Cardiac calsequestrin (Casq2) is thought to be the key sarcoplasmic reticulum (SR) Ca 2+ storage protein essential for SR Ca 2+ release in mammalian heart. Human CASQ2 mutations are associated with catecholaminergic ventricular tachycardia. However, homozygous mutation carriers presumably lacking functional Casq2 display surprisingly normal cardiac contractility. Here we show that Casq2-null mice are viable and display normal SR Ca 2+ release and contractile function under basal conditions. The mice exhibited striking increases in SR volume and near absence of the Casq2-binding proteins triadin-1 and junctin; upregulation of other Ca 2+ -binding proteins was not apparent. Exposure to catecholamines in Casq2-null myocytes caused increased diastolic SR Ca 2+ leak, resulting in premature spontaneous SR Ca 2+ releases and triggered beats. In vivo, Casq2-null mice phenocopied the human arrhythmias. Thus, while the unique molecular and anatomic adaptive response to Casq2 deletion maintains functional SR Ca 2+ storage, lack of Casq2 also causes increased diastolic SR Ca 2+ leak, rendering Casq2-null mice susceptible to catecholaminergic ventricular arrhythmias.

We isolated a total of 3 3 10 8 guard cell protoplasts from 22,000 Arabidopsis thaliana plants and identified 1734 unique proteins using three complementary proteomic methods: protein spot identification from broad and narrow pH range twodimensional (2D) gels, and 2D liquid chromatography-matrix assisted laser desorption/ionization multidimensional protein identification technology. This extensive single-cell-type proteome includes 336 proteins not previously represented in transcriptome analyses of guard cells and 52 proteins classified as signaling proteins by Gene Ontology analysis, of which only two have been previously assessed in the context of guard cell function. THIOGLUCOSIDE GLUCOHYDROLASE1 (TGG1), a myrosinase that catalyzes the production of toxic isothiocyanates from glucosinolates, showed striking abundance in the guard cell proteome. tgg1 mutants were hyposensitive to abscisic acid (ABA) inhibition of guard cell inward K + channels and stomatal opening, revealing that the glucosinolate-myrosinase system, previously identified as a defense against biotic invaders, is required for key ABA responses of guard cells. Our results also suggest a mechanism whereby exposure to abiotic stresses may enhance plant defense against subsequent biotic stressors and exemplify how enhanced knowledge of the signaling networks of a specific cell type can be gained by proteomics approaches.

Background-Calmodulin kinase (CaMK) II is linked to arrhythmia mechanisms in cellular models where repolarization is prolonged. CaMKII upregulation and prolonged repolarization are general features of cardiomyopathy, but the role of CaMKII in arrhythmias in cardiomyopathy is unknown. Methods and Results-We studied a mouse model of cardiac hypertrophy attributable to transgenic (TG) overexpression of a constitutively active form of CaMKIV that also has increased endogenous CaMKII activity. ECG-telemetered TG mice had significantly more arrhythmias than wild-type (WT) littermate controls at baseline, and arrhythmias were additionally increased by isoproterenol. Arrhythmias were significantly suppressed by an inhibitory agent targeting endogenous CaMKII. TG mice had longer QT intervals and action potential durations than WT mice, and TG cardiomyocytes had frequent early afterdepolarizations (EADs), a hypothesized mechanism for triggering arrhythmias. EADs were absent in WT cells before and after isoproterenol, whereas EAD frequency was unaffected by isoproterenol in TG mice. L-type Ca 2ϩ channels (LTTCs) can activate EADs, and LTCC opening probability (Po) was significantly higher in TG than WT cardiomyocytes before and after isoproterenol. A CaMKII inhibitory peptide equalized TG and WT LTCC Po and eliminated EADs, whereas a peptide antagonist of the Na ϩ /Ca 2ϩ exchanger current, also hypothesized to support EADs, was ineffective. Conclusions-These findings support the hypothesis that CaMKII is a proarrhythmic signaling molecule in cardiac hypertrophy in vivo. Cellular studies point to EADs as a triggering mechanism for arrhythmias but suggest that the increase in arrhythmias after ␤-adrenergic stimulation is independent of enhanced EAD frequency.

SummaryOpen Stomata 1 (OST1) (SnRK2.6 or SRK2E), a serine/threonine protein kinase, is a positive regulator in abscisic acid (ABA)-mediated stomatal response, but OST1-regulation of K + and Ca 2+ currents has not been studied directly in guard cells and it is unknown whether OST1 activity is limiting in ABA-mediated stomatal responses.We employed loss-of-function and gain-of-function approaches to study native ABA responses of Arabidopsis guard cells. We performed stomatal aperture bioassays, patch clamp analyses and reactive oxygen species (ROS) measurements.ABA inhibition of inward K + channels and light-induced stomatal opening are reduced in ost1 mutants while transgenic plants overexpressing OST1 show ABA hypersensitivity in these responses. ost1 mutants are insensitive to ABA-induced stomatal closure, regulation of slow anion currents, Ca 2+-permeable channel activation and ROS production while OST1 overexpressing lines are hypersensitive for these responses, resulting in accelerated stomatal closure in response to ABA. Overexpression of OST1 in planta in the absence of ABA application does not affect basal apertures or ion currents. Moreover, we demonstrate the physical interaction of OST1 with the inward K + channel KAT1, the anion channel SLAC1, and the NADPH oxidases AtrbohD and AtrbohF. Our findings support OST1 as a critical limiting component in ABA regulation of stomatal apertures, ion channels and NADPH oxidases in Arabidopsis guard cells.