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PURPOSE: The goal of this research was to explore the role of miR-24-3p in heart failure (HF), with a focus on its impact on the specificity protein 1 (Sp1)/phosphoinositide 3-kinase (PI3K) pathway. METHODS: HF rat and HF cell models were established using doxorubicin(Dox). Cardiac function was assessed through echocardiography, while histological changes were observed via hematoxylin-eosin (HE) staining. To further investigate the underlying mechanisms, HF cell models were treated with either an Sp1 inhibitor or a PI3K inhibitor. Additionally, models with miR-24-3p overexpression or silencing were constructed. N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were determined by ELISA. Cell apoptosis was evaluated using TUNEL staining, and lactate dehydrogenase (LDH) levels were measured by colorimetry. Reactive oxygen species (ROS) production was analyzed using flow cytometry. Related gene and protein expressions were assessed via qRT-PCR and Western blotting. Finally, the relationship between miR-24-3p and Sp1 was confirmed through dual-luciferase assays. RESULTS: Dox treatment increased the left ventricular internal diameter (LVIDd) while decreasing ejection fraction (EF) and fractional shortening (FS), leading to disorganized cardiomyocyte arrangement, cellular edema, and necrosis in rats. In HF rats, NT-proBNP, Caspase-3, and miR-24-3p expression levels were elevated, whereas Sp1 and PI3K mRNA and protein expression levels were decreased. Similarly, Dox-induced damage in H9c2 cardiomyocytes resulted in increased NT-proBNP, apoptosis, Caspase-3, LDH, ROS, and miR-24-3p expression, along with decreased Sp1 and PI3K expression. Treatment with either Sp1 or PI3K inhibitors exacerbated the Dox-induced cardiomyocyte damage, further elevating NT-proBNP, apoptosis, Caspase-3, LDH, ROS, and miR-24-3p expression levels. Notably, Sp1 inhibition reduced PI3K expression, and PI3K inhibition, in turn, suppressed Sp1 expression. Overexpression of miR-24-3p worsened Dox-induced cardiomyocyte damage, characterized by increased NT-proBNP, apoptosis, Caspase-3, LDH, and ROS expression, alongside reduced Sp1 and PI3K expression. In contrast, silencing miR-24-3p mitigated these detrimental effects and increased Sp1 and PI3K expression. Dual-luciferase assays confirmed that miR-24-3p directly targets Sp1. CONCLUSION: Dox induces cardiomyocyte damage, impairs cardiac function, and promotes cardiomyocyte apoptosis and oxidative stress. Silencing miR-24-3p offers a protective effect by activating the Sp1/PI3K signaling pathway in heart failure.

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The E-twenty-six variant 1 (ETV1)-dependent transcriptome plays an important role in atrial electrical and structural remodelling and the occurrence of atrial fibrillation (AF), but the underlying mechanism of ETV1 in AF is unclear. In this study, cardiomyocyte-specific ETV1 knockout (ETV1f/fMyHCCre/+, ETV1-CKO) mice were constructed to observe the susceptibility to AF and the underlying mechanism in AF associated with ETV1-CKO mice. AF susceptibility was examined by intraesophageal burst pacing, induction of AF was increased obviously in ETV1-CKO mice than WT mice. Electrophysiology experiments indicated shortened APD50 and APD90, increased incidence of DADs, decreased density of ICa,L in ETV1-CKO mice. There was no difference in VINACT,1/2 and VACT,1/2, but a significantly longer duration of the recovery time after inactivation in the ETV1-CKO mice. The recording of intracellular Ca2+ showed that there was significantly increased in the frequency of calcium spark, Ca2+ transient amplitude, and proportion of SCaEs in ETV1-CKO mice. Reduction of Cav1.2 rather than NCX1 and SERCA2a, increase RyR2, p-RyR2 and CaMKII was reflected in ETV1-CKO group. This study demonstrates that the increase in calcium spark and SCaEs corresponding to Ca2+ transient amplitude may trigger DAD in membrane potential in ETV1-CKO mice, thereby increasing the risk of AF.

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OBJECTIVE: The spontaneous action potential of isolated sinoatrial node (SAN) cells is regulated by a coupled-clock system of two clocks: the calcium clock and membrane clock. However, it remains unclear whether calcium clock inhibitors have a direct effect on the membrane clock. The purpose of this study was to investigate the direct effect of cyclopiazonic acid (CPA), a selective calcium clock inhibitor, on the function of the membrane clock of SAN cells. METHODS: at SAN cells were isolated by trypsinization and identified based on morphology and electrophysiology. If and HCN currents were recorded via patch clamp technique. The expression of the HCN channel protein was determined by Western blotting analysis. RESULTS: The diastolic depolarization rate of spontaneous action potentials and the current densities of If were reduced by exposure to 10 µM CPA. The inhibitory effect of CPA was concentration-dependent with an IC50 value of 16.3 µM and a Hill coefficient of 0.98. The effect of CPA on If current was also time-dependent, and the If current amplitude was partially restored after washout. Furthermore, the steady-state activation curve of the If current was shifted to a negative potential, indicating that channel activation slowed down. Finally, the protein expression of HCN4 in HEK293 cells was markedly downregulated by CPA. CONCLUSIONS: These results indicate that the direct inhibition effect of CPA on the If current in SAN cells is both concentration- and time-dependent. The underlying mechanisms may involve slowing down steady-state activation and the downregulation of pacemaker channel protein expression.

