Insulin and contraction increase GLUT4 translocation in skeletal muscle via distinct signaling mechanisms. Akt substrate of 160 kDa (AS160) mediates insulin-stimulated GLUT4 translocation in L6 myotubes, presumably through activation of Akt. Using in vivo, in vitro, and in situ methods, insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR all increased AS160 phosphorylation in mouse skeletal muscle. Insulin-stimulated AS160 phosphorylation was fully blunted by wortmannin in vitro and in Akt2 knockout (KO) mice in vivo. In contrast, contraction-stimulated AS160 phosphorylation was only partially decreased by wortmannin and unaffected in Akt2 KO mice, suggesting additional regulatory mechanisms. To determine if AMPK mediates AS160 signaling, we used AMPK ␣2-inactive (␣2i) transgenic mice. AICARstimulated AS160 phosphorylation was fully inhibited, whereas contraction-stimulated AS160 phosphorylation was partially reduced in the AMPK ␣2i transgenic mice. Combined AMPK ␣2 and Akt inhibition by wortmannin treatment of AMPK ␣2 transgenic mice did not fully ablate contraction-stimulated AS160 phosphorylation. Maximal insulin, together with either AICAR or contraction, increased AS160 phosphorylation in an additive manner. In conclusion, AS160 may be a point of convergence linking insulin, contraction, and AICAR signaling. While Akt and AMPK ␣2 activities are essential for AS160 phosphorylation by insulin and AICAR, respectively, neither kinase is indispensable for the entire effects of contraction on AS160 phosphorylation.

LKB1 is a tumor suppressor that may also be fundamental to cell metabolism, since LKB1 phosphorylates and activates the energy sensing enzyme AMPK. We generated muscle-specific LKB1 knockout (MLKB1KO) mice, and surprisingly, found that a lack of LKB1 in skeletal muscle enhanced insulin sensitivity, as evidenced by decreased fasting glucose and insulin concentrations, improved glucose tolerance, increased muscle glucose uptake in vivo, and increased glucose utilization during a hyperinsulinemic-euglycemic clamp. MLKB1KO mice had increased insulin-stimulated Akt phosphorylation and a >80% decrease in muscle expression of TRB3, a recently identified Akt inhibitor. Akt/TRB3 binding was present in skeletal muscle, and overexpression of TRB3 in C2C12 myoblasts significantly reduced Akt phosphorylation. These results demonstrate that skeletal muscle LKB1 is a negative regulator of insulin sensitivity and glucose homeostasis. LKB1-mediated TRB3 expression provides a novel link between LKB1 and Akt, critical kinases involved in both tumor genesis and cell metabolism.LKB1 is a serine/threonine kinase that links a diverse array of cellular processes, including cancer, cellular polarity, and metabolism. Originally identified as the tumor suppressor protein mutated in Peutz-Jeghers syndrome (17, 21), LKB1 has since been shown to regulate polarity in a number of systems, including Caenorhabditis elegans (48), Drosophila melanogaster (28), Xenopus (33), and mammalian cells (3). Biochemically, LBK1 can phosphorylate and activate at least 13 members of the AMP-activated protein kinase (AMPK) subfamily of protein kinases (20, 27) when associated with two regulatory proteins essential for catalytic activity, STE20-related adapter protein and mouse protein 25 (MO25) (15). AMPK, the most studied of LKB1's downstream substrates, is a conserved serine/threonine kinase that functions in the regulation of energy metabolism (16,18,29). Studies with cell culture (15,40,51) or conditional knockouts of LKB1 in skeletal muscle (38), cardiac muscle (39), or liver (41) have found that LKB1 regulates AMPK activity both in vitro and in vivo. Other than AMPK and the microtubule affinity-regulating kinase (MARK) proteins, which have been implicated in the control of cellular polarity (9), relatively little is known about the function of the AMPK-related kinases. The well-established role of AMPK in metabolism, however, directly implicates LKB1 in the maintenance of energy balance.The protein kinase Akt functions both in the control of cell proliferation and as a critical node in insulin signaling, appearing to mediate most of the metabolic effects of insulin (44). Akt is activated by phosphorylation of Thr 308 within the T loop of the catalytic domain and Ser 473 , located in a C-terminal, noncatalytic region of the enzyme (1). A mammalian homolog of D. melanogaster tribbles, TRB3, was recently identified as a negative regulator of Akt activity in human embryonic kidney 293 (HEK293) cells and mouse liver (10). In HEK293 cells and liver, TRB3 bind...

