A Resident-Led Firearm Curriculum for Pediatrics Residents Improves Safe Storage Counseling.

OBJECTIVE: To develop and implement a resident-led firearm safety curriculum, delivered to pediatrics residents, and to evaluate outcomes. STUDY DESIGN: A firearm safety curriculum was developed in 2019-2020 at a single academic center, using Kern's framework and cognitive load theory. The curriculum was organized using the "Be SMART" firearm safety model. Sessions were led by resident peers. The content included workshops on firearm safety counseling, advocacy training, and a gun lock program in collaboration with the local police department. Content was integrated into existing residency didactic curriculum. Impact was measured by a pre/posttest knowledge assessment and a systematic chart review. RESULTS: The curriculum was provided to 41/66 (62%) pediatrics residents. Knowledge improved (67% to 77% correct) when comparing pre-intervention with post-intervention. A total of 1477 charts were reviewed. Compared with a historical cohort, participants more often asked about presence of a firearm (27% vs 69%, P < .0001) and counseled on firearm safety (9% vs 25%, P < .0001). In the post-intervention timeframe, 25% of eligible families were provided a gun lock. CONCLUSIONS: A firearm safety curriculum designed by pediatrics residents and administered to their peers resulted in a statistically significant improvement in inquiries about firearm ownership and safety counseling in an urban tertiary care continuity clinic. These results demonstrate the promising outcomes of a firearm safety program developed by residents and delivered to peers.

The adenosine-dependent angiogenic switch of macrophages to an M2-like phenotype is independent of interleukin-4 receptor alpha (IL-4Rα) signaling.

Murine macrophages are activated by interferon-γ (IFN-γ) and/or Toll-like receptor (TLR) agonists such as bacterial endotoxin (lipopolysaccharide [LPS]) to express an inflammatory (M1) phenotype characterized by the expression of nitric oxide synthase-2 (iNOS) and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-12. In contrast, Th2 cytokines IL-4 and IL-13 activate macrophages by inducing the expression of arginase-1 and the anti-inflammatory cytokine IL-10 in an IL-4 receptor-α (IL-4Rα)-dependent manner. Macrophages activated in this way are designated as "alternatively activated" (M2a) macrophages. We have shown previously that adenosine A2A receptor (A(2A)R) agonists act synergistically with TLR2, TLR4, TLR7, and TLR9 agonists to switch macrophages into an "M2-like" phenotype that we have termed "M2d." Adenosine signaling suppresses the TLR-dependent expression of TNF-α, IL-12, IFN-γ, and several other inflammatory cytokines by macrophages and induces the expression of vascular endothelial growth factor (VEGF) and IL-10. We show here using mice lacking a functional IL-4Rα gene (IL-4Rα(-/-) mice) that this adenosine-mediated switch does not require IL-4Rα-dependent signaling. M2d macrophages express high levels of VEGF, IL-10, and iNOS, low levels of TNF-α and IL-12, and mildly elevated levels of arginase-1. In contrast, M2d macrophages do not express Ym1, Fizz1 (RELM-α), or CD206 at levels greater than those induced by LPS, and dectin-1 expression is suppressed. The use of these markers in vivo to identify "M2" macrophages thus provides an incomplete picture of macrophage functional status and should be viewed with caution.

Type 1 inositol (1,4,5)-trisphosphate receptor activates ryanodine receptor 1 to mediate calcium spark signaling in adult mammalian skeletal muscle.

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.

Analysis of osmotic stress induced Ca2+ spark termination in mammalian skeletal muscle.

Ca2+ sparks represent synchronous opening of the ryanodine receptor (RyR) Ca2+ release channels located at the sarcoplasmic reticulum (SR) membrane. Whereas a quantal nature of Ca2+ sparks has been defined in cardiac muscle, the regulation of Ca2+ sparks in skeletal muscle has not been well-studied. Osmotic-stress applied to an intact skeletal muscle fiber can produce brief Ca2+ sparks and prolonged Ca2+ burst events. Here, we show that termination of Ca2+ bursts occurs in a step wise and quantal manner. Ca2+ burst events display kinetic features that are consistent with the involvement of both stochastic attrition and coordinated closure of RyR channels in the termination of SR Ca2+ release. Elemental unitary transition steps could be defined with a mean deltaF/F0 of approximately 0.28. corresponding to the gating of 1-2 RyR channels. Moreover, the amplitude of the elemental transition steps declines at the later stage of the burst event. In tandem Ca2+ burst events where two Ca2+ bursts occur at the same position within a fiber in rapid succession, the trailing event is consistently of lower amplitude than the initial event. These two complementary results suggest that SR Ca2+ release may be associated with local depletion of SR Ca2+ stores in mammalian skeletal muscle.

Recombinant MG53 protein modulates therapeutic cell membrane repair in treatment of muscular dystrophy.

