Calcium-mediated DAD in membrane potentials and triggered activity in atrial myocytes of ETV1f / fMyHCCre /+ mice.

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

Telomerase Reverse Transcriptase and p53 Regulate Mammalian Peripheral Nervous System and CNS Axon Regeneration Downstream of c-Myc.

Although several genes have been identified to promote axon regeneration in the CNS, our understanding of the molecular mechanisms by which mammalian axon regeneration is regulated is still limited and fragmented. Here by using female mouse sensory axon and optic nerve regeneration as model systems, we reveal an unexpected role of telomerase reverse transcriptase (TERT) in regulation of axon regeneration. We also provide evidence that TERT and p53 act downstream of c-Myc to control sensory axon regeneration. More importantly, overexpression of p53 in sensory neurons and retinal ganglion cells is sufficient to promote sensory axon and optic never regeneration, respectively. The study reveals a novel c-Myc-TERT-p53 signaling pathway, expanding horizons for novel approaches promoting CNS axon regeneration.SIGNIFICANCE STATEMENT Despite significant progress during the past decade, our understanding of the molecular mechanisms by which mammalian CNS axon regeneration is regulated is still fragmented. By using sensory axon and optic nerve regeneration as model systems, the study revealed an unexpected role of telomerase reverse transcriptase (TERT) in regulation of axon regeneration. The results also delineated a c-Myc-TERT-p53 pathway in controlling axon growth. Last, our results demonstrated that p53 alone was sufficient to promote sensory axon and optic nerve regeneration in vivo Collectively, the study not only revealed a new mechanisms underlying mammalian axon regeneration, but also expanded the pool of potential targets that can be manipulated to enhance CNS axon regeneration.

Inhibition of PTEN activity promotes IB4-positive sensory neuronal axon growth.

Traumatic nerve injuries have become a common clinical problem, and axon regeneration is a critical process in the successful functional recovery of the injured nervous system. In this study, we found that peripheral axotomy reduces PTEN expression in adult sensory neurons; however, it did not alter the expression level of PTEN in IB4-positive sensory neurons. Additionally, our results indicate that the artificial inhibition of PTEN markedly promotes adult sensory axon regeneration, including IB4-positive neuronal axon growth. Thus, our results provide strong evidence that PTEN is a prominent repressor of adult sensory axon regeneration, especially in IB4-positive neurons.

c-Myc controls the fate of neural progenitor cells during cerebral cortex development.

The anatomical structure of the mammalian cerebral cortex is the essential foundation for its complex neural activity. This structure is developed by proliferation, differentiation, and migration of neural progenitor cells (NPCs), the fate of which is spatially and temporally regulated by the proper gene. This study was used in utero electroporation and found that the well-known oncogene c-Myc mainly promoted NPCs' proliferation and their transformation into intermediate precursor cells. Furthermore, the obtained results also showed that c-Myc blocked the differentiation of NPCs to postmitotic neurons, and the expression of telomere reverse transcriptase was controlled by c-Myc in the neocortex. These findings indicated c-Myc as a key regulator of the fate of NPCs during the development of the cerebral cortex.

TGFß1 and HGF regulate CTGF expression in human atrial fibroblasts and are involved in atrial remodelling in patients with rheumatic heart disease.

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

Regulation of adult mammalian intrinsic axonal regeneration by NF-κB/STAT3 signaling cascade.

The inflammatory response is a critical regulator for the regeneration of axon following nervous system injury. Nuclear factor-kappa B (NF-κB) is characteristically known for its ubiquitous role in the inflammatory response. However, its functional role in adult mammalian axon growth remains elusive. Here, we found that the NF-κB signaling pathway is activated in adult sensory neurons through peripheral axotomy. Furthermore, inhibition of NF-κB in peripheral sensory neurons attenuated their axon growth in vitro and in vivo. Our results also showed that NF-κB modulated axon growth by repressing the phosphorylation of STAT3. Furthermore, activation of STAT3 significantly promoted adult optic nerve regeneration. Taken together, the findings of our study indicated that NF-κB/STAT3 cascade is a critical regulator of intrinsic axon growth capability in the adult nervous system.

Calcium/calmodulin-dependent protein kinase II regulates mammalian axon growth by affecting F-actin length in growth cone.

While axon regeneration is a key determinant of functional recovery of the nervous system after injury, it is often poor in the mature nervous system. Influx of extracellular calcium (Ca2+ ) is one of the first phenomena that occur following axonal injury, and calcium/calmodulin-dependent protein kinase II (CaMKII), a target substrate for calcium ions, regulates the status of cytoskeletal proteins such as F-actin. Herein, we found that peripheral axotomy activates CaMKII in dorsal root ganglion (DRG) sensory neurons, and inhibition of CaMKII impairs axon outgrowth in both the peripheral and central nervous systems (PNS and CNS, respectively). Most importantly, we also found that the activation of CaMKII promotes PNS and CNS axon growth, and regulatory effects of CaMKII on axon growth occur via affecting the length of the F-actin. Thus, we believe our findings provide clear evidence that CaMKII is a critical modulator of mammalian axon regeneration.

