Deficiency of LncRNA-CIRBIL promotes J-wave syndrome by enhancing transmural heterogeneity of Ito current

doi:10.21203/rs.3.rs-2171528/v1

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Deficiency of LncRNA-CIRBIL promotes J-wave syndrome by enhancing transmural heterogeneity of I_to current

https://doi.org/10.21203/rs.3.rs-2171528/v1

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Transmural heterogeneity of I_to current is a major cause of J-wave syndrome (JWS), while the underlying molecular mechanisms remain elusive. The present study aims to explore the influence of Cardiac Injury-Related Bclaf1-Interacting LncRNA (lncCIRBIL) on cardiac J-wave syndrome and to delineate the molecular mechanisms.

The plasma level of lncCIRBIL was reduced in JWS patients and cold-induced JWS mice. Knockout of lncCIRBIL increased the frequency of J-wave and the susceptibility to ventricular arrhythmia in mice. The transmural difference of KCND2 and I_to currents were dramatically increased in the right ventricle, but not the left ventricle of lncCIRBIL-KO mice. In contrast, cardiomyocyte-specific transgenic overexpression of lncCIRBIL produced the opposite effects. The human homologous conserved fragment of lncCIRBIL (hcf-CIRBIL) reduced I_to, downregulated action potential notch and prolonged APD₂₀ in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). LncCIRBIL titrates the transmural heterogeneity of KCND2 by regulating UPF1 mediated mRNA decay. Inhibition of lncCIRBIL promoted J-wave syndrome by enhancing the transmural heterogeneity of I_to in the right ventricle. These findings imply that lncCIRBIL represents a potential therapeutic target for J-wave syndrome.

Health sciences/Cardiology/Cardiovascular biology/Cardiovascular diseases/Arrhythmias/Ventricular fibrillation

Biological sciences/Biochemistry/Ion channels/Potassium channels

lncRNA

J-wave syndrome

KCND2

UPF1

arrhythmia

J-wave syndrome (JWS) is characterized by distinctive J-waves on the electrocardiogram1 and closely associated with an increased risk for life-threatening ventricular arrhythmias2. J-wave is formed by the notch on action potential of ventricular myocytes, which mainly mediated by the transmural difference of transient outward potassium current (Ito) and sodium current (INa). J-wave is often recorded in Brugada (BrS) and early repolarization (ERS) syndromes3. Ito is predominantly expressed in the subepicardial myocytes and creates a transmural voltage gradient between epicardial and endocardial myocytes in the earliest phase of the action potential, which is especially significant in the right ventricle and underlies the formation of J-waves4. Loss-of-function mutations of sodium SCN5A channel in patients with idiopathic ventricular fibrillation demonstrates prominent J-wave on the ECG5. Certain pathological conditions such as hypothermia or ischaemia in patients are associated with electrocardiographic J-wave and increased risk of arrhythmia6, 7. However, although progression on the mechanisms of J-wave syndrome has been continuously achieved in recent year, the precise molecular mechanisms remain to be elusive. Long noncoding RNAs (lncRNAs) are single-strand RNAs that are longer than 200 nucleotides with no protein-coding ability. LncRNAs play key roles in the regulation of various cardiac diseases, including myocardial infarction8, heart failure9 and cardiac arrhythmia10, 11. LncRNAs participate in the regulation of diverse electrophysiological properties of cardiomyocytes including excitation conduction12, membrane repolarization13 and automaticity14. The dysregulation of lncRNAs promotes the genesis of arrhythmias by altering the expression and function of related ion channels15. Currently, the role of lncRNA in J-wave syndrome is still unknown. In a previous study, we discovered a cardioprotective lncRNA, named cardiac injury-related bclaf1-inhibiting lncRNA(lncCIRBIL)16. When recording electrocardiographs, we accidentally observed prominent J-waves in lncCIRBIL knockout mice. Therefore, we conducted the present study to explore the specific role of lncCIRBIL in J-wave syndrome and to decipher the underlying molecular mechanisms. Our data revealed that lncCIRBIL regulates the formation of J-wave by modulating Up-frameshift protein1 (UPF1) mediated KCND2 mRNA degradation and the transmural heterogeneity of Ito in the right ventricle.