Variants in SCN10A, which encodes a voltage-gated sodium channel, are associated with alterations of cardiac conduction parameters and the cardiac rhythm disorder Brugada syndrome; however, it is unclear how SCN10A variants promote dysfunctional cardiac conduction. Here we showed by high-resolution 4C-seq analysis of the Scn10a-Scn5a locus in murine heart tissue that a cardiac enhancer located in Scn10a, encompassing SCN10A functional variant rs6801957, interacts with the promoter of Scn5a, a sodium channel-encoding gene that is critical for cardiac conduction. We observed that SCN5A transcript levels were several orders of magnitude higher than SCN10A transcript levels in both adult human and mouse heart tissue. Analysis of BAC transgenic mouse strains harboring an engineered deletion of the enhancer within Scn10a revealed that the enhancer was essential for Scn5a expression in cardiac tissue. Furthermore, the common SCN10A variant rs6801957 modulated Scn5a expression in the heart. In humans, the SCN10A variant rs6801957, which correlated with slowed conduction, was associated with reduced SCN5A expression. These observations establish a genomic mechanism for how a common genetic variation at SCN10A influences cardiac physiology and predisposes to arrhythmia.

Cardiac conduction system (CCS) disease, which results in disrupted conduction and impaired cardiac rhythm, is common with significant morbidity and mortality. Current treatment options are limited, and rational efforts to develop cell-based and regenerative therapies require knowledge of the molecular networks that establish and maintain CCS function. Recent genome-wide association studies (GWAS) have identified numerous loci associated with adult human CCS function, including TBX5 and SCN5A. We hypothesized that TBX5, a critical developmental transcription factor, regulates transcriptional networks required for mature CCS function. We found that deletion of Tbx5 from the mature murine ventricular conduction system (VCS), including the AV bundle and bundle branches, resulted in severe VCS functional consequences, including loss of fast conduction, arrhythmias, and sudden death. Ventricular contractile function and the VCS fate map remained unchanged in VCS-specific Tbx5 knockouts. However, key mediators of fast conduction, including Na v 1.5, which is encoded by Scn5a, and connexin 40 (Cx40), demonstrated Tbx5-dependent expression in the VCS. We identified a TBX5-responsive enhancer downstream of Scn5a sufficient to drive VCS expression in vivo, dependent on canonical T-box binding sites. Our results establish a direct molecular link between Tbx5 and Scn5a and elucidate a hierarchy between human GWAS loci that affects function of the mature VCS, establishing a paradigm for understanding the molecular pathology of CCS disease. IntroductionThe cardiac conduction system (CCS) consists of a network of specialized cardiomyocytes that generate and propagate the electrical impulses that organize cardiac contraction. The CCS is composed of the slowly propagating atrial nodes, including the sinoatrial (SA) and atrioventricular (AV) nodes, and the rapidly propagating ventricular conduction system (VCS), including the AV (His) bundle and right and left bundle branches. The VCS is uniquely adapted for fast conduction in order to rapidly transmit the electrical impulse governing ventricular contraction from the AV node to the ventricular apex. Disorders of the VCS are common, carry significant morbidity, and are poorly understood from a molecular perspective.The transcriptional networks required to maintain function of the adult CCS are undefined. Our current understanding of the molecular mediators of CCS function stems largely from heritable monogenic disorders and mouse models that have identified a limited number of genes essential for maintaining cardiac rhythm, most of which encode ion channels and their interacting partners (reviewed in ref. 1). Similar approaches have also begun to uncover the transcriptional networks required for CCS development (reviewed in ref.2). Recent genome-wide association studies (GWAS) have identified loci implicated in ECG interval variation (3-8), providing candidate genes with potentially important roles in CCS function in the general population. Specifically, numerous loci near genes e...

