BACKGROUND AND OBJECTIVES:MicroRNAs (miRNAs) are small, non-coding RNAs that control gene expression patterns by inducing the degradation of messenger RNAs (mRNAs). Furthermore, miRNAs are known to play an important role in the pathogenesis of atrial fibrillation (AF). AF is typically diagnosed using an electrocardiogram. However, this study investigated whether specific miRNAs could be involved in alleviating AF by regulating aquaporin 4 (AQP4). METHODS:HL-1 cells were transfected with either miRNA negative control (NC) or miR-140-5p, followed by incubation with or without tachypacing (TP) condition. We investigated the protein expression of calcium-handling and inflammation-related proteins in control, control + miR-NC, control + miR-140-5p, TP, and TP + miR-140-5p groups by Western blotting. Also, the relative mRNA expression of AQP4 was determined through real-time polymerase chain reaction. RESULTS:Compared to the control, miR-140-3p was increased in the TP-induced AF group. Additionally, AQP4 protein expression and mRNA level were increased in the AF group along with inflammation-related proteins toll-like receptor 4, nucleotide-binding domain-like receptor protein 3, ERK, AKT, and interleukin-1β. The increase in such proteins was mitigated through miR-140-5p treatment. In accordance with these results, calcium-handling protein markers CaMKII, phospholamban, and ryanodine receptor 2 gene were also increased in the AF group and alleviated with miR-140-5p treatment. CONCLUSIONS:miR-140-5p is engaged in suppressing the expression of AQP4 in TP-induced AF HL-1 cells. In doing so, miR-140-5p reduced the risk of AF by suppressing inflammation and phosphorylation of Ca-handling proteins.

Proton therapy is increasingly used to treat pediatric and adult brain tumors, but there is still uncertainty surrounding the biological effects of protons on the heart. Also, the molecular and functional responses to proton irradiation are still unknown. This study investigates the effect of protons on cardiac disease by comparing their effects on the hearts of rats exposed to hypergravity. A total of 20 Sprague Dawley rats were tested, including a group that was irradiated with 0.1 Gy of protons to the heart, a group exposed to hypergravity, a group exposed to both protons and hypergravity, and a control group. Changes in AQP4, calcium homeostasis, and fibrosis-related markers were investigated using Western blotting, immunohistochemistry, etc. The proton-irradiated group showed no changes compared to the control group. In rats exposed to hypergravity, the cardiac fibrosis markers TGF-ꞵ1, MMP9, and MMP2 were increased. On the other hand, the group exposed to hypergravity followed by proton irradiation tended to display a significant decrease in these markers. Along with reduced fibrosis-related markers, the consistent tendency was also confirmed in the cardiac calcium homeostasis-related proteins and AQP4 through Western blotting. In summary, our findings indicate that rats subjected to hypergravity experienced both cardiac hypertrophy and fibrosis, while proton therapy appeared to mitigate the effects of cardiac disease. These results suggest that proton therapy prevents heart disease triggered by hypergravity, providing insights for protecting astronauts’ cardiovascular health.

Background: The frequency and prevalence of atrial fibrillation (AF) and heart failure are expected to increase over time, which are expected to result in enormous medical expenses and socioeconomic losses. Previous studies have confirmed that SGLT2 inhibitor (SGLT2i) reduces cardiovascular mortality and hospitalization rates in heart failure patients. Also, follow-up studies have been reported that the use of SGLT2i reduces atrial fibrillation, and its administration is recommended in clinical practice guidelines. In spite of beneficial effects of SGLT2i on atrial fibrillation, the exact mechanism by which SGLT2i reduces atrial fibrillation has not been revealed. Methods: We performed experiments by creating an AF cell model using Ang II and an AF animal model using Acetylcholin-Calcium chloride. Fibrosis markers and calcium regulatory proteins were confirmed by Western blot, and the occurrence and burden of AF were evaluated by using a 1F catheter in animal models. Results: In our cell studies and animal experiments, the inhibitory effect of SGLT2i administartion on atrial fibrillation was confirmed. First, fibrosis markers tend to reduce in atrial fibrillation models treated with SGLT2i, compared to non-treated atrial fibrillation models. In addition, the phosphorylation of the calcium handling proteins, such as CaMKII, RyR2, and PLB, increased in the AF group, whereas the degree of phosphorylation in SGLT2i treated group decreased to the phosphorylation level in control group. Conclusion: In AF-induced cell and animal models, the effect of SGLT2i was shown to be highly effective against atrial fibrillation. In particular, the results indicate that administration of SGLT2i before inducing atrial fibrillation could bring out the preventive and therapeutic effects through amelioration of fibrosis and reduction of calcium handling proteins phosphorylation.

