The goal of the present study was a comprehensive investigation of LCC RFI in atrial myocytes. The study was performed on voltage‐clamped rabbit atrial myocytes using a double pulse protocol with variable diastolic intervals in cells held at physiological holding potentials, with intact intracellular Ca2+ release, and preserved Na+ current and Na+/Ca2+ exchanger (NCX) activity. We demonstrate that the kinetics of RFI of LCCs are co‐regulated by several factors including resting membrane potential, [Ca2+]i, Na+ influx, and activity of CaMKII. In addition, activation of CaMKII resulted in increased ICa amplitude at higher pacing rates. Pharmacological inhibition of NCX failed to have any significant effect on RFI, indicating that impaired removal of Ca2+ by NCX has little effect on LCC recovery. Finally, RFI of intracellular Ca2+ release was substantially slower than LCC RFI, suggesting that inactivation kinetics of LCC do not significantly contribute to the beat‐to‐beat refractoriness of SR Ca2+ release. The study demonstrates that CaMKII and intracellular Ca2+ dynamics play a central role in modulation of LCC activity in atrial myocytes during increased workloads that could have important consequences under pathological conditions such as atrial fibrillations, where Ca2+ cycling and CaMKII activity are altered.Adaptation of the myocardium to varying workloads critically depends on the recovery from inactivation (RFI) of L‐type Ca 2+ current that serves as the trigger for Ca2+ release and cardiac contraction. Unlike in ventricular cells, recovery from inactivation of L‐type Ca2+ channels in atrial myocytes has not been investigated in depth. We demonstrate how the kinetics of recovery from inactivation of L‐type Ca2+ channels in rabbit atrial myocytes are regulated by resting membrane potential, Ca2+/calmodulin‐dependent kinase II, intracellular Ca2+ concentration, Na+influx, and activity of Na+/Ca2+ exchanger.Adaptation of the heart to different workloads depends on the recovery from inactivation of the L‐type Ca L‐type Ca2+ current (ICa) not only contributes to the generation of the action potential (AP), but also participates in signaling pathways that modulate multiple cellular functions such as mitochondrial energy production, gene transcription, and cell death  by Ca2+‐ induced Ca2+ release (CICR) that leads to the activation of myofilaments and contraction of the heart. In cardiac myocytes Ca2+ homeostasis is tightly controlled by the regulation of Ca2+ influx via ICa and extrusion from the cell by Na+/Ca2+ exchanger (NCX), as well as Ca2+ release from and uptake into the SR. LCC activation by depolarization of membrane voltage (Vm) is followed by their inactivation that is both Vm and Ca2+ dependent. Upon ICa activation and subsequent SR Ca2+ release the local [Ca2+] in close vicinity of LCCs is elevated which leads to Ca2+ binding to calmodulin that is pre‐bound to the channel, resulting in Ca2+‐dependent inactivation (CDI) of LCCs   of ICa. RFI refers to the process that readies LCCs for the next heartbeat. Most previous studies have investigated RFI of LCCs in ventricular myocytes providing valuable insights into the mechanisms of RFI regulation. In atrial cells, however, LCC RFI remains poorly investigated. Although regulation of LCCs in atrial and ventricular myocytes share similarities, atrial and ventricular excitation‐contraction coupling (ECC) reveal substantial differences  plays an essential role in LCC CDF , 1000 I.U./l Heparin; pH 7.4 with NaOH), the heart was perfused with minimal essential medium Eagle (MEM) solution containing 20 µM Ca2+ and 22.5 µg/ml Liberase TH (Roche Diagnostic Corporation) for ~25 min at 37˚C. The left atrium was dissected from the heart and minced, filtered, and washed in MEM solution containing 50 µM Ca2+ and 10 mg/ml bovine serum albumin. Isolated cells were washed and kept in MEM solution with 50 µM Ca2+ at room temperature (20–24˚C) and were used within 1–8 h after isolation.Atrial myocytes were isolated from male New Zealand White rabbits  and Envigo (12 rabbits)). All procedures and protocols were approved by the Institutional Animal Care and Use Committee of Rush University and comply with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Rabbits were anesthetized with an intravenous injection of sodium pentobarbital (100 mg/kg) and heparin (1000 I.U./kg). Hearts were excised, mounted on a Langendorff apparatus and retrogradely perfused via the aorta. After an initial 5 min perfusion with oxygenated Ca2.2ICa was recorded from single atrial myocytes in the whole‐cell ruptured patch clamp configuration using Axopatch 200A and 200B patch clamp amplifiers, the Axon Digidata 1440A, and 1550B interfaces and pCLAMP 10.3 software (Molecular Devices). Current recordings were low‐pass filtered at 5 kHz and digitized at 10 kHz. For ICa measurements patch clamp electrodes were pulled from borosilicate glass capillaries (WPI) with a horizontal puller P‐97 (Sutter Instruments) and filled with internal solution containing (in mM): 130 Cs+ glutamate, 10 NaCl, 10 CsCl, 0.33 MgCl2, 4 MgATP, and 10 Hepes with pH adjusted to 7.2 with CsOH. Internal solutions were filtered through 0.22‐μm pore filters. Electrode resistance was 1.5–3 MΩ when filled with internal solution. In some experiments 5 mM EGTA or 10 mM BAPTA were added to the internal solution for increased Ca buffering. Nominally applied resting voltage and voltage steps were corrected for a junction potential error of −10 mV. Series resistance was compensated to 70%–80%. The external solution contained (in mM): 135 Na+ glutamate, 4 CsCl, 2 CaCl2, 1 MgCl2, 10 Hepes, 10 D‐glucose, pH 7.4 with NaOH. All experiments were performed at room temperature (20–24°C). For Na+‐free experiments all Na+ in the external solution was substituted by N‐methyl‐D‐glucamine and in the internal solution NaCl was replaced by CsCl.1–S2) protocol. Atrial cells were held at −90 mV resting potential and depolarized to 0 mV for 400 ms during S1 and S2 voltage steps. Both S1 and S2 steps were preceded by 50 ms prepulses to −60 mV to inactivate Na+ current. The time interval between S1 and S2 steps is termed here as diastolic interval (DI) and was varied from 50 to 1050 ms. Each S1–S2 pair was preceded by six depolarizing steps to 0 mV for 400 ms at 1 Hz to ensure consistent ICa recordings and SR Ca2+ loading. The ratio of ICa amplitudes observed during S2 to that evoked by S1 (S2/S1) was used as a measure of the recovery of the channels from inactivation. RFI was quantified by the time constant (τRFI) of S1/S2 dependence on DI. τRFI was calculated by fitting recovery curves to a single exponential association equation (GraphPad Prism 5): Y = Y0+ (Ymax − Y0)*(1 − exp(−(t − t0)/τRFI)) where, t0 was set to 50 ms, Ymax was set to ≤1. Y represents S2/S1,Y0 is the average S2/S1 value between t = 0 and t0. The goodness of the fit was assessed by the standard deviation of the residuals /(n‐k); where residual is the vertical distance of a measured data point from the fit line, n is the number of data points, k is the number of parameters fit by regression, n−k equals to the number of degrees of freedom of the regression). Only τRFI obtained from fits with SDR < 0.15 were included in the statistical analysis. In addition to τRFI we also provide time to 50% (t50) and 80% (t80) recovery of ICa as a quantitative measure of RFI.Recovery from inactivation was monitored using a two square voltage step  or Cal520 potassium salt  via the patch pipette. Fluo‐4 and Cal520 fluorescence were excited at 485 nm with a Xe arc lamp and [Ca2+]i‐dependent fluorescence signals were collected at 515 nm using a photomultiplier tube. Background‐subtracted fluorescence emission signals (F) were normalized to diastolic fluorescence (F0) recorded under steady‐state conditions at the beginning of an experiment, and changes of [Ca2+]i are presented as changes of F/F0. Data recording and digitization were achieved using the Axon Digidata 1440A interface and pCLAMP 10.3 software. Fluorescence signals were low‐pass filtered at 50 Hz.In a series of experiments i in atrial myocytes play an important role in LCC adaptation to changes in heart rate by permitting enhanced Ca2+ influx at faster heart rates through shortening of LCC refractoriness.Because of the CaA Figure . The slo3.3Vh   Figure  and EGTA3.4INa was not suppressed allowing physiological Na+ influx and normal activity of NCX. In order to compare our results with previous studies in which RFI of LCC was investigated under conditions of full or partial INa inhibition, we performed a series of experiments by blocking INa with lidocaine or TTX  or eliminating INa by Na+ substitution in external and internal solutions with N‐Methyl‐D‐glucamine and CsCl, respectively. Figure + from the internal and external solutions  resulted in impaired LCC RFI with significantly slower kinetics. Mean ICa density in Na‐free conditions was 4.86 ± 0.26 pA/pF (n = 14) and was essentially identical with ICa in control . Additionally, slower RFI kinetics were also observed in cells where INa was suppressed by lidocaine   and 191 ± 19 ms . Similarly, kinetics of RFI were also slowed by the application of 1 µM TTX. TTX at 1 µM is expected to block late INa and to have just a partial inhibitory effect on peak INa (30%–50%)  compared with effects of Na+‐free conditions or lidocaine application. The mean ICa densities in control and in presence of TTX (paired measurements) were 6.56 ± 0.66 and 5.76 ± 0.74 pA/pF, respectively. Lidocaine at concentrations used here could potentially have a minor blocking effect on LCCs . Also, the effects of lidocaine were in‐line with observations in Na‐free conditions and TTX results, thus a potential lidocaine effect on LCCs is unlikely to affect our conclusions.Under our control conditions ) Figure . Mean τRINa inhibition with TTX. In cardiomyocytes dialyzed with the CaMKII blocker AIP, 1 µM TTX failed to further slow RFI . Subsequent exposure to TTX  resulted in τRFI of 210 ± 26 ms which was not significantly different from the effect of AIP alone (p = 0.1176). In summary, our results show that both pharmacological blocker of Na+ channels and substitution of Na+ resulted in slower LCC recovery.We also tested for a possible interplay between CaMKII and I Figure . In this+ substitution can also affect NCX function. Therefore, in the next set of experiments we explored a potential contribution of NCX to RFI directly.Pharmacological Na channel inhibition and Na3.5+ substantially slowed recovery of LCCs indirectly suggests the possibility of an involvement of NCX. Therefore, to further investigate the effect of NCX activity on RFI of LCCs we used pharmacological NCX blockers. Preincubation of atrial myocytes with the NCX blocker ORM‐10103  which is likely a consequence of increased [Ca2+]i and enhanced CDF of ICa  which preferentially blocks reverse mode of NCX  while the average τ of CaT recovery was 302 ± 45 ms (n = 12). Furthermore, at the level of individual cells only a weak correlation was found between the time constants of CaT and LCC RFI .In a subset of cells we simultaneously measured e Figure . RFI of e Figure  was quan1 Figure . Kinetice Figure . In thisI Figure . These r44.1Ca, especially at increased pacing frequencies. LCC RFI was extensively studied in ventricular myocytes, however, much less is known about the specific properties of LCC recovery in atrial cells. While ECC in atria and ventricular myocytes share similarities, there are also substantial differences. First, atrial and ventricular cells differ in t‐tubule network organization and density. T‐tubules, invaginations of plasma membrane that penetrate deep into ventricular cells, permit rapid AP propagation into the cell interior and assure cell wide synchronous Ca2+ release. In atria, however, the t‐tubule system is not well developed or even entirely lacking, and the location of LCCs is generally restricted to the cell periphery  Namiki et al.  in rabbit ventricular myocytes. In addition, consistent with our observations  use of depolarized holding potentials of −60 to −40 mV that completely inactivates INa, however RFI of LCCs is Vm dependent and recovery at depolarized Vh is considerably slower  use of pharmacological INa blockers. In this study we applied 50 ms prepulses to −60 mV during which INa is activated and subsequently Na+ channels remain refractory during the CaI recordings. This approach has some benefits as it allows to investigate ICa properties using physiological holding potentials, and with no pharmacological INa blocker present potential unwanted unspecific drug effects are precluded. [Ca2+] in the dyadic cleft is governed by several processes including Ca2+ influx through LCCs, CICR from SR, intracellular Ca2+ buffering, Ca2+ diffusion, Ca2+ reuptake into the SR, and Ca2+ removal by NCX  LCC RFI was slowed by intracellular Ca2+ buffering and depended on resting membrane potential; (3) suppression of INa by intra‐ and extracellular Na+ substitution or using INa blockers led to a substantial slowing of RFI of LCCs; (4) at variance with previous reports that removal of Ca2+ by NCX modulates LCC RFI, NCX blockers used in our study failed to have any significant effect on RFI, indicating that impaired removal of Ca2+ by NCX has little effect on LCC recovery. Finally, (5) recovery from inactivation of intracellular Ca2+ release was substantially slower than that of LCCs, suggesting that in atrial cells inactivation kinetics of LCC do not significantly contribute to the refractoriness of SR Ca2+ release.The goal of the present study was to characterize the mechanism of RFI of LCCs in atrial myocytes. The main novel findings are as follows: (1) with The authors declare that there is no conflict of interest.E.M‐H, L.A.B, and G.K contributed to conception and design of the experiments, analysis and interpretation of the results. E.M‐H and G.K collected data. E.M‐H, L.A.B, and G.K contributed to writing of the manuscript and approved the final version of the manuscript.


Our prior studies have characterized the participation of histone demethylase KDM3A in diabetic vascular remodeling, while its roles in myocardial ischemia/reperfusion (I/R) injury (MIRI) remain to be illustrated. Here we show that KDM3A was significantly downregulated in rat I/R and cellular hypoxia/reoxygenation (H/R) models. Subsequently, gain- and loss-of-function experiments were performed to investigate the effects of KDM3A in the settings of MIRI. KDM3A knockout exacerbated cardiac dysfunction and cardiomyocytes injury both in vivo and in vitro. The deteriorated mitochondrial apoptosis, reactive oxygen species, and inflammation were simultaneously observed. Conversely, KDM3A overexpression developed the ameliorated alternations in MIRI. Mechanistically, the MIRI-alleviating effects of KDM3A were associated with the enhancement of ETS1 expression. ChIP-PCR affirmed that KDM3A bound to the ETS1 promoter and removed dimethylation of histone H3 lysine 9 (H3K9me2), thus promoting ETS1 transcription. Our findings suggest that KDM3A is available for alleviating multi-etiologies of MIRI through the regulation of ETS1. Prevention of cardiac injury requires a deeper mechanistic understanding of ischemia/reperfusion (I/R) episodes. Here, the authors find that the epigenetic modifier KDM3A plays a crucial role in myocardial I/R injury through its activation of the gene ETS1 and suggest boosting KDM3A expression could be a potential treatment strategy. Timely revascularization, despite the benefits of limiting ischaemic injury and infarct expansion, governs the key endpoints that not only give rise to ischemia/reperfusion (I/R) episodes but also progress to chronic cardiac impairment6. Since achieving success in reperfusion to dampen ischemia, the focus of current experimental and clinical efforts has shifted towards the limitations of myocardial I/R injury (MIRI) to optimize the benefits of revascularization10. Although not completely clear, current investigations indicate that MIRI aetiologies consist of diverse cellular events with respect to apoptosis, reactive oxygen species (ROS), and inflammation13. Intriguingly, accumulating studies have illustrated that the dynamic regulation of epigenetic processes, such as histone methylation and acetylation, might be closely involved in these pathophysiologic progressions in response to MIRI14. Therefore, additional interference underlying epigenome-evoked multipathogenesis has the capacity to elicit promising targets and potential therapeutic avenues against MIRI.Epidemiological data from the World Health Organization demonstrate that approximately of 18.6 million patients who succumb to coronary vascular disease annually19. In contrast to histone methylation20, the modulation of histone demethylation with respect to MIRI occurrence is poorly investigated21. Our prior study confirmed that KDM3A-mediated H3K9me2 demethylation plays a crucial role in diabetic vascular remodeling19. The benefits derived from KDM3A inhibition on high insulin-induced vascular smooth cells malfunctions have also been shown, manifesting as a therapeutic approach due to pleiotropic potencies in the regulation of inflammation, apoptosis, and ROS16. However, little is known about whether and/or how KDM3A participates in cardiac damage, especially for MIRI. Recently, emerging studies have revealed that methylation is an important component of the epigenetic machinery in diabetic cardiomyopathy22, myocardial infarction23, and cardiac hypertrophy25. For instance, Mathias et al. suggested that KDM3A is significantly induced in failing myocardium, wherein its elevation potential but the inhibition negates the correlations with ANP and BNP transcription26. The potential mechanisms are ascribed at least in part to the constitutive alternation of histone methylation states on lysine residues (K), especially for the H3K9me2, involving multiple pathological events such as apoptosis and ROS generation23. As such the aberrant histone methylations implicated in KDM3A during MIRI have begun to gain attention based on the effects on H3K9me2. Given that H3K9me2 acts as a specific catalytic substrate of KDM3A, it is of interest to explore the underlying contributions of KDM3A in MIRI.Lysine specific demethylase 3 A  is a well-known epigenetic activator functioning in target gene transcription via removal of the suppressive histone dimethylation mark of histone H3 at lysine 9 (H3K9me2) and is one member of the Jumonji C (JmjC) domain-containing dioxygenases (JMJC demethylases)31. It has been reported that KDM3A-mediated transactivation of ETS1 occurs via a decrease in H3K9me2 is linked to cancer metastasis18. Specifically, Bian et al. demonstrated that mitochondrial apoptosis is markedly worsened in response to an absence of ETS1, increasing cardiac vulnerability to I/R injury31. However, the comprehensive roles of ETS1 in MIRI are far from clear, and more importantly, there is no evidence on whether KDM3A exerts cardioprotective effects against I/R in an ETS1-dependent manner. In the present study, by utilizing gain- and loss-of-functional approaches, we identified KDM3A as a novel protective regulator against MIRI both in vivo and in vitro. The mechanism underlay epigenetic modification of ETS1 via repression of histone methylation marker H3K9me2 on the promoter, thereby directly involving in the cell-fate outcome like survival, mitochondria-relevant apoptosis, ROS and inflammation. Thus, KDM3A emerges as a potent therapeutic candidate for the prevention of MIRI.Erythroblastosis virus E26 oncogene homologue 1 (ETS1) has