Mitsugumin 53 (MG53), a muscle-specific TRIM family protein, is an essential component of the cell membrane repair machinery. Here, we examined the translational value of targeting MG53 function in tissue repair and regenerative medicine. Although native MG53 protein is principally restricted to skeletal and cardiac muscle tissues, beneficial effects that protect against cellular injuries are present in nonmuscle cells with overexpression of MG53. In addition to the intracellular action of MG53, injury to the cell membrane exposes a signal that can be detected by MG53, allowing recombinant MG53 protein to repair membrane damage when provided in the extracellular space. Recombinant human MG53 (rhMG53) protein purified from Escherichia coli fermentation provided dose-dependent protection against chemical, mechanical, or ultraviolet-induced damage to both muscle and nonmuscle cells. Injection of rhMG53 through multiple routes decreased muscle pathology in the mdx dystrophic mouse model. Our data support the concept of targeted cell membrane repair in regenerative medicine, and present MG53 protein as an attractive biological reagent for restoration of membrane repair defects in human diseases.

Regulation of Macrophage Polarization and Wound Healing.

BACKGROUND: Macrophages (Mφs) participate in wound healing by coordinating inflammatory and angiogenic processes. Mφs respond to environmental cues by adopting either "classically" activated (M1) proinflammatory or "alternatively" activated (M2a, M2b, M2c, M2d) wound healing phenotypes. THE PROBLEM: Mφ polarization is essential for wound healing and aberrations in this process are linked to several pathologies. It is important to elucidate molecular mechanisms underlying Mφ polarization. BASIC/CLINICAL SCIENCE ADVANCES: Mφs are categorized as proinflammatory (M1) or anti-inflammatory/wound healing (M2). M1 Mφs are observed in initial tissue damage responses, are induced by exogenous pathogen-associated molecular patterns or endogenous damage-associated molecular patterns, and exhibit increased phagocytosis and pro-inflammatory cytokine production, facilitating innate immunity and wound debridement. M2 Mφs predominate later in repair, express vascular endothelial growth factor, transforming growth factor beta, and interleukin 10 (IL-10), are activated by varied stimuli, assist in the resolution of inflammation, and promote tissue formation and remodeling. Recent work has characterized a novel "M2d" phenotype resulting from adenosine-dependent "switching" of M1 Mφs that exhibits a pattern of marker expression that is distinct from canonical IL-4/IL-13-dependent M2a Mφs. Recent studies have demonstrated important roles for specific transcriptional elements in M1 and M2a Mφ polarization, notably members of the interferon regulatory factor family interferon regulatory factor 5 (IRF5) and IRF4, respectively. The role of these IRFs in M2d polarization and wound healing remains to be determined. CLINICAL CARE RELEVANCE: Knowledge of microenvironmental signals and molecular mechanisms that mediate Mφ polarization should permit their manipulation to regulate important physiological processes and resolve pathological conditions. CONCLUSION: Proper Mφ polarization is essential to effective wound healing, and distinct phenotypes, such as the angiogenic M2d Mφ, may be of critical importance to this process. The IRF5 transcription factor has been shown to play a key role in M1 Mφ activation and the Jumonji domain containing-3-IRF4 pathway has been implicated in M2 Mφ activation.

Disrupted membrane structure and intracellular Ca²âº signaling in adult skeletal muscle with acute knockdown of Bin1.

Efficient intracellular Ca²âº ([Ca²âº]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca²âº homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short -hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca²âº release. Voltage-induced Ca²âº released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca²âº current and SR Ca²âº transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca²âº sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca²âº sparks' amplitude that was attributed to decreased total Ca²âº stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca²âº]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy.

MG53 constitutes a primary determinant of cardiac ischemic preconditioning.

BACKGROUND: Ischemic heart disease is the greatest cause of death in Western countries. The deleterious effects of cardiac ischemia are ameliorated by ischemic preconditioning (IPC), in which transient ischemia protects against subsequent severe ischemia/reperfusion injury. IPC activates multiple signaling pathways, including the reperfusion injury salvage kinase pathway (mainly PI3K-Akt-glycogen synthase kinase-3beta [GSK3beta] and ERK1/2) and the survivor activating factor enhancement pathway involving activation of the JAK-STAT3 axis. Nevertheless, the fundamental mechanism underlying IPC is poorly understood. METHODS AND RESULTS: In the present study, we define MG53, a muscle-specific TRIM-family protein, as a crucial component of cardiac IPC machinery. Ischemia/reperfusion or hypoxia/oxidative stress applied to perfused mouse hearts or neonatal rat cardiomyocytes, respectively, causes downregulation of MG53, and IPC can prevent ischemia/reperfusion-induced decrease in MG53 expression. MG53 deficiency increases myocardial vulnerability to ischemia/reperfusion injury and abolishes IPC protection. Overexpression of MG53 attenuates whereas knockdown of MG53 enhances hypoxia- and H(2)O(2)-induced cardiomyocyte death. The cardiac protective effects of MG53 are attributable to MG53-dependent interaction of caveolin-3 with phosphatidylinositol 3 kinase and subsequent activation of the reperfusion injury salvage kinase pathway without altering the survivor activating factor enhancement pathway. CONCLUSIONS: These results establish MG53 as a primary component of the cardiac IPC response, thus identifying a potentially important novel therapeutic target for the treatment of ischemic heart disease.

