Abstract-Cardiac arrhythmia is a common and often lethal manifestation of many forms of heart disease. Gap junction remodeling has been postulated to contribute to the increased propensity for arrhythmogenesis in diseased myocardium, although a causative role in vivo remains speculative. By generating mice with cardiac-restricted knockout of connexin43 (Cx43), we have circumvented the perinatal lethal developmental defect associated with germline inactivation of this gap junction channel gene and uncovered an essential role for Cx43 in the maintenance of electrical stability. Mice with cardiac-specific loss of Cx43 have normal heart structure and contractile function, and yet they uniformly (28 of 28 conditional Cx43 knockout mice observed) develop sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age. Optical mapping of the epicardial electrical activation pattern in Cx43 conditional knockout mice revealed that ventricular conduction velocity was significantly slowed by up to 55% in the transverse direction and 42% in the longitudinal direction, resulting in an increase in anisotropic ratio compared with control littermates (2.1Ϯ0.

BACKGROUNDTransthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODSAfter conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTSPreclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONSIn a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.

Abstract-Connexin43 (Cx43), the predominant ventricular gap junction protein, is critical for maintaining normal cardiac electrical conduction, and its absence in the mouse heart results in sudden arrhythmic death. The mechanisms linking reduced Cx43 abundance in the heart and inducibility of malignant ventricular arrhythmias have yet to be established. In this report, we investigate arrhythmic susceptibility in a murine model genetically engineered to express progressively decreasing levels of Cx43. Progressively older cardiac-restricted Cx43 conditional knockout (CKO) mice were selectively bred to produce a heart-specific Cx43-deficient subline ("O-CKO" mice) in which the loss of Cx43 in the heart occurs more gradually. O-CKO mice lived significantly longer than the initial series of CKO mice but still died suddenly and prematurely. At 25 days of age, cardiac Cx43 protein levels decreased to 59% of control values (PϽ0.01), but conduction velocity was not significantly decreased and no O-CKO mice were inducible into sustained ventricular tachyarrhythmias. By 45 days of age, cardiac Cx43 abundance had decreased in a heterogeneous fashion to 18% of control levels, conduction velocity had slowed to half of that observed in control hearts, and 80% of O-CKO mice were inducible into lethal tachyarrhythmias. Enhanced susceptibility to induced arrhythmias was not associated with altered invasive hemodynamic measurements or changes in ventricular effective refractory period. Thus, moderately severe reductions in Cx43 abundance are associated with slowing of impulse propagation and a dramatic increase in the susceptibility to inducible ventricular arrhythmias. Key Words: connexin43 Ⅲ arrhythmia Ⅲ electrophysiology Ⅲ heart Ⅲ mice V entricular tachyarrhythmias are a frequent cause of sudden cardiac death in ischemic and nonischemic heart disease. Despite intense investigation, molecular mechanisms underlying the propensity of diseased myocardium to initiate and propagate lethal arrhythmias are incompletely understood. In recent years, a number of genetically engineered murine models have been developed to explore the pathophysiology of arrhythmogenesis. Although many such mice display increases in the frequency of spontaneous or inducible ventricular ectopy, in almost all cases this activity is self-limited and has not been shown to be the proximate cause of death. 1-4 Several other mutant mouse models are able to support sustained ventricular arrhythmias, but these require provocative stimuli, such as anesthesia or exercise with administration of adrenergic agents. [5][6][7][8] To date, only the heartspecific connexin43 (Cx43) conditional knockout (CKO) mouse has been shown to die prematurely from spontaneous sustained ventricular tachyarrhythmias. 9 Because of the arrhythmic propensity of the Cx43 CKO mouse and the ease of inducible sustained arrhythmias, 10 it has served as an ideal model for the study of basic mechanisms of arrhythmia.In myopathic hearts, abnormal expression of Cx43 (gap junction remodeling) may contri...

