comorbidities.

Hyperglycemia repression of miR-24 coordinately upregulates endothelial cell expression and secretion of von Willebrand factor.

An elevated level of von Willebrand factor (VWF) in diabetic patients is associated with increased risk of thrombotic cardiovascular events. The underlying mechanism of how VWF expression is upregulated in diabetes mellitus is poorly understood. We now report that hyperglycemia-induced repression of microRNA-24 (miR-24) increases VWF expression and secretion in diabetes mellitus. In diabetic patients and diabetic mouse models (streptozotocin/high-fat diet-induced and db/db mice), miR-24 is reduced in both tissues and plasma. Knockdown of miR-24 in mice leads to increased VWF mRNA and protein levels and enhanced platelet tethering (spontaneous thrombosis). miR-24 tightly controls VWF levels through pleiotropic effects, including direct binding to the 3' untranslated region of VWF and targeting FURIN and the histamine H1 receptor, known regulators of VWF processing and secretion in endothelial cells. We present a novel mechanism for miR-24 downregulation through hyperglycemia-induced activation of aldose reductase, reactive oxygen species, and c-Myc. These findings support a critical role for hyperglycemic repression of miR-24 in VWF-induced pathology. miR-24 represents a novel therapeutic target to prevent adverse thrombotic events in patients with diabetes mellitus.

Regulation of VWF expression, and secretion in health and disease.

PURPOSE OF REVIEW: Von Willebrand factor (VWF) is a large multidomain, multimeric glycoprotein that plays an essential role in regulating the balance between blood clotting and bleeding. Aberrant VWF regulation can lead to a spectrum of diseases extending from bleeding disorders [Von Willebrand disease (VWD)] to aberrant thrombotic thrombocytopenic purpura (TTP). Understanding the biology of VWF expression and secretion is essential for developing novel targeted therapies for VWF-related hemostasis disorders. RECENT FINDINGS: A number of recent elegant in-vitro and in-vivo studies will be highlighted, including the discovery of intronic splicing in the VWF gene, microRNA-regulated VWF gene expression, and syntaxin binding protein and autophagy mediated VWF secretion. Compared with the already established critical role of VWF in VWD and TTP pathophysiology, additional clinical studies have clarified and reinforced the association of elevated plasma levels of VWF with an increased risk of stroke, myocardial infarction, venous thrombosis, and diabetic thrombotic complications. Moreover, experimental mouse models of ischemic stroke and myocardial infarction have further supported VWF as a potential therapeutic target. SUMMARY: VWF biosynthesis, maturation, and secretion is a complex process, which mandates tight regulation. Significant progress has been made in our understandings of VWF expression and secretion and its association with thrombotic diseases, contributing to the development of novel targeting VWF drugs for prevention and treatment of deficient and enhanced hemostasis.

Mechanism of von Willebrand factor scissile bond cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13).

The platelet-tethering function of von Willebrand factor (VWF) is proteolytically regulated by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which cleaves the Tyr1605-Met1606 (P1-P1') bond in the VWF A2 domain. To date, most of the functional interactions between ADAMTS13 and VWF that have been characterized involve VWF residues that are C terminal to the scissile bond. We now demonstrate that the substrate P3 position in VWF, Leu1603, is a critical determinant of VWF proteolysis. When VWF Leu1603 was substituted with Ser, Ala, Asn, or Lys in a short VWF substrate, VWF115, proteolysis was either greatly reduced or ablated (up to 400-fold reduction in k(cat)/K(m)). As Leu1603 must interact with residues proximate to the Zn(2+) ion coordinated in the active center of ADAMTS13, we sought the corresponding S3 interacting residues. Substitution of 10 candidate residues in the metalloprotease domain of ADAMTS13 identified two spatially separated clusters centered on Leu198 or Val195 (acting with Leu232 and Leu274, or with Leu151, respectively), as possible subsites interacting with VWF. These experimental findings using the short VWF115 substrate were replicated using full-length VWF. It is hypothesized that VWF Leu1603 interacts with ADAMTS13 Leu198/Leu232/Leu274 and that Val195/Leu151 may form part of a S1 subsite. The recognition of VWF Leu1603 by ADAMTS13, in conjunction with previously reported remote exosites C terminal of the cleavage site, suggests a mechanism whereby the VWF P1-P1' scissile bond is brought into position over the active site for cleavage. Together with recently characterized remote exosite interactions, these findings provide a general framework for understanding the ADAMTS family substrate interactions.

