Calcium channel ß3 subunit regulates ATP-dependent migration of dendritic cells.

Migratory dendritic cells (migDCs) continuously patrol tissues and are activated by injury and inflammation. Extracellular adenosine triphosphate (ATP) is released by damaged cells or actively secreted during inflammation and increases migDC motility. However, the underlying molecular mechanisms by which ATP accelerates migDC migration is not understood. Here, we show that migDCs can be distinguished from other DC subsets and immune cells by their expression of the voltage-gated calcium channel subunit ß3 (Cavß3; CACNB3), which exclusively facilitates ATP-dependent migration in vitro and during tissue damage in vivo. By contrast, CACNB3 does not regulate lipopolysaccharide-dependent migration. Mechanistically, CACNB3 regulates ATP-dependent inositol 1,4,5-trisphophate receptor-controlled calcium release from the endoplasmic reticulum. This, in turn, is required for ATP-mediated suppression of adhesion molecules, their detachment, and initiation of migDC migration. Thus, Cacnb3-deficient migDCs have an impaired migration after ATP exposure. In summary, we identified CACNB3 as a master regulator of ATP-dependent migDC migration that controls tissue-specific immunological responses during injury and inflammation.

Fractional Flow Reserve-Negative High-Risk Plaques and Clinical Outcomes After Myocardial Infarction.

Importance: Even after fractional flow reserve (FFR)-guided complete revascularization, patients with myocardial infarction (MI) have high rates of recurrent major adverse cardiovascular events (MACE). These recurrences may be caused by FFR-negative high-risk nonculprit lesions. Objective: To assess the association between optical coherence tomography (OCT)-identified high-risk plaques of FFR-negative nonculprit lesions and occurrence of MACE in patients with MI. Design, Setting, and Participants: PECTUS-obs (Identification of Risk Factors for Acute Coronary Events by OCT After STEMI [ST-segment elevation MI] and NSTEMI [non-STEMI] in Patients With Residual Non-flow Limiting Lesions) is an international, multicenter, prospective, observational cohort study. In patients presenting with MI, OCT was performed on all FFR-negative (FFR > 0.80) nonculprit lesions. A high-risk plaque was defined containing at least 2 of the following prespecified criteria: (1) a lipid arc at least 90°, (2) a fibrous cap thickness less than 65 µm, and (3) either plaque rupture or thrombus presence. Patients were enrolled from December 14, 2018, to September 15, 2020. Data were analyzed from December 2, 2022, to June 28, 2023. Main Outcome and Measure: The primary end point of MACE, a composite of all-cause mortality, nonfatal MI, or unplanned revascularization, at 2-year follow-up was compared in patients with and without a high-risk plaque. Results: A total of 438 patients were enrolled, and OCT findings were analyzable in 420. Among included patients, mean (SD) age was 63 (10) years, 340 (81.0) were men, and STEMI and non-STEMI were equally represented (217 [51.7%] and 203 [48.3%]). A mean (SD) of 1.17 (0.42) nonculprit lesions per patient was imaged. Analysis of OCT images revealed at least 1 high-risk plaque in 143 patients (34.0%). The primary end point occurred in 22 patients (15.4%) with a high-risk plaque and 23 of 277 patients (8.3%) without a high-risk plaque (hazard ratio, 1.93 [95% CI, 1.08-3.47]; P = .02), primarily driven by more unplanned revascularizations in patients with a high-risk plaque (14 of 143 [9.8%] vs 12 of 277 [4.3%]; P = .02). Conclusions and Relevance: Among patients with MI and FFR-negative nonculprit lesions, the presence of a high-risk plaque is associated with a worse clinical outcome, which is mainly driven by a higher number of unplanned revascularizations. In a population with a high recurrent event rate despite physiology-guided complete revascularization, these results call for research on additional pharmacological or focal treatment strategies in patients harboring high-risk plaques.

Intra-pituitary follicle-stimulating hormone signaling regulates hepatic lipid metabolism in mice.