been well-recognized as a multifaceted mediator of inflammation, apoptosis, and ROSh after reperfusion compared to the sham-operated group. Accordingly, double IF for KDM3A (green) and α-actinin (red) further revealed decreased expression and cytoplasmic/nuclear location of KDM3A in I/R hearts relative to sham hearts . These findings confirm that KDM3A overexpression ameliorates I/R-induced cardiac dysfunction, infarct size enlargement, and myocardial necrosis in vivo.To examine the potential functions of KDM3A in MIRI, intramyocardial infection with AdKDM3A or AdGFP was performed before establishing the MIRI model. The hallmarks of MIRI were assessed by functional and morphological alterations. As shown in Fig. c release, Bax/Bcl-2 ratio, and cleaved caspase-9/332. To determine the effect of KDM3A on mitochondria-associated apoptotic cascades, TUNEL staining for apoptotic cells in parallel with TEM detection and ATP content assay for mitochondrial structure and function were measured respectively after MIRI. Moreover, we assessed the protein expressions of cytoplasmic/mitochondrial cytochrome c, Bax, Bcl-2, cleaved caspase-9, and cleaved caspase-3. As shown in Fig. c, Bax, and cleaved caspase-9/3 and increased mitochondrial cytochrome c and Bcl-2, were substantially observed after AdKDM3A delivery in MIRI .Apoptosis originating from mitochondrial malfunction is widely regarded as a potent indicator of MIRI, accompanied by disruption of mitochondrial structure/function and increased levels of cytochrome 33. To further reveal KDM3A’s participation in modulating ROS formation, in situ DHE staining together with the activity of antioxidative SOD and the content of the oxidative marker MDA were determined essentially. As shown in Fig. 2- generation in AdKDM3A-overexpressing rats relative to the I/R controls 24 h after reperfusion. In parallel, limited ROS activity as manifested by decreased MDA and augmented SOD was also verified . Furthermore, concentrations of the well-known proinflammatory mediators (IL-6 and TNF-α) remained dramatically lower levels in the AdKDM3A + I/R group than in the I/R group . Notably, no significant differences were observed in apoptosis-/ROS-/inflammation-associated parameters between the I/R and the AdGFP +I/R groups (p > 0.05). Thus, the above gain-of-function evidence implies that KDM3A upregulation serves as a pivotal agent in suppressing apoptosis, ROS, and inflammation, in which it confers multiple protective effects against MIRI.ROS generation and inflammation also act as preferential agents in response to myocardial I/R insult despite apoptotic cascadesKDM3A−/− rat strain was generated using the well-identified CRISPR/Cas9 technology  were subjected to an I/R procedure or sham operation. Interestingly, KDM3A−/− hearts exhibited drastic increases in myocardial infarct size  whether KDM3A was involved in MIRI; if so, (2) how it impacts multiple pathogeneses of MIRI, yielding protective effects and (3) to uncover the underlying mechanism, particularly ETS1, anchoring the I/R-inhibitory roles of KDM3A.Apoptosis-, ROS- and inflammation-induced myocardial loss represents the most pivotal agents in MIRI36. Diverse pharmacological and surgical therapies could efficiently reverse ischemia, whereas ineluctable I/R damage paradoxically counteracts reperfusion advances and strikingly facilitates deleterious cardiac performance36. Although therapeutic methods for minimizing MIRI are currently finite, emerging evidence indicates that the vigorous stimulation of apoptotic cascades, secondary to incipient mitochondrial malfunction, induces critical mechanisms that aggravate myocardial loss in I/R settings35. Among these factors, the evoked Bax/Bcl-2 ratio cooperatively results in the formation of mitochondrial pores, parallel to the aggravated cytochrome c release and an irreversible apoptotic cascade35. Moreover, ROS accumulation originating from mitochondrial impairments can also subsequently amplifies cytotoxic outcomes10. Once oxidative stress disrupts the balance between antioxidant defence and ROS in favour of the latter, it constitutes a crucial aetiological component of MIRI10. Excessive ROS elicits an imbalanced redox status, particularly as reflected by the inactivation of the antioxidant enzyme SOD and increased MDA levels33. Furthermore, inflammation has been proposed to aggravate MIRI5. Consistently, our data revealed that I/R leads to disturbed mitochondrial structure and function in concert with elevated inflammation, ROS, and the mitochondria-associated apoptotic cascade. Interestingly, KDM3A overexpression ameliorated these abnormalities, while KDM3A knockout exacerbated these abnormalities both in vivo and in vitro. Therefore, we believe that KDM3A protects cardiomyocytes from I/R injury particularly by interfering with ROS, apoptosis, and inflammation. Interventions involved in multiple pathogenesis, more specifically for KDM3A, may glean unrecognized modalities and provide impetus in mitigating MIRI.AMI is one of the leading causes of morbidity and mortality worldwide20. Specific alternations of histones affect the chromatin architecture switch between opening and condensation, regulating target gene activation or repression beyond genomics25. Among these, histone H3 is one of the four categories of core histones. The activities of histone demethylases (HDMs), including LSD1 and the JMJC domain-containing proteins37,and histone methyltransferases (HMTs), including SUV39H1, G9a, and others38 remove or deposit methyl groups at lysine 9 of H3 (H3K9) from pathogenic gene promoters respectively, that are strictly relevant to facilitating or repressing target genes accordingly. In contrast to the well-identified contributions of HDMs and HMTs in diabetes and cardiac hypertrophy39, little is known regarding their properties in MIRI. However, the discovery of HMTs, i.e., SUV39H1, G9a, Smyd2, and others, has substantially shifted this paradigm. For instance, a promising observation based on the selective knockout of caveolin-1 was verified in a prior study regarding the capacity of worsened outcome after IPC during MIRI by stimulating G9a40. Erlinge et al. recently provided direct clues that IPC within MIRI confers epigenetic inhibition of Mtor and causal enhancement of autophagy through G9a-dependent H3K9me2 augmentation20. A similar finding underlying the ROS- and apoptosis-suppressed actions as a result of SUV39H1 silencing or inhibition is observed both in MIRI and AMI, and potential mechanisms include the SIRT1 trans-repression by erasing H3K9me3 from its promoter and the subsequent normalization of SIRT1 in cardiomyocytes23. In terms of the opposite biochemical action of HDMs relative to HMTs, it is tempting to speculate the possible roles of HDMs in response to MIRI, although they are not entirely clear. In the present study, we provided evidence for the first time that KDM3A, a member of the JMJC domain-containing proteins that belong to the HDMs family, was sufficient to recapitulate multiple protective potencies by removing the repressive H3K9me2 histone mark. This study suggests that histone demethylation seems to be another crucial pathogenetic mechanism irrespective of histone transmethylation during MIRI. Another critical goal of future research is to mechanistically identify direct or indirect sensors in response to KDM3A that regulate KDM3A to convey pleiotropic protection against MIRI.Epigenetic modulations that occur within MIRI principally involve DNA methylation, noncoding RNAs and posttranslational modifications, such as histone methylation28, renal injury in salt-sensitive hypertension41 and I/R disorder31. For instance, ETS1 stimulation is responsible for apoptotic limitation in renal I/R29. Diminished ROS generation and inflammation in Ang II-induced vascular remodeling were observed in ETS1-deficient mice30. On the other hand, miRNAs, which are epigenetic mediators, have been suggested to regulate ETS1 in various of ways. Gareri et al. demonstrated that miR-125a-5p modulates the phenotypic switch of vascular smooth cells by directly targeting ETS142, and other miRNAs, such as miR-221, appear to modify ox-LDL-induced endothelial apoptosis by affecting ETS143. Nevertheless, the involvement of ETS1 in cardiac I/R has received less attention. A recent report demonstrated that a lack of ETS1 provides the possibility of reducing mitochondrial apoptosis under MIRI, as significantly characterized by alterations in of Bax, Bcl-2, and caspase-331. Because of the compelling roles of ETS1 in MIRI and the potential of ETS1 to be a KDM3A-targeted gene, and the versatile activities of ETS1 in inflammation, ROS, and apoptosis, we assessed possible correlations between ETS1 and KDM3A in MIRI. As expected, our study found that KDM3A directly targeted ETS1 by binding to its promoter region, in concert with the abrasion of H3K9me2. More intriguingly, KDM3A overexpression resulted in higher ETS1 transcription in concomitant with reduced apoptosis, ROS, and inflammation, while KDM3A knockdown reversed these alterations. In addition, by applying ETS1-siRNA to the H/R-exposed cardiomyocytes, KDM3A overexpression blunted the protective outcomes and restored apoptosis, ROS, and inflammation. Therefore, our findings reinforce that the protective roles of KDM3A against MIRI are, at least partially, ascribed to the epigenetic transactivation of ETS1 and that the exact participation of ETS1 in MIRI is constitutively expanded, in addition to the known apoptosis-limited effect. More studies are still warranted to explore whether there are other direct targets of KDM3A irrespective of the ETS1 in MIRI.To clarify the mechanisms underpinning the I/R-reduced roles of KDM3A, ETS1 drew our great attention. ETS1 is a member of the ETS family featuring a large group