Brazilin-Ce nanoparticles attenuate inflammation by de/anti-phosphorylation of IKKß.

Inflammation is associated with a series of diseases like cancer, cardiovascular disease and infection, and phosphorylation/dephosphorylation modification of proteins are important in inflammation regulation. Here we designed and synthesized a novel Brazilin-Ce nanoparticle (BX-Ce NPs) using Brazilin, which has been used for anti-inflammation in cardiovascular diseases but with narrow therapeutic window, and Cerium (IV), a lanthanide which has the general activity in catalyzing the hydrolysis of phosphoester bonds, to conferring de/anti-phosphorylation of IKKß. We found that BX-Ce NPs specifically bound to Asn225 and Lys428 of IKKß and inhibited its phosphorylation at Ser181, contributing to appreciably anti-inflammatory effect in cellulo (IC50 = 2.5 µM). In vivo mouse models of myocardial infarction and sepsis also showed that the BX-Ce NPs significantly ameliorated myocardial injury and improved survival in mice with experimental sepsis through downregulating phosphorylation of IKKß. These findings provided insights for developing metal nanoparticles for guided ion interfere therapy, particularly synergistically target de/anti-phosphorylation as promising therapeutic agents for inflammation and related diseases.

Unraveling the scissile bond: how ADAMTS13 recognizes and cleaves von Willebrand factor.

von Willebrand factor (VWF) is a large adhesive glycoprotein with established functions in hemostasis. It serves as a carrier for factor VIII and acts as a vascular damage sensor by attracting platelets to sites of vessel injury. VWF size is important for this latter function, with larger multimers being more hemostatically active. Functional imbalance in multimer size can variously cause microvascular thrombosis or bleeding. The regulation of VWF multimeric size and platelet-tethering function is carried out by ADAMTS13, a plasma metalloprotease that is constitutively active. Unusually, protease activity of ADAMTS13 is controlled not by natural inhibitors but by conformational changes in its substrate, which are induced when VWF is subject to elevated rheologic shear forces. This transforms VWF from a globular to an elongated protein. This conformational transformation unfolds the VWF A2 domain and reveals cryptic exosites as well as the scissile bond. To enable VWF proteolysis, ADAMTS13 makes multiple interactions that bring the protease to the substrate and position it to engage with the cleavage site as this becomes exposed by shear. This article reviews recent literature on the interaction between these 2 multidomain proteins and provides a summary model to explain proteolytic regulation of VWF by ADAMTS13.

Molecular insight into arrhythmogenic cardiomyopathy caused by DSG2 mutations.

Mutant desmoglein 2 (DSG2) is the second most common pathogenic gene in arrhythmogenic cardiomyopathy (ACM), accounting for approximately 10% of ACM cases. In addition to common clinical and pathological features, ACM caused by mutant DSG2 has specific characteristics, manifesting as left ventricle involvement and a high risk of heart failure. Pathological studies have shown extensive cardiomyocyte necrosis, infiltration of immune cells, and fibrofatty replacement in both ventricles, as well as abnormal desmosome structures in the hearts of humans and mice with mutant DSG2-related ACM. Although desmosome dysfunction is a common pathway in the pathogenesis of mutant DSG2-related ACM, the mechanisms underlying this dysfunction vary among mutations. Desmosome dysfunction induces cardiomyocyte injury, plakoglobin dislocation, and gap junction dysfunction, all of which contribute to the initiation and progression of ACM. Additionally, dysregulated inflammation, overactivation of transforming growth factor-beta-1 signaling and endoplasmic reticulum stress, and cardiac metabolic dysfunction contribute to the pathogenesis of ACM caused by mutant DSG2. These features demonstrate that patients with mutant DSG2-related ACM should be managed individually and precisely based on the genotype and phenotype. Further studies are needed to investigate the underlying mechanisms and to identify novel therapies to reverse or attenuate the progression of ACM caused by mutant DSG2.

