This study reports a new mechanism of cAMP mediated relaxation of Ca2+sensitized force, in smooth muscle (SM) through Epac, a GTP exchange factor for the small GTPase Rap1 which results in suppression of RhoA activity. We find that Epac selective cAMP analogue, 8‐pCPT‐2′‐O‐Me‐cAMP (007), significantly reduced agonist‐induced contractile force, in both intact and permeabilized vascular, gut and airway SM. Responses to 007 were independent of PKA and PKG. Activation of Epac resulted in increased Rap1·GTP accompanied by a significant decrease in RhoA activity and reductions in phosphorylation of RLC20 and MLCP. Transcriptional regulation of SM α‐actin and SM22, known to be regulated by RhoA, was also significantly decreased by activation of Epac. Forskolin, the phosphodiesterase inhibitor IBMX and isoproterenol significantly increased Rap1·GTP in rat aortic SM cells. Over‐expression of wild‐type Epac but not dominant negative Epac1R279E increased Rap1 activation after 007 stimulation. LPA‐induced activation of RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts. All together, our findings show a novel signaling mechanism whereby activation of Epac via cAMP results in PKA independent, Rap1 dependent Ca2+ desensitization of force in SM.

DAPK1 and ZIPK (also called DAPK3) are closely related serine/threonine protein kinases that regulate programmed cell death and phosphorylation of non-muscle and smooth muscle myosin. We have developed a fluorescence linked enzyme chemoproteomic strategy (FLECS) for the rapid identification of inhibitors for any element of the purinome and identified a selective pyrazolo[3,4-d]pyrimidinone (HS38) that inhibits DAPK1 and ZIPK in an ATP-competitive manner at nanomolar concentrations. In cellular studies, HS38 decreased RLC20 phosphorylation. In ex vivo studies, HS38 decreased contractile force generated in mouse aorta and rabbit ileum, and calyculin A stimulated arterial muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor also promoted relaxation in Ca2+-sensitized vessels. A close structural analogue (HS43) with 5-fold lower affinity for ZIPK produced no effect on cells or tissues. These findings are consistent with a mechanism of action wherein HS38 specifically targets ZIPK in smooth muscle. The discovery of HS38 provides a lead scaffold for the development of therapeutic agents for smooth muscle related disorders and a chemical means to probe the function of DAPK1 and ZIPK across species.

Krüppel-like factor 4 (Klf4) is a transcription factor involved in differentiation and proliferation in multiple tissues. We demonstrated previously that tamoxifen-induced deletion of the Klf4 gene in mice accelerated neointimal formation but delayed down-regulation of smooth muscle cell differentiation markers in carotid arteries following injury. To further determine the role of Klf4 in the cardiovascular system, we herein derived mice deficient for the Klf4 gene in smooth and cardiac muscle using the SM22␣ promoter (SM22␣-CreKI ؉ /Klf4 loxP/loxP mice). SM22␣-CreKI؉ /Klf4 loxP/loxP mice were born at the expected Mendelian ratio, but they gradually died after birth. Although ϳ40% of SM22␣-CreKI ؉ /Klf4 loxP/loxP mice survived beyond postnatal day 28, they exhibited marked growth retardation. In wild-type mice, Klf4 was expressed in the heart from late embryonic development through adulthood, whereas it was not expressed in smooth muscle. No changes were observed in morphology or expression of smooth muscle cell differentiation markers in vessels of SM22␣-CreKI ؉ /Klf4 loxP/loxP mice. Of interest, cardiac output was significantly decreased in SM22␣-CreKI ؉ /Klf4 loxP/loxP mice, as determined by magnetic resonance imaging. Moreover, a lack of Klf4 in the heart resulted in the reduction in expression of multiple cardiac genes, including Gata4. In vivo chromatin immunoprecipitation assays on the heart revealed that Klf4 bound to the promoter region of the Gata4 gene. Results provide novel evidence that Klf4 plays a key role in late fetal and/or postnatal cardiac development.

In the canonical model of smooth muscle (SM) contraction, the contractile force is generated by phosphorylation of the myosin regulatory light chain (RLC20) by the myosin light chain kinase (MLCK). Moreover, phosphorylation of the myosin targeting subunit (MYPT1) of the RLC20 phosphatase (MLCP) by the RhoA-dependent ROCK kinase, inhibits the phosphatase activity and consequently inhibits dephosphorylation of RLC20 with concomitant increase in contractile force, at constant intracellular [Ca2+]. This pathway is referred to as Ca2+-sensitization. There is, however, emerging evidence suggesting that additional Ser/Thr kinases may contribute to the regulatory pathways in SM. Here, we report data implicating the p90 ribosomal S6 kinase (RSK) in SM contractility. During both Ca2+- and agonist (U46619) induced SM contraction, RSK inhibition by the highly selective compound BI-D1870 (which has no effect on MLCK or ROCK) resulted in significant suppression of contractile force. Furthermore, phosphorylation levels of RLC20 and MYPT1 were both significantly decreased. Experiments involving the irreversible MLCP inhibitor microcystin-LR, in the absence of Ca2+, revealed that the decrease in phosphorylation levels of RLC20 upon RSK inhibition are not due solely to the increase in the phosphatase activity, but reflect direct or indirect phosphorylation of RLC20 by RSK. Finally, we show that agonist (U46619) stimulation of SM leads to activation of extracellular signal-regulated kinases ERK1/2 and PDK1, consistent with a canonical activation cascade for RSK. Thus, we demonstrate a novel and important physiological function of the p90 ribosomal S6 kinase, which to date has been typically associated with the regulation of gene expression.

Background: Epicardial cells are a potential source of progenitor cells for revascularization of the injured heart. Results: Decreased p63RhoGEF and GEF-H1 and increased Epac, p190RhoGAP, and Rnds activities suppress RhoA signaling in epicardial progenitors. Conclusion:The embryonic epicardial progenitor state is maintained by signaling pathways that control RhoA activity. Significance: Manipulation of these signaling molecules might promote cardiac revascularization.