Doxorubicin (DOX) as a chemotherapeutic agent can cause mitochondrial dysfunction and heart failure. COX5A has been described as an important regulator of mitochondrial energy metabolism. We investigate the roles of COX5A in DOX-induced cardiomyopathy and explore the underlying mechanisms. C57BL/6J mice and H9c2 cardiomyoblasts were treated with DOX, and the COX5A expression was assessed. An adeno-associated virus serum type 9 (AAV9) and lenti-virus system were used to upregulate COX5A expression. Echocardiographic parameters, morphological and histological analyses, transmission electron microscope and immunofluorescence assays were used to assess cardiac and mitochondrial function. In a human study, we found that cardiac COX5A expression was dramatically decreased in patients with end-stage dilated cardiomyopathy (DCM) compared to the control group. COX5A was significantly downregulated following DOX stimulation in the heart of mice and H9c2 cells. Reduced cardiac function, decreased myocardium glucose uptake, mitochondrial morphology disturbance, reduced activity of mitochondrial cytochrome c oxidase (COX) and lowered ATP content were detected after DOX stimulation in mice, which could be significantly improved by overexpression of COX5A. Overexpression of COX5A effectively protected against DOX-induced oxidative stress, mitochondrial dysfunction and cardiomyocyte apoptosis in vivo and in vitro. Mechanistically, the phosphorylation of Akt (Thr308) and Akt (Ser473) were also decreased following DOX treatment, which could be reserved by the upregulation of COX5A. Furthermore, PI3K inhibitors abrogated the protection effects of COX5A against DOX-induced cardiotoxicity in H9c2 cells. Thus, we identified that PI3K/Akt signaling was responsible for the COX5A-mediated protective role in DOX-induced cardiomyopathy. These results demonstrated the protective effect of COX5A in mitochondrial dysfunction, oxidative stress, and cardiomyocyte apoptosis, providing a potential therapeutic target in DOX-induced cardiomyopathy.

BACKGROUND: Ischemia-reperfusion injury compromises revascularization strategies for myocardial infarction and contributes to cardiac microvascular disorders. This study aimed to investigate the role of the sGC (soluble guanylate cyclase)–cGMP–PKG (protein kinase G) pathway in cardiac microvascular reperfusion injury with a focus on ferroptosis. METHODS: Key genes in the sGC-cGMP-PKG pathway were analyzed at different reperfusion times using bulk and single-cell mRNA sequencing. Endothelial cell (EC) specific conditional GUCY1A1 (guanylate cyclase soluble subunit alpha 1) knockout mice (GUCY1A1 ^flox/flox /-CreERT2) and adeno-associated virus transfer-induced EC-specific GUCY1A1-overexpressing mice were assessed for cardiac microvascular reperfusion injury. LDHA (lactate dehydrogenase A) and GPX4 (glutathione peroxidase 4) phosphorylation sites were identified by mass spectrometry and mutationally inactivated via the CRISPR–Cas9 (clustered regularly interspaced short palindromic repeats and their associated protein 9) system. Protein interactions and chaperone-mediated autophagy of GPX4 were detected using coimmunoprecipitation assays. RESULTS: GUCY1A1 was decreased in the EC group after cardiac ischemia-reperfusion injury. EC-specific knockout of GUCY1A1 further reduced microvascular perfusion, increased the no-reflow area, and enlarged the infarction area in the acute phase of ischemia-reperfusion injury, ultimately aggravating cardiac dysfunction and structural remodeling in the chronic phase. In contrast, GUCY1A1 overexpression or its activator, vericiguat, alleviated microvascular dysfunction via the suppression of endothelial ferroptosis; the effects were majorly dependent on PKG activity. Mechanistically, PKG phosphorylated LDHA at threonine 95 and activated the LDHA moonlighting kinase function to phosphorylate GPX4, resulting in reduced chaperone-mediated autophagy-dependent degradation of GPX4 and ferroptosis. In human ischemic cardiomyopathy, GUCY1A1 expression, LDHA phosphorylation at threonine 95, and GPX4 phosphorylation at serine 131 were negatively associated with lipid peroxidation and cardiac fibrosis, suggesting that this pathway was involved in the pathogenesis of cardiac ischemia-reperfusion injury. CONCLUSIONS: These findings indicate that the compromise of the sGC-cGMP-PKG pathway is associated with reduced phosphorylation of LDHA and GPX4, and that GUCY1A1 activation may be considered as a strategy to alleviate endothelial ferroptosis and cardiac microvascular reperfusion injury.

Background: The perturbation of fatty acid metabolism in heart failure (HF) has been a critical issue. It is unclear whether the amounts of circulating carnitines will benefit primary etiology diagnosis and prognostic prediction in HF. This study was designed to assess the diagnostic and prognostic values of serum carnitine profiles between ischemic and non-ischemic derived heart failure. Methods: HF patients (non-ischemic dilated cardiomyopathy: DCM-HF, n = 98; ischemic heart disease: IHD-HF, n = 63) and control individuals (n = 48) were enrolled consecutively. The serum carnitines were quantitatively measured using the UHPLC-MS/MS method. All patients underwent a median follow-up of 28.3 months. Multivariate Cox regression analysis was performed during the prognosis evaluation. Results: Amongst 25 carnitines measured, all of them were increased in HF patients, and 20 acylcarnitines were associated with HF diagnosis independently. Seven acylcarnitines were confirmed to increase the probability of DCM diagnosis independently. The addition of isobutyryl-L-carnitine and stearoyl-L-carnitine to conventional clinical factors significantly improved the area under the receiver operating characteristic curve (ROC) from 0.771 to 0.832 (p = 0.023) for DCM-HF diagnosis (calibration test for the composite model: Hosmer-Lemeshow χ² = 7.376, p = 0.497 > 0.05). Using a multivariate COX survival analysis adjusted with clinical factors simultaneously, oleoyl L-carnitine >300 nmol/L (HR = 2.364, 95% CI = 1.122–4.976, p = 0.024) and isovaleryl-L-carnitine 420 nmol/L, succinyl carnitine >60 nmol/L and isovaleryl-L-carnitine <100 nmol/L increased the risk of HF rehospitalization independently. Conclusions: Serum carnitines could not only serve as diagnostic and predictive biomarkers in HF but also benefit the identification of HF primary etiology and prognosis.