LncCIRBIL deficiency is associated with significant J-point elevation and increased arrhythmia susceptibility

We firstly assessed the ECG waveforms of lncCIRBIL knockout (lncCIRBIL-KO) mice. J-wave was more frequently presented in lncCIRBIL-KO mice (20 of 22) than in wild-type (WT) mice (13 of 23) (Fig. 1a, b). We calculated the derivative of J-wave downward deflection and found that it was more negative in lncCIRBIL-KO mice than WT controls (Fig. 1c). As accentuated J-waves were closely associated with increased arrhythmia vulnerability¹⁷, we then performed programmed electrical stimulation to test ventricular arrhythmia susceptibility in lncCIRBIL-KO mice. Ventricular tachycardia (VT) and ventricular fibrillation (VF) were inducible in 11 of 14 lncCIRBIL-KO mice, while only 2 of 14 WT mice (Fig. 1d, e). The VT/VF duration was longer in lncCIRBIL-KO mice relative to WT controls (Fig. 1f).

The gradient change of potassium and sodium current were shown to contribute to the development of J-wave syndrome¹⁸. We therefore examined the transmural expression changes of KCND2, KCND3, KCNIP2 and SCN5A in the right and left ventricles of WT and lncCIRBIL-KO mice. The transmural difference of KCND2 was dramatically increased in the right ventricle of lncCIRBIL-KO mice, while KCND3, KCNIP2 and SCN5A were not altered (Fig. 1g-j). In constract, lncCIRBIL deletion produced no significant influence on transmural differences of KCND2, KCNIP2, KCND3 and SCN5A mRNAs in the left ventricle (Fig. 1k-n). We then examined lncCIRBIL level in the subepi- and subendocardium of right and left ventricles in WT mice. It is much higher in subepicardium than subendocardium in right ventricle of WT mice, while no gradient expression of lncCIRBIL was detected in the left ventricle (Fig. 1o). We also evaluated the influence of lncCIRBIL deletion on cardiac morphology by H.E staining (Supplementary Fig. 3a-c). Moreover, lncRNA deficiency elicited no detectable influence on ejection fraction (EF) and fractional shortening (FS) in mice (Supplementary Fig. 3d-f). These data indicated that lncCIRBIL deficiency elevated J-wave and promoted arrhythmia susceptibility possibly by enhancing the transmural heterogeneity of KCND2 in the right ventricle.

Transmural differences in transient outward current (I_to) are amplified in lncCIRBIL-KO mice

We then performed voltage-clamp recording to further validate the influence of lncCIRBIL knockout on transmural difference of I_to currents in the right ventricle. The data showed that I_to was larger in subepicardial than subendocardial myocytes of the right ventricle in WT mice, and the difference was further amplified in lncCIRBIL-KO mice (Fig. 2a-c). Consistently, the protein level of Kv4.2 was higher in subepi- than subendo cardiomyocytes of right ventricle and the difference was boosted by lncCIRBIL knockout (Fig. 2d). In line with the effects of lncCIRBIL deficiency on I_to, a further shorter APD was recorded in myocytes from subepicardium than subendocardium in right ventricle of lncCIRBIL-KO mice than WT controls (Fig. 2e-h). LncCIRBIL knockout produced no difference in action potential amplitude (APA), resting membrane potential (RP) and overshoot (OS) of right ventricular myocytes (Fig. 2i-k). No significant differences in action potential and I_to were observed between subendo- and subepi- cardiomyocytes of the left ventricle from lncCIRBIL-KO and WT mice (Supplementary Fig. 4a-c). These data indicated that lncCIRBIL deficiency enhanced the transmural difference of I_to in the right ventricle.

Transgenic overexpression of lncCIRBIL reduced J-wave and the transmural difference of I_to in the right ventricle

We then employed cardiac-specific lncCIRBIL transgenic overexpression (lncCIRBIL-TG) mice to further elucidate the influence of lncCIRBIL on J-wave. LncCIRBIL overexpression significantly reduced the frequency of J-waves (Fig. 3a- c). The subepicardial and endocardial differences of I_to currents in the right ventricle were dramatically reduced in lncCIRBIL-TG than WT mice (Fig. 3d-f). Consistently, lncCIRBIL overexpression significantly reduced the levels of KCND2 mRNA and Kv4.2 protein in subepicardium of right ventricle and abolished their transmural differences (Fig. 3g-i). In line with the effects of lncCIRBIL overexpression on I_to, the APD differences between subepicardium and subendocardium of the right ventricle were diminished in lncCIRBIL-TG mice (Fig. 3j-m). LncCIRBIL overexpression produced no influence on action potential amplitude, resting membrane potential and overshoot of right ventricular myocytes (Fig. 3n-p).

