Spontaneous resolution of traumatic bronchial tear after thoracic crush injury.

Traumatic bronchial tears are rare life-threatening injuries. Here, we report a 28-year old male who presented after sustaining a crush injury to his thoracic cavity, resulting in a spiral left mainstem bronchial tear secondary to high intraluminal pressure. While preparing for surgery, a preoperative bronchoscopy found that the bronchial tear had re-approximated and effectively sealed the laceration. No operative intervention was performed and the patient subsequently underwent a full recovery. While most bronchial tears undergo surgical intervention, our report describes the successful management of a bronchial tear injury with a non-operative approach and supportive care.

Heparin-Induced Thrombocytopenia After Mitral Valve Replacement.

Background: Heparin-induced thrombocytopenia (HIT) is a rare autoimmune reaction that involves a decrease in platelet count following heparin exposure and can be associated with life-threatening thrombosis. Because of their prolonged heparin exposure, patients undergoing cardiac surgery are at risk of HIT, with an incidence of 0.1% to 3%. Case Report: A 65-year-old male with severe mitral regurgitation and preoperative ejection fraction of 20% to 25% underwent mitral valve bioprosthetic replacement with coronary artery bypass graft surgery. Heparin anticoagulation was started on postoperative day (POD) 1. Respiratory failure resulted in prolonged mechanical ventilation and heparinization without the ability to initiate warfarin. While the patient was on heparin, his platelet count declined on POD 2 and then steadily increased to above the preoperative level on POD 7. On POD 10, the patient's platelet count dramatically decreased, and on POD 13 he developed acute common femoral artery occlusion necessitating embolectomy. Intraoperative transesophageal echocardiography revealed heavy thrombus burden across the mitral bioprosthesis. HIT was confirmed with a positive heparin-induced platelet antibody and serotonin release assay. Heparin was stopped and argatroban initiated. The patient underwent reoperative bioprosthetic mitral valve replacement on POD 18 using bivalirudin intraoperatively. Despite resolution of HIT, the patient developed sepsis and died on POD 59. Conclusion: The diagnosis of HIT is challenging in patients who undergo cardiopulmonary bypass. Platelet counts often decrease 40% to 60% during the first 72 hours postoperatively, and the frequency of nonspecific anti-platelet factor 4/heparin antibody formation is high. These findings can mask early signs of HIT and delay diagnosis.

Pharmacogenetic guidelines and decision support tools for depression treatment: application to late-life.

Late-life depression (LLD) is a major depressive disorder that affects someone after the age of 60 years. LLD is frequently associated with inadequate response and remission from antidepressants, in addition to polypharmacy. Pharmacogenetics offers a promising approach to improve clinical outcomes in LLD via new discoveries determining the genetic basis of response rates and side effects, as well as the development of tailored pharmacogenetic-based decision support tools. This invited review evaluates the LLD pharmacogenetic evidence base and the extent to which this was incorporated into existing commercial decision support tools and clinical pharmacogenetic guidelines.

Structural mechanism of ligand activation in human calcium-sensing receptor.

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca(2+) homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca(2+) and PO4(3-) ions. Both ions are crucial for structural integrity of the receptor. While Ca(2+) ions stabilize the active state, PO4(3-) ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.

hERG 1a LQT2 C-terminus truncation mutants display hERG 1b-dependent dominant negative mechanisms.

BACKGROUND: The human ether-à-go-go-related gene (hERG 1a) potassium channel is critical for cardiac repolarization. hERG 1b, another variant subunit, co-assembles with hERG 1a, modulates channel biophysical properties and plays an important role in repolarization. Mutations of hERG 1a lead to type 2 long QT syndrome (LQT2), and increased risk for fatal arrhythmias. The functional consequences of these mutations in the presence of hERG 1b are not known. OBJECTIVE: To investigate whether hERG 1a mutants exert dominant negative gating and trafficking defects when co-expressed with hERG 1b. METHODS: Electrophysiology, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments in HEK293 cells and guinea pig cardiomyocytes were used to assess the mutants on gating and trafficking. Mutations of 1a-G965X and 1a-R1014X, relevant to gating and trafficking were introduced in the C-terminus region. RESULTS: The hERG 1a mutants when expressed alone did not result in decreased current amplitude. Compared to wild-type hERG 1a currents, 1a-G965X currents were significantly larger, whereas those produced by the 1a-R1014X mutant were similar in magnitude. Only when co-expressed with wild-type hERG 1a and 1b did a mutant phenotype emerge, with a marked reduction in surface expression, current amplitude, and a corresponding positive shift in the V1/2 of the activation curve. Co-immunoprecipitation and FRET assays confirmed association of mutant and wild-type subunits. CONCLUSION: Heterologously expressed hERG 1a C-terminus truncation mutants, exert a dominant negative gating and trafficking effect only when co-expressed with hERG 1b. These findings may have potentially profound implications for LQT2 therapy.

Rad and Rem are non-canonical G-proteins with respect to the regulatory role of guanine nucleotide binding in Ca(V)1.2 channel regulation.