Intermittent hypoxia (IH) has been extensively studied in recent years, demonstrating adverse and beneficial effects on several physiological systems. However, the precise mechanism underlying its cardiac effects on the heart remains unclear. This study aims to explore the effect of treatment on atrial fibrillation under IH conditions, providing data that can potentially be used in the treatment of heart disease. An atrial fibrillation (AF) model was induced by injecting monocrotaline (MCT, 60 mg/kg) into rats. The study included 32 rats divided into four groups: Control, Control + IH, AF, and AF + IH. We evaluated molecular changes associated with AF using ELISA and Western blot and performed electrophysiological experiments to evaluate AF. Arrhythmia-related calcium and fibrosis markers were investigated. Phosphorylation levels of CaMKII, Phospholamban, and RyR2 all increased in the AF group but decreased in the IH-exposed group. Additionally, fibrosis marker expressions such as SMA, MMP2, MMP9, and TGF-β increased in the AF group but were significantly downregulated with IH treatment. Connexin 43 and AQP4 expression were restored in the IH-treated group. These findings suggest that IH may prevent AF by downregulating the expression of calcium-handling proteins and fibrosis-associated proteins in an AF-induced rat model.

One of the main challenges with implantable biomedical devices is the replacement surgeries of depleted batteries for elderly patients. This not only poses medical risks but also results in financial burdens. Consequently, there is a growing need for self-powered in vivo electronics that can convert the biomechanical movements of organs into usable electric energy. Notably, a previously reported in vivo flexible energy harvester generated relatively low current output from a living porcine heart, restricting its ability to operate self-powered electronic devices. In this study, a high current output implantable flexible energy harvester was demonstrated by adopting a highly-piezoelectric single crystal Pb(In1/2Nb1/2)O3-Pb(Mg1/3Nb2/3)O3-PbTiO3 (PIN-PMN-PT) film. The in vivo flexible energy harvester generated a short-circuit output current of 20 mu A (corresponding to a current density of 3.08 mu A/mm3) from the contraction and relaxation of a porcine heart. This recorded in vivo output current density was attributed to its outstanding piezoelectric charge coefficient of the single crystal PIN-PMNPT, optimized Ni stressor assisted exfoliation as well as the implementation of a metal-insulator-metal (MIM) structure. Moreover, the flexible energy harvester was also demonstrated as a self-powered cardiac sensor to monitor irregular heartbeats by observing heart rate variations due to drug administration in a porcine heart. Additionally, output performance evaluation was conducted with the chest closed to further assess the feasibility of practical in vivo harvesting. Finally, the single crystal harvester exhibited biocompatibility, showing no signs of cytotoxicity based on cell viability assessments and histological characterizations. These results underscore the potential of the flexible PIN-PMN-PT energy harvesting device as a sustainable power source for self-powered in vivo biomedical electronics.

MicroRNAs (miRNAs) are small non‑coding RNAs that control patterns of gene expression by inducing the degradation of mRNAs. In addition, miRNAs are known to serve an important role in the pathogenesis of atrial fibrillation (AF). In general, AF is diagnosed using electrocardiography. However, the present study investigated whether specific miRNAs derived from microarray analysis of human urine could regulate AF through the inhibition of calcium handling protein phosphorylation in an AF model. Microarray analysis of the transcriptome in the human urine of patients with paroxysmal supraventricular tachycardia and AF revealed that 7 differentially expressed miRNAs were significantly downregulated (miR‑3613, 6763, 423, 3162, 1180, 6511, 3197) in patients with AF. In addition, quantitative PCR results demonstrated that collagen I, collagen III, fibronectin and TGF‑β, which are fibrosis‑related genes, were upregulated in patients with AF. Furthermore, fibrosis‑related genes were upregulated in angiotensin II‑induced atrial myocytes, which demonstrated that these genes may be targets of miR‑423. In the AF cell model transfected with miR‑423, the expression of calcium handling proteins, including phosphorylated calmodulin‑dependent protein kinase II, was reduced. The transfection of miR‑423 attenuated damage to cardiac cells caused by calcium handling proteins. The findings highlight the importance of calcium handling protein phosphorylation changes in fibrosis‑induced AF and support miR‑423 detection in human urine as a potential novel approach of AF diagnosis.