LncCIRBIL binds to UPF1 to regulate KCND2 mRNA stability

Next, we explored the potential mechanism by which lncCIRBIL regulates Kv4.2 expression. As lncCIRBIL located in the cytosol¹⁶ and changes in lncCIRBIL expression altered the level of KCND2 mRNA, we hypothesized that lncCRIBIL may affect the stability of KCND2 mRNA by interacting with certain protein in the cytoplasm. Interestingly, the mRNA degradation-related protein upstream frameshift proteins 1 (UPF1) was among the lncCIRBIL associated proteins we identified by RNA pulldown assay and mass spectrometry (MS) in the previous study¹⁶, which may be a potential target molecule contributing to the reduction of KCND2 mRNA. To test this possibility, we firstly validated the binding between lncCIRBIL and UPF1 by RNA pulldown and RNA immunoprecipitation assays. We found that the sense, but not the antisense sequence of lncCIRBIL successfully pulled down UPF1 (Fig. 4a). Meanwhile, UPF1 antibody precipitated lncCIRBIL (Fig. 4b).

We then evaluated whether UPF1 was responsible for the degradation of KCND2 mRNA and how the binding of lncCIRBIL to UPF1 influenced this process. We found that the decay half-life of KCND2 mRNA was significantly increased when UPF1 was silenced (Fig. 4c). Additionally, when lncCIRBIL was silenced in neonatal mouse ventricular myocytes, the decay half-life of KCND2 mRNA was significantly prolonged (Fig. 4d); however, overexpression of lncCIRBIL significantly shortened the decay half-life of KCND2 mRNA (Fig. 4e).

The UPF1 antibody precipitated KCND2 mRNA in WT mice, but not in lncCIRBIL knockout mice (Fig. 4f, g). To further examine the direct binding between lncCIRBIL and KCND2 mRNA, we conducted RNA-RNA pull-down using biotin pull-down assay followed by RT-qPCR. It showed that lncCIRBIL can pull down KCND2 mRNA either in the presence of UPF1 or not (Fig. 4h-j). These data indicated that UPF1 was recruited to KCND2 mRNA depending on lncCIRBIL, and the binding of lncCIRBIL and KCND2 mRNA was independent of UPF1.

UPF1 normally requires decapping of the 5' end of mRNA to initiate mRNA decay¹⁹. We further found that the decapping level of KCND2 mRNA was significantly reduced in lncCIRBIL-KO mice, while it was dramatically elevated in lncCIRBIL-TG mice (Fig. 4k). Since UPF1 plays a key role in nonsense-mediated mRNA decay (NMD), we tested whether KCND2 transcripts are subject to NMD. We used translation inhibitor Cycloheximide (CHX) to block the NMD process and found that CHX didn’t affect KCND2 transcript levels (Fig. 4l), indicating that UPF1 mediated KCND2 mRNA degradation should be in a non-NMD manner. Additionally, the expression of UPF1 did not change in the endocardium and epicardium of the right ventricle from WT and lncCIRBIL-KO mice (Fig. 4m). These data indicated that lncCIRBIL recruits UPF1 to regulate KCND2 mRNA stability in a non-NMD pathway.

Effects of human homologous conserved fragment of lncCIRBIL (hcf-CIRBIL) on KCND2

LncCIRBIL is an intergenic lncRNA of 862 nucleotides (nts) long that locates on chromosome 6 (Supplementary Fig. 5). By blasting this sequence with human genome, we found a conservative sequence motif (407 nts) of human lncCIRBIL (hcf-CIRBIL) (Supplementary Fig. 5a, b). Intriguingly, the plasma level of hcf-CIRBIL was lower in patients with J-wave syndrome than in control subjects (Fig. 5a). Furthermore, hcf-CIRBIL successfully pulled down UPF1 in human AC16 cells and in mouse heart (Fig. 5b). Overexpressing hcf-CIRBIL in AC16 cells significantly decreased the protein and mRNA levels of KCND2 (Fig. 5c), while knocking down of hcf-CIRBIL significantly upregulated the protein and mRNA levels of KCND2 (Fig. 5d).