Inter-organ communication is a major hallmark of health and is often orchestrated by hormones released by the anterior pituitary gland. Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to regulate gonadal function and control fertility. Whether FSH and LH also act on organs other than the gonads is debated. Here, we find that gonadotrope depletion in adult female mice triggers profound hypogonadism, obesity, glucose intolerance, fatty liver, and bone loss. The absence of sex steroids precipitates these phenotypes, with the notable exception of fatty liver, which results from ovary-independent actions of FSH. We uncover paracrine FSH action on pituitary corticotropes as a mechanism to restrain the production of corticosterone and prevent hepatic steatosis. Our data demonstrate that functional communication of two distinct hormone-secreting cell populations in the pituitary regulates hepatic lipid metabolism.

TRPM7 kinase is required for insulin production and compensatory islet responses during obesity.

Most overweight individuals do not develop diabetes due to compensatory islet responses to restore glucose homeostasis. Therefore, regulatory pathways that promote ß cell compensation are potential targets for treatment of diabetes. The transient receptor potential cation channel subfamily M member 7 protein (TRPM7), harboring a cation channel and a serine/threonine kinase, has been implicated in controlling cell growth and proliferation. Here, we report that selective deletion of Trpm7 in ß cells disrupted insulin secretion and led to progressive glucose intolerance. We indicate that the diminished insulinotropic response in ß cell-specific Trpm7-knockout mice was caused by decreased insulin production because of impaired enzymatic activity of this protein. Accordingly, high-fat-fed mice with a genetic loss of TRPM7 kinase activity displayed a marked glucose intolerance accompanied by hyperglycemia. These detrimental glucoregulatory effects were engendered by reduced compensatory ß cell responses because of mitigated protein kinase B (AKT)/ERK signaling. Collectively, our data identify TRPM7 kinase as a potentially novel regulator of insulin synthesis, ß cell dynamics, and glucose homeostasis under obesogenic diet.

IP3-dependent Ca2+ signals are tightly controlled by Cavß3, but not by Cavß1, 2 and 4.

Independent of its function as a subunit of voltage-gated Ca2+ channels, the Cavß3 subunit desensitizes fibroblasts and pancreatic ß-cells to low concentrations of inositol-1,4,5-trisphosphate (IP3). This alters agonist-induced Ca2+ signaling and cellular functions, for example, insulin secretion and wound healing. A total of four Cavß subunits exist, Cavß1, Cavß2, Cavß3, and Cavß4. To investigate whether the other Cavß subunits, like Cavß3, can desensitize cells to IP3 and thereby modulate Ca2+ signaling, we expressed the cDNAs of Cavß1, Cavß2, Cavß3, and Cavß4 in COS-7 cells lacking endogenous Cavß proteins. ATP stimulation of these cells results in the release of Ca2+ from intracellular stores. This receptor-mediated Ca2+ release is significantly decreased by Cavß3 but not by Cavß1, Cavß2, and Cavß4. Electrophysiological recordings of voltage-dependent Ca2+ currents from fibroblasts show a small Ca2+ current, the amplitude of which is slightly but not significantly smaller in fibroblasts from Cavß2 gene-deficient animals than in fibroblasts from wild-type animals. Compared with fibroblasts from wild-type animals, Ca2+ release is not significantly increased in Cavß2-deficient fibroblasts, in contrast to Ca2+ release in Cavß3-deficient fibroblasts. In summary, our results show that desensitization of cells to low concentrations of IP3 is a specific property of Cavß3 that is not shared by other Cavß subunits.

Cavß3 Regulates Ca2+ Signaling and Insulin Expression in Pancreatic ß-Cells in a Cell-Autonomous Manner.

Voltage-gated Ca2+ (Cav) channels consist of a pore-forming Cavα1 subunit and auxiliary Cavα2-δ and Cavß subunits. In fibroblasts, Cavß3, independent of its role as a Cav subunit, reduces the sensitivity to low concentrations of inositol-1,4,5-trisphosphate (IP3). Similarly, Cavß3 could affect cytosolic calcium concentration ([Ca2 +]) in pancreatic ß-cells. In this study, we deleted the Cavß3-encoding gene Cacnb3 in insulin-secreting rat ß-(Ins-1) cells using CRISPR/Cas9. These cells were used as controls to investigate the role of Cavß3 on Ca2+ signaling, glucose-induced insulin secretion (GIIS), Cav channel activity, and gene expression in wild-type cells in which Cavß3 and the IP3 receptor were coimmunoprecipitated. Transcript and protein profiling revealed significantly increased levels of insulin transcription factor Mafa, CaMKIV, proprotein convertase subtilisin/kexin type-1, and nitric oxide synthase-1 in Cavß3-knockout cells. In the absence of Cavß3, Cav currents were not altered. In contrast, CREB activity, the amount of MAFA protein and GIIS, the extent of IP3-dependent Ca2+ release and the frequency of Ca2+ oscillations were increased. These processes were decreased by the Cavß3 protein in a concentration-dependent manner. Our study shows that Cavß3 interacts with the IP3 receptor in isolated ß-cells, controls IP3-dependent Ca2+-signaling independently of Cav channel functions, and thereby regulates insulin expression and its glucose-dependent release in a cell-autonomous manner.