An evaluation of the clinical efficacy of the application of 28mm cryoballoon for linear ablation of left atrial apex combined with enlarged pulmonary vein vestibule ablation for persistent atrial fibrillation.

OBJECTIVE: to retrospectively investigate the efficacy and safety of the application of 28 mm cryoballoon for pulmonary vein electrical isolation (PVI) combined with top left atrial linear ablation and pulmonary vein vestibular expansion ablation for persistent atrial fibrillation. METHODS: From July 2016 to December 2020, 413 patients diagnosed with persistent atrial fibrillation were evaluated, including 230 (55.7%) in the PVI group (PVI only) and 183 (44.3%) in the PVIPLUS group (PVI plus ablation of the left atrial apex and pulmonary vein vestibule). The safety and efficacy of the two groups were retrospectively analyzed. RESULTS: The AF/AT/AFL-free survival rates at 6, 12, 18, 24 and 30 months after procedure was 86.6%, 72.6%, 70.0%, 61.1% and 56.3% in the PVI group and 94.5%, 87.0%, 84.1%, 75.0% and 67.9% in the PVIPLUS group, respectively. At 30 months after procedure, the AF/AT/AFL-free survival rate was significantly higher in the PVIPLUS group than in the PVI group (P = 0.036; HR:0.63; 95% CI:0.42 to 0.95). CONCLUSION: The application of 28-mm cryoballoon for pulmonary vein electrical isolation combined with linear ablation of the left atrial apex and expanded ablation of the pulmonary vein vestibule improves the outcome of persistent atrial fibrillation.

[Effect of hepatocyte growth factor and transforming growth factor-ß(1) on atrial fibroblasts fibrosis].

OBJECTIVE: To investigate the effect of hepatocyte growth factor (HGF) and transforming growth factor-ß(1) (TGFß(1)) on the expression of α-smooth muscle actin (α-SMA) and collagen I in human atrial fibroblast in vitro, and to explore the possible molecular mechanism of atrial fibrosis in patients with atrial fibrillation (AF). METHODS: Human atrial fibroblast, isolated from aseptic right atrial appendage tissues of 10 sinus rhythm (SR) and 10 chronic atrial fibrillation (CAF) patients, were cultured with HGF and TGFß(1). mRNA expressions of collagen I and α-SMA were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), the protein expression of α-SMA was determined by immunofluorescence and Western blot. RESULTS: (1) Compared with SR group, left atrium was significantly dilated in CAF group (t = 2.692, P < 0.05), the mRNA expression of collagen I and α-SMA of atrial fibroblasts were significantly upregulated (all P < 0.01), mRNA expression of collagen I was positively correlated with left atrial dimension (LAD) (r = 0.836, P = 0.014), AF duration (r = 0.739, P = 0.045) and α-SMA mRNA level (r = 0.886, P = 0.012). (2) Compared with SR group, the expression of α-SMA protein in CAF atrial fibroblasts were significantly increased (P < 0.01). (3) TGFß(1) further stimulated while HGF significantly attenuated the expression of collagen I and α-SMA in CAF atrial fibroblasts (all P < 0.01). CONCLUSIONS: Increasing expression of collagen I and α-SMA in human atrial fibroblasts might promote atria remodeling leading to the development and sustaining of AF. HGF is involved in the negative regulation on the expression of α-SMA and collagen I.

Efficacy of extended antrum ablation based on substrate mapping plus pulmonary vein isolation in the treatment of atrial fibrillation.

INTRODUCTION AND OBJECTIVES: Pulmonary vein isolation (PVI) technique has become the cornerstone of atrial fibrillation (AF) catheter ablation. The objective of this study was to assess the efficacy and safety of extended antrum ablation based on electrophysiological substrate mapping plus PVI in AF patients who underwent cryoballoon ablation. METHODS: In this observational study, a total of 121 paroxysmal AF patients and 80 persistent AF patients who did not achieve the procedure endpoint after cryoballoon ablation received extra extended antrum ablation (EAA) based on electrophysiological substrate mapping via radiofrequency ablation (EAA group). As a control group (PVI group), among paroxysmal AF and persistent AF patients, we conducted a propensity score-matched cohort, in whom only PVI was completed. RESULTS: The average follow-up time was 15.27±7.34 months. Compared with PVI group, paroxysmal AF patients in the EAA group had a significantly higher rate of AF-free survival (90.1% vs. 80.2%, p=0.027) and AF, atrial flutter, or atrial tachycardia (AFLAT) -free rate survival (89.3% vs. 79.3%, p=0.031). Persistent AF patients in the EAA group also had a significantly higher rate of AF-free survival (90.0% vs. 75.0%, p=0.016) and AFLAT-free survival (88.8% vs. 75.0%, p=0.029) than PVI group. Complication rates did not significantly differ between both groups, in either paroxysmal AF or persistent AF patients. CONCLUSION: Our findings demonstrate that extra extended antrum ablation based on electrophysiological substrate mapping is effective and safe. Moreover, the strategy can improve the outcome of AF cryoablation.