We also explored the influence of lncCIRBIL on KCND2 in hiPSC-CM. Knockdown of hcf-CIRBIL increased I_to (Fig. 6a, b), enlarged the action potential notch and shortened APD₂₀ (Fig. 6c-f). Conversely, overexpression of hcf-CIRBIL reduced I_to (Fig. 6g-h), downregulated action potential notch and prolonged APD₂₀ (Fig. 6i-l). Transfection of hcf-CIRBIL significantly reduced the decay half-life of KCND2 mRNA (Fig. 6m). These data imply that the hcf-CIRBIL is critical in regulating I_to currents and may participate in J-wave syndrome.

Transgenic lncCIRBIL overexpression alleviated cold-related J-wave syndrome and ventricular arrhythmia susceptibility in mice

To further explore the functional role of lncCIRBIL in JWS, we established a cold-induced JWS model by exposing mice to cold temperature (4 ℃±1℃) for 6 weeks. The plasma level of lncCIRBIL was significantly lower in mice exposed to cold (4 ℃±1℃) than that in room temperature (26℃±1℃) (Fig. 7a). Cold temperature exposure significantly increased the ratio of J-point elevation, with 11 of 13 at 4±1℃ and 3 of 11 at 26 ℃±1℃ in WT mice. Cardiomyocyte transgenic overexpression of lncCIRBIL significantly inhibited J-point elevation, only 3 in 10 CIRBIL-TG mice at 4±1 ℃ (Fig. 7b-d). Furthermore, the occurrence rate of ventricular arrhythmia was significantly increased in mice exposed to cold, which was dramatically alleviated by overexpression of lncCIRBIL (Fig. 7e-g). Moreover, exposure to cold temperature significantly increased the level of Kv4.2 and KCND2 mRNA in subepi- than subendo-myocardium of right ventricle (Fig. 7h-j). These data indicated that lncCIRBIL overexpression can prevent cold exposure induced J-point elevation.

In this study, we found that lncCIRBIL deficiency enhanced the transmural difference of I_to in the right ventricle and increased the frequency of J-wave and arrhythmia susceptibility. Transgenic overexpression of lnCIRBIL in mice mitigated cold-induced J-wave and I_to transmural heterogeneity. Mechanistically, lncCIRBIL promoted the degradation of KCND2 mRNA by binding to UPF1, which led to the increase of I_to. These findings imply that lncCIRBIL is a critical regulator of J-wave syndrome and is closely related to ventricular arrhythmia predisposition.

The special role of lncRNAs in ventricular and atrial arrhythmias has been gradually elucidated by recent studies²⁰. Zhang et al. showed that lncCCRR inhibited the occurrence of ventricular arrhythmia by improving connexin43 expression in gap junction and cardiac conduction¹². LncR-KCNA2as upregulation decreases KCNA2 mRNA and Kv1.2 protein levels in rats with chronic heart failure²⁰. LncRNA TCONS-00106987 promotes atrial electrical remodeling during atrial fibrillation by sponging miR-26 to regulate KCNJ2¹⁰. In this study, we found that both the frequency of J-wave and the induction rate of ventricular arrhythmia significantly increased in lncCIRBIL knockout mice. Consistently, the plasma lncCIRBIL level in patients with J-wave syndrome was much lower than non-J-wave subjects. This observation revealed a new avenue concerning the role of lncRNA in cardiac electrophysiology.