Cytotoxicity, metabolism, and isozyme mapping of the synthetic cannabinoids JWH-200, A-796260, and 5F-EMB-PINACA studied by means of in vitro systems.

Intake of synthetic cannabinoids (SC), one of the largest classes of new psychoactive substances, was reported to be associated with acute liver damage but information about their hepatotoxic potential is limited. The current study aimed to analyze the hepatotoxicity including the metabolism-related impact of JWH-200, A-796260, and 5F-EMB-PINACA in HepG2 cells allowing a tentative assessment of different SC subclasses. A formerly adopted high-content screening assay (HCSA) was optimized using a fully automated epifluorescence microscope. Metabolism-mediated effects in the HCSA were additionally investigated using the broad CYP inhibitor 1-aminobenzotriazole. Furthermore, phase I metabolites and isozymes involved were identified by in vitro assays and liquid chromatography-high-resolution tandem mass spectrometry. A strong cytotoxic potential was observed for the naphthoylindole SC JWH-200 and the tetramethylcyclopropanoylindole compound A-796260, whereas the indazole carboxamide SC 5F-EMB-PINACA showed moderate effects. Numerous metabolites, which can serve as analytical targets in urine screening procedures, were identified in pooled human liver microsomes. Most abundant metabolites of JWH-200 were formed by N-dealkylation, oxidative morpholine cleavage, and oxidative morpholine opening. In case of A-796260, most abundant metabolites included an oxidative morpholine cleavage, oxidative morpholine opening, hydroxylation, and dihydroxylation followed by dehydrogenation. Most abundant 5F-EMB-PINACA metabolites were generated by ester hydrolysis plus additional steps such as oxidative defluorination and hydroxylation. To conclude, the data showed that a hepatotoxicity of the investigated SC cannot be excluded, that metabolism seems to play a minor role in the observed effects, and that the extensive phase I metabolism is mediated by several isozymes making interaction unlikely.

Identification of anatomic risk factors for acute coronary events by optical coherence tomography in patients with myocardial infarction and residual nonflow limiting lesions: rationale and design of the PECTUS-obs study.

INTRODUCTION: In patients with myocardial infarction, the decision to treat a nonculprit lesion is generally based on its physiological significance. However, deferral of revascularisation based on nonischaemic fractional flow reserve (FFR) values in these patients results in less favourable outcomes compared with patients with stable coronary artery disease, potentially caused by vulnerable nonculprit lesions. Intravascular optical coherence tomography (OCT) imaging allows for in vivo morphological assessment of plaque 'vulnerability' and might aid in the detection of FFR-negative lesions at high risk for recurrent events. METHODS AND ANALYSIS: The PECTUS-obs study is an international multicentre prospective observational study that aims to relate OCT-derived vulnerable plaque characteristics of nonflow limiting, nonculprit lesions to clinical outcome in patients with myocardial infarction. A total of 438 patients presenting with myocardial infarction (ST-elevation myocardial infarction and non-ST-elevation myocardial infarction) will undergo OCT-imaging of any FFR-negative nonculprit lesion for detection of plaque vulnerability. The primary study endpoint is a composite of major adverse cardiovascular events (all-cause mortality, nonfatal myocardial infarction or unplanned revascularisation) at 2-year follow-up. Secondary endpoints will be the same composite at 1-year and 5-year follow-up, target vessel failure, target vessel revascularisation, target lesion failure and target lesion revascularisation. ETHICS AND DISSEMINATION: This study has been approved by the Medical Ethics Committee of the region Arnhem-Nijmegen. The results of this study will be disseminated in a main paper and additional papers with subgroup analyses. TRIAL REGISTRATION NUMBER: NCT03857971.