This study aimed to investigate the prognostic implications of increased post-procedural cardiac troponin levels in patients undergoing elective percutaneous coronary intervention (PCI) and to define the threshold of prognostically relevant periprocedural myocardial injury (PMI). A total of 3249 patients with normal baseline troponin levels referred for elective PCI were enrolled and followed up for a median period of 20 months. The primary endpoint was major adverse cardiovascular events (MACEs) comprising all-cause death, myocardial injury (MI), and ischaemic stroke. Post-PCI high-sensitivity cardiac troponin T (hs-cTnT) >99% upper reference limit (URL) occurred in 78.3% of the patients and did not increase the risk of MACEs [adjusted hazard ratio (adHR) 1.00, 95% confidence interval (CI) 0.58–1.74, P = 0.990], nor did ‘major PMI’, defined as post-PCI hs-cTnT >5× URL (adHR 1.30, 95% CI 0.76–2.23, P = 0.340). Post-PCI troponin >8× URL, with an incidence of 15.2%, started to show an association with a higher risk of MACEs (adHR 1.89, 95% CI 1.06–3.37, P = 0.032), mainly driven by myocardial infarction (adHR 2.38, 95% CI 1.05–5.38, P = 0.037) and ischaemic stroke (adHR 3.35, 95% CI 1.17–9.64, P = 0.025). In patients with normal baseline troponin values undergoing elective PCI, PMI defined as hs-cTnT >8× URL after PCI was more appropriate for identifying patients with an increased risk of MACEs, which may help guide clinical practice in this population. In multivariate regression model, high-sensitivity cardiac troponin T above 8 × URL was associated with increased risk of longterm adverse clinical outcomes.

Periprocedural myocardial injury (PMI) is a common complication of percutaneous coronary intervention (PCI) associated with poor prognosis. Inflammation has been demonstrated to exert a crucial role in PMI. However, how the inflammation is initiated or sustained in PMI remains elusive. RNA-seq in peripheral blood mononuclear cells (PBMCs) from 3 Non-PMI and 6 PMI patients was performed with subsequent bioinformatics analysis. RNA-seq results were verified in a patient cohort. We also established the coronary microembolization (CME) mice model to mimic PMI. The activity of caspase-1 in PBMCs was detected by flow cytometry. The levels of interleukin (IL)-1β, IL-18 and cardiac troponin in plasma were measured by enzyme-linked immunosorbent assay. We identified a total of 901 differentially expressed genes (DEGs) between Non-PMI and PMI patients. These DEGs participated in several inflammation-related processes. NOD-like receptor signaling pathway was significantly enriched in pathway analysis. All the key genes composed in the NLRP3 inflammasome, including NLRP3, PYCARD, CASP1 and IL1B, were upregulated in PMI patients. The activation of NLRP3 inflammasome was then verified by increased activity of caspase-1 in PBMCs, and elevated levels of IL-1β and IL-18 in plasma in PMI patients. Spearman analysis confirmed tight correlations between caspase-1 activity, IL-1β, IL-18 levels and troponin T level. In addition, caspase-1 activity, IL-1β and IL-18 levels were also enhanced in CME mice. We discovered that NLRP3 inflammasome was involved in PMI, thus providing evidence supporting the therapeutic value of NLRP3 inflammasome-targeted strategies in PMI.

Background. Periprocedural myocardial injury (PMI) is a most common complication of percutaneous coronary intervention (PCI). Microembolization and inflammation underlying PMI could lead to coronary microvascular dysfunction (CMD) and vice versa. Reactive hyperemia index (RHI) assessed by peripheral artery tonometry (PAT) has been considered as a noninvasive method to assess endothelial function and CMD, which could be useful to predict PMI. Methods. 268 patients suspected with stable coronary artery disease (CAD) and scheduled for elective coronary angiography were enrolled. RHI was measured by using the Endo-PAT2000™ device before angiography. The association among RHI, PMI, and cardiovascular events was further assessed. Results. In this cohort, 189 patients (70.5%) were diagnosed with CAD and 119 patients (44.4%) underwent drug-eluting stent (DES) implantation. Compared with patients without CAD, CAD patients had lower RHI (1.88 ± 0.55 vs. 2.02 ± 0.58, ). Patients with PMI had a lower RHI before angiography (1.75 ± 0.37 vs. 1.95 ± 0.50, ). Receiver operating characteristic curve analysis of RHI revealed an area under the curve (AUC) of 0.61, with a sensitivity of 62.7% and specificity of 50.0% to predict PMI. Moreover, we found that CAD patients with RHI ≤ 1.81 had a higher incidence of composite cardiac events after stenting (adjusted hazard ratio (HR) 3.31, 95% confidence interval (CI) 1.07–10.22, ). Conclusions. RHI assessment through PAT could be a promising method to predict PMI before the procedure. RHI is associated with increased risk of long-term adverse cardiac events after DES implantation.