Rad and Rem are Ras-like G-proteins linked to diverse cardiovascular functions and pathophysiology. Understanding how Rad and Rem are regulated is important for deepened insights into their pathophysiological roles. As in other Ras-like G-proteins, Rad and Rem contain a conserved guanine-nucleotide binding domain (G-domain). Canonically, G-domains are key control modules, functioning as nucleotide-regulated switches of G-protein activity. Whether Rad and Rem G-domains conform to this canonical paradigm is ambiguous. Here, we used multiple functional measurements in HEK293 cells and cardiomyocytes (Ca(V)1.2 currents, Ca(2+) transients, Ca(V)ß binding) as biosensors to probe the role of the G-domain in regulation of Rad and Rem function. We utilized Rad(S105N) and Rem(T94N), which are the cognate mutants to Ras(S17N), a dominant-negative variant of Ras that displays decreased nucleotide binding affinity. In HEK293 cells, over-expression of either Rad(S105N) or Rem(T94N) strongly inhibited reconstituted Ca(V)1.2 currents to the same extent as their wild-type (wt) counterparts, contrasting with reports that Rad(S105N) is functionally inert in HEK293 cells. Adenovirus-mediated expression of either wt Rad or Rad(S105N) in cardiomyocytes dramatically blocked L-type calcium current (I(Ca,L)) and inhibited Ca(2+)-induced Ca(2+) release, contradicting reports that Rad(S105N) acts as a dominant negative in heart. By contrast, Rem(T94N) was significantly less effective than wt Rem at inhibiting I(Ca,L) and Ca(2+) transients in cardiomyocytes. FRET analyses in cardiomyocytes revealed that both Rad(S105N) and Rem(T94N) had moderately reduced binding affinity for Ca(V)ßs relative to their wt counterparts. The results indicate Rad and Rem are non-canonical G-proteins with respect to the regulatory role of their G-domain in Ca(V)1.2 regulation.

LQT1 mutations in KCNQ1 C-terminus assembly domain suppress IKs using different mechanisms.

AIMS: Long QT syndrome 1 (LQT1) mutations in KCNQ1 that decrease cardiac IKs (slowly activating delayed rectifier K(+) current) underlie ventricular arrhythmias and sudden death. LQT1 mutations may suppress IKs by preventing KCNQ1 assembly, disrupting surface trafficking, or inhibiting gating. We investigated mechanisms underlying how three LQT1 mutations in KCNQ1 C-terminus assembly domain (R555H/G589D/L619M) decrease IKs in heterologous cells and cardiomyocytes. METHODS AND RESULTS: In Chinese hamster ovary (CHO) cells, mutant KCNQ1 + KCNE1 channels either produced no currents (G589D/L619M) or displayed markedly reduced IKs with a right-shifted voltage-dependence of activation (R555H). When co-expressed with wild-type (wt) KCNQ1, the mutant KCNQ1s displayed varying intrinsic dominant-negative capacities that were affected by auxiliary KCNE1. All three mutant KCNQ1s assembled with wt KCNQ1 as determined by fluorescence resonance energy transfer (FRET). We developed an optical quantum dot labelling assay to measure channel surface density. G589D/R555H displayed substantial reductions in surface density, which were either partially (G589D) or fully (R555H) rescued by wt KCNQ1. Unexpectedly, L619M showed no trafficking defect. In adult rat cardiomyocytes, adenovirus-expressed homotetrameric G589D/L619M + KCNE1 channels yielded no currents, whereas R555H + KCNE1 produced diminished IKs with a right-shifted voltage-dependence of activation, mimicking observations in CHO cells. In contrast to heterologous cells, homotetrameric R555H channels showed no trafficking defect in cardiomyocytes. CONCLUSION: Distinct LQT1 mutations in KCNQ1 assembly domain decrease IKs using unique combinations of biophysical and trafficking mechanisms. Functional deficits in IKs observed in heterologous cells are mostly, but not completely, recapitulated in adult rat cardiomyocytes. A 'methodological chain' combining approaches in heterologous cells and cardiomyocytes provides mechanistic insights that may help advance personalized therapy for LQT1 mutations.

Manipulating L-type calcium channels in cardiomyocytes using split-intein protein transsplicing.

Manipulating expression of large genes (>6 kb) in adult cardiomyocytes is challenging because these cells are only efficiently transduced by viral vectors with a 4-7 kb packaging capacity. This limitation impedes understanding structure-function mechanisms of important proteins in heart. L-type calcium channels (LTCCs) regulate diverse facets of cardiac physiology including excitation-contraction coupling, excitability, and gene expression. Many important questions about how LTCCs mediate such multidimensional signaling are best resolved by manipulating expression of the 6.6 kb pore-forming α1C-subunit in adult cardiomyocytes. Here, we use split-intein-mediated protein transsplicing to reconstitute LTCC α1C-subunit from two distinct halves, overcoming the difficulty of expressing full-length α1C in cardiomyocytes. Split-intein-tagged α1C fragments encoding dihydropyridine-resistant channels were incorporated into adenovirus and reconstituted in cardiomyocytes. Similar to endogenous LTCCs, recombinant channels targeted to dyads, triggered Ca(2+) transients, associated with caveolin-3, and supported ß-adrenergic regulation of excitation-contraction coupling. This approach lowers a longstanding technical hurdle to manipulating large proteins in cardiomyocytes.