TRIC channels are essential for Ca2+ handling in intracellular stores.

Cell signalling requires efficient Ca2+ mobilization from intracellular stores through Ca2+ release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca2+ release. Ca2+ release channels were cloned more than 15 years ago, whereas the molecular identity of putative counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are differentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysiological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca2+ handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K+ permeability, as well as altered Ca2+ 'spark' signalling and voltage-induced Ca2+ release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchronization with Ca2+ release from intracellular stores.

Systemic ablation of RyR3 alters Ca2+ spark signaling in adult skeletal muscle.

Ca2+ sparks are localized intracellular Ca2+ release events from the sarcoplasmic reticulum in muscle cells that result from synchronized opening of ryanodine receptors (RyR). In mammalian skeletal muscle, RyR1 is the predominant isoform present in adult skeletal fibers, while some RyR3 is expressed during development. Functional studies have revealed a differential role for RyR1 and RyR3 in the overall Ca2+ signaling in skeletal muscle, but the contribution of these two isoforms to Ca2+ sparks in adult mammalian skeletal muscle has not been fully examined. When enzyme-disassociated, individual adult skeletal muscle fibers are exposed to an osmotic shock, the resting fiber converts from a quiescent to a highly active Ca2+ release state where Ca2+ sparks appear proximal to the sarcolemmal membrane. These osmotic shock-induced Ca2+ sparks occur in ryr3(-/-) muscle with a spatial distribution similar to that seen in wild type muscle. Kinetic analysis reveals that systemic ablation of RyR3 results in significant changes to the initiation, duration and amplitude of individual Ca2+ sparks in muscle fibers. These changes may reflect the adaptation of the muscle Ca2+ signaling or contractile machinery due to the loss of RyR3 expression in distal tissues, as biochemical assays identify significant changes in expression of myosin heavy chain protein in ryr3(-/-) muscle.

The presenilin-2 loop peptide perturbs intracellular Ca2+ homeostasis and accelerates apoptosis.

In cells undergoing apoptosis, a 22-amino-acid presenilin-2-loop peptide (PS2-LP, amino acids 308-329 in presenilin-2) is generated through cleavage of the carboxyl-terminal fragment of presenilin-2 by caspase-3. The impact of PS2-LP on the progression of apoptosis, however, is not known. Here we show that PS2-LP is a potent inducer of the mitochondrial-dependent cell death pathway when transduced as a fusion protein with HIV-TAT. Biochemical and functional studies demonstrate that TAT-PS2-LP can interact with the inositol 1,4,5-trisphosphate receptor and activate Ca(2+) release from the endoplasmic reticulum. These results indicate that PS2-LP-mediated alteration of intracellular Ca(2+) homeostasis may be linked to the acceleration of apoptosis. Therefore, targeting the function of PS2-LP could provide a useful therapeutic tool for the treatment of cancer and degenerative diseases.

Sites of protein kinase A activation of the human ClC-2 Cl(-) channel.

Human ClC-2 Cl(-) (hClC-2) channels are activated by protein kinase A (PKA) and low extracellular pH(o). Both of these effects are prevented by the PKA inhibitor, myristoylated PKI. The aims of the present study were to identify the PKA phosphorylation site(s) important for PKA activation of hClC-2 at neutral and low pH(o) and to examine the relationship between PKA and low pH(o) activation. Recombinant hClC-2 with point mutations of consensus phosphorylation sites was prepared and stably expressed in HEK-293 cells. The responses to forskolin plus isobutylmethylxanthine at neutral and acidic pH(o) were studied by whole cell patch clamp in the presence and absence of phosphatase inhibitors. The double phosphorylation site (RRAT655(A) plus RGET691(A)) mutant hClC-2 lost PKA activation and low pH(o) activation. Either RRAT or RGET was sufficient for PKA activation of hClC-2 at pH(o) 7.4, as long as phosphatase inhibitors (cyclosporin A or endothal) were present. At pH(o) 6 only RGET was needed for PKA activation of hClC-2. Low pH(o) activation of hClC-2 Cl(-) channel activity was PKA-dependent, retained in RGET(A) mutant hClC-2, but lost in RRAT(A) mutant hClC-2. RRAT655(D) mutant hClC-2 was constitutively active and was further activated by PKA at pH(o) 7.4 and 6.0, consistent with the above findings. These results show that activation of hClC-2 is differentially regulated by PKA at two sites, RRAT655 and RGET691. Either RRAT655 or RGET691 was sufficient for activation at pH(o) 7.4. RGET, but not RRAT, was sufficient for activation at pH(o) 6.0. However, in the RGET691(D) mutant, there was PKA activation at pH(o) 6.0.