of transcription factors. In a previous study, KDM3A- mediated transactivation of ETS1 was shown to occur via the H3K9me2 decrease in the genomic region flanking the promoter and was especially linked to cancer development. Notably, many studies to date have verified ETS1 as a multifunctional mediator implicated in vascular remodeling19. For instance, KDM3A inhibition is considered a promising avenue for the restoration of vascular function in diabetes-mediated metabolic memory. In this study, the Rho/ROCK2 and AngII/AGTR1/MAPKs  signaling pathways were identified as two of the KDM3A-mediated effectors, and AGTR1/ROCK2 acts as a KDM3A-targeted gene by controlling H3K9me2 in their proximal promoters. In addition, KDM3A acts as an upstream modulator of the MAKP/NF-κB pathways in high insulin-induced vascular smooth muscle cell malfunctions primarily by disrupting inflammation, apoptosis, and ROS. Therefore, this investigation elucidated KDM3A’s biological participation in vascular injury with the connections to canonical signaling pathways and the innovative targets. However, the exact impacts and potential mechanisms of KDM3A in cardiac insult, especially for MIRI are less exploited. Recently, Mathias and coworkers revealed that KDM3A is upregulated in maladaptive cardiac remodeling, and KDM3A correlates positively with increased ANP and BNP expression and inversely with H3K9me2 in the promoter region of ANP26. Connecting these data with current findings, such cardiac-influenced consequences further expand the biochemical actions of KDM3A across cardiovascular disease, albeit with divergent results between pathological cardiac hypertrophy and MIRI.The pleiotropic activities of KDM3A in the regulation of migration, proliferation, apoptosis, inflammation, and ROS have been elucidated in our previous studies44, it was of appealing interest to exploit their epigenetic crosstalk in regulating MIRI based on the existing I/R-regulatory roles of miRNA-22, which seems to permit unrecognized patterns in the prevention of I/R injury.In summary, our findings raise hope for prospective approaches against MIRI through KDM3A-dependent ETS1 enhancement by impacting multipathogenetic episodes. However, further explorations are still warranted to highlight worthwhile targets directly or indirectly regulated by KDM3A and other types of posttranslational modifications cooperatively. For instance, considering that KDM3A has been verified as a potential target of miRNA-2219. Small interfering RNA (siRNA) against ETS1 (siETS1) and negative control siRNA (siCtrl) were designed and synthesized by RiboBio . Two kinds of KDM3A primary antibodies were purchased from Abcam  (ab106456) for western blotting assay and Santa Cruz  (sc-376608X) for ChIP-PCR detection, respectively. Primary antibodies used in the experiments were purchased from the following suppliers: H3K9me2 (ab1220), Bax (ab32503), Bcl-2 (ab196495), cleaved caspase-9 (ab52298), cleaved caspase-3 (ab2302), cytoplasmic cytochrome c (ab13575), mitochondrial cytochrome c (ab13575), VDAC1 (ab15895) and GAPDH (ab37168) were purchased from Abcam; ETS1 was purchased from Proteintech Group, Inc (12118-1 AP). The secondary antibodies, either HRP-linked goat anti-mouse IgG or goat anti-rabbit IgG, were obtained from BIOSS . Foetal bovine serum (FBS) was purchased from Hyclone . Cell culture reagents and other chemicals were purchased from Sigma  unless otherwise specified. Enhanced chemiluminescence reagents and apoptosis detection kits were obtained from BD Biotechnology. DHE reagent was obtained from Beyotime Biotechnology. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc.Adenovirus encoding KDM3A-shRNA (AdshKDM3A) or KDM3A (AdKDM3A) was provided by Genechem . AdshRNA or AdGFP was used as a negative control. These viruses were used in our previous studies and were verified to be efficient for the induction of KDM3A knockdown or overexpression both in vivo and in vitroh light/dark cycle), and ad libitum access to food and water. For genetic deletion of KDM3A, CRISPR/Cas9 genome-editing technology45 was used to generate KDM3A-knockout (KDM3A-KO) (hereafter referred to as KDM3A−/−) rats (SD background). SD rats were obtained from the Vital River . One single guide RNA (sgRNA) flanked exon 5 of KDM3A gene in rat was designed and created. When purified Cas9 mRNA and sgRNA were mixed and microinjected into embryos, PCR products were TA cloned and sequenced to define the exacted indel mutations of generated founders. The primers were listed as follows: KDM3A F: 5’-TCCCTGGAGTTGCAGTAGTTT-3’; KDM3A R: 5’-AGGTCTATCACTGGCTAACTCA-3’. Out of the overall 29 embryos, 8 pups were generated in which three founders were measured to be multiple mutated allele  and identified as mosaics. Founder #70-13 was chosen as the breeding line and mated with the WT (KDM3A+/+) SD rat strain to generate F1 heterozygote (KDM3A+/−). The homozygous KDM3A−/− strain was obtained through sibling mating of heterozygous F1 offspring and was identified by sequencing chromatograms of PCR products. The western blotting assay was performed to validate KDM3A protein expression. To induce myocardial overexpression of KDM3A in vivo, male SD rats  were purchased from Vital River Laboratories . Cardiac-specific KDM3A overexpression in vivo was garnered by intramyocardial injection of AdKDM3A33. Male SD rats  were purchased from Vital River Laboratories . The intramyocardial injection of 100 ul solution of AdKDM3A (1 × 1010 pfu/ml) were performed at five separate sites as depicted in our previous description. Three days later, the MIRI model was operated as following delineations and as our prior experiments. The experiments and all animal care outlined in our study were performed in adherence with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and were approved by the Institutional Animal Care and Use Committee of Wuhan University.All Sprague-Dawley (SD) rats were bred in a standard environment with controlled temperature (20-25 °C), humidity (40–60%), light conditions  (Sigma). Adequate anaesthesia was confirmed by the loss of a toe pinch reflex. A left parasternal incision was performed after rats were anaesthetized. The left anterior descending artery was then ligated for 30 min, and 24 h reperfusion was subsequently performed. The chest cavity, muscle and skin were closed in layers. Myocardial tissues from the infarct area (IA) and area at risk (AAR) (5 mm around the IA) were collected to ensure detection after I/R procedures. Sham-operated rats underwent the same procedures without left anterior descending artery occlusion or reperfusion.The myocardial I/R model was generated32. In brief, paraffin-embedded sections were incubated in PBS containing 10% goat serum for 1 h and consecutively incubated with primary antibodies against KDM3A  and α-actinin . Next, the sections were incubated with appropriate secondary antibodies, including the FITC-conjugated goat anti-rabbit IgG  and Cy3-conjugated goat anti-mouse IgG . The 4’,6-Diamidino-2-phenylindole  was used to stain the cell nuclei. Images were acquired using a fluorescence microscope  in at least 5 random fields per sample. Staining per section was estimated in a blinded method (400× magnification). In addition, the same methods were performed in primary cardiomyocytes cultured on 6-well chamber slides as in previous demonstrations.Expression of KDM3A in rat hearts was observed by immunofluorescence46. LV M-mode tracing at mid-papillary levels was measured and averaged for the left ventricular ejection fraction (LVEF) and fractional shortening (LVFS).Echo assay was performed to determine cardiac left ventricular (LV) functions after 24 h after reperfusion32 by Evans blue and TTC staining. Each section was imaged using a microscope camera and quantified using Image-Pro Plus 6.0 . The percentage of myocardial infarction was calculated using the following formula: IA/total left ventricles × 100%.Twenty-four hours following I/R injury, the left anterior descending artery was religated at the same site of prior occlusion, and myocardial IA was determined7. Anti-α-actinin antibody, red fluorescent dye, and DAPI were used to specifically identify cardiomyocytes, apoptotic nuclei, and total nuclei, respectively. TUNEL-positive nuclei (red) and total cells (blue) were imaged using an Olympus DX51 fluorescent microscope  in at least five randomly selected fields per section (400× magnification). The apoptotic index is expressed as the ratio of apoptotic cells to total myocytes of DAPI-stained nuclei.Paraffin sections were stained using an in situ TUNEL detection kit  to identify apoptotic cells36.Ventricular specimens were collected from the apex territory to the left anterior descending artery occlusion region at 24 h after reperfusion. The dissected heart tissue in 1–2 mm wide blocks was immersed in 4% glutaraldehyde overnight, After the samples were postfixed in 1% osmium tetroxide for 1 h, the sections were dehydrated in a graded ethanol series to 100% and embedded in epoxy resin. Ultrathin sections of 80 nm thick were observed using a JEM-1400 transmission electron microscope (TEM) . Mitochondria were imaged at high magnification (×6000)46. In brief, hearts from anesthetized rats were collected, intersected into pieces and digested with 0.125% trypsin and 0.08% collagenase II. After cells were pre-plated for 90 min, nonadherent cells were collected and re-seeded into 6-well plates at a density of 1 × 106 cells/well. Then, the cells were cultured in complete DMEM/F12 medium consisted of 10% FBS and 1% penicillin/streptomycin for 48 h before the following treatments. For adenoviral infection, primary NRCMs were incubated with Ad-KDM3A/Ad-GFP at a multiplicity of infection (MOI) of 50 or Ad-shKDM3A/Ad-shRNA at an MOI of 50 for 4 h. siETS1 or siCtrl (as a control) transfection was performed in line with the instructions suggested by the manufacturer. For experimental H/R injury, isolated primary cardiomyocytes were seeded into culture plates, and fresh medium was added prior to each procedures. An H/R model was established as previously demonstrated. Briefly, hypoxia was induced in an anaerobic incubator chamber  suffused with a gas mixture composed of 95% N2 and 5% CO2 at 37 °C, and cells were incubated with serum- and glucose-deprived medium for 4 h. To mimic reperfusion, the medium was replaced with 10% DMEM/F12 medium and cells were removed to normoxic condition containing 95% O2 and 5% CO2 for 24 h to establish reoxygenation. Meanwhile, control cells were maintained in normoxic incubator for equivalent durations.Neonatal rat cardiomyocytes (NRCMs) were isolated from the hearts of SD rats at 1–3 d after birth and cultured in DEEM/F12 medium47 . The results were determined in international units per litre. The levels of proinflammatory mediators such as IL-6/TNF-α in cardiomyocytes were detected using commercial ELISA kits  according to the manufacturer’s instructions.To assay necrotic enzymes released by cardiomyocytes, blood samples and cellular supernatants were collected for the biological analysis of LDH and CK-MB using commercial analytical kits36. In brief, cells were homogenized in lysis buffer and centrifuged at 12,000 g for 10 min at 4 °C. The collected supernatant was mixed with assessment reagent, and a microplate reader was used to measure ATP content on a luminescence luminometer. The levels of ATP in heart tissues were measured in the same way.ATP content was detected using a bioluminescent assay kit  according to the instruction2- in particular) in NRCMs, cells were labeled with a dihydroethidium (DHE) probe  and subjected to flow cytometry assay 48. To further detect intercellular superoxide generation and lipid peroxidation induced by H/R injury, the MDA concentration and SOD enzymatic activity in NRCMs were measured using the same methods in cardiac tissue, in accordance with the descriptions of commercial kits 48. To evaluate tissue generation of ROS, left ventricular samples (5-μm sections) were probed with DHE (2 mmol/L) for 1 h at room temperature in the dark as previously introduced41. The slides were visualized by fluorescence microscopy , and the mean fluorescence intensity (MFI) of each slide was quantified by Image-Pro Plus software version 6.0.Isolated primary cardiomyocytes were seeded into 96-well plates, and cell viability was assessed using 3--2,5-diphenyltertrazolium bromide (MTT) reagent . Cell viability was expressed as a percentage of the controls. Flow cytometry was used to estimate apoptotic cells. The cells underwent staining with AV-APC and 7-AAD dyes, followed by fluorescence-activated cell sorting on a flow cytometric assay . Apoptotic cells were defined as those that were AV-APC-positive and 7-AAD-negative cells. To determine the levels of ROS  was utilized to obtain cytosolic and mitochondrial fractions. Consistent with the manufacturer’s instructions, cells or fresh tissue were collected, homogenized in ice-cold lysis buffer I and centrifuged for 5 min at 1000 g. Then, the supernatant was added to 500 μl of mitochondrial isolation reagent followed by centrifugation at 15,000 g for 20 min. The supernatant containing the cytosolic fraction was harvested, and the precipitate was washed with rinsing buffer. After centrifugation at 15,000 g for 10 min, the supernatant was discarded, and ice-cold lysis buffer II was chosen to resuspend the pellet. After 15 min of centrifugation , the supernatant  was obtained. Protein levels of cytochrome 19. In brief, treated cardiomyocytes were fixed and cross-liked with 1% formaldehyde for 20 min at room temperature. After terminating cross-linking by 0.125 M glycine for 5 min, cell lysates were sonicated into chromatin fragments of approximately 400 bp in length. The samples were subjected to immunoprecipitation using 5 μg of antibody against H3K9me2 (Abcam) or nonspecific IgG control (Santa Cruz) in the presence of magnetic beads conjugated with secondary antibody. Immune complexes and input were washed and eluted with buffer. Then, ChIP-enriched DNA fragments were assayed by real-time PCR using primers close to the promoter sites on the ETS1 with GAPDH used as a control. The primer sequences were listed as follows: ETS1, ; GAPDH, .ChIP was performed, and ChIP-enriched DNA was analysed by real-time PCR19. In brief, total RNA from cardiac tissue was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Two micrograms of mRNA were reverse-transcribed into cDNA using a cDNA synthesis kit (Fermentas). RT-PCR was performed using a SYBR green/fluorescein qPCR Master Mix kit (Fermentas) with an ABI Prism 7500 system. Data were normalized to β-actin to indicate relative expression levels. The sequence-specific primers used are listed as follows:Quantitative RT-PCR (qRT-PCR) were was performed as previously demonstratedKDM3A, F: 5′-TGAGGGCCTCTGTGAAATGT-3′, R: 5′- GGAAGCACTGATTTGGCACA -3′;ETS-1, F: 5′- CCCAGAATCCCGTTACACCT-3′, R: 5′- GTGTCTGTCTGGAGAGGGTC -3′.19. After the loading concentration was essentially normalized to 50 μg per well, extracted proteins were separated by 8–12% SDS-PAGE and then transferred onto PVDF membranes (Merck Millipore). Next, the membranes were blocked in 5% skim milk powder dissolved in Tris-buffered saline containing 0.1% Tween-20, washed three times and subsequently probed with the corresponding primary antibodies at the recommended dilution overnight at 4 °C. After incubation with horseradish peroxidase-conjugated secondary antibodies for 60 min, the bands visualized using enhanced chemiluminescence reagent. GAPDH was used as a loading control for whole cell lysate and cytosolic protein, and VDAC1 was used as a loading control for the targets in mitochondria.For western blotting assays, frozen heart tissue was lysedp values between two groups were calculated using the Student’s t-test. Comparisons of more than two groups were assessed by one-way analysis of variance (ANOVA) and a post hoc Tukey’s test. Statistical significance was assigned at a p value less than 0.05. All results were independently reproduced at least three times with similar results.The numerical data were represented as the mean ± SEM. Statistical Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Entering Mentoring training curriculum framework, this report provides a brief synopsis and key takeaways from the 2019 NIH-ASCB Accomplishing Career Transition (ACT) workshop, “Introduction to Effective Mentorship for Scientists” for 30 senior postdoctoral and early-career faculty researchers from historically underrepresented racial and ethnicity backgrounds. In addition, effective strategies and best practices to enhance STEM mentoring for early-career researchers are provided, which have practical applications for diverse mentoring relationships across disciplines, career stages, and mentee types.Diversifying the scientific workforce remains a national priority due to the continued lack of representation from underrepresented individuals in STEM fields. Quality mentoring has been identified as a stimulus to enhance not only research success, but also recruitment and retention of underrepresented groups pursuing STEM careers. Utilizing the  Scientific investigators are always seeking more effective strategies to build diverse research teams and increase research productivity . While tRecent reports have suggested that quality mentoring can positively impact both lab dynamics and research success. Specifically, positive mentoring experiences have been linked to increased productivity, career satisfaction, and research success of trainees –11. For Entering Mentoring [While the importance of mentoring is widely recognized, few structured training interventions exist for (1) novice mentors to acquire evidence-based skills or (2) experienced mentors to develop new approaches and innovative mentoring practices –20. Furtentoring  mentor tentoring –20. Ther1. Describing the core elements of mentoring, including maintaining effective communication, establishing expectations, and addressing equity and inclusion.2. Clearly articulating expectations for the mentoring relationship.3. Practicing effective strategies for improving communication.4. Recognizing the impact of biases, prejudices, privilege, and power on the mentor-mentee relationship and acquiring skills to manage them.5. Identifying the roles mentors play in the overall professional development of their mentees and work-life integration using Stewart Friedman’s Total Leadership model .In this report, key insights and discussion that emerged from this mentoring workshop are highlighted to foster and cultivate effective mentor-mentee relationships for early-career STEM researchers. Moreover, these strategies and mentoring principles can be practically applied across various disciplines and career stages of mentoring relationships to enhance mentor engagement, mentee success, and support scientific workforce diversity.Effective mentorship can fulfill several critical purposes, including career advancement, role modeling, and psychosocial functions . In partAdditionally, STEM mentees have reported a lack of sensitivity to the importance of race, gender, and class in the mentoring relationship . Often, The benefits of mentoring in the growth and advancement of scholars and scientists are widely documented , 40. ForAn ideal mentoring network should consist of near-peer, peer, and established professional mentors. Near-peer mentoring is especially beneficial for many trainees who experience challenges when transitioning into higher education, i.e. acclimatization to the new environment and academic expectations. For