GJA1 (also known and referred to here as connexin 43 and abbreviated CX43) is the predominant testicular gap junction protein, and CX43 may regulate Sertoli cell maturation and spermatogenesis. We hypothesized that lack of CX43 would inhibit Sertoli cell differentiation and extend proliferation. To test this, a Sertoli cell-specific Cx43 knockout (SC-Cx43 KO) mouse was generated using Cre-lox technology. Immunohistochemistry indicated that CX43 was not expressed in the Sertoli cells of SC-Cx43 KO mice, but was normal in organs such as the heart. Testicular weight was reduced by 41% and 76% in SC-Cx43 KO mice at 20 and 60 days, respectively, vs. wild-type (wt) mice. Seminiferous tubules of SC-Cx43 KO mice contained only Sertoli cells and actively proliferating early spermatogonia. Sertoli cells normally cease proliferation at 2 wk of age in mice and become terminally differentiated. However, proliferating Sertoli cells were present in SC-Cx43 KO but not wt mice at 20 and 60 days of age. Thyroid hormone receptor alpha (THRA) is high in proliferating Sertoli cells, then declines sharply in adulthood. Thra mRNA expression was increased in 20-day SC-Cx43 KO vs. wt mice, and it showed a trend toward an increase in 60-day mice, indicating that loss of CX43 in Sertoli cells inhibited their maturation. In conclusion, we have generated mice lacking CX43 in Sertoli cells but not other tissues. Our data indicate that CX43 in Sertoli cells is essential for spermatogenesis but not spermatogonial maintenance/proliferation. SC-Cx43 KO mice showed continued Sertoli cell proliferation and delayed maturation in adulthood, indicating that CX43 plays key roles in Sertoli cell development.

Exposure of blood to tissue factor (TF) activates the extrinsic (TF:FVIIa) and intrinsic (FVIIIa:FIXa) pathways of coagulation. In this study, we found that mice expressing low levels of human TF (Ϸ1% of wild-type levels) in an mTF ؊/؊ background had significantly shorter lifespans than wild-type mice, in part, because of spontaneous fatal hemorrhages. All low-TF mice exhibited a selective heart defect that consisted of hemosiderin deposition and fibrosis. Direct intracardiac measurement demonstrated a 30% reduction (P < 0.001) in left ventricular function in 8-month-old low-TF mice compared with age-matched wild-type mice. Mice expressing low levels of murine FVII (Ϸ1% of wild-type levels) exhibited a similar pattern of hemosiderin deposition and fibrosis in their hearts. In contrast, FIX ؊/؊ mice, a model of hemophilia B, had normal hearts. Cardiac fibrosis in low-TF and low-FVII mice appears to be caused by hemorrhage from cardiac vessels due to impaired hemostasis. We propose that TF expression by cardiac myocytes provides a secondary hemostatic barrier to protect the heart from hemorrhage. E xpression of tissue factor (TF) by adventitial fibroblasts and vascular smooth muscle cells surrounding blood vessels provides a hemostatic barrier that activates coagulation when vascular integrity is disrupted (1). TF is also expressed by cardiac muscle but not by skeletal muscle (1). TF functions as the high-affinity cellular receptor for FVII͞VIIa (2). The coagulation protease cascades are comprised of the extrinsic (TF:FVIIa) and intrinsic (FVIIIa:FIXa) pathways, which together maintain hemostasis (3).Many murine models of coagulation have been generated that provide new insights into the role of the various procoagulant and anticoagulant proteins in hemostasis (4). For instance, FV Leiden/Leiden mice, which express an FV variant that is resistant to inactivation by activated protein C, and TM Pro/Pro mice, which express a mutated version of thrombomodulin (TM) with reduced thrombin binding, both exhibit prothrombotic phenotypes with increased fibrin deposition in select tissues (5-7). Mice with prohemorrhage phenotypes include models of hemophilia A (FVIII Ϫ/Ϫ ) and B (FIX Ϫ/Ϫ ), as well as fibrinogen-deficient mice (Fbg Ϫ/Ϫ ) and thrombocytopenic mice (NF-E2 Ϫ/Ϫ ) (8-12). Mice with complete deficiencies in TF, FVII, FX, FV, and prothrombin die in utero or shortly after birth (4). We and others have generated mice expressing low levels (Ͻ0.1-1% of wild-type levels) of human TF, murine FVII, and murine FV (13-15). We have shown that low-TF mice have impaired uterine hemostasis (16). A similar phenotype is observed with low-FVII mice.In this study, we performed a detailed characterization of low-TF mice. These mice exhibited shorter lifespans than wildtype mice. Histological analysis of various tissues of low-TF mice revealed hemosiderin deposition and fibrosis selectively in their hearts. Our data suggest that cardiac fibrosis in low-TF mice is caused by hemorrhage from cardiac vessels due to impaired hemostasis. M...