BACKGROUND AND OBJECTIVES:Ambient particulate matter (PM) in real urban air pollution (RUA) is an environmental health risk factor associated with increased cardiac events. This study investigated the threshold level to induce arrhythmia, as well as arrhythmogenic mechanism of RUA that mainly consisted of PM <2.5 μm in aerodynamic diameter close to ultrafine particles. METHODS:RUA was artificially produced by a lately developed pyrolysis based RUA generator. C57BL/6 mice were divided into 4 groups: a control group (control, n=12) and three groups with exposure to RUA with the concentration of 200 μg/m³ (n=12), 400 μg/m³ (n=12), and 800 μg/m³ (n=12). Mice were exposed to RUA at each concentration for 8 hr/day and 5 day/week to mimic ordinary human activity during 3 weeks. RESULTS:The QRS and QTc intervals, as well as intracellular Ca2+ duration, apicobasal action potential duration (APD) gradient, fibrosis, and inflammation of left ventricle of mouse hearts were increased dose-dependently with the increase of RUA concentration, and significantly increased at RUA concentration of 400 μg/m³ compared to control (all p<0.001). In mice exposed to RUA concentration of 800 μg/m³, spontaneous ventricular arrhythmia was observed in 42%, with significant increase of inflammatory markers, phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII), and phospholamban (PLB) compared to control. CONCLUSIONS:RUA could induce electrophysiological changes such as APD and QT prolongation, fibrosis, and inflammation dose-dependently, with significant increase of ventricular arrhythmia at the concentration of 400 μg/m³. RUA concentration of 800 μg/m³ increased phosphorylation of CaMKII and PLB.

Exosomes serve important functions in cell-to-cell communication and biological functions by serving as a delivery cargo shuttle for various molecules. The application of an improved delivery method for microRNAs (miRNAs/miRs) may enhance their potential as a therapeutic tool in cardiac diseases. Thus, the present study investigated whether human peripheral blood-derived exosomes may be used as a delivery cargo system for miRNAs, and whether the delivery of miR-21 using a human peripheral blood derived-exosome may influence the degree of remodeling following myocardial infarction (MI). In H9C2 and HL-1 cells, miR-21 expression was successfully regulated by treatment with human peripheral blood derived-exosomes loaded with an miR-21 mimic or inhibitor compared with untreated cells. In addition, the mRNA and protein expression levels of SMAD family member 7 (Smad7), phosphatase and tensin homolog (PTEN) and matrix metalloproteinase 2 (MMP2), which are involved in cardiac fibrosis, were associated with the uptake of miR-21 mimic- or inhibitor-loaded exosomes. Similarly, the in vivo mRNA and protein expression of Smad7, PTEN and MMP2 were altered following treatment with miR-21 mimic- or inhibitor-loaded exosomes. Furthermore, miR-21 mimic-loaded exosomes enhanced fibrosis, whereas miR-21 inhibitor-loaded exosomes reduced fibrosis in a mouse MI model. These results suggested that miRNA-loaded human peripheral blood derived-exosomes may be used as a therapeutic tool for cardiac diseases.