MicroRNA-targeting in spermatogenesis: Over-expressions of microRNA-23a/b-3p and its affected targeting of the genes ODF2 and UBQLN3 in spermatozoa of patients with oligoasthenozoospermia.

BACKGROUND: Male infertility is a multifactorial syndrome with diverse phenotypic representations. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in the post-transcriptional regulation of gene expression. Altered abundance levels of ODF2 and UBQLN3 have been reported in patients with different spermatogenic impairments. However, the transcriptional regulation of these two genes by miR-23a/b-3p is still unclear. OBJECTIVES: To investigate experimentally whether miR-23a/b-3p targets the genes ODF2 and UBQLN3 and whether this targeting impacts abundance levels of ODF2 and UBQLN3 in patients with oligoasthenozoospermia. MATERIALS AND METHODS: A total of 92 men attending a fertility clinic were included in the study, including 46 oligoasthenozoospermic men and 46 age-matched normozoospermic volunteers who served as controls. Reverse transcription-quantitative PCR (RT-qPCR), Western blot, and dual-luciferase (Firefly-Renilla) assays were used to validate the miRNAs and their target genes. RESULTS: RT-qPCR revealed that miR-23a/b-3p was more abundant and ODF2 and UBQLN3 targets were less abundant in men with impaired spermatogenesis. Besides, Western blot shows that ODF2 and UBQLN3 protein levels were reduced in men with impaired spermatogenesis. In silico prediction and dual-luciferase assays revealed that potential links exist between the higher abundance level of miR-23a/b-3p and the lower abundance level of ODF2 and UBQLN3 targets. Mutations in the miR-23a/b-3p-binding site within the 3'UTRs (3'untranslated regions) of ODF2 and UBQLN3 genes resulted in abrogated responsiveness to miR-23a/b-3p. Correlation analysis showed that sperm count, motility, and morphology were negatively correlated with miR-23a/b-3p and positively correlated with the lower abundance level of UBQLN3, while ODF lower abundance level was positively correlated with sperm motility. CONCLUSION: Findings indicate that the higher abundance level of miR-23a/b-3p and the lower abundance level of ODF2 and UBQLN3 targets are associated with oligoasthenozoospermia and male subfertility.

Disturbed Presynaptic Ca2+ Signaling in Photoreceptors in the EAE Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a demyelinating disease caused by an auto-reactive immune system. Recent studies also demonstrated synapse dysfunctions in MS patients and MS mouse models. We previously observed decreased synaptic vesicle exocytosis in photoreceptor synapses in the EAE mouse model of MS at an early, preclinical stage. In the present study, we analyzed whether synaptic defects are associated with altered presynaptic Ca2+ signaling. Using high-resolution immunolabeling, we found a reduced signal intensity of Cav-channels and RIM2 at active zones in early, preclinical EAE. In line with these morphological alterations, depolarization-evoked increases of presynaptic Ca2+ were significantly smaller. In contrast, basal presynaptic Ca2+ was elevated. We observed a decreased expression of Na+/K+-ATPase and plasma membrane Ca2+ ATPase 2 (PMCA2), but not PMCA1, in photoreceptor terminals of EAE mice that could contribute to elevated basal Ca2+. Thus, complex Ca2+ signaling alterations contribute to synaptic dysfunctions in photoreceptors in early EAE.

Control of Insulin Release by Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels.