In the uterus, the formation of new maternal blood vessels in the stromal compartment at the time of embryonic implantation is critical for the establishment and maintenance of pregnancy. Although uterine angiogenesis is known to be influenced by the steroid hormones estrogen (E) and progesterone (P), the underlying molecular pathways remain poorly understood. Here, we report that the expression of connexin 43 (Cx43), a major gap junction protein, is markedly enhanced in response to E in uterine stromal cells surrounding the implanted embryo during the early phases of pregnancy. Conditional deletion of the Cx43 gene in these stromal cells and the consequent disruption of their gap junctions led to a striking impairment in the development of new blood vessels within the stromal compartment, resulting in the arrest of embryo growth and early pregnancy loss. Further analysis of this phenotypical defect revealed that loss of Cx43 expression resulted in aberrant differentiation of uterine stromal cells and impaired production of several key angiogenic factors, including the vascular endothelial growth factor (Vegf). Ablation of CX43 expression in human endometrial stromal cells in vitro led to similar findings. Collectively, these results uncovered a unique link between steroid hormone-regulated cell-cell communication within the pregnant uterus and the development of an elaborate vascular network that supports embryonic growth. Our study presents the first evidence that Cx43-type gap junctions play a critical and conserved role in modulating stromal differentiation, and regulate the consequent production of crucial paracrine signals that control uterine neovascularization during implantation.

Background-Heterogeneous remodeling of gap junctions is observed in many forms of heart disease. The consequent loss of synchronous ventricular activation has been hypothesized to result in diminished cardiac performance. To directly test this hypothesis, we designed a murine model of heterogeneous gap junction channel expression. Methods and Results-We generated chimeric mice formed from connexin43 (Cx43)-deficient embryonic stem cells and wild-type or genetically marked ROSA26 recipient blastocysts. Chimeric mice developed normally, without histological evidence of myocardial fibrosis or hypertrophy. Heterogeneous Cx43 expression resulted in conduction defects, however, as well as markedly depressed contractile function. Key Words: ion channels Ⅲ contractility Ⅲ conduction Ⅲ genes Ⅲ arrhythmia G ap junctions play a pivotal role in the coordinated excitation of the heart. 1 Formed from the assembly of a family of proteins known as connexins, gap junction channels in the myocardium are preferentially targeted to the intercalated disks between adjacent myocytes and account in part for anisotropic conduction in normal cardiac tissue. 2 In a wide spectrum of cardiac disease states, abnormalities in connexin subcellular localization is observed. [3][4][5][6][7][8][9][10] Increasing experimental evidence, including recent murine genetic studies, links this pathological remodeling of gap junctions to cardiac conduction abnormalities and rhythm disturbances. 8,11,12 Remodeling of gap junctions is often focal, a spatial pattern that might result in discrete areas of conduction defects within the myocardium. 7,13 Although experimental data are lacking, it has been hypothesized that remodeling could disrupt wave-front propagation, interfere with coordination of myocyte contraction, and diminish contractile performance. 7,14 Accordingly, in this study, we designed a model of heterogeneous gap junction expression in the heart to test the hypothesis that loss of synchronous ventricular activation diminishes cardiac contractile performance. We generated chimeric mice, formed from connexin 43 (Cx43)-deficient embryonic stem (ES) cells and wild-type or genetically marked ROSA26 recipient blastocysts, to model the heterogeneous pattern of gap junction expression typical of diseased myocardium. In contrast to the uniform conduction slowing in Cx43 conditional knockout mice, 12 optical maps of chimeric hearts with voltage-sensitive dye showed highly irregular epicardial activation profiles. Moreover, chimeric mice developed significant contractile defects, supporting the hypothesis that loss of synchronous electrical activation of the ventricular myocardium from gap junction remodeling leads to systolic dysfunction. Methods Gene Targeting and Production of Cx43؊/؊ Chimeric MiceThe generation of Cx43 ϩ/flox ES cells (129/Sv) harboring a "floxed" Cx43 allele has been described previously. Western Blot AnalysisWestern blot analyses were performed with a rabbit polyclonal antibody to Cx43 and a mouse monoclonal antibody to tubulin (...

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21 cip1. and p16 INK4a , and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.