Quantification of Cardiomyocyte Contraction In Vitro and Drug Screening by MyocytoBeats.

Cardiomyocyte contractility is the crucial feature of heart function. Quantifying cardiomyocyte contraction in vitro is essential for disease phenotype characterization, mechanism illumination, and drug screening. Although many experimental methods have been employed to determine contraction dynamics in vitro, a time-saving and easy-to-use software is still needed to be developed. We presented a reliable tool, named MyocytoBeats, to measure cardiomyocyte contraction by processing recorded videos. Analysis results by MyocytoBeats of various experimental models have shown a significant linear relationship with another validated software. We also performed pharmacology screen in the platform, and astragaloside IV was identified to stabilize the frequency and amplitude of cardiomyocyte in the arrhythmia model. MyocytoBeats is a high-performance tool for generating cardiomyocyte contraction data of vitro study and shows a great potential in cardiac pharmacology study.

A transient wave of Bhlhe41+ resident macrophages enables remodeling of the developing infarcted myocardium.

The immune system plays a critical role during myocardial injury, contributing to repair and remodeling post myocardial infarction (MI). The myocardial infarct and border zone exhibit high heterogeneity, in turn leading to reconstructing macrophage subsets and specific functions. Here we use a combination of single-cell RNA sequencing, spatial transcriptomes, and reporter mice to characterize temporal-spatial dynamics of cardiac macrophage subtype in response to MI. We identify that transient appearance of monocyte-derived Bhlhe41+ Mφs in the "developing" infarct zone peaked at day 7, while other monocyte-derived macrophages are identified in "old" infarct zone. Functional characterization by co-culture of Bhlhe41+ Mφs with cardiomyocytes and fibroblasts or depletion of Bhlhe41+ Mφs unveils a crucial contribution of Bhlhe41+ Mφs in suppression of myofibroblast activation. This work highlights the importance of Bhlhe41+ Mφ phenotype and plasticity in preventing excessive fibrosis and limiting the expansion of developing infarct area.

AP-1 activation mediates post-natal cardiomyocyte maturation.

AIMS: Post-natal maturation of mammalian cardiomyocytes proceeds rapidly after birth, with most of the myocytes exiting cell cycle, becoming binucleated, and adopting oxidative phosphorylation as the primary metabolic route. The triggers and transcriptional programmes regulating cardiomyocyte maturation have not been fully understood yet. We performed single-cell RNA-Seq in post-natal rat hearts in order to identify the important factors for this process. METHODS AND RESULTS: Single-cell RNA-Seq profiling was performed of post-natal Day 1 and Day 7 rat hearts, and we found that members of the activating protein 1 (AP-1) transcription factors showed a transient up-regulation in the maturing cardiomyocytes, suggesting their functional involvement in the process. Activating members of the AP-1 family by palmitate or adrenergic stimulation inhibited cardiomyocyte cytokinesis and promoted cardiomyocyte maturation. In contrast, knocking down AP-1 members Atf3 and Jun promoted cardiomyocyte cytokinesis, reduced polyploidy, and inhibited maturation. Mechanistically, RNA-Seq results and rescue experiments indicated that AP-1 members activate the expression of fatty acid metabolic genes to promote cardiomyocyte maturation. Finally, intraperitoneal injection of AP-1 inhibitor T-5224 in neonatal mice inhibits cardiomyocyte maturation in vivo. CONCLUSION: Our results are the first evidence implicating AP-1 transcription factors in post-natal cardiomyocyte maturation both in vitro and in vivo, which expand our understanding of the molecular mechanism of cardiomyocyte maturation, and may lead to novel therapies to treat congenital heart diseases.

Large animal models of cardiac ischemia-reperfusion injury: Where are we now?