BACKGROUND/AIMS: The release of insulin in response to increased levels of glucose in the blood strongly depends on Ca2+ influx into pancreatic beta cells by the opening of voltage-gated Ca2+ channels. Transient Receptor Potential Melastatin 3 proteins build Ca2+ permeable, non-selective cation channels serving as pain sensors of noxious heat in the peripheral nervous system. TRPM3 channels are also strongly expressed in pancreatic beta cells that respond to the TRPM3 agonist pregnenolone sulfate with Ca2+ influx and increased insulin release. Therefore, we hypothesized that in beta cells TRPM3 channels may contribute to pregnenolone sulfate- as well as to glucose-induced insulin release. METHODS: We used INS-1 cells as a beta cell model in which we analysed the occurrence of TRPM3 isoformes by immunoprecipitation and western blotting and by cloning of RT-PCR amplified cDNA fragments. We applied pharmacological as well as CRISPR/Cas9-based strategies to analyse the interplay of TRPM3 and voltage-gated Ca2+ channels in imaging experiments (FMP, Fura-2) and electrophysiological recordings. In immunoassays, we examined the contribution of TRPM3 channels to pregnenolone sulfate- and glucose-induced insulin release. To confirm our findings, we generated beta cell-specific Trpm3-deficient mice and compared their glucose clearance with the wild type in glucose tolerance tests. RESULTS: TRPM3 channels triggered the activity of voltage-gated Ca2+ channels and both channels together contributed to insulin release after TRPM3 activation. Trpm3-deficient INS-1 cells lacked pregnenolone sulfate-induced Ca2+ signals just like the pregnenolone sulfate-induced insulin release. Both, glucose-induced Ca2+ signals and the glucose-induced insulin release were strongly reduced. Accordingly, Trpm3-deficient mice displayed an impaired decrease of the blood sugar concentration after intraperitoneal or oral administration of glucose. CONCLUSION: The present study suggests an important role for TRPM3 channels in the control of glucose-dependent insulin release.

Why endogenous TRPV6 currents are not detectable-what can we learn from bats?

TRPV6 is a calcium selective TRP channel and is expressed in many species. TRPV6 transcripts are abundantly expressed in few tissues but strangely channel properties are only accessible to electrophysiological recordings after overexpression whereas in native tissue functional channel currents seem not to be detectable. Another exceptional property of human and mouse TRPV6 proteins is that the initiation of translation starts from a non-canonical ACG triplet which is translated as methionine. This triplet is located 120 bp upstream of the first in-frame AUG codon of the human/mouse TRPV6 mRNA. In contrast, the TRPV6 gene of bats is initiated from an AUG triplet at the corresponding position of the human ACG. On the basis of these structural nucleotide differences between human and bats we studied the role of the absolute N-Terminus for the regulation of translation by developing chimera and mutants of human/bat TRPV6 channels. The human sequence which is located downstream of the initiation codon slows down ribosomal scanning in 3' direction. We suggest that the mechanism involves most likely the deceleration of ribosome scanning by stem-loop formation and the use of the common initiator tRNA, tRNAiMet, which is placed onto the inappropriate ACG codon resulting in low protein synthesis. The reduced translation efficiency is important to protect TRPV6 expressing cells from toxic calcium overload. The regulation of the TRPV6 translation in bats may be an adaptation to low calcium amounts present in the natural nutrition. In addition, we show that also the GFP protein can be controlled using the translational mechanism of human TRPV6.

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the ß3 subunit of voltage-gated calcium channels (Cavß3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cavß3-deficient mice (Cavß3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cavß3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cavß3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cavß3-deficient mice or cells might also be applicable for specific molecules other than Cavß3.

Identification of Inhibitory Ca2+ Binding Sites in the Upper Vestibule of the Yeast Vacuolar TRP Channel.

By vacuolar patch-clamp and Ca2+ imaging experiments, we show that the yeast vacuolar transient receptor potential (TRPY) channel 1 is activated by cytosolic Ca2+ and inhibited by Ca2+ from the vacuolar lumen. The channel is cooperatively affected by vacuolar Ca2+ (Hill coefficient, 1.5), suggesting that it may accommodate a Ca2+ receptor that can bind two calcium ions. Alanine scanning of six negatively charged amino acid residues in the transmembrane S5 and S6 linker, facing the vacuolar lumen, revealed that two aspartate residues, 401 and 405, are essential for current inhibition and direct binding of 45Ca2+. Expressed in HEK-293 cells, a significant fraction of TRPY1, present in the plasma membrane, retained its Ca2+ sensitivity. Based on these data and on homology with TRPV channels, we conclude that D401 and D405 are key residues within the vacuolar vestibule of the TRPY1 pore that decrease cation access or permeation after Ca2+ binding.

IP3 Receptor-Dependent Cytoplasmic Ca2+ Signals Are Tightly Controlled by Cavß3.