Atrial fibrillation (AF), the most common type of cardiac arrhythmia, is thought to be regulated by changes in microRNA (miRNA) expression. However, the evidence for this is inconsistent. The high stability and expression of circulating exosomal miRNAs may allow their use as candidate biomarkers. For the discovery phase, exosomes were isolated from the serum of patients with supraventricular tachycardia (SVT) as the controls (n = 5) and with paroxysmal AF (n = 4) and persistent AF (n = 5) for microarray analysis of miRNAs. Forty-five miRNAs were expressed significantly higher (>1.5-fold) in patients with persistent AF, but not in patients with paroxysmal AF, relative to the levels in patients with SVT control. Notably, expression of 5 miRNAs (miRNA-103a, -107, -320d, -486, and let-7b) was elevated by more than 4.5-fold in patients with persistent AF. For the validation phase, miRNAs were analyzed using quantitative RT-PCR analysis in exosomes from the serum of patients with SVT control (n = 20) and patients with persistent AF (n = 40). These miRNAs and their target genes were involved in atrial function and structure, oxidative stress, and fibrosis pathways. These findings suggest that serum exosomal miRNAs might be used as novel biomarkers to reflect the progression of AF.Mun, D., Kim, H., Kang, J.-Y., Park, H., Park, H., Lee, S.-H., Yun, N., Joung, B. Expression of miRNAs in circulating exosomes derived from patients with persistent atrial fibrillation.

Exosomes serve important functions in cell‑to‑cell communication and biological functions by serving as a delivery cargo shuttle for various molecules. The application of an improved delivery method for microRNAs (miRNAs/miRs) may enhance their potential as a therapeutic tool in cardiac diseases. Thus, the present study investigated whether human peripheral blood‑derived exosomes may be used as a delivery cargo system for miRNAs, and whether the delivery of miR‑21 using a human peripheral blood derived‑exosome may influence the degree of remodeling following myocardial infarction (MI). In H9C2 and HL‑1 cells, miR‑21 expression was successfully regulated by treatment with human peripheral blood derived‑exosomes loaded with an miR‑21 mimic or inhibitor compared with untreated cells. In addition, the mRNA and protein expression levels of SMAD family member 7 (Smad7), phosphatase and tensin homolog (PTEN) and matrix metalloproteinase 2 (MMP2), which are involved in cardiac fibrosis, were associated with the uptake of miR‑21 mimic‑ or inhibitor‑loaded exosomes. Similarly, the in vivo mRNA and protein expression of Smad7, PTEN and MMP2 were altered following treatment with miR‑21 mimic‑ or inhibitor‑loaded exosomes. Furthermore, miR‑21 mimic‑loaded exosomes enhanced fibrosis, whereas miR‑21 inhibitor‑loaded exosomes reduced fibrosis in a mouse MI model. These results suggested that miRNA‑loaded human peripheral blood derived‑exosomes may be used as a therapeutic tool for cardiac diseases. [the original article was published in International Journal of Molecular Medicine 43: 2319‑2328, 2019; DOI:10.3892/ijmm.2019.4150].

Purpose: Bioactive molecules critical to intracellular signaling are contained in extracellular vesides (EVs) and have cardioprotective effects in ischemia/reperfusion (IR) injured hearts. This study investigated the mechanism of the cardioprotective effects of EVs derived from hypoxia-preconditioned human mesenchymal stem cells (MSCs). Materials and Methods: EV solutions (0.4 mu g/mu L) derived from normoxia-preconditioned MSCs (EVNM) and hypoxia-preconditioned MSCs (EVHM) were delivered in a rat IR injury model. Successful EV delivery was confirmed by the detection of PKH26 staining in hearts from EV-treated rats. Results: EVHM significantly reduced infarct size (24 +/- 2% vs. 8 +/- 1%, p<0.001), and diminished arrhythmias by recovering electrical conduction, I-Na current, and Cx43 expression. EVHM also reversed reductions in Wntl and beta-catenin levels and increases in GSK3 beta induced after IR injury. miRNA-26a was significantly increased in EVHM, compared with EVNM, in real-time PCR. Finally, in in vitro experiments, hypoxia-induced increases in GSK3 beta expression were significantly reduced by the overexpression of miRNA-26a. Conclusion: EVHM reduced IR injury by suppressing GSK3 beta expression via miRNA-26a and increased Cx43 expression. These findings suggest that the beneficial effect of EVHM related with Wnt signaling pathway.