Large animal models of cardiac ischemia-reperfusion are critical for evaluation of the efficacy of cardioprotective interventions prior to clinical translation. Nonetheless, current cardioprotective strategies/interventions formulated in preclinical cardiovascular research are often limited to small animal models, which are not transferable or reproducible in large animal models due to different factors such as: (i) complex and varied features of human ischemic cardiac disease (ICD), which are challenging to mimic in animal models, (ii) significant differences in surgical techniques applied, and (iii) differences in cardiovascular anatomy and physiology between small versus large animals. This article highlights the advantages and disadvantages of different large animal models of preclinical cardiac ischemic reperfusion injury (IRI), as well as the different methods used to induce and assess IRI, and the obstacles faced in using large animals for translational research in the settings of cardiac IR.

Anti-inflammatory effect of Danhong injection through inhibition of GSDMD-mediated pyroptosis.

BACKGROUND: Pyroptosis is an inflammatory form of cell death that has been implicated in various infectious and non-infectious diseases. Gasdermin family proteins are the key executors of pyroptotic cell death, thus they are considered as novel therapeutic targets for inflammatory diseases. However, only limited gasdermin specific inhibitors have been identified to date. Traditional Chinese medicines have been applied in clinic for centuries and exhibit potential in anti-inflammation and anti-pyroptosis. We attempted to find candidate Chinese botanical drugs which specifically target gasdermin D (GSDMD) and inhibit pyroptosis. METHODS: In this study, we performed high-throughput screening using a botanical drug library to identify pyroptosis specific inhibitors. The assay was based on a cell pyroptosis model induced by lipopolysaccharides (LPS) and nigericin. Cell pyroptosis levels were then evaluated by cell cytotoxicity assay, propidium iodide (PI) staining and immunoblotting. We then overexpressed GSDMD-N in cell lines to investigate the direct inhibitory effect of the drug to GSDMD-N oligomerization. Mass spectrometry studies were applied to identify the active components of the botanical drug. Finally, a mouse model of sepsis and a mouse model of diabetic myocardial infarction were constructed to verify the protective effect of the drug in disease models of inflammation. RESULTS: High-throughput screening identified Danhong injection (DHI) as a pyroptosis inhibitor. DHI remarkably inhibited pyroptotic cell death in a murine macrophage cell line and bone marrow-derived macrophages. Molecular assays demonstrated the direct blockade of GSDMD-N oligomerization and pore formation by DHI. Mass spectrometry studies identified the major active components of DHI, and further activity assays revealed salvianolic acid E (SAE) as the most potent molecule among these components, and SAE has a strong binding affinity to mouse GSDMD Cys192. We further demonstrated the protective effects of DHI in mouse sepsis and mouse myocardial infarction with type 2 diabetes. CONCLUSION: These findings provide new insights for drug development from Chinese herbal medicine like DHI against diabetic myocardial injury and sepsis through blocking GSDMD-mediated macrophage pyroptosis.

Structural repurposing of SGLT2 inhibitor empagliflozin for strengthening anti-heart failure activity with lower glycosuria.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been reapproved for heart failure (HF) therapy in patients with and without diabetes. However, the initial glucose-lowering indication of SGLT2i has impeded their uses in cardiovascular clinical practice. A challenge of SGLT2i then becomes how to separate their anti-HF activity from glucose-lowering side-effect. To address this issue, we conducted structural repurposing of EMPA, a representative SGLT2 inhibitor, to strengthen anti-HF activity and reduce the SGLT2-inhibitory activity according to structural basis of inhibition of SGLT2. Compared to EMPA, the optimal derivative JX01, which was produced by methylation of C2-OH of the glucose ring, exhibited weaker SGLT2-inhibitory activity (IC50 > 100 nmol/L), and lower glycosuria and glucose-lowering side-effect, better NHE1-inhibitory activity and cardioprotective effect in HF mice. Furthermore, JX01 showed good safety profiles in respect of single-dose/repeat-dose toxicity and hERG activity, and good pharmacokinetic properties in both mouse and rat species. Collectively, the present study provided a paradigm of drug repurposing to discover novel anti-HF drugs, and indirectly demonstrated that SGLT2-independent molecular mechanisms play an important role in cardioprotective effects of SGLT2 inhibitors.