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The serine/threonine kinase Akt/PKB plays diverse roles in cells, and genetic studies have indicated distinct roles for the three Akt isoforms expressed in mammalian cells and tissues. Akt2 is a key signaling intermediate for insulin-stimulated glucose uptake and glycogen synthesis in skeletal muscle. Akt2 has also been shown to be activated by exercise and muscle contraction in both rodents and humans. In this study, we used Akt2 knockout mice to explore the role of Akt2 in exercise-stimulated glucose uptake and glycogen synthesis as well as intracellular signaling pathways that regulate glycogen metabolism in skeletal muscle. We found that Akt2 deficiency does not affect basal or exercise-stimulated glucose uptake or intracellular glycogen content in the soleus muscle. In addition, lack of Akt2 did not result in alterations in basal Akt Thr308 or basal and contraction-stimulated glycogen synthase kinase-3β (GSK-3β) Ser9 phosphorylation, glycogen synthase phosphorylation, or glycogen synthase activity. In contrast, in situ contraction failed to elicit normal increases in Akt T-loop Thr308 phosphorylation and GSK-3α Ser21 phosphorylation in tibialis anterior muscles from Akt2-deficient animals. Our data establish a key role for Akt2 in the regulation of GSK-3α Ser21 phosphorylation with contraction and add genetic evidence to support the separation of the intracellular pathways regulated by insulin and exercise that converge on glucose uptake and glycogen synthesis in skeletal muscle.

BACKGROUND: Postpartum hemorrhage is a leading cause of maternal mortality. Antifibrinolytic therapy has the potential to influence outcomes in postpartum hemorrhage, but the incidence of elevated fibrinolytic activity in postpartum hemorrhage is unknown. METHODS: We retrospectively reviewed thromboelastography (TEG) results obtained for postpartum hemorrhage from 118 deliveries at The University of Chicago. TEG results were obtained as part of our postpartum hemorrhage protocol when blood loss exceeded 500 mL after vaginal delivery or 1000 mL after cesarean delivery. Our primary outcome was the incidence of elevated fibrinolytic activity, which we predefined as clot lysis ≥3% at 30 minutes (Ly30) on kaolin TEG. Platelet-mediated clot retraction can also lead to an elevated Ly30 on kaolin TEG. Therefore, to distinguish between fibrinolysis and clot retraction, we evaluated clot lysis using functional fibrinogen TEG, which contains a platelet inhibitor. We considered a kaolin TEG Ly30 ≥3% in conjunction with a nonzero functional fibrinogen TEG Ly30 suggestive of elevated fibrinolytic activity. We also recorded quantitative blood loss, primary etiology of hemorrhage, standard laboratory measurements of coagulation, and demographic and obstetric characteristics of the study population. RESULTS: The median kaolin TEG Ly30 was 0.2% (interquartile range: 0%–0.8%). Fifteen of 118 women (12.7%; 95% confidence interval, 7.9%–19.9%) had kaolin TEG Ly30 values ≥3%. Of 15 patients with elevated Ly30 values, functional fibrinogen TEG Ly30 was available for 13, of which none demonstrated detectable clot lysis. CONCLUSIONS: Our observation that none of the patients in our sample with kaolin TEG Ly30 values ≥3% had a nonzero functional fibrinogen TEG Ly30 value suggests that the observed elevations in kaolin TEG Ly30 may have been secondary to platelet-mediated clot retraction as opposed to fibrinolysis. Platelet-mediated clot retraction should be distinguished from fibrinolysis when assayed using viscoelastic techniques in postpartum hemorrhage. Further research is necessary to determine the optimal methods to assess fibrinolytic activity in postpartum hemorrhage.

Rationale: The heartbeat is organized by the cardiac conduction system (CCS), a specialized network of cardiomyocytes. Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat. Distinct node versus VCS physiology has been recognized for more than a century, but the molecular basis of this regional patterning is not well understood. Objective: To study the genetic and genomic mechanisms underlying node versus VCS distinction and investigate rhythm consequences of failed VCS patterning. Methods and Results: Using mouse genetics, we found that the balance between T-box transcriptional activator, Tbx5, and T-box transcriptional repressor, Tbx3, determined the molecular and functional output of VCS myocytes. Adult VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into slow, nodal-like cells based on molecular and functional criteria. In these cases, gene expression profiling showed diminished expression of genes required for VCS-specific fast conduction but maintenance of expression of genes required for nodal slow conduction physiology. Action potentials (APs) of Tbx5-deficient VCS myocytes adopted nodal-specific characteristics, including increased AP duration and cellular automaticity. Removal of Tbx5 in-vivo precipitated inappropriate depolarizations in the atrioventricular (His)-bundle associated with lethal ventricular arrhythmias. TBX5 bound and directly activated cis-regulatory elements at fast conduction channel genes required for fast physiological characteristics of the VCS action potential, defining the identity of the adult VCS. Conclusions: The CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS gene regulatory network (GRN) allowed nodal physiology to emerge, providing a plausible molecular mechanism for some lethal ventricular arrhythmias.