Programmed cell death 5 (PDCD5) has been associated with human cancers as a regulator of cell death; however, the role of PDCD5 in the endothelium has not been revealed. Thus, we investigated whether PDCD5 regulates protein kinase B (PKB/AKT)-endothelial nitric oxide synthase (eNOS)-dependent signal transduction in the endothelium and affects atherosclerosis. Endothelial-specific PDCD5 knockout mice showed significantly reduced vascular remodeling compared with wild-type (WT) mice after partial carotid ligation. WT PDCD5 competitively inhibited interaction between histone deacetylase 3 (HDAC3) and AKT, but PDCD5(L6R), an HDAC3-binding-deficient mutant, did not. Knockdown of PDCD5 accelerated HDAC3-AKT interaction, AKT and eNOS phosphorylation, and nitric oxide (NO) production in human umbilical vein endothelial cells. Moreover, we found that serum PDCD5 levels reflect endothelial NO production and are correlated with diabetes mellitus, high-density lipoprotein cholesterol, and coronary calcium in human samples obtained from the cardiovascular high-risk cohort. Therefore, we conclude that PDCD5 is associated with endothelial dysfunction and may be a novel therapeutic target in atherosclerosis.

Purpose: The mechanisms underlying repolarization abnormalities during pregnancy are not fully understood. Although maternal serotonin (5-hydroxytryptamine, 5-HT) production is an important determinant for normal fetal development in mice, its role in mothers remains unclear. We evaluated the role of serotonin in ventricular repolarization in mice hearts via 5Htr3 receptor (Htr3a) and investigated the mechanism of QT-prolongation during pregnancy. Materials and Methods: We measured current amplitudes and the expression levels of voltage-gated K+ (Kv) channels in freshly-isolated left ventricular myocytes from wild-type non-pregnant (WT-NP), late-pregnant (WT-LP), and non-pregnant Htr3a homozygous knockout mice (Htr3a(-/-)-NP). Results: During pregnancy, serotonin and tryptophan hydroxylase 1, a rate-limiting enzyme for the synthesis of serotonin, were markedly increased in hearts and serum. Serotonin increased Kv current densities concomitant with the shortening of the QT interval in WT-NP mice, but not in WT-LP and Htr3a(-/-)-NP mice. Ondansetron, an Htr3 antagonist, decreased Kv currents in WT-LP mice, but not in WT-NP mice. Kv4.3 directly interacted with Htr3a, and this binding was facilitated by serotonin. Serotonin increased the trafficking of Kv4.3 channels to the cellular membrane in WT-NP. Conclusion: Serotonin increases repolarizing currents by augmenting Kv currents. Elevated serotonin levels during pregnancy counterbalance pregnancy-related QT prolongation by facilitating Htr3-mediated Kv currents.

Naturally occurring RNA carriers such as exosomes might be an untapped source of effective delivery vehicles. However, if exosomes are to be exploited for therapeutic applications, they must target specific tissues or cell types to avoid off-target effects. This study evaluated whether genetic modification of exosomes could enhance exosome delivery to heart cells and heart tissue without toxicity. Exosomes expressing cardiac-targeting peptide (CTP)-Lamp2b on the exosomal membrane (CTP-Exo) were generated by introducing vectors encoding CTP-Lamp2b into HEK 293 cells. The expression of CTP-Lamp2b peptide on exosomes was stabilized by attaching glycosylation sequences. Exosomes expressing only Lamp2b on exosomal membranes (CTL-Exo) were generated as a control. The in vitro and in vivo uptake of CTL-Exo and CTP-Exo was evaluated in cell lines and mice. Both exosomes were delivered to HEK 293 and H9C2 cells. The delivery of the exosome was not different between CTP-Exo and CTL-Exo in HEK 293 cells, whereas the delivery of CTP-Exo was 16% greater than that of CTL-Exo in H9C2 cells (P = 0.047). Cell viability was maintained at almost 100% with different dosages of both CTL-Exo and CTP-Exo. Moreover, compared with CTL-Exo, the in vivo delivery of exosomes to the hearts of mice was increased by 15% with CTP-Exo (P = 0.035). The delivery to livers and spleens was not different between the two exosomes. Genetic modification of exosomes by expressing CTP-Lamp2b on the exosomal membrane enhanced exosome delivery to heart cells and the heart tissue. These results suggested that CTP-Exo might be used as a therapeutic tool for heart disease.