Voltage-gated calcium channels (Cavs) are major Ca2+ entry pathways in excitable cells. Their ß subunits facilitate membrane trafficking of the channel's ion-conducting α1 pore and modulate its gating properties. We report that one ß subunit, ß3, reduces Ca2+ release following stimulation of phospholipase C-coupled receptors and inositol 1,4,5-trisphosphate (IP3) formation. This effect requires the SH3-HOOK domain of Cavß3, includes physical ß3/IP3 receptor interaction, and prevails when agonist-induced IP3 formation is bypassed by photolysis of caged IP3. In agreement with ß3 acting as a brake on Ca2+ release, fibroblast migration is enhanced in vitro, and in vivo, closure of skin wounds is accelerated in the absence of ß3. To mediate specific physiological responses and to prevent Ca2+ toxicity, cytoplasmic Ca2+ signals must be tightly controlled. The described function of ß3, unrelated to its function as a Cav subunit, adds to this tight control.

Protein kinase A regulates C-terminally truncated CaV 1.2 in Xenopus oocytes: roles of N- and C-termini of the α1C subunit.

KEY POINTS: ß-Adrenergic stimulation enhances Ca2+ entry via L-type CaV 1.2 channels, causing stronger contraction of cardiac muscle cells. The signalling pathway involves activation of protein kinase A (PKA), but the molecular details of PKA regulation of CaV 1.2 remain controversial despite extensive research. We show that PKA regulation of CaV 1.2 can be reconstituted in Xenopus oocytes when the distal C-terminus (dCT) of the main subunit, α1C , is truncated. The PKA upregulation of CaV 1.2 does not require key factors previously implicated in this mechanism: the clipped dCT, the A kinase-anchoring protein 15 (AKAP15), the phosphorylation sites S1700, T1704 and S1928, or the ß subunit of CaV 1.2. The gating element within the initial segment of the N-terminus of the cardiac isoform of α1C is essential for the PKA effect. We propose that the regulation described here is one of two or several mechanisms that jointly mediate the PKA regulation of CaV 1.2 in the heart. ABSTRACT: ß-Adrenergic stimulation enhances Ca2+ currents via L-type, voltage-gated CaV 1.2 channels, strengthening cardiac contraction. The signalling via ß-adrenergic receptors (ß-ARs) involves elevation of cyclic AMP (cAMP) levels and activation of protein kinase A (PKA). However, how PKA affects the channel remains controversial. Recent studies in heterologous systems and genetically engineered mice stress the importance of the post-translational proteolytic truncation of the distal C-terminus (dCT) of the main (α1C ) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT-truncated CaV 1.2 in Xenopus oocytes, which previously failed with the non-truncated α1C . cAMP and the purified catalytic subunit of PKA, PKA-CS, injected into intact oocytes, enhanced CaV 1.2 currents by ∼40% (rabbit α1C ) to ∼130% (mouse α1C ). PKA blockers were used to confirm specificity and the need for dissociation of the PKA holoenzyme. The regulation persisted in the absence of the clipped dCT (as a separate protein), the A kinase-anchoring protein AKAP15, and the phosphorylation sites S1700 and T1704, previously proposed as essential for the PKA effect. The CaV ß2b subunit was not involved, as suggested by extensive mutagenesis. Using deletion/chimeric mutagenesis, we have identified the initial segment of the cardiac long-N-terminal isoform of α1C as a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α1C subunit, is one of several mechanisms underlying the overall PKA action on CaV 1.2 in the heart. We hypothesize that PKA is acting on CaV 1.2, in part, by affecting a structural 'scaffold' comprising the interacting cytosolic N- and C-termini of α1C .

Comparative assessment of the antirestenotic efficacy of two paclitaxel drug-eluting balloons with different coatings in the treatment of in-stent restenosis.