Inhibitory Effects of Cyclopiazonic Acid on the Pacemaker Current in Sinoatrial Nodal Cells.

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

Different intervals of ovum pick-up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos.

The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used as cytoplast donors. The rates of development of the reconstituted embryos to the blastocyst stage were 23.1% (Group I), 15.0% (Group II), 10.9% (Group III), 4.9% (Group IV), and 29.0% (Group V). The results indicate that the developmental potential of follicles from the same living donors were different when different intervals of OPU were adopted and early atretic follicles from the second follicular wave had higher competence to support the early development of cloned bovine embryos.

Differential Roles of Glycogen Synthase Kinase 3 Subtypes Alpha and Beta in Cortical Development.

Glycogen synthase kinases 3 (GSK3) α and ß are expressed in the nervous system, and disruption of GSK3 signaling has been implicated in a wide range of neurodevelopmental and psychiatric disorders. Although several studies have established a role of GSK3 signaling in the nervous system, much less is known about isoform-specific functions. Here, we have examined the role of GSK3α and GSK3ß in the developing neocortex by performing in utero electroporation with specific small interfering RNAs targeting each isoform. We found that depletion of either GSK3α or GSK3ß commonly promoted the proliferation of neural progenitor cells in the ventricular zone, but at later stages, knocking down of each isoform resulted in distinct outcomes. In particular, the transformation of radial progenitors to intermediate progenitor cells was promoted in GSK3α-depleted cells, but markedly prevented in GSK3ß-depleted cells. Moreover, knocking down of GSK3ß but not GSK3α prevented the generation of upper-layer Cux1+ neurons. Consistent with the distinct outcomes, protein levels of c-Myc and ß-catenin, well-known substrates of GSK3, were differentially affected by depletion of GSK3α and GSK3ß. Together, these results suggest that GSK3α and GSK3ß might play distinct roles in the genesis and differentiation of neuronal lineage cells during neocortex development by differential regulation of downstream signaling pathways.

[Establish a transgenic mice model harboring structural genes of hepatitis C virus].

OBJECTIVES: To establish an animal model of HCV transgenic mice to elucidate the pathogenesis of hepatitis C virus infection and function of the viral structural proteins. METHODS: Structural gene of HCV were amplified and recombined into eukaryotic expression vectors, pcDNA4HisMax and pMT/BiP/V5-His A, after their expressive activity was confirmed to detect the structural protein in the transfected COS7 and S2 cells by Western blot. The fertilized expression element, which contained CMV or pMT promoter, structural gene of HCV and polyadenylation signal sequence, was microinjected into 1736 C57BL/6 mouse fertilized ova. The ova were then replanted into the oviducts of 69 pseudopregnant recipient mice. RESULTS: Twenty-five recipient mice were impregnated and later produced 105 newborns; 49 of them died from unknown causes and 57 survived. After the specific HCV structural genes were identified by PCR and Southern blot hybridization, 26 founders were obtained; among them 10 were stable expression mice and 16 were the inducible ones. The rate of founders developed from implanted embryos was only 1.50%. Through hybridization with normal mice, 58 hybrid mice have been obtained at present. CONCLUSION: Two kinds of different transgenic mice of HCV were developed; one is of stable expression, and the other is inducible. This transgenic mice model may create an opportunity for studying the function of the structural gene of HCV and elucidate its pathogenicity.

Generation and characterization of transgenic mice expressing tamoxifen-inducible cre-fusion protein specifically in mouse liver.