undergraduate and graduate students, near-peer mentoring is an established relationship between students, in which one student is 1–4 years senior to the other student, on similar academic tracts, and within the same institution and/or laboratory environment . Due to Similarly, peer-mentoring, also called horizontal mentoring, is a valuable type of mentorship between students or student groups who are new to a given field, subject matter, or campus. Peer-mentoring has been shown to accelerate academic and professional development of individuals by providing reciprocal support and collaboration . MoreoveFurther, mentees should be encouraged and supported in creating a developmental network of established professional mentors, beyond their primary mentors. This will usually involve informal mentoring pairs, where established professionals work to understand the mentee’s overall professional, research, or careers goals in order to support the mentee in achieving a specific goal(s). These mentors are both accessible and committed to helping the mentee, while also providing a different perspective from the primary mentor. Developmental networks have also been shown to be beneficial for mentors as these mentoring networks can provide support for both the mentee and primary mentor if difficulties arise . TherefoAn important role of mentors is to foster the professional development of their mentees. In STEM fields, mentees often train for a specified duration with their primary mentors, followed by employment or additional training. To support discussions regarding professional development, the Individual Development Plan (IDP) is a highly recommended, yet often underutilized tool. First developed for postdoctoral fellows in 2002 , IDPs haIn addition, a significant benefit of having mentors is access to resources and networking opportunities. Networking entails establishing enduring contacts that can boost one’s STEM career. While networking is often considered within the framework of a job search, it is also critical for sustained success in STEM fields for both mentors and mentees. Specifically, networking opportunities can boost visibility of mentees while increasing contact with established and potential collaborators for mentors. Networking can also provide a source of external career advisors for mentees.In a successful mentoring relationship, mentors and mentees should establish regular meetings to get to know each other, which can be conducted either formally or informally, and clearly express their expectations of the mentoring relationship. This can be done by documenting short- (1 year), mid- (2–5 years), and long- (> 5 years) term professional goals as part of a written career development plan or mentoring compact , 56. MenTo aid in clarifying and developing your mentoring approach and style, there are several tools available, including, but not limited to, a mentor philosophy or mentoring action plan . By deveEntering Research and Mentoring Up, have been developed to support mentees as they learn to mentor up at all career stages [When faced with challenges in a mentoring relationship, it is important for the mentor to first recognize when an issue arises and identify its source. Due to the inherent power dynamic in mentoring relationships, it can be difficult for mentees to see problems and subsequently approach their mentors to address these issues. However, the mentee shares responsibility to nurture and proactively engage in the mentoring relationship by managing or “mentoring” up . As mutur stages –67.When issues are present, it is imperative that the mentor and mentee engage in a difficult conversation to keep the mentoring relationship on track. There are several strategies to approach these conversations –70. FirsThis report describes best practices and resources to support effective mentorship of STEM researchers. These evidence-based strategies, such as developing mentoring networks and addressing culture and diversity, have been shown to positively impact mentoring relationships across career stages, disciplines, and mentee types. Therefore, by leveraging available mentoring resources  when initiating new mentoring partnerships, early-career mentors are enabled to implement specific actions to enhance their mentoring practices and principles with diverse mentees, thus contributing to a skilled, diverse, and sustainable scientific workforce.


Network pharmacology further revealed 33 key targets and 75 KEGG pathways as possible pathways and targets involved in WK-mediated treatment, with the CaMKII/CNCA1C/Ca2+ pathway being the most significantly affected. This finding was validated using an AC-induced rat arrhythmias model. Pretreatment with Wenxin Keli reduced the malignant arrhythmias and shortened RR, PR, and the QT interval. Wenxin Keli exerted some antiarrhythmic effects by inhibiting p-CaMKII and intracellular Ca2+ transients and overexpressing CNCA1C. Thus, suppressing SR Ca2+ release and maintaining intracellular Ca2+ balance may be the primary mechanism of Wenxin Keli against arrhythmia. In view of the significance of CaMKII and NCX identified in this experiment, we suggest that CaMKII and NCX are essential targets for treating arrhythmias.This study employed a systems pharmacology approach to identify the active compounds and action mechanisms of Wenxin Keli for arrhythmia treatment. Sixty-eight components identified  Arrhythmias are a group of conditions that cause the heart to beat irregularly . It may Codonopsis pilosula (Franch.) Nannf., Panax notoginseng (Burkill) F.H.Chen, Nardostachys jatamansi (D.Don) DC., Amber, and Polygonatum kingianum Collett & Hemsl. It is reported to be effective in arrhythmias treatment  is handy in arrhythmia treatment. TCM is widely and frequently used in China and abroad . Wenxin reatment  and is treatment . It alsoreatment . NeverthThis study employed a systems pharmacology approach to investigate the pharmacological mechanisms of WK . The potThe UPLC/Q-TOF-MS was employed to analyze aqueous solutions and serum components of WK qualitatively. The identified components were regarded as the potential active ingredients of WK.in silico approach was employed to identify the target proteins for the possible active ingredients (components identified from 2.1.1) of WK. Predictive models including the TCMSP database (http://ibts.hkbu.edu.hk/LSP/tcmsp.php), STITCH (http://stitch.embl.de/) (http://www.swisstargetprediction.ch/), Target-Prediction (http://prediction.charite.de/index.php?site=chemdoodle_search_target), and DrugBank (http://www.drugbank.ca/) were used to predict the target proteins of WK.Target Proteins of WK: TCM formulas can effectively prevent the devastating effects of complex diseases through the synergistic effects of multiple compounds and targets . Therefombl.de/) , Swiss Thttp//triad.fsm.it/cardmoc/).Target Proteins of Arrhythmia: Targets related to arrhythmia were obtained from “The gene connection for the heart” database  network based on the candidate compounds of WK and the potential targets to understand the complex interaction of compounds and their corresponding targets at a systems level.Protein-protein Interaction (PPI) Network: Networks are used to view global relationships between nodes. In this study, a PPI network was used to identify potential target proteins associated with arrhythmia based on the arrhythmia targets identified in 2.1.2 using the Cytoscape 3.8.1 Bisogenet app .Experimental approaches for determining the targets of candidate drugs are costly, labor-intensive, and time-consuming. In this study, a systems pharmacology approach was used to determine the key targets through which WK exerts an antiarrhythmic effect. The Merge tool of the Cytoscape 3.8.1 app was employed to determine the common targets of the C-T network and PPI network. They were regarded as the key targets by WK in arrhythmia treatment.https://david.ncifcrf.gov/). An incorporated arrhythmia pathway was then assembled based on the basic pathway information by picking out the closely linked pathways related to arrhythmia pathology.Signaling pathways are essential components of the system pharmacology that link receptor-ligand interactions to pharmacodynamics outputs . An incoAcetonitrile  and formic acid  were purchased from Fisher Scientific . Purified water was acquired from a Milli-Q system . Medlab RU/4C50R multi-channel physiological signal acquisition and processing system were purchased from Shanghai Tongyu Teaching Instrument Manufacturing Co., Ltd (batch No. 17060155). WK was purchased from Shandong Buchang Pharmaceutical Co., Ltd, Tianjin, China . Aconitine (AC) was sourced from Baoji Chenguang Biotechnology Co., Ltd., Shanxi, China (batch No. ha001198). Pentobarbital sodium was purchased from Baoji Chenguang Biotechnology Co., Ltd., Shanxi, China (batch No. B21882). In the same line, the p-CaMKII rabbit antibody was sourced from Abcam (ab32678), the GAPDH rabbit antibody from Hangzhou Xianzhi biology Co., Ltd, China (AB-P-R 001), and the HRP labeled Goat anti-rabbit second antibody from Wuhan bode Bioengineering Co., Ltd, China (BA1054). The cDNA synthesis kit (K1622) was purchased from ThermoFisher Scientific, United States.WK (5 g) was dissolved in 20.6 ml purified water (Nongfu Spring) and left to stand for 1 h. The solution was then filtered through four layers of gauze to filter out the dregs. The filtrate was subsequently centrifuged for 10 min at 10,000 rpm and 4°C, and 300 ml of the supernatant was stored at −20°C. The supernatant was diluted four times and filtered through a 0.22 μm filter membrane before injection for UPLC/Q-TOF-MS determination.Blood samples (0.5 ml) were collected into serum tubes from the eye sockets of rats after 0.5, 1, 1.5, and 2 h of WK administration followed by immediate