Flexible piezoelectric energy harvesters have been regarded as an overarching candidate for achieving self-powered electronic systems for environmental sensors and biomedical devices using the self-sufficient electrical energy. In this research, we realize a flexible high-output and lead-free piezoelectric energy harvester by using the aerosol deposition method and the laser lift-off process. We also investigated the comprehensive biocompatibility of the lead-free piezoceramic device using ex-vivo ionic elusion and in vivo bioimplantation, as well as in vitro cell proliferation and histologic inspection. The fabricated LiNbO3-doped (K,Na)NbO3 (KNN) thin film-based flexible energy harvester exhibited an outstanding piezoresponse, and average output performance of an open-circuit voltage of ∼130 V and a short-circuit current of ∼1.3 μ A under normal bending and release deformation, which is the best record among previously reported flexible lead-free piezoelectric energy harvesters. Although both the KNN and Pb(Zr,Ti)O3 (PZT) devices showed short-term biocompatibility in cellular and histological studies, excessive Pb toxic ions were eluted from the PZT in human serum and tap water. Moreover, the KNN-based flexible energy harvester was implanted into a porcine chest and generated up to ∼5 V and 700 nA from the heartbeat motion, comparable to the output of previously reported lead-based flexible energy harvesters. This work can compellingly serve to advance the development of piezoelectric energy harvesting for actual and practical biocompatible self-powered biomedical applications beyond restrictions of lead-based materials in long-term physiological and clinical aspects.

Continuous monitoring of an arterial pulse using a pressure sensor attached on the epidermis is an important technology for detecting the early onset of cardiovascular disease and assessing personal health status. Conventional pulse sensors have the capability of detecting human biosignals, but have significant drawbacks of power consumption issues that limit sustainable operation of wearable medical devices. Here, a self-powered piezoelectric pulse sensor is demonstrated to enable in vivo measurement of radial/carotid pulse signals in near-surface arteries. The inorganic piezoelectric sensor on an ultrathin plastic achieves conformal contact with the complex texture of the rugged skin, which allows to respond to the tiny pulse changes arising on the surface of epidermis. Experimental studies provide characteristics of the sensor with a sensitivity (≈0.018 kPa-1 ), response time (≈60 ms), and good mechanical stability. Wireless transmission of detected arterial pressure signals to a smart phone demonstrates the possibility of self-powered and real-time pulse monitoring system.

Background The Htr3a antagonist, ondansetron, has been reported to prolong the QT interval and induce Torsades de pointes in the treatment of postoperative nausea and vomiting. To explore the mechanisms underlying these findings, we examined the effects of ondansetron on the mouse cardiac voltage-gated K+ (Kv) channel. Methods and Results Ondansetron increased QT intervals in late pregnant (LP) mice. We measured the Kv channels in freshly isolated left ventricular (LV) myocytes from non-pregnant (NP) and late pregnant (LP) mice, using patch-clamp electrophysiology. Ondansetron blocked Kv current at a dose of 50 μM, and reduced the amplitude of peak current densities in a dose-dependent manner (0, 1, 5, 50 μM), in LP but not in NP mice. In contrast, serotonin and the Htr3 agonist, m-CPBG, increased Kv current densities in NP, but not in LP mice. Interestingly, during pregnancy, serum serotonin levels were markedly increased, suggesting the saturation of the effect of serotonin. Immunostaning data showed that Kv4.3 protein and Htr3a co-localize at the membrane and t-tubule of cardiomyocytes. Moreover, Kv4.3 membrane trafficking was enhanced in response to Htr3a-mediated serotonin stimulation in NP, but not in LP mice. Membrane analysis showed that serotonin enhances Kv4.3 membrane trafficking in NP, but not LP mice. Conclusion Ondansetron reduced Kv current densities, and reduced the Kv4.3 membrane trafficking in LP mouse ventricular cardiomyocytes. This data suggests that QT prolongation by ondansetron is mediated by the reduction of Kv current densities and Kv4.3 membrane trafficking. Key words: Ondansetron; Serotonin Receptor Type 3; Pregnancy; Voltage-Gated K+ (Kv) Current; Membrane Trafficking

Background: Exosome contains and delivers bioactive molecults critical to intracellular signaling. However, the role and mechanism of exosome which regulates molecular interactions in atrial fibrillation (AF) was not revealed. This study evaluated whether exosome from AF patients can effect