J-wave syndrome has been associated with the disturbance of various ionic channels, including I_Na, I_to and I_CaL. In a landmark study Yan et al. showed that heterogeneous distribution of I_to mediated the J-wave, which is more significant in the right ventricle. They provided the first direct evidence in support of the hypothesis that spike-and-dome morphology of the action potential across the ventricular wall underlies the manifestation of ECG J-wave⁴. I_to-mediated action potential notch in the epicardium but not endocardium may produce a transmural voltage gradient during early ventricular repolarization that could register as a J-wave or J-point elevation on the ECG²¹. In the current study, we found that I_to current increased and action potential duration shortened in the right epicardium than endocardium of lncCIRBIL knockout mice. The same pattern of expression changes in KCND2 mRNA and Kv4.2 protein were obtained. In hiPS-CM, silencing of lncCIRBIL shortened the action potential duration and obvious notches on action potential was observed, indicating that disrupting of right ventricle I_to transmural heterogeneity by lncCIRBIL deficiency underlies J-wave syndrome.

One key mechanism for lncRNAs to exert their biological function is to directly bind to certain protein and interfere with its function²². UPF1 is well known as a key factor in a variety of RNA decay pathways²³. Recent study has shown that UPF1 also modulates normal mRNAs in some diseases. In cytoplasm, UPF1 is critical for NMD, a conserved mRNA surveillance pathway that degrades abnormal mRNA containing a premature translation termination codon. In addition to NMD, UPF1 can promote other mRNA decay pathways mediated by various RNA-binding proteins, such as staufen, glucocorticoid receptor, tudor-staphylococcal/micrococcal-like nuclease and regnase-1²⁴. More recently, it was reported that UPF1 promotes rapid degradation of m6A-containing RNAs²⁵. LncRNAs also regulate the post-transcriptional processes of mRNA degradation²⁶. For example, lncRNA 1/2-sbsRNAs induces mRNA decay through its sequence complementarity with specific target mRNAs and the recruitment of STAU1²⁷. LncRNA ICR²⁸ and SNHG5²⁹ promote mRNA stabilization via partial base-pairing with ICAM-1 and SPATS2 mRNA, respectively. In addition, the function of UPF1 in concert with lncRNA to regulate mRNA stability has been reported. Lee et al. found that linc-ASEN cooperates with UPF1 to regulate p21 mRNA stability by binding to the p21 mRNA 3’UTR³⁰.

In this study, we found that lncCIRBIL can directly bind to UPF1 and mediate the degradation of KCND2 mRNA. LncCIRBIL played a critical role in guiding the UPF1 to the KCND2 mRNA, ultimately influencing KCND2 mRNA stability. LncCIRBIL deficiency led to the dissociation of UPF1 from KCND2 mRNA, resulting in decreased decapping at 5’end 7-methyl-guanosine (m⁷G) cap in the KCND2 mRNA and increased the stability of KCND2 mRNA.

J-waves were demonstrated to be augmented by hypothermia. Yamaki et al. showed that a low room temperature can trigger J-wave syndromes, and the associated enhancement of ventricular arrhythmia vulnerability can be prevented by using I_to blockers and management of room temperature³¹. In this study, we found that overexpression of lncCIRBIL alleviated cold exposure (4℃±1℃) induced increase in incidence rate of J-waves and ventricular arrythmia susceptibility, indicating that manipulation of lncCIRBIL level may be beneficial in intervening the development of J-wave syndrome.

The present study revealed that lncCIRBIL is a critical regulator of cardiac J-wave syndrome. Deficiency of lncCIRBIL boosted the transmural heterogeneity of I_to via inhibiting UPF1 mediated degradation of KCND2 mRNA, which finally promoted J-wave formation and increased arrhytmia susceptibility. The findings highlight the novel molecular mechanism of J-wave syndrome and imply the clinical relevance of lncCIRBIL.

Human plasma samples. The human plasma samples used in this study were obtained from 10 J-wave patients and 10 non-J-wave patients, and the J-wave was diagnosed based on the electrocardiogram. The samples were collected in the First Affiliated Hospital of the Harbin Medical University (Harbin, China) from March 2021 to September 2021. The use of human samples was approved by the Institutional Review Board of College of Pharmacy, Harbin Medical University (IRB2007821) and complies with the requirements of the Declaration of Helsinki. Written informed consent of each participant was obtained.

Animals. The generation of lncCIRBIL cardiac-specific transgenic overexpression and knockout mice has been described previously¹⁶. Male adult mice (weighing 22–26 g) were used in the present study. Mice were kept with the standard animal room conditions (Temperature, 22℃; Humidity, 55% − 60%). All experiments were in accordance with the guiding principles for the care and use of laboratory animals in Harbin Medical University, and approved by the Ethics Committee for Animal Experimentation of School of Pharmacy, Harbin Medical University.

