Perceived Stress, Grit, and Self-Care Behaviors in First-Year Medical Students.

Medical students experience more stress than the general population, which over time can cause mental and physical disease, including burnout. Identifying factors impacting stress during early medical training could inform strategies to minimize its impacts throughout training and in clinical practice. This study surveyed 238 first-year osteopathic medical students to assess stress (Perceived Stress Scale; PSS), grit, sleep quality (Pittsburgh Sleep Quality Index; PSQI), physical activity (Godin-Shephard Leisure-Time Physical Activity Score; LTPA), and nutrition habits (Rapid Eating Assessment for Participants; REAP) within the first 2 weeks of starting medical school and again 10 weeks later. Incomplete responses were removed, leaving 204 study participants. We observed statistically significant decreases in grittiness (∆grit = -2.230%, P = .002) and physical activity (∆LTPA = -22.147%, P < .0001), while perceived stress (∆PSS = 34.548%, P < .0001) and poor sleep quality (∆PSQI% = 19.853, P < .0001) increased. Correlation analyses identified the strongest relationships were between ∆PSS vs ∆PSQI (r = .47, P < .0001) and ∆PSS vs ∆LTPA (r = -.20, P < .01). Multivariable linear regression analysis isolated ∆PSQI (P < .0001) and ∆LTPA (P = .012) as statistically significant predictors of ∆PSS. These results suggest early, repeated curricular interventions focused on physical activity and sleep hygiene may help students better manage stress during medical education.

A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation.

Regulators of G Protein Signaling (RGS proteins) inhibit G protein-coupled receptor (GPCR) signaling by accelerating the GTP hydrolysis rate of activated Gα subunits. Some RGS proteins exert additional signal modulatory functions, and RGS12 is one such protein, with five additional, functional domains: a PDZ domain, a phosphotyrosine-binding domain, two Ras-binding domains, and a Gα·GDP-binding GoLoco motif. RGS12 expression is temporospatially regulated in developing mouse embryos, with notable expression in somites and developing skeletal muscle. We therefore examined whether RGS12 is involved in the skeletal muscle myogenic program. In the adult mouse, RGS12 is expressed in the tibialis anterior (TA) muscle, and its expression is increased early after cardiotoxin-induced injury, suggesting a role in muscle regeneration. Consistent with a potential role in coordinating myogenic signals, RGS12 is also expressed in primary myoblasts; as these cells undergo differentiation and fusion into myotubes, RGS12 protein abundance is reduced. Myoblasts isolated from mice lacking Rgs12 expression have an impaired ability to differentiate into myotubes ex vivo, suggesting that RGS12 may play a role as a modulator/switch for differentiation. We also assessed the muscle regenerative capacity of mice conditionally deficient in skeletal muscle Rgs12 expression (via Pax7-driven Cre recombinase expression), following cardiotoxin-induced damage to the TA muscle. Eight days post-damage, mice lacking RGS12 in skeletal muscle had attenuated repair of muscle fibers. However, when mice lacking skeletal muscle expression of Rgs12 were cross-bred with mdx mice (a model of human Duchenne muscular dystrophy), no increase in muscle degeneration was observed over time. These data support the hypothesis that RGS12 plays a role in coordinating signals during the myogenic program in select circumstances, but loss of the protein may be compensated for within model syndromes of prolonged bouts of muscle damage and repair.

Regulation of the subcellular localization of the G-protein subunit regulator GPSM3 through direct association with 14-3-3 protein.

G-protein signaling modulator-3 (GPSM3), also known as G18 or AGS4, is a member of the Gα(i/o)-Loco (GoLoco) motif containing proteins. GPSM3 acts through its two GoLoco motifs to exert GDP dissociation inhibitor activity over Gα(i) subunits; recently revealed is the existence of an additional regulatory site within GPSM3 directed toward monomeric Gß subunits during their biosynthesis. Here, using in silico and proteomic approaches, we have found that GPSM3 also interacts directly with numerous members of the 14-3-3 protein family. This interaction is dependent on GPSM3 phosphorylation, creating a mode II consensus 14-3-3 binding site. 14-3-3 binding to the N-terminal disordered region of GPSM3 confers stabilization from protein degradation. The complex of GPSM3 and 14-3-3 is exclusively cytoplasmic, and both moieties mutually control their exclusion from the nucleus. Phosphorylation of GPSM3 by a proline-directed serine/threonine kinase and the resultant association of 14-3-3 is the first description of post-translational regulation of GPSM3 subcellular localization, a process that likely regulates important spatio-temporal aspects of G-protein-coupled receptor signaling modulation by GPSM3.