BACKGROUND/OBJECTIVES: Preclinical investigations have suggested that coating technology is crucial for the efficacy of drug-eluting balloons (DEB). Aim of this study is to compare the antirestenotic efficacy of two paclitaxel DEB with different coatings in the treatment of in-stent restenosis (ISR) by means of a morphological and functional assessment. METHODS: In a single center, prospective, non-randomized study, the shellac-paclitaxel coated DIOR, and the urea-paclitaxel coated IN.PACT Falcon were compared in the setting of ISR. Quantitative angiography, fractional flow reserve (FFR), and optical coherence tomography (OCT) were performed at baseline, postprocedure and 6-month follow-up. Main endpoints were QCA, FFR and OCT-based parameters of restenosis. RESULTS: Forty-five patients were included, 20 (44 %) received treatment with the DIOR and 25 (56 %) with the IN.PACT Falcon. Angiographic and device success were 100 and 90 % for the DIOR, and 100 and 92 % for the IN.PACT Falcon, respectively. After 6-months, in-segment late lumen loss (-0.03 ± 0.43 vs. 0.36 ± 0.48 mm, p = 0.014) and diameter stenosis (30.7 ± 16.2 vs. 41.3 ± 22.6 %, p = 0.083) were lower for the IN.PACT Falcon. FFR distal of the stent was significantly higher in the IN.PACT Falcon group (0.92 ± 0.07 vs. 0.84 ± 0.13, p = 0.029) and in-stent FFR gradient was lower (0.05 ± 0.05 vs. 0.13 ± 0.12, p = 0.002). Between postprocedure and follow-up, a 16 % decrease in neointimal volume was observed for the IN.PACT Falcon, while a 30 % increase was observed for the DIOR (p = 0.006). CONCLUSIONS: The IN.PACT Falcon DEB showed higher antirestenotic efficacy than the DIOR in the treatment of ISR, demonstrating that DEB with an excipient-based coating is not equally effective.

Primary percutaneous coronary intervention by drug-eluting balloon angioplasty: the nonrandomized fourth arm of the DEB-AMI (drug-eluting balloon in ST-segment elevation myocardial infarction) trial.

OBJECTIVE: To evaluate a paclitaxel drug-eluting balloon (DEB) only strategy in primary percutaneous coronary intervention (PPCI), aiming at a comparison with bare metal stent (BMS) alone, DEB followed by BMS, and paclitaxel eluting stent (PES), as assessed in the randomized Drug Eluting Balloon in Acute ST-Segment Elevation Myocardial Infarction (DEB-AMI) trial. BACKGROUND: DEB-only seems an attractive strategy in PPCI, as it obviates the risk of stent thrombosis. METHODS: This study is a prospective registry with the same inclusion/exclusion criteria used in the DEB-AMI trial, as it constitutes the fourth, nonrandomized, treatment arm of this trial. Patients presenting with ST-elevation myocardial infarction were allocated to DEB-only (DIOR II, Eurocor GmbH, Bonn, Germany) after successful thrombus aspiration and predilatation. Primary endpoint was 6-month angiographic in-balloon/stent late-luminal loss (LLL). Secondary endpoints were in-balloon/stent binary restenosis and major adverse cardiac events (MACE: death, myocardial infarction, target-vessel revascularization). RESULTS: Forty patients underwent PPCI by DEB-only. Procedural success was achieved in 97.5% with bail-out stenting required in 10.0% of procedures. In DEB-only, LLL was 0.51 ± 0.59 mm as compared to 0.74 ± 0.57 mm in BMS (P = 0.44), 0.64 ± 0.56 mm in DEB+BMS (P = 0.88) and 0.21 ± 0.32 mm in PES (P < 0.01); in-balloon/stent binary restenosis rates were 22.2%, 23.8% (P = 0.67), 28.6% (P = 0.97), and 4.5% (P = 0.07), respectively; and MACE rates were 17.5%, 23.5% (P = 0.20), 20.0% (P = 0.26), and 4.1% (P = 0.90), respectively. No acute or late thrombotic events occurred in the DEB-only group. CONCLUSIONS: PPCI by DEB-only in selected patients yielded an angiographic outcome comparable to BMS alone and DEB followed by BMS. PES proved angiographic superiority to DEB-only. DEB-only is therefore a potential treatment alternative during PPCI in patients with contra-indications to drug-eluting stents.

Emerging Alternative Functions for the Auxiliary Subunits of the Voltage-Gated Calcium Channels.

Voltage gated calcium channels (Cav) are composed of up to five proteins: The ion conducting pore subunit α1 and the auxiliary subunits α2, δ, ß, and γ. Recent reports show that Cavα1 and Cavß comprise the calcium channel core complex and that ß, α2 δ and γ may serve additional roles that are independent of the Cavα1 subunit. This short review will summarize these emerging functions.