centrifugation at 3,500 rpm for 15 min at 4 C. The extracted serum was then subjected to UPLC/Q-TOF-MS determination.18 column  at a column temperature of 45°C and 5 μl injection volume. The sample was eluted at a flow rate of 0.3 ml min−1 in a gradient elution program of A (0.1% formic acid: water) and B (0.1% acetonitrile: water): 0–6 min (98–78% A); 6–10 min (78–70% A); 10–14 min (70–62% A); 14–18 min (62–62% A) 18–22 min (62–40% A); 22–26 min (40–0% A); 26–28 min (0–0% A); 28–29 min (0–98% A) 29–30 min (98–98% A). MS detection was performed on a high definition MS (HDMS) system  with negative and positive electrospray (ESI) modes. Its sufficient sensitivity ensured that as many putative compounds as possible were identified. The optimized operating parameters were capillary voltage; 3.0 kV(Positive)/2.0 kV(Negative), source temperature; 120°C (Positive)/110°C (Negative), desolvation gas temperature; 450°C, desolvation gas (N2) flow rate; 800 L/h, collision energy (CE); 25 V, scanning time; 0.1 s, and scanning time interval; 0.02 s. Leucine-cerebral peptide solution (Waters) at a concentration of 200 pg/ml was used as the Lock-Spray calibration solution to ensure the accuracy and repeatability of the mass-to-charge ratio. The exact mass-to-charge ratios in positive and negative ion modes were [M+H]− = 556.2771 and [M-H]− = 554.2615, respectively. The data acquisition range and time were m/z 50–1000 and 0–30 min, respectively.Chromatographic separation was performed on an ACQUITY UPLC BEH CClean grade male Sprague Dawley  rats aged 6–7 weeks and weighing 220 ± 20 g were obtained from the Experimental Animal Center of the Institute of health and environmental medicine, Academy of Military Medical Sciences, PLA, [license no. SYXK(Jin) 2014-0002]. All the animal-based experiments were approved by the Animal Care Committee of the Institute of Radiation Medicine of the Chinese Academy of Medical Sciences and performed following the relevant guidelines and regulations. Efforts were also made to minimize animal suffering. All rats were housed in the specific pathogen-free (SPF) animal laboratory of the experimental animal center of the Institute of Radiation Medicine Chinese Academy of Medical Sciences at a temperature of 23°C ± 2°C and 35% ± 5% relative humidity. The animals were acclimatized to the environment for 1 week with free access to a standard pellet diet before initiating the experiments. The rats were randomly divided into six groups, each comprising eight animals: the control (C) group, model (M) group, low-dose WK-treated (LD) group, medium-dose WK-treated (MD) group, high-dose WK-treated (HD) group, and the amiodarone (A) group. Intragastric administration of WK was once a day for 14 consecutive days in groups LD (2.41 g/kg/d), MD (4.82 g/kg/d), and HD (9.64 g/kg/d) . Rats in group A were administrated with amiodarone (40 mg/kg), while those in groups C and M were administrated with an equal volume of distilled water.The rats were intraperitoneally anesthetized with 0.6% pentobarbital 30 mg/kg an hour after the last WK administration. They were then fixed on the plank in a prone position, followed by the insertion of electrodes into the subcutaneous tissue of the limb. The ECG parameters were subsequently recorded in each group using a Medlab RU/4C50R multi-channel physiological signal acquisition and processing system via a standard limb lead II. Rats in the other five groups, except those in group C, were subsequently injected with 0.001% AC 40 µg/kg after ECG stabilization to establish the arrhythmia model. The injection was through the caudal vein at a rate of 0.2 ml/min. Equal volumes of 0.9% normal saline were injected into rats in group C. The ECG of the rats was then observed in real-time for 20 min after the heart rhythm had normalized. The parameters measured included the initial time of arrhythmia and the types, occurrence, and duration of various VAs.All animals were euthanized after ECG detection, and their hearts were immediately harvested and stored in liquid nitrogen awaiting WB and qRt-PCR analysis.The proteins were separated on a 10% SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were subsequently incubated with p-CaMKII rabbit antibody at 4°C overnight and then washed thrice with Tris-buffered saline (TBS) containing Tween 20 (TBST) to wash off the excess antibodies. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. ECL visualization was then performed, followed by calculating the gray values using the Image J software . The intensity of the target proteins was normalized to that of an internal reference to determine their relative expression level.2+ pathway was validated using Real-time Quantitative PCR (RT-qPCR) analysis. The total RNA of the myocardial pieces was first isolated using the Trizol reagent , following the manufacturer’s instructions. The RNA samples (5 μg) were then reverse-transcribed to complementary DNA (cDNA) using a cDNA synthesis kit, followed by quantitative Rt-PCR on a 7500 Fast Real-Time PCR System (Applied Biosystems). The primer pair used was CAMKⅡ forward; 5′-CAAGTTCATCGAGGTCACCAC-3′ and reverse; 5′-ATACACAGCTCTCGTCCTCTG-3′, with RNA U6 as the internal control. The miR-1 specific primer sequences for quantitative real-time PCR were used for TaqMan MicroRNA Assays . The expression levels of the miR-1 were subsequently normalized to U6 RNA expression levels and then calculated using the 2−ΔΔCt method  with a ×40 magnification, 1.3 numerical aperture oil immersion objective, and axial resolutions of 1.5 μm. Fluo-4 AM: Fluo-4AM+8μlDMSO+2μlF-127 was first prepared and then diluted 1,000 times using 1 ml DMem+1 μl Fluo-4. Cardiomyocytes were then incubated with 500–10,002 μl Fluo-4 AM  for 20 min at 37°C and recorded in normal Tyrode’s solution. Fluo-4 was excited at 488nm, followed by measurement of the fluorescence emission at 505 nm. Images were acquired in the line-scan (X-T) mode with 512 pixels  per line at a rate of 3 ms per scan. The Ca2+ transients were then analyzed using a modified version of the MATLAB program, and their fluorescence emission intensity was expressed as F/F0, where F0 was the basal fluorescence intensity level. The recording was performed at 35°C.Calcium imaging was carried out at room temperature within 6 h after isolation of cardiomyocytes from rats in the control group . Ca2+ trt-test, while those between multiple groups were analyzed using one-way analysis of variance . p < 0.05 indicated significant differences between groups.Data were expressed as the means ± SEM. Differences between the two groups were analyzed using the Student’s two-tailed in vitro and in vivo, which were regarded as the potential active components in this study  revealed 61 targets related to arrhythmia  and 429 compound-target interactions. The mean degree value (the number of targets associated with it) of the candidate compounds was 41. Twelve compounds possessed a degree larger than 14 (2-fold degree median), indicating that they regulated multiple targets to exert varying therapeutic effects. Specially, five compounds, including Atractylenolide II, Tanshinone IIA, ginsenoside Rh2, Ginsenoside Rg1, and Jatamansinol, acted on 28, 24, 21, 20, and 16 targets, respectively. The compounds were regarded as the crucial active compounds for WK because of their prime positions in the network.PPI network of arrhythmia: There were 1395 targets closely related to known arrhythmia targets. They were regarded to be indirectly related to arrhythmia.Analysis of the common targets of WK and arrhythmia revealed 33 key targets . These tp-values. 2+ of the calcium signaling pathway were thus selected as the observation index in this study based on these findings combined with other relevant literature.The pathway information of the 33 key targets revealed 75 KEGG pathways, including the calcium, oxytocin, adrenergic signaling in cardiomyocytes, adipocytokine, and dilated cardiomyopathy signaling pathways. The pathways were closely associated with arrhythmias  based on2+ current (LTCC) plays a crucial role in the ECC. In the same line, membrane depolarization in ventricular myocytes during the action potential (AP) causes synchronous activation of multiple LTCCs, consequently triggering the release of Ca2+ from the sarcoplasmic reticulum (SR). These occurrences increase the intracellular Ca2+ that initiates contraction. Ca2+ influx via the LTCC provides a multifunctional signal that triggers muscle contraction, controls AP, and regulates gene expression (2+ leakage (2+ leak in return initiates early afterdepolarizations (EADs)  is an essential aspect of arrhythmia pathophysiology. The L-type Capression . As such leakage . The dias (EADs)  and delas (EADs) .2+ pathway. The assumption is that WK suppresses CaMKII phosphorylation and CACNA1C activation and antagonizes the intracellular Ca2+ overload to avoid the occurrence of EADs and DADs. CaMKII, CACNA1C, and Ca2+ concentration were thus selected as the observation indices of this study.Therefore, we hypothesized that WK could inhibit arrhythmias by regulating the CaMKII/CACNA1C/Ca1) Mortality: There were no deaths in groups C, HD, and A during the experiments. However, two animals in group M and one animal in the groups LD and MD died. The animals died of ventricular fibrillation (VF) after pleomorphic ventricular tachycardia (Polymorphic VT). Nonetheless, the ECG recording time of the dead rats was very short and thus could not be compared with that of other rats. As such, we only analyzed the ECG of the surviving rats. All groups had different degrees of arrhythmia except group C.p > 0.05; p < 0.01; 2) The initial time and duration of arrhythmias: There were insignificant differences in the initial time and duration of arrhythmias among the five experimental groups (3) Types