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OBJECTIVE: This study aimed to investigate the effects of transforming growth factor ß1 (TGF ß1) and hepatocyte growth factor (HGF) on the expression of connective tissue growth factor (CTGF) in human atrial fibroblasts, and to explore the relationship of these factors in atrial fibrosis and atrial anatomical remodelling (AAR) of patients with atrial fibrillation (AF). METHODS: Fresh right auricular appendix tissue of 20 patients with rheumatic heart disease undergoing valve replacement surgery was collected during surgeries, 10 patients had sinus rhythm(SR), and 10 patients had chronic atrial fibrillation (CAF). Atrial fibroblasts were then cultured from the tissues with differential attachment technique and treated with either TGFß1 (10 ng/mL) or HGF (100 ng/mL). CTGF mRNA levels were measured by RT-PCR, and CTGF protein content was determined using immunofluorescence and Western blotting assays. RESULTS: CAF group had higher left atrial diameters (LADs) and higher CTGF mRNA expression in atrial fibroblasts compared with SR group. The CTGF protein content in CAF group was higher than that of SR group and positively correlated with LAD and AF duration. After CAF group was treated with TGFß1, CTGF mRNA and protein expression were significantly down-regulated, whereas when treated with HGF, expression was up-regulated compared with SR group. CONCLUSIONS: Increased CTGF expression was associated with enlarged LAD, atrial fibrosis and AAR in patients with AF. TGFß1 and HGF regulate CTGF expression in human atrial fibroblasts with up-regulation of mRNA and down-regulation of protein, therefore, either promote or inhibit atrial fibrosis, which could be related to the incidence and persistence of AF.

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Aim: We investigated the underlying mechanisms in atrial fibrillation (AF) associated with R33Q mutation and Ca2+-triggered activity. Methods and Results: We examined AF susceptibility with intraesophageal burst pacing in the sarcoplasmic reticulum (SR) Ca2+ leak model calsequestrin 2 R33Q (Casq2R33Q/R33Q) mice. Atrial trigger appeared in R33Q mice but not WT mice (17.24%, 5/29 vs. 0.00%, 0/32, P < 0.05). AF was induced by 25 Hz pacing in R33Q mice (48.27%, 14/29 vs. 6.25%, 2/32, P < 0.01). The mice were given 1.5 mg/kg isoproterenol (Iso), and the incidences of AF increased (65.51%, 19/29 vs. 9.21%, 3/32, P < 0.01). Electrophysiology experiments and the recording of intracellular Ca2+ indicated significant increases in the Ca2+ sparks (5.24 ± 0.75 100 µM-1.s-1 vs. 0.29 ± 0.04 100 µM-1.s-1, n = 20, P < 0.05), intracellular free Ca2+ (0.238 ± 0.009 µM vs. 0.172 ± 0.006 µM, n = 20, P < 0.05), Ca2+ wave (11.74% vs. 2.24%, n = 20, P < 0.05), transient inward current (ITi) (-0.56 ± 0.02 pA/pF vs. -0.42 ± 0.01 pA/pF, n = 10, P < 0.05), and oscillation in membrane potentials (10.71%, 3/28 vs. 4.16%, 1/24, P < 0.05) in the R33Q group, but there was no significant difference in the L-type calcium current. These effects were enhanced by Iso, and the inhibition of calmodulin-dependent protein kinase II (CaMKII) by 1 µM KN93 reversed the effects of Iso on Ca2+ sparks (5.01 ± 0.66 100 µm-1.s-1 vs. 11.33 ± 1.63 100 µm-1.s-1, P < 0.05), intracellular Ca2+ (0.245 ± 0.005 µM vs. 0.324 ± 0.008 µM, P < 0.05), Ca2+ wave (12.35% vs. 17.83%, P < 0.05), ITi (-0.61 ± 0.02 pA/pF vs. -0.78 ± 0.03 pA/pF, n = 10, P < 0.05), and oscillation in membrane potential (17.85% 5/28 vs. 32.17% 9/28, P < 0.05). The reduction of ryanodine receptor 2 (RyR2) stable subunits (Casq2, triadin, and junctin) rather than RYR2 and the increase in CaMKII, phosphor-CaMKII, phosphor-RyR2 (Ser 2814), SERCA, and NCX1.1 was reflected in the R33Q group. Conclusion: This study demonstrates that the increase in spontaneous calcium elevations corresponding to ITi that may trigger the oscillation in membrane potentials in the R33Q group, thereby increasing the risk of AF. The occurrence of spontaneous calcium elevations in R33Q atrial myocytes is due to the dysfunction of RyR2 stable subunits, CaMKII hyperactivity, and CaMKII-mediated RyR phosphorylation. An effective therapeutic strategy to intervene in Ca2+-induced AF associated with the R33Q mutation may be through CaMKII inhibition.

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Brugada syndrome (BrS), which causes arrhythmias that lead to sudden cardiac death, is linked to loss-of-function mutations that affect sodium channels. Here, we investigate the rescue effect of alpha-allocryptopine (All) from Chinese herbal medicine in a T353I mutation of SCN5A, which combines trafficking abnormalities with Brugada syndrome. SCN5A-T353I expressed in HEK293 cells showed a small peak current (I(peak)) of only 59.6% of WT and an observably sustained current (I(sus)). We found that All strongly enhanced the I(peak) of the T353I channel by enhancing the plasma membrane (PM) expression of Nav1.5 and rescued defective trafficking after co-incubation with HEK293 cells that carry mutation channel 24 h. It is also beneficial to increase the I(peak) of the T353I mutation by All by prolonging the closed-state inactivation (CSI) process and shortening the recovery from inactivation of the T353I mutation. Interestingly, the I(sus) of T353I was significantly inhibited by All, which reduces the occurrence of LQT syndrome 3 (LQT3). We provide evidence that All can rescue the trafficking deficiencies and restore the cellular electrophysiological characteristics of SCN5A-T353I. This feature of All may benefit patients with the BrS-associated Nav1.5 channel and might have other potential therapeutic effects.

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