AIM: To establish transgenic mice expressing tamoxifen-inducible Cre-ERt recombinase specifically in the liver and to provide an efficient animal model for studying gene function in the liver and creating various mouse models mimicking human diseases. METHODS: Alb-Cre-ERt transgenic mice were produced by microinjecting the construct with Cre-ERt fusion gene of DNA fragments into fertilized eggs derived from inbred C57BL/6 strain. Transgenic mice were identified by using PCR and Southern blotting. Expression of Cre-ERt fusion gene was analyzed in the liver, kidney, brain and lung from F1 generation transgenic mice at 8 weeks of age by reverse transcription (RT)-PCR. RESULTS: Four hundred and fourteen fertilized eggs of C57 BL/6 mice were microinjected with recombinant Alb-Cre-ERt DNA fragments, and 312 survival eggs injected were transferred to the oviducts of 12 pseudopregnant recipient mice, 6 of 12 recipient mice became pregnant and gave birth to 44 offsprings. Of the 44 offsprings, two males and one female carried the hybrid Cre-ERt fusion gene. Three mice were determined as founders, and were back crossed to set up F1 generations with other inbred C57BL/6 mice. Transmission of Cre-ERt fusion gene in F1 offspring followed Mendelian rules. The expression of Cre-ERt mRNA was detected only in the liver of F1 offspring from two of three founder mice. CONCLUSION: Transgenic mice expressing tamoxifen-inducible Cre-ERt recombinase under control of the liver-specific promoter are preliminary established.

Isolation and characterization of human anti-VEGF165 monoclonal antibody with anti-tumor efficacy from transgenic mice expressing human immunoglobulin loci.

The purpose of this study was to prepare a fully human anti-VEGF (vascular endothelial growth factor) monoclonal antibody with anti-tumor activity from five-feature mice which express human immunoglobin loci. Four hybridomas secreting mAb stably were isolated successfully. Some characters such as isotypes, cross-reactivity, inhibition on the binding of hVEGF to VEGFR-2, dissociation constants and the idiotypic characteristic were determined. Proliferation of T24 and Ls-174-T cell line and nude mice bearing human colorectal cancer were used to evaluate therapeutic effects and safety of this mAb. Pharmacokinetics data shows the half life of this mAb was about 5 days after a single intravenous injection. These results suggest the fully human anti-VEGF mAb maybe safe and efficient for cancer treatment.

[Knockout serum replacement improves establishment efficiency of C57BL/6J mouse embryonic stem cell line].

AIM: To eliminate the influence of serum on self-renewal of embryonic stem cells (ESCs), knockout serum replacement (KSR), a defined formulation, was used to replace serum for the establishment of C57BL/6J mouse ESC line. METHODS: C57BL/6J mouse blastocysts collected at 3.5 days post coitum (d.p.c.) were cultured in the medium supplemented with KSR. In control experiment, KSR was substituted by fetal bovine serum (FBS). When ESC line was established, the morphology of ESCs, the expression of alkaline phosphatase and oct-4, and the karyotype and differentiating ability of ESCs were analyzed. RESULTS: 13 blastocysts were cultured in the medium supplemented with KSR and one ESC line (MES-1) was established with a normal and stable XX karyotype after cultured for more than 20 passages, and then the high expression of alkaline phosphatase and oct-4 was detected. When cultured in suspension, MES-1 formed embryoid bodies. When inoculated subcutaneously into nude mice, MES-1 formed teratoma. After injected into ICR mouse blastocysts collected at 3.5 d.p.c., MES-1 incorporated into the inner cell mass of the host blastocyst and contributed to the development of a chimera. In control experiment, no ESC lines were cultured for more than 3 passages. CONCLUSION: KSR can be efficiently used to isolate and culture C57BL/6J mouse ESCs, which can eliminate traditional prescreening of FBS suitable for isolation and culture of ESCs.

Secondary SCNT doubles the pre-implantation development rate of reconstructed interspecies embryos by using cytoplasts of Sannen dairy goat ova.

The aim of this study was to investigate whether ova of Sannen goat could support the pre-implantation development of interspecies embryos constructed through somatic cell nucleus transfer (SCNT) embryos and whether secondary SCNT (SSCNT) could improve the pre-implantation development of those embryos. The primary SCNT (PSCNT) embryos were produced by using Sannen goat ovum cytoplasts as recipients and fibroblast cells, derived from human, rabbit and Boer goat skins, as nucleus donors. The blastomeres of 8 to 16 cells stage of PSCNT embryos were subsequently used as nucleus donor cells and Sannen goat ovum cytoplasts as recipients to evaluate the effect of SSCNT on the pre-implantation development rate of these reconstructed interspecies embryos. Our results indicate that the pre-implantation development rates of SSCNT embryos reconstructed using these three different blastomeres are almost twice of that of corresponding PSCNT embryos (human, 15.8% vs. 7.8%; rabbit, 27.9% vs. 12.5%; Boer goat 55.3% vs. 24.5%; P < 0.05 in all three cases). The time durations that embryos need for the serial events of remodeling and reprogramming to take place vary, depending on the animal species of nucleus donors. These data suggest that remodeling and reprogramming of donor nucleus may be enhanced by prolonged exposure of donor nucleus to maternal cytoplast. We conclude that Sannen goat cytoplast can support the pre-implantation development of embryos constructed with nuclei from various donors, including fibroblasts of human, rabbit and Boer goat; and the somatic nucleus derived from different species requires more time to achieve its reprogramming necessary for pre-implantation development.