of arrhythmias: VT and bigeminy were observed in the five experimental groups. However, VP was observed in groups M, LD, and A, normal ECG was observed in groups M, LD, and HD, while VF was observed in group LD .4) ECG parameters: Compared with ECG parameters before the experiment, R-R, P-R, and Q-T interval did not change in groups C and M. However, they were prolonged in groups LD, MD, and A, and shortened in group HD . In the p < 0.05). Notably, group HD had a significantly lower level of p-CaMKII than group C (p < 0.01), suggesting that a high WK dose markedly inhibited the expression of CaMKII  than group C, suggesting that AC could inhibit CACNA1C to induce arrhythmia. Compared with group M, the expression of CACNA1C increased in all groups except group LD after treatment of WK and amiodarone, with group HD exhibiting the most significant increase (p < 0.01). Notably, group HD had a significantly higher level of CACNA1C than group C (p < 0.01), suggesting that a high WK dose markedly increased the expression of CACNA1C  to evaluate the time-course effects of WK on intracellular Ca2+ homeostasis. WK significantly reduced the resting Ca2+ ratio and the amplitude of Ca2+ ratio within 5 min after adding WK (p < 0.01). WK also induced a decrease in the cytoplasmic Ca2+ ratio within 5 min in a time-dependent manner  (1) Promote Naolarized . Large Nies (TA) , therebyies (TA) .2+ Overload: Abundant Na+ influx into the cytosol causes Ca2+ overload via sequential activation of electrogenic Na+-Ca2+ exchanger (NCX), thus inducing DAD and TA  Lead to CaD and TA . As a reuinidine  in clini+ Currents: AC significantly increases Ca2+ influx, inhibits outward K+ currents, prolongs repolarization, and produces DAD and reentry, resulting in arrhythmias (3) Inhibition of Outward Khythmias .2+ influx. It triggers the release of Ca2+ via the Ca2+ release channels, ryanodine receptors (RyRs) of the SR. During normal AP, early LTCC peaks trigger robust SR release followed by partial inactivation because of two processes: Ca2+-and voltage-dependent inactivation. LTCC regulates AP repolarization and is vital in developing cardiac arrhythmias such as VF, the leading cause of sudden cardiac death (2+ elevation (2+ influx through LTCC (4) Inhibition of LTCC: LTCC is activated upon membrane depolarization and Caac death . Studiesac death . The effac death . Moreovelevation . In contugh LTCC . These d2+ overload and apoptosis via the p38 signaling pathway (5) Others: AC also stimulates the vagus nerve and inhibits the sinoatrial node and conduction system, resulting in a slowed heart rate and conduction block . Moreove pathway .AC is a major bioactive diterpenoid alkaloid derived from aconitum plants. It has been used in the past as an antipyretic, analgesic, antirheumatic drug, and a neurotransmission inhibitor . However, and VF . AC-indu, and VF , decreas, and VF , and leaIn the present study, the antagonistic effects of WK on AC-induced arrhythmia were observed in rats. ECG recording experiments demonstrated that AC administration decreased the heart rate and prolonged the Q-T intervals, leading to VP, coupled rhythm, and tachycardia. These occurrences ultimately induced VF and rat mortality. These findings are consistent with those of 2+ homeostasis, resulting in excitation-contraction coupling. Ventricular AP starts with a sodium (Na+) influx through voltage-gated Na+ channels that depolarize the cell. Voltage-sensitive LTCC is activated at a certain threshold, allowing the influence of Ca2+ into the cytosol, which triggers a much larger Ca2+ release from the SR, the main intracellular Ca2+ storage organelle  and is the fundamental link between electrical and mechanical activation in the heart  that pumps Ca2+ back into the SR stores. It is also caused by Ca2+ extrusion and releases into the extracellular space through NCX, which exchanges three Na+ ions entering for one Ca2+ ion leaving the cell . SERCA2a takes up approximately 63% of cytosolic Ca2+, while NCX extrudes 37% of Ca2+ in humans  and intracellular Carganelle . Notably+ sparks . They orhe heart . The cytthe cell . The NCXn humans . The depcroscopy . In this2+ flowing into LTCC through mycoplasma voltage gating triggers SR to release large amounts of Ca2+. This myocardial contraction and blood drawing process driven by Ca2+ is called ECC  Ca2+ channel family. It is structurally homologous to voltage-gated Na+ channels and belongs to a family of six membrane-spanning voltage-gated ion channels. CACNA1C, CACNB2, and CACNA2D1 genes encode the pore-forming a-subunit (Cav1.2) and the b2 and a2d ancillary subunits that form the cardiac Ca2+ channel, which generates the LTCC  for relaxation to occur in normal conditions. This phenomenon consequently takes Ca2+ back into the SR. NCX operates in both the Ca2+ efflux and influx (or reverse) modes, depending on the internal and external concentrations of Na+ and Ca2+. INa is elevated in AC-induced myocytes, while NCX works almost exclusively in the Na+ extrusion mode, thus significantly increasing the amount of Ca2+ influx. This influx raises the cellular and SR Ca2+ content, resulting in larger Ca2+ transients. In contrast, other studies postulate that SR Ca2+ release modulates the sarcolemmal ICaL, suggesting a retrograde in communication between the SR and the sarcolemmal LTCC in cardiac ECC  (ICaL must inhibit the transportation of Cadiac ECC . MoreoveCACNA1C) . NotablyCACNA1C) . StudiesCACNA1C) .2+ overload is not achieved by increasing LTCC. However, these findings contrasted those of 2+ release channel and LTCC. In this study, WK significantly reduced intracellular Ca2+ transients, thus confirming that the increased expression of NCX and the presence of Ca2+ influx mode were the primary causes of AC-induced arrhythmia. AC also increased the expression of p-CaMKⅡ, thus promoting the release of Ca2+ from SR. The Ca2+ release of SR inhibited LTCC because of the retrograde communication between them. QRT-PCR results of CACNA1C also confirmed these findings. Similarly, laser-scanning confocal calcium imaging directly demonstrated the inhibitory effect of WK on intracellular Ca2+ concentration. Maintaining intracellular Ca2+ balance may thus be the primary mechanism of WK against arrhythmia.This study showed that AC has a significant inhibitory effect on LTCC, indicating that AC-induced intracellular Ca1) It is a good model for TCM research: The approach also detected the chemical components of WK besides the anti-arrhythmia mechanism and correlated the potential active components with their pharmacodynamic mechanisms. The research model was more systematic and complete, thus proving a good model for TCM research.2) It provides a research direction of antiarrhythmic effects of TCM: Besides the most relevant calcium signaling pathway, other signaling pathways were also detected, thus providing a good direction for subsequent studies. The pathways included the oxytocin signaling pathway, adrenergic signaling in cardiomyocytes, adipocytokine signaling pathway, and dilated cardiomyopathy associated with the metabolism of adrenaline, angiotensin, fats, glycometabolism, apoptotic factors, and other arrhythmia-related factors. These findings demonstrate that TCM treatment of diseases has a multi-target comprehensive effect, thus further proving that systems pharmacology is an effective method in TCM study.3) Construction of appropriate disease model: The present study used AC to construct a pure arrhythmia rat model instead of the toxic arrhythmia that causes myocardial infarction and heart failure. The pure arrhythmia model was more conductive to reveal the pathogenesis of arrhythmia because it did not interfere with the complex pathological mechanisms.2+ transients. CaMKII and NCX play a vital role in Ca2+ balance. As such, blocking CaMKII and NCX can be a safe and effective strategy against arrhythmias.4) New antiarrhythmic strategy: This study demonstrated that WK had therapeutic effects in AC-induced rat arrhythmias by inhibiting p-CaMKⅡ, over-expressing CNCA1C, and inhibiting intracellular Cain vivo and in vitro as the potential active components of WK. Among them, 33 key targets were screened based on their corresponding targets of WK potential active ingredients and targets of arrhythmia. These targets were regarded as the antiarrhythmic targets of WK . Moreover, AC-induced SR Ca2+ leakage through the RyR2 channel further inhibits LTCC (Carability . AC actirhythmia . The intrhythmia . There iits LTCC .2+ transients and overexpressing CNCA1C, thus suppressing SR Ca2+ release, which is the most probable mechanism of WK inhibition of the Ca2+ overload. Maintaining intracellular Ca2+ balance is, therefore, an essential mechanism of WK against arrhythmia. The antiarrhythmic mechanism of WK also suggests that TCM has a multi-target comprehensive effect against diseases. Notably, high dose WK has the best impact and should thus be highly considered during clinical treatment.However, pretreatment with WK lowers mortality, lessens malignant arrhythmias, and shortens RR, PR, and QT intervals than AC-induced rats. WK induces some antiarrhythmic effects by inhibiting p-CaMKⅡ and intracellular Ca2+ oscillations. As such, its activation is directly linked to increased APD, EADs, and arrhythmias (2+ homeostatic proteins involved in E-C are CaMKII targets (2+ to activate CaMKII, which then binds to and phosphorylates β2a to trigger EADs. Therefore, CaMKII can serve as a novel therapeutic target for arrhythmias.CaMKII activity reflects the frequency of cytosolic Cahythmias . The Ca2 targets . APD pro+ accumulation and reversal, leading to a high cytoplasmic Ca2+ influx.Similarly, NCX blocking is a promising safe and effective strategy against repolarization-dependent arrhythmias because it causes intracellular Na