G-protein signaling modulator-3 regulates heterotrimeric G-protein dynamics through dual association with Gß and Gαi protein subunits.

Regulation of the assembly and function of G-protein heterotrimers (Gα·GDP/Gßγ) is a complex process involving the participation of many accessory proteins. One of these regulators, GPSM3, is a member of a family of proteins containing one or more copies of a small regulatory motif known as the GoLoco (or GPR) motif. Although GPSM3 is known to bind Gα(i)·GDP subunits via its GoLoco motifs, here we report that GPSM3 also interacts with the Gß subunits Gß1 to Gß4, independent of Gγ or Gα·GDP subunit interactions. Bimolecular fluorescence complementation studies suggest that the Gß-GPSM3 complex is formed at, and transits through, the Golgi apparatus and also exists as a soluble complex in the cytoplasm. GPSM3 and Gß co-localize endogenously in THP-1 cells at the plasma membrane and in a juxtanuclear compartment. We provide evidence that GPSM3 increases Gß stability until formation of the Gßγ dimer, including association of the Gß-GPSM3 complex with phosducin-like protein PhLP and T-complex protein 1 subunit eta (CCT7), two known chaperones of neosynthesized Gß subunits. The Gß interaction site within GPSM3 was mapped to a leucine-rich region proximal to the N-terminal side of its first GoLoco motif. Both Gß and Gα(i)·GDP binding events are required for GPSM3 activity in inhibiting phospholipase-Cß activation. GPSM3 is also shown in THP-1 cells to be important for Akt activation, a known Gßγ-dependent pathway. Discovery of a Gß/GPSM3 interaction, independent of Gα·GDP and Gγ involvement, adds to the combinatorial complexity of the role of GPSM3 in heterotrimeric G-protein regulation.

Regulator of G-protein signaling 14 (RGS14) is a selective H-Ras effector.

BACKGROUND: Regulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Galpha-mediated GTP hydrolysis ("GTPase-accelerating proteins" or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Galpha GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras. METHODOLOGY/PRINCIPAL FINDINGS: Full-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co-transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor-mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling. CONCLUSIONS/SIGNIFICANCE: In cells, RGS14 facilitates the formation of a selective Ras.GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras-binding domain architecture with RGS14.

Epac and phospholipase Cepsilon regulate Ca2+ release in the heart by activation of protein kinase Cepsilon and calcium-calmodulin kinase II.

Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)epsilon that plays a critical role in maximal beta-adrenergic receptor (betaAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLCepsilon phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCepsilon-stimulated Rap1 GEF activity are both required for PLCepsilon-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCepsilon significantly enhances Rap activation in response to betaAR stimulation in the heart. Downstream of PLCepsilon hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both betaAR- and Epac-stimulated increases in CICR in PLCepsilon+/+ myocytes but had no effect in PLCepsilon-/- myocytes. betaAR and Epac activation caused membrane translocation of PKCepsilon in PLCepsilon+/+ but not PLCepsilon-/- myocytes and small interfering RNA-mediated PKCepsilon knockdown significantly inhibited both betaAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited betaAR- and Epac-mediated CICR in PLCepsilon+/+ but not PLCepsilon-/- myocytes. Epac activation increased CamKII Thr286 phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser2815) and phospholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and betaAR-stimulated peak L-type current density are similar in PLCepsilon+/+ and PLCepsilon-/- myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLCepsilon, PKCepsilon, and CamKII downstream of betaAR activation.

Phospholipase Cepsilon is a nexus for Rho and Rap-mediated G protein-coupled receptor-induced astrocyte proliferation.

