The present study aimed to assess the protective effect of epigallocatechingallate (EGCG) against myocardial injury in a mouse model of heart failure and to determine the mechanism underlying regulation of the transforming growth factor‑β1/mothers against decapentaplegic homolog 3 (TGF‑β1/Smad3) signaling pathway. Mouse models of heart failure were established. Alterations in ejection fraction, left ventricular internal diastolic diameter (LVIDd) and left ventricular internal systolic diameter (LVIDs) were measured by echocardiography. Pathological alterations of myocardial tissue were determined by hematoxylin and eosin, and Masson staining. The levels of serum brain natriuretic peptide (BNP), N‑terminal‑proBNP, interleukin (IL)‑1β, IL‑6, tumor necrosis factor‑α, malondialdehyde, superoxide dismutase and glutathione peroxidase were detected with ELISA. Expression of collagen I, collagen III were detected by western blotting and reverse transcription quantitative polymerase chain reaction. Transforming growth factor‑β1 (TGF‑β1), Smad3, phosphorylated (p)‑Smad3, apoptosis regulator BAX (Bax), caspase‑3 and apoptosis regulator Bcl2 in mouse cardiac tissue were measured by western blotting. P‑smad3 and TGF‑β1 were measured by immunofluorescence staining. EGCG reversed the alterations in LVIDd and LVIDs induced by establishment of the model of heart failure, increased ejection fraction, inhibited myocardial fibrosis, attenuated the oxidative stress, inflammatory and cardiomyocyte apoptosis and lowered the expression levels of collagen I and collagen III. Following treatment with TGF‑β1 inhibitor, the protective effect of EGCG against heart failure was attenuated. The results of the present study demonstrated that EGCG can inhibit the progression and development of heart failure in mice through inhibition of myocardial fibrosis and reduction of ventricular collagen remodeling. This protective effect of EGCG is likely mediated through inhibition of TGF‑β1/smad3 signaling pathway.

Objectives. We aimed to observe the protective effect of κ opioid receptor (κ‐OR) agonist on myocardial injury in heart failure (HF) rats and its effect on Ca²⁺‐SERCA2a and to explore the regulatory mechanism with the Nrf2/HO‐1 signaling pathway. Methods. 50 Sprague‐Dawley rats were randomly divided into the following groups: the sham operation group (sham group), HF model group (HF group), HF+κ‐OR agonist U50488 group (HU group), HF+U50488H+novel calmodulin‐dependent protein kinase II (CaMKII) agonist (oleic acid) (HUO group), and HF+U50488H+Nrf2 inhibitor (HUM group). The HF rat’s model was established through surgical ligation of the left anterior descending coronary artery and the exhausting swimming exercise. After that, rat’s cardiac function was monitored by echocardiography. HE and MASSON staining was used to detect the myocardial injury, and TUNEL staining was used to detect the myocardial apoptosis. ELISA was performed to detect the biomarkers of oxidative stress. Moreover, the distribution of reactive oxygen species (ROS) and Nrf2 was detected under immunofluorescence. The expression of sarco/endoplasmic reticulum calcium (Ca²⁺) ATPase (SERCA) 2a, calmodulin, endoplasmic reticulum stress‐ (ERS‐) related proteins, and Nrf2/HO‐1 signaling pathway‐related proteins were detected by Western Blotting. Results. κ‐OR agonist U50488H can significantly enhance rat’s cardiac function, reduce the injury and apoptosis of myocardial cells, and alleviate endoplasmic reticulum stress injury in HF rats via upregulating the SERCA2a expression and inhibiting the Ca²⁺ influx. Furthermore, U50488H could also inhibit the phosphorylation of CaMKII and cAMP‐response element binding protein (CREB). Additionally, administration of CaMKII‐specific agonist could partially block the therapeutic effect of κ‐OR agonist on the myocardium of HF rats. Interestingly, the antagonist of Nrf2 could also significantly reverse the therapeutic effect of κ‐OR agonist. Therefore, these results suggested that the effect of U50488H on HF rats is dependent on regulating CaMKII phosphorylation and activating the Nrf2/HO‐1 pathway. Conclusion. κ‐OR agonists U50488H can improve ERS in cardiomyocytes and relieve myocardial injury in HF rats through activating the Nrf2/HO‐1 pathway and regulating Ca²⁺‐SERCA2a to inhibit Ca²⁺ influx.

Familial renal glucosuria (FRG) is characterized by persistent glucosuria despite normal serum glucose and the absence of overt tubular dysfunction. Variants in solute carrier family 5 (sodium–glucose cotransporter), member 2 (SLC5A2) have been reported in FRG patients. However, the functional and expression-related consequences of such variants have been scarcely investigated. In the current study, we studied five FRG families and identified six missense mutations, including four novel variants (c.1051T>C/.(C351R), c.1400T>C/p.(V467A), c.1420G>C/p.(A474P), c.1691G>A/p.(R564Q); RNA not analyzed) and two variants that had been previously reported (c.294C>A/p.(F98L), c.736C>T/p.(P246S); RNA not analyzed). The probands were either heterozygous or compound heterozygous for SLC5A2 variants and had glucosuria of 5.9%–19.6 g/day. Human 293 cells were transfected with plasmid constructs to study the expression and function of SLC5A2 variants in vitro. Western blotting revealed that the expression levels of SLC5A2–351R-GFP, SLC5A2–467A-GFP, SLC5A2–474P-GFP, and SLC5A2–564Q-GFP were significantly decreased compared with wild-type SLC5A2-GFP (37%–55%). Confocal microscopy revealed that three variants (c.1400T>C, c.1420G>C, c.1691G>A) resulted in a loss of the punctate membrane pattern typical of wild-type SLC5A2. All variants had a significantly lower transport capacity in than the wild-type control. The current study provides a starting point to further investigate the molecular mechanism of SLC5A2 in FRG families and provides functional clues for antidiabetes drugs.

Population aging has led to increased sick sinus syndrome (SSS) incidence; however, no effective and safe medical therapy has been reported thus far. Yixin‐Fumai granules (YXFMs), a Chinese medicine granule designed for bradyarrhythmia treatment, can effectively increase SSS patients’ heart rate. Senescence‐induced sinoatrial node (SAN) degeneration is an important part of SSS pathogenesis, and older people often show high levels of oxidative stress; reactive oxygen species (ROS) accumulation in the SAN causes abnormal SAN pacing or conduction functions. The current study observed the protective effects of YXFMs on senescent SAN and explored the relationship between the NRF‐2/HO‐1 pathway, SHOX2, and T‐type calcium channels. We selected naturally senescent C57BL/6 mice with bradycardia to simulate SSS; electrocardiography, Masson’s trichrome staining, and DHE staining were used to assess SAN function and tissue damage. Immunofluorescence staining and Western blotting were used to assay related proteins. In vitro, we treated human‐induced pluripotent stem cell‐derived atrial myocytes (hiPSC‐AMs) and mouse atrial myocyte‐derived cell line HL‐1 with D‐galactose to simulate senescent SAN‐pacemaker cells. CardioExcyte96 was used to evaluate the pulsatile function of the hiPSC‐AMs, and the mechanism was verified by DCFH‐DA, immunofluorescence staining, RT‐qPCR, and Western blotting. The results demonstrated that YXFMs effectively inhibited senescence‐induced SAN hypofunction, and this effect possibly originated from scavenging of ROS and promotion of NRF‐2, SHOX2, and T‐type calcium channel expression. In vitro experiment results indicated that ML385, si‐SHOX2, LDN193189, and Mibefradil reversed YXFMs’ effects. Moreover, we, for the first time, found that ROS accumulation may hinder SHOX2 expression; YXFMs can activate SHOX2 through the NRF‐2/HO‐1 pathway‐mediated ROS scavenging and then regulate CACNA1G through the SHOX2/BMP4/GATA4/NKX2‐5 axis, improve T‐type calcium channel function, and ameliorate the SAN dysfunction. Finally, through network pharmacology and molecular docking, we screened for the most stable YXFMs compound that docks to NRF‐2, laying the foundation for future studies.

With the population aging, age-related sinoatrial node dysfunction (SND) has been on the rise. Sinoatrial node (SAN) degeneration is an important factor for the age-related SND development. However, there is no suitable animal modeling method in this field. Here, we investigated whether D-galactose could induce SAN degeneration and explored the associated mechanism. In vivo, twelve C57BL/6 mice were divided into Control and D-galactose group to receive corresponding treatments. Senescence was confirmed by analyzing the hair and weight; cardiac function was evaluated through echocardiography, cerebral blood flux and serum-BNP; the SAN function was evaluated by electrocardiogram; fibrotic change was evaluated by Masson's trichrome staining and oxidative stress was assessed through DHE staining and serum indicators. Mechanism was verified through immunofluorescence-staining and Western blotting. In vitro, mouse-atrial-myocytes were treated with D-galactose, and edaravone was utilized as the ROS scavenger. Senescence, oxidative stress, proliferation ability and mechanism were verified through various methods, and intuitive evidence was obtained through electrophysiological assay. Finally, we concluded that D-galactose can be used to induce age-related SND, in which oxidative stress plays a key role, causing PITX2 ectopic expression and downregulates SHOX2 expression, then through the downstream GATA4/NKX2-5 axis, results in pacing-related ion channels dysfunction, and hence SND development.