Assessment of susceptibility to ventricular arrhythmias. Mice were lightly anesthetized by intraperitoneal injection of 2% avertin (0.1 mL/10 g body weight). After anesthesia, an eight-electrode catheter (1.1F, Octapolar EP catheter; NY, Ithaca, USA) was inserted into the right ventricle via a right jugular vein. Programmed ventricular stimulation was performed by delivering ten stimuli (S1*10), followed by two additional stimuli (S2 and S3). Ventricular arrhythmia was defined as rapid non-sinus rhythm ventricular activation lasting for three or more beats.

ECG recordings. Mice were lightly anesthetized by intraperitoneal injection of 2% avertin (0.1 mL/10 g body weight) and placed in the supine position on a warming pad. Electrodes were inserted subcutaneously in the limbs and connected to an ECG amplifier.

Echocardiography. Mice were lightly anesthetized by intraperitoneal injection of 2% avertin (0.1 mL/10 g body weight) and placed in the supine or lateral position on a warming pad. Cardiac diameter and function were evaluated by M-mode echocardiography using a Vinno6 Imaging System (Vinno, China). The short-axis view was used to obtain the M-mode tracings of the left ventricle (LV) and the ultrasound beam was perpendicular to the left ventricle at the midpapillary level to determine EF, FS, wall thickness, left ventricular diameter and left ventricular volume. The mean LV dimension was derived from a minimum of six consecutive cardiac cycles per heart.

Isolation of Ventricular Myocytes. Mice were injected intraperitoneally with sodium heparin 15 min before euthanasia. Hearts were extracted via a median thoracotomy, cannulated and retrogradely perfused in a langendorff apparatus with Tyrode solution for 5 min at 37°C. Tyrode solution contained (mM): 123 NaCl, 5.4 KCl, 1.0 MgCl2, 0.33 NaH2PO4, 10 HEPES, and 10 glucoses; pH 7.4 and equilibrated with 100% O2. The perfusion was continued for 19 min with 25 mL of the same solution containing Type II collagenase (1 mg/mL). After the tissue was softened, the subendocardial and subepicardial layers were dissected from the left and right ventricular free wall. Tissue pieces were gently minced into small chunks and then stored in Tyrode solution with 200 µM CaCl2 and 1% BSA. Single rod-shaped cells were used for experiments.

Whole-cell Patch-clamp technique. Action potentials and ionic currents were recorded using the whole-cell patch clamp technique (Axopatch 700B amplifier). Action potentials were recorded at 36 ± 0.2°C in Tyrode solution. Pipettes were filled with solutions contained (mM): 130 K-glutamate; 1 MgCl2; 5 NaCl; 15 KCl; 5 MgATP ;1 CaCl2; 5 EGTA; 10 HEPES; pH 7.2 (KOH). Potassium current was recorded with external solution (mM): 138 NaCl; 5.4 KCl; 10 HEPES; 1 MgCl2; 10 Glucose; 1.8 CaCl2; 0.02 Nifedipine; pH 7.30 and pipette solution (mM): 130 L-glutamic acid; 1 MgCl2; 5 NaCl; 15 KCl; 5 MgATP; 1 CaCl2; 5 EGTA; 10 HEPES; pH 7.2). Signals were filtered at 1 kHz and data were acquired by A/D conversion (Digidata 1440, Axon Instrument).

Isolation and culture of neonatal mouse cardiomyocytes. The hearts were isolated from 1-day-old neonatal mice and washed in PBS. After adding trypsin (Beyotime, Shanghai, China) and PBS, shake them in 4°C for 10 ~ 12 hours. Then the trypsin was removed and the mixture of Collagenase type II (Gibco Invitrogen, Carlsbad, CA) and DMEM (Biological Industries, Haemek, Israel) was added. Shake them in 37°C for 10 min and collect the lysates. Repeat this step until all hearts are fully digested. After centrifugation at 1500 g for 5 min, the precipitated cells were resuspended in DMEM buffer supplemented with 5% fetal bovine serum (Biological Industries, Haemek, Israel), and 0.8% penicillin and streptomycin (Beyotime, Shanghai, China). The cells were pre-plated and cultured in humidified 5% CO2 incubator at 37°C for 1.5 h. The dissociated cardiomyocytes were then collected and plated for another 48 h before use for subsequent experiments.