Phospholipase Cepsilon (PLCepsilon) has been suggested to transduce signals from small GTPases, but its biological function has not yet been clarified. Using astrocytes from PLCepsilon-deficient mice, we demonstrate that endogenous G protein-coupled receptors (GPCRs) for lysophosphatidic acid, sphingosine 1-phosphate, and thrombin regulate phosphoinositide hydrolysis primarily through PLCepsilon. Stimulation by lysophospholipids occurs through G(i), whereas thrombin activates PLC through Rho. Further studies reveal that PLCepsilon is required for thrombin- but not LPA-induced sustained ERK activation and DNA synthesis, providing a novel mechanism for GPCR and Rho signaling to cell proliferation. The requirement for PLCepsilon in this pathway can be explained by its role as a guanine nucleotide exchange factor for Rap1. Thus, PLCepsilon serves to transduce mitogenic signals through a mechanism distinct from its role in generation of PLC-derived second messengers.

Epac-mediated activation of phospholipase C(epsilon) plays a critical role in beta-adrenergic receptor-dependent enhancement of Ca2+ mobilization in cardiac myocytes.

Recently we demonstrated that PLC(epsilon) plays an important role in beta-adrenergic receptor (betaAR) stimulation of Ca(2+)-induced Ca(2+) release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of betaAR involving the cAMP-dependent Rap GTP exchange factor, Epac, and PLC(epsilon) regulates CICR in cardiac myocytes. To demonstrate a role for Epac in the stimulation of CICR, cardiac myocytes were treated with an Epac-selective cAMP analog, 8-4-(chlorophenylthio)-2'-O-methyladenosine-3',5'-monophosphate (cpTOME). cpTOME treatment increased the amplitude of electrically evoked Ca(2+) transients, implicating Epac for the first time in cardiac CICR. This response is abolished in PLC(epsilon)(-/-) cardiac myocytes but rescued by transduction with PLC(epsilon), indicating that Epac is upstream of PLC(epsilon). Furthermore, transduction of PLC(epsilon)(+/+) cardiac myocytes with a Rap inhibitor, RapGAP1, significantly inhibited isoproterenol-dependent CICR. Using a combination of cpTOME and PKA-selective activators and inhibitors, we have shown that betaAR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/(epsilon) pathway. We also show that Epac/PLC(epsilon)-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca(2+) clearance mechanisms. These data define a novel endogenous PKA-independent betaAR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLC(epsilon) that enhances intracellular Ca(2+) release in cardiac myocytes.

Phospholipase C epsilon modulates beta-adrenergic receptor-dependent cardiac contraction and inhibits cardiac hypertrophy.

Phospholipase C (PLC) epsilon is a recently identified enzyme regulated by a wide range of molecules including Ras family small GTPases, Rho A, Galpha(12/13), and Gbetagamma with primary sites of expression in the heart and lung. In a screen for human signal transduction genes altered during heart failure, we found that PLCepsilon mRNA is upregulated. Two murine models of cardiac hypertrophy confirmed upregulation of PLCepsilon protein expression or PLCepsilon RNA. To identify a role for PLCepsilon in cardiac function and pathology, a PLCepsilon-deficient mouse strain was created. Echocardiography indicated PLCepsilon(-/-) mice had decreased cardiac function, and direct measurements of left ventricular contraction demonstrated that PLCepsilon(-/-) mice had a decreased contractile response to acute isoproterenol administration. Cardiac myocytes isolated from PLCepsilon(-/-) mice had decreased beta-adrenergic receptor (betaAR)-dependent increases in Ca2+ transient amplitudes, likely accounting for the contractile deficiency in vivo. This defect appears to be independent from the ability of the betaAR system to produce cAMP and regulation of sarcoplasmic reticulum Ca2+ pool size. To address the significance of these functional deficits to cardiac pathology, PLCepsilon(-/-) mice were subjected to a chronic isoproterenol model of hypertrophic stress. PLCepsilon(-/-) mice were more susceptible than wild-type littermates to development of hypertrophy than wild-type littermates. Together, these data suggest a novel PLC-dependent component of betaAR signaling in cardiac myocytes responsible for maintenance of maximal contractile reserve and loss of PLCepsilon signaling sensitizes the heart to development of hypertrophy in response to chronic cardiac stress.