Recent genomic studies have identified subtypes of uterine leiomyoma (LM) with distinctive genetic alterations. Here we report the elucidation of the biological characteristics of the two most prevalent LM subtypes, MED12 mutant (MED12-LM) and HMGA2-overexpressing (HMGA2-LM) LM. Since each tumor carries only one genetic alteration, both subtypes are considered to be monoclonal. Approximately 90% of cells in HMGA2-LM were smooth muscle cells (SMC) with HMGA2 overexpression. In contrast, MED12-LM consisted of similar numbers of SMC and non-SMC, which were mostly tumor-associated fibroblasts (TAF). Paradoxically, TAF carried no mutations in MED12, suggesting an interaction between SMC and TAF to coordinate their growth. The higher amount of ECM in MED12-LM than HMGA2-LM was partially due to the high concentration of collagen-producing TAF. SMC growth in a xenograft assay was driven by progesterone in both LM subtypes. In contrast, TAF in MED12-LM proliferated in response to estradiol, whereas progesterone had no effect. The high concentration of estrogen-responsive TAF in MED12-LM explains the inconsistent discoveries between in vivo and in vitro studies on the mitogenic effect of estrogen and raises questions regarding the accuracy of previous studies utilizing MED12-LM cell culture. In addition, the differential effects of estradiol and progesterone on these LM subtypes emphasize the importance of subtypes and genotypes in designing non-surgical therapeutic strategies for LM.

). In the present studies, activation of type I␤ PKG by either autophosphorylation or cGMP-binding alone causes (i) an electronegative charge shift on ion exchange chromatography, (ii) a similar increase (ϳ3.5 Å) in the Stokes radius as determined by gel filtration chromatography, and (iii) a similar decrease in the mobility of the enzyme on native gel electrophoresis. Consistent with these results, cGMP binding increases the rate of phosphoprotein phosphatase-1 catalyzed dephosphorylation of PKG which is autophosphorylated only at Ser-63 (not activated); however, dephosphorylation of PKG that is highly autophosphorylated (activated) is not stimulated by cGMP. The combined results suggest that activation of PKG by either autophosphorylation or cGMP binding alone produces a similar apparent elongation of the enzyme, implying that either process activates the enzyme by a similar molecular mechanism.Protein phosphorylation plays key roles in regulating protein function in myriad biological processes. Activation of the protein kinases that catalyze these protein phosphorylations is certainly one of the major mechanisms by which cellular functions are controlled. A particular protein kinase not only phosphorylates one or more cellular proteins (heterophosphorylation), 1 but it commonly phosphorylates itself as well, a process termed autophosphorylation. Protein kinase autophosphorylation is functionally important, since it frequently alters kinase function, e.g. by increasing the catalytic activity, increasing the affinity for allosteric ligand binding, or increasing the kinase binding to cellular proteins such as those containing SH2 domains. Many protein kinases are activated by either allosteric ligand binding or autophosphorylation (1). In some cases, ligand binding stimulates the rates of both autophosphorylation and heterophosphorylation. Furthermore, autophosphorylation of some protein kinases increases both the binding affinity for regulatory ligand(s) and the kinase catalytic activity. Therefore, in these instances ligand binding and autophosphorylation act in concert to produce an enhanced activation. The mechanisms of activation of protein kinases by these two processes are still unknown. Although these processes seem quite different, it seems reasonable that similar molecular perturbations may be involved to produce the final activation state for each process.Recently, activation of cGMP-dependent protein kinase (PKG) 2 by cGMP binding was shown to cause a conformational change in the enzyme (2, 3). The results show that an increased net negative surface charge and elongation of the enzyme occurs when PKG binds cGMP. These effects are apparently associated with a conformational change that relieves the interaction of the autoinhibitory domain with the catalytic site, thereby activating the protein kinase. Like many other protein kinases, PKG and type II cAMP-dependent protein kinase (PKA) undergo autophosphorylation, and this process affects the kinetic properties of each enzyme (4 -18). The type I␣ or ty...

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Recent evidence suggests that neuronal Na channels (nNas) contribute to catecholamine-promoted delayed afterdepolarizations (DADs) and catecholaminergic polymorphic ventricular tachycardia (CPVT). The newly identified overlap between CPVT and long QT (LQT) phenotypes has stoked interest in the cross-talk between aberrant Na and Ca handling and its contribution to early afterdepolarizations (EADs) and DADs. Here, we used Ca imaging and electrophysiology to investigate the role of Na and Ca handling in DADs and EADs in wild-type and cardiac calsequestrin (CASQ2)-null mice. In experiments, repolarization was impaired using 4-aminopyridine (4AP), whereas the L-type Ca and late Na currents were augmented using Bay K 8644 (BayK) and anemone toxin II (ATX-II), respectively. The combination of 4AP and isoproterenol prolonged action potential duration (APD) and promoted aberrant Ca release, EADs, and DADs in wild-type cardiomyocytes. Similarly, BayK in the absence of isoproterenol induced the same effects in CASQ2-null cardiomyocytes. In vivo, it prolonged the QT interval and, upon catecholamine challenge, precipitated wide QRS polymorphic ventricular tachycardia that resembled human torsades de pointes. Treatment with ATX-II produced similar effects at both the cellular level and in vivo. Importantly, nNa inhibition with riluzole or 4,9-anhydro-tetrodotoxin reduced the incidence of ATX-II-, BayK-, or 4AP-induced EADs, DADs, aberrant Ca release, and VT despite only modestly mitigating APD prolongation. These data reveal the contribution of nNas to triggered arrhythmias in murine models of LQT and CPVT-LQT overlap phenotypes. We also demonstrate the antiarrhythmic impact of nNa inhibition, independent of action potential and QT interval duration, and provide a basis for a mechanistically driven antiarrhythmic strategy.

The voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (NaV1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for NaV1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal NaV1.6 antibody and undertook super-resolution microscopy studies of NaV1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (NaV1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of NaV1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel NaV1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the NaV1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.