AC16 cell culture. AC16 human adult ventricular cardiomyocyte cell line was grown as previously described³².The cells were maintained in DMEM F-12(#SH30023.01; HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1% fungizone (#C0222; Beyotime, Shanghai, China), and grown at 37℃ in a humid atmosphere of 5% CO2 + 95% air until they had reached 70–80% confluence. Cells were incubated under hypoxia for 12 h and then collected for RT-PCR.

Preparation of iPS-CM. Human iPSCs were purchased form NanJing Helps Company. Briefly, the cells were maintained in E8 medium supplemented with 10 µM Y-27632 in a 37°C incubator with 5% CO2. Culture dishes were coated with 1% Matrigel at least 30 minutes before cell seeding. Every 4 days, the iPSCs were subcultured using Accutase at 37°C for 3–5 minutes, and then seeded onto Matrigel-coated cell culture plates. The supernatant of iPSCs was derived from the mTeSR1 medium cultured with iPSCs for 1 day. The supernatant was filtered (using a 0.22µm filter) to remove dead cells and cell debris. The supernatant was then stored at − 80°C for at least 2 weeks. The iPSC supernatant was mixed with DMEM-HG at a ratio of 1:2 to obtain iPS-CM.

Cell transfection. Transfections were carried out using lipofectamine 2000 reagent (Invitrogen, Carlsbad, America) or X-treme gene siRNA transfection reagent (Roche, Basel, Switzerland) according to the manufacturer’s protocol. The sequences used in this study are shown in Online Table Ⅱ.

RNA extraction and qRT-PCR. Total RNA was extracted from cells or tissues by using Trizol reagent (Invitrogen, Carlsbad, America) according to the manufacturer’s protocol and cDNA was synthesized using the Trans-Script Allin-one First-strand cDNA Synthesis Supermix for qPCR Kit (TransGen Biotech, Beijing, China). Real-time quantitative PCR was performed useing the SYBR Green Master (Roche, Basel, Switzerland), and cDNA was amplified using the primers listed in Online Table Ⅲ. The relative expression levels were calculated based on Ct values and were normalized to GAPDH.

mRNA stability assay. To detect the effects of lncCIRBIL and UPF1 on KCND2 mRNA stability, Actinomycin D (Act D) (Selleckchem, Houston, TX, USA) was added into cells with a final concentration of 5 µg/mL. Total RNA was extracted after 0, 1, 2, 3, and 4 hours of incubation with Actinomycin D and used for qRT-PCR.

Decapping Analysis. Decapped mRNA was quantified as previously described³³. Briefly, 10 µg of total RNA was treated with RNaseOUT and ligated with the rP5_RND primer (Online Table Ⅲ), which binds specifically to 5′ ends lacking the cap structure and presenting a phosphate group. Afterwards, 1 µg were used for cDNA synthesis, and qRT-PCR was performed using the primers indicated in Online Table Ⅲ. Values were normalized to total mRNA expression in each sample.

RNA-RNA pulldown assay. RNA-RNA pulldown assay was carried out employing an optimized streptavidin-binding RNA aptamer (a4×S1m tag)³⁴. Briefly, full-length lncCIRBIL was cloned into pcDNA3 backbone and followed by the 4×S1m tag. S1m-plasmids with and without lncCIRBIL were digested overnight at 37℃ by EcoRV-HF (New England Biolabs, MA, United Statesand). The linearized plasmids were transcribed into RNA probes in vitro by T7 RNA polymerase (Roche, Mannheim, Germany). The lncCIRBIL RNA probe and control were individually incubated with streptavidin beads (Invitrogen, Shanghai, China) for 2 h at 4℃ under rotation to couple together. Neonatal mouse ventricular myocytes were harvested and re-suspended with lysis bufferA (150 mM KCl; 25 mM Tris, pH 7.4; 5 mM EDTA; 0.5% NP40; 0.5 mM DTT; 100 U/mL RNase inhibitor). Subsequently, the cell samples were sonicated gently on ice and then centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was precleared with streptavidin Dynabeads for 2 h at 4℃ under rotation and followed by taking 10% volume of cell lysis as input. Meanwhile, the beads were coupled by RNA probe and then incubated with pre-cleared cell lysis supernatant for 2 h at 4℃ under rotation to assemble RNA-RNA complexes. Subsequently, the lncCIRBIL-KCND2 mRNA complexes conjugated with streptavidin beads were eluted employing 20 mM biotin (Roche, Mannheim, Germany). Finally, all input and the eluted samples were suppled Trizol and extracted RNA to evaluate LncCIRBIL-mediated RNA binding affinity.

Western blot. Ventricular myocardium was lysed in RIPA buffer (Beyotime, Beijing, China) in the presence of protease inhibitor cocktail (Roche, Basel, Switzerland) at 4°C. Lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Pall Corporation, Mexico, USA) with Trans-Blot Turbo system (Bio-Rad). The membranes were blocked in Blocking Buffer (GenScript, Piscataway, USA). The primary anti-Kv4.2 (Proteintech, Chicago, USA), anti-Upf1 (Abcam, Cambridge, UK) and anti-GAPDH (Proteintech, Chicago, USA) were used. After washing with PBST (phosphate-buffered saline (PBS) with Tween), the membranes were incubated with the secondary antibody at room temperature for 60 min. The western blot bands were captured and analyzed by ODYSSEY machine (LI-COR, American).

RNA-binding protein immunoprecipitation. RIP was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) according to the manufacturer’s instructions, followed by real-time RT-PCR using the SYBR Green Master (Roche, Basel, Switzerland).

RNA pulldown assay. LncCIRBIL was in vitro synthesized and labeled with biotin (Roche Diagnostics GmbH, Lot: 31385421, Mannheim, Germany). Then the biotinylated RNAs were incubated with cell extracts, and pulled down with streptavidin beads. The mixture was shaken violently at a shaking table for 1 h and then centrifuged. The supernatant was discarded and the RNA-binding-protein complex was obtained by elution and precipitation. The lncRNA-interacting proteins were further separated by SDS-PAGE. Western blot was used to analyze the bound proteins. The antisense RNA served as a control.

Statistical analysis. All data were expressed as mean ± standard deviation. Two groups were compared by analysis of unpaired Student’s t-test, and for all electrophysiological parameters, these criteria were met. The fisher exact test was used to compare categoric variables. To determine main and interaction effects of site (subepicardium versus subendocardium) and genotype (WT versus CIRBIL-KO/CIRBIL-TG), 2-way ANOVAs were performed, followed by Bonferroni-corrected t-tests. Level of significance was set at P < 0.05. Statistical analysis of all data was performed by using GraphPad prism 8.0 software.

Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data Availability

The data that support the findings of this study and unique materials are available from the corresponding authors upon reasonable request. The lncCIRBIL microarray data are deposited to the Gene Expression Omnibus (GSE) with the accession number

GSE161151.

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Acknowledgements

This work was supported by National Natural Science Foundation of China (U21A20339 to B. Y.; 82270320 to D. Y.; 82070344, 81870295 to Z. P.), Heilongjiang Touyan Innovation Team Program and CAMS Innovation Fund for Medical Sciences (CIFMS, 2019-I2M-5-078 to B. Y.) and HMU Marshal Initiative Funding (HMUMIF-21017 to Z.P).

Author contributions

X.J., W.M., Y.Z., H.G., D.Y., and Y.G. performed experiments, analyzed data, and prepared the manuscript. D.L., S.L., J.S., and J.L. provided experimental guidance and participated in discussions. J.M. and L.Z. helped perform experiments and collect data. Y.L., B.Y., and Y.L. oversaw the project and proofread the manuscript. Z.P. designed the project, oversaw the experiments, and prepared the manuscript.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains supplementary material

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Correspondence and requests for materials should be addressed to Yue Li, Baofeng Yang or Zhenwei Pan.

There is NO Competing Interest.

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Deficiency of LncRNA-CIRBIL promotes J-wave syndrome by enhancing transmural heterogeneity of I_to current

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