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Role of the Endothelial Inositol 1,4,5-Trisphosphate Receptor in Blood Pressure Regulation
Wednesday, February 15, 2017 cardiac stem cells was identified. These cells are positive for W8B2 marker (called also mesenchymal stem cell antigen-1). W8B2 positive cardiac stem cells (W8B2þ CSCs) exhibit a strong therapeutic potential when transplanted into a chronic myocardial infarction rat model. However, the functional characterization (electrophysiology and calcium signaling) of these cells has not been studied yet. We first establish the conditions of isolation and expansion of W8B2þ CSCs from human heart biopsies by magnetic cell sorting system followed by flow cytometry cell sorting. These cells have a self-renewal capacity demonstrated by their ability to form colonies. In addition, our preliminary results of RT-qPCR show an induction of transcripts encoding contractile proteins such as actin and cardiac troponin-T after in vitro differentiation of W8B2þ CSCs. This differentiation is accompanied by the appearance of cyclic calcium activity assessed by the calcium protein sensor GCaMP protein. The analysis of calcium activity shows that calcium oscillations profile change during differentiation. Using patch-clamp in whole cell configuration, we show for the first time the electrophysiological signing of BKCa channel. In addition, RT-PCR analysis reveals the presence of KCNMA1 (BKCa) mRNA in W8B2þ CSCs. Interestingly, BKCa channel inhibition by paxilline decreases cell proliferation in a concentration-dependent manner and abolishes calcium activity after W8B2þ CSCs differentiation. Taken together, our results are consistent with an important role of BKCa channels on cell cycle progression and on calcium activity during stem cell differentiation. 2649-Pos Board B256 Novel Microarchitecture Induces Functional Remodeling of the Calcium Signaling Mechanisms in Restructured IPSC-Cardiomyocytes Nicole Silbernagel1, Benjamin Richter2, Mona Jaggy3, Sarah Bertels2, Martin Bastmeyer3, Nina D. Ullrich1. 1 University of Heidelberg, Heidelberg, Germany, 2Cell- and Neurobiology, Zoological Institute, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany, 3Cell- and Neurobiology, Zoological Institute, Institute of Functional Interfaces (IFG), Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. New promising therapeutic strategies for regeneration of the diseased heart focus on cardiomyocytes derived from pluripotent stem cells (iPSC-CM). Despite their cardiogenic properties, a well-known drawback of these cells is their immature structural and functional phenotype in contrast to adult cardiomyocytes. A characteristic feature of iPSC-CMs is the lack of a defined microarchitecture, which results in disorganized myofilament arrangement, altered electrophysiological characteristics and desynchronized calcium signals. As a consequence, iPSC-CMs exhibit spontaneous contractile activity and inefficient force production. The aim of this study was to test the hypothesis that a change in cell morphology influences the Ca2þ-handling properties of iPSC-CMs leading to an improved functional phenotype comparable to adult cardiomyocytes. Using the method of direct laser writing, 3D-scaffolds of different geometries were produced from photolabile polyethylene glycol for single cell analysis. Cells were seeded in predesigned rectangular or hexagonal shapes and compared with control cells growing without any spatial limitations. Immunocytochemical investigations of the sarcomeric units revealed strong parallel alignment of myofilaments in rectangular scaffolds, while hexagonal-shaped cells exhibited diffuse myofilament orientation similar to control cells. Combining the techniques of whole-cell voltage clamp and confocal Ca2þ imaging using fluo-4, we discovered that despite similar L-type Ca2þ current densities (in pA/pF: control 12.250.9, rectangle 11.751.4, hexagon 10.651.4) the fast Ca2þ-dependent current inactivation (t1) was significantly slower in rectangular cells due to structural cellular reorganization compared to hexagonal and control cells (in ms: control 11.351.1, rectangle 28.657.5, hexagon 16.751.9). Furthermore, spontaneous Ca2þ transients in rectangular-shaped cells showed a reduction in the peak-to-peak interval variation indicating enhanced signaling maturation (mean variance: control 0.2150.07, rectangle 0.1750.12; range: 1.61 vs. 1.05, respectively). In conclusion our data demonstrate that structural remodeling of iPSC-CMs by distinct cell shapes is paralleled by functional adaptations resulting in maturation of the Ca2þ handling machinery and contractile activity. 2650-Pos Board B257 Role of the Endothelial Inositol 1,4,5-Trisphosphate Receptor in Blood Pressure Regulation Qi Yuan. Physiology, Columbia University, New York, NY, USA. Endothelial cells (EC) are critical mediators of blood pressure regulation, primarily via the generation and release of vasorelaxants including nitric oxide. Nitric oxide (NO) is produced in EC by endothelial nitric oxide synthase (eNOS) which is activated both by calcium (Ca2þ)-dependent and independent
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pathways. Here we report that intracellular Ca2þ release from the endoplasmic reticulum (ER) via the type 1 inositol 1,4,5-trisphosphate receptor (IP3R) is required for Ca2þ-dependent eNOS activation. EC-specific IP3R1 knockout (EC IP3R1-/-) mice develop severe hypertension (HTN) by 3 months of age and blunted vasodilation in response to acetylcholine (ACh). eNOS activity is reduced in both isolated IP3R1 deficient murine EC, and in human EC following IP3R1 knock-down. IP3R1 resides upstream of calcineurin, a Ca2þ/calmodulin activated serine/threonine protein phosphatase. The calcineurin/NFAT pathway was less active and eNOS levels are decreased in IP3R1 deficient EC. The calcineurin inhibitor cyclosporin A (CsA) reduced eNOS activity and vasodilation following ACh stimulation. Our results demonstrate that IP3R1 provides the Ca2þ necessary for EC-mediated vasorelaxation and the maintenance of normal blood pressure. 2651-Pos Board B258 Tissue-Specific Mitochondrial Decoding of Cytoplasmic Ca2D Signals is Controlled by the Stoichiometry of MICU1/2 and MCU Melanie Paillard1, Gyo¨rgy Csorda´s1, Gergo¨ Szanda2, T€unde Golena´r1, ´ da´m Barto´k1, Cynthia Moffat1, Erin L. Seifert1, Valentina Debattisti1, A Andra´s Sp€at2, Gyo¨rgy Hajno´czky1. 1 MitoCare Center, Pathology, Thomas Jefferson University, Philadelphia, PA, USA, 2Department of Physiology, Semmelweis University, Budapest, Hungary. Mitochondrial Ca2þ uptake through the uniporter is central to oxidative metabolism and cell signaling. However, the mechanisms underlying its adaptation to the tissue-specific differences have remained elusive. Both mitochondrial Ca2þ uptake kinetics and mRNA level for the pore forming unit of the uniporter (MCU) and the Ca2þ sensing regulators (MICUs) are tissue-specific. We have investigated if the mitochondrial Ca2þ uptake differences are determined by tissue-specific stoichiometry between MICUs and MCU. In fluorometric measurements of mitochondrial Ca2þ uptake, heart mitochondria displayed a decreased threshold and lesser cooperativity compared to liver mitochondria. Additionally, NAD(P)H elevation was detectable after exposure to moderate [Ca2þ] elevations only in heart mitochondria. Assessment of more direct mitochondrial Ca2þ uptake by 45Ca sequestration showed that liver mitochondria has a higher threshold and Ca2þ-dependence for uniporter activation than heart and skeletal muscle from the same donor mouse. Only the MICU1 to MCU protein expression ratio showed a complementing pattern with the mitochondrial Ca2þ uptake phenotype: lower MICU1 to MCU ratio in heart and skeletal muscle correlated with lower threshold and cooperativity of the uniporter than in liver. Increasing the MICU1 protein level in HEKs cells led to a higher MICU1 amount pulled down by MCU, showing that the abundance of MICU1 relative to MCU is reflected directly in the association between MICU1 and MCU. Overexpression of MICU1 in the heart using AAV9-MICU1 tail-vein injection increased the MICU1 to MCU ratio and caused conversion to liver-like mitochondrial Ca2þ uptake phenotype, further leading to a contractile dysfunction of the heart. In summary, our work identifies the MICU1 to MCU ratio as a physiological regulator of the mitochondrial Ca2þ uptake, contributing to the tissue-specific decoding of [Ca2þ]c oscillations towards differentially regulating oxidative metabolism in each tissue. 2652-Pos Board B259 Interactions between Transmembrane Helices in Orai1 Regulate CRAC Channel Gating Priscilla S.-W. Yeung, Megumi Yamashita, Murali Prakriya. Department of Pharmacology, Northwestern University, Chicago, IL, USA. Ca2þ release-activated Ca2þ (CRAC) channels are a family of plasma membrane-localized Ca2þ channels that are activated in response to depletion of ER Ca2þ stores, a common occurrence following stimulation of a variety of cell surface receptors. CRAC channels are assembled from two proteins: Orai1, the pore-forming subunit, and STIM1, the ER Ca2þ sensor and CRAC channel activator. Each channel consists of six Orai1 molecules with the transmembrane (TM) helices arranged in concentric rings around the pore-lining TM1 segments. The current model of channel gating postulates that the Orai1 C-terminus recruits STIM1 to the channel while STIM1 binding to the Orai1 N-terminus is exclusively responsible for channel gating. Mutations at the N-terminus abolish gating, which was attributed to a defect in STIM1 binding at the N-terminus. However, several recent studies have suggested a role for the Orai1 C-terminus in gating while calling into question the relevance of the STIM1 binding site at the N-terminus. The increasingly evident role of the C-terminus in channel activation has led us explore a conformational wave that propagates from the Orai1 C-terminus through the TMs to open the pore. In order to locate key interactions between the TMs that are involved in pore opening, we performed a cysteine scan of all four TMs using patch-clamp analysis and
537a
pathways. Here we report that intracellular Ca2þ release from the endoplasmic reticulum (ER) via the type 1 inositol 1,4,5-trisphosphate receptor (IP3R) is required for Ca2þ-dependent eNOS activation. EC-specific IP3R1 knockout (EC IP3R1-/-) mice develop severe hypertension (HTN) by 3 months of age and blunted vasodilation in response to acetylcholine (ACh). eNOS activity is reduced in both isolated IP3R1 deficient murine EC, and in human EC following IP3R1 knock-down. IP3R1 resides upstream of calcineurin, a Ca2þ/calmodulin activated serine/threonine protein phosphatase. The calcineurin/NFAT pathway was less active and eNOS levels are decreased in IP3R1 deficient EC. The calcineurin inhibitor cyclosporin A (CsA) reduced eNOS activity and vasodilation following ACh stimulation. Our results demonstrate that IP3R1 provides the Ca2þ necessary for EC-mediated vasorelaxation and the maintenance of normal blood pressure. 2651-Pos Board B258 Tissue-Specific Mitochondrial Decoding of Cytoplasmic Ca2D Signals is Controlled by the Stoichiometry of MICU1/2 and MCU Melanie Paillard1, Gyo¨rgy Csorda´s1, Gergo¨ Szanda2, T€unde Golena´r1, ´ da´m Barto´k1, Cynthia Moffat1, Erin L. Seifert1, Valentina Debattisti1, A Andra´s Sp€at2, Gyo¨rgy Hajno´czky1. 1 MitoCare Center, Pathology, Thomas Jefferson University, Philadelphia, PA, USA, 2Department of Physiology, Semmelweis University, Budapest, Hungary. Mitochondrial Ca2þ uptake through the uniporter is central to oxidative metabolism and cell signaling. However, the mechanisms underlying its adaptation to the tissue-specific differences have remained elusive. Both mitochondrial Ca2þ uptake kinetics and mRNA level for the pore forming unit of the uniporter (MCU) and the Ca2þ sensing regulators (MICUs) are tissue-specific. We have investigated if the mitochondrial Ca2þ uptