Wang, Wei

Background. Na + /Ca 2+ exchanger (NCX) is essential in tuning intracellular Ca 2+ and the development of heart failure (HF). We reported that loss of Junctophilin 2 (JPH2) in adult mouse hearts leads to reduced NCX activity in addition to hyperactive RyR2s and more arrhythmogenic Ca 2+ waves. NCX has been found to co-localize with RyR2 recently. However, what is the physiological significance of junctional NCX and whether the reduced junctional NCX activity following JPH2 knockdown contributes to the arrhythmogenesis is still unknown. Hypothesis. Dysfunction of Junctional NCX in response to JPH2 knockdown alters spatial properties of Ca 2+ sparks and contributes to arrhythmogenesis. Methods. Tamoxifen-treated MCM and MCM-shJPH2 (KD) mice were studied. Contractile function was measured by echocardiography. Open probability of single RyR2 was evaluated by lipid bilayer. Ca 2+ spark were assessed with confocal microscopy. NCX activity was measured by exponential fitting of the declining phase of caffeine-induced Ca 2+ transient. Results. JPH2 knockdown increased spark size (MCM: 2.55 ± 0.14; KD: 4.25 ± 0.16, P < 0.001), suggesting an increased occurrence of proarrhythmic macrosparks. Cardiac Contractile function (EF% ) is negatively correlated with Ca 2+ spark size (n = 8, P < 0.05). Increased Open probability of RyR2 (MCM: 0.038 ± 0.018 KD: 0.274 ± 0.142, P < 0.01) is unlikely the reason for the ~70% increase in spark size, since it is not observed in 3 other mouse models with hyperactive RyR2s but normal JPH2 levels (RyR2:S2814D, mdx and FKBP12.6 -/- ). Loss of JPH2 also slows Ca 2+ efflux via NCX by ~50% (MCM: 2.70 ± 0.23 vs. KD: 5.11 ± 0.61 s, P < 0.01) without altering NCX expression level. Spark size and NCX activity are negatively correlated (n = 8 mice, P < 0.05), and partial inhibition (~30%) of NCX with 1 mM Cd 2+ significantly increased spark size by 14.0 ± 2.8% (n = 8, P <0.05) in wt mice. Conclusion. For the first time we showed that reduced NCX activity increases spatial properties of Ca 2+ sparks and thus arrhythmogenesis. Since reduced JPH2 is associated with HF and NCX plays more important role in human than in murine, reduced junctional NCX activity following loss of JPH2 could be a novel mechanism underlying the development of HF and associated ventricular arrhythmias.

Abstract Histological and lineage immunofluorescence examination revealed that healthy conducting airways of humans and animals harbor sporadic poorly differentiated epithelial patches mostly in the dorsal noncartilage regions that remarkably manifest squamous differentiation. In vitro analysis demonstrated that this squamous phenotype is not due to intrinsic functional change in underlying airway basal cells. Rather, it is a reversible physiological response to persistent Wnt signaling stimulation during de novo differentiation. Squamous epithelial cells have elevated gene signatures of glucose uptake and cellular glycolysis. Inhibition of glycolysis or a decrease in glucose availability suppresses Wnt-induced squamous epithelial differentiation. Compared with pseudostratified airway epithelial cells, a cascade of mucosal protective functions is impaired in squamous epithelial cells, featuring increased epithelial permeability, spontaneous epithelial unjamming, and enhanced inflammatory responses. Our study raises the possibility that the squamous differentiation naturally occurring in healthy airways identified herein may represent “vulnerable spots” within the airway mucosa that are sensitive to damage and inflammation when confronted by infection or injury. Squamous metaplasia and hyperplasia are hallmarks of many airway diseases, thereby expanding these areas of vulnerability with potential pathological consequences. Thus, investigation of physiological and reversible squamous differentiation from healthy airway basal cells may provide critical knowledge to understand pathogenic squamous remodeling, which is often nonreversible, progressive, and hyperinflammatory.

Expression silencing of junctophilin-2 (JPH2) in mouse heart leads to ryanodine receptor type 2 (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ leak and rapid development of heart failure. The mechanism and physiological significance of JPH2 in regulating RyR2-mediated SR Ca2+ leak remains elusive. We sought to elucidate the role of JPH2 in regulating RyR2-mediated SR Ca2+ release in the setting of cardiac failure. Cardiac myocytes isolated from tamoxifen-inducible conditional knockdown mice of JPH2 (MCM-shJPH2) were subjected to confocal Ca2+ imaging. MCM-shJPH2 cardiomyocytes exhibited an increased spark frequency width with altered spark morphology, which caused increased SR Ca2+ leakage. Single channel studies identified an increased RyR2 open probability in MCM-shJPH2 mice. The increase in spark frequency and width was observed only in MCM-shJPH2 and not found in mice with increased RyR2 open probability with native JPH2 expression. Na+/Ca2+-exchanger (NCX) activity was reduced by 50% in MCM-shJPH2 with no detectable change in NCX expression. Additionally, 50% inhibition of NCX through Cd2+ administration alone was sufficient to increase spark width in myocytes obtained from wild-type mice. Additionally, superresolution analysis of RyR2 and NCX colocalization showed a reduced overlap between RyR2 and NCX in MCM-shJPH2 mice. In conclusion, decreased JPH2 expression causes increased SR Ca2+ leakage by directly increasing open probability of RyR2 and by indirectly reducing junctional NCX activity through increased dyadic cleft Ca2+. This demonstrates two novel and independent cellular mechanisms by which JPH2 regulates RyR2-mediated SR Ca2+ leak and heart failure development.

Background— Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca 2+ channels and Ca 2+ release channels on sarcoplasmic reticulum within junctional membrane complexes. Although previous studies revealed a loss of junctional membrane complexes and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in junctional membrane complex formation and the Ca 2+ -induced Ca 2+ release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy. Methods and Results— To elucidate the role of JPH2 in the heart, we developed a novel approach to conditionally reduce JPH2 protein levels using RNA interference. Cardiac-specific JPH2 knockdown resulted in impaired cardiac contractility, which caused heart failure and increased mortality. JPH2 deficiency resulted in loss of excitation-contraction coupling gain, precipitated by a reduction in the number of junctional membrane complexes and increased variability in the plasmalemma–sarcoplasmic reticulum distance. Conclusions— Loss of JPH2 had profound effects on Ca 2+ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca 2+ release channels in cardiac myocytes. Thus, our novel approach of cardiac-specific short hairpin RNA–mediated knockdown of junctophilin-2 has uncovered a critical role for junctophilin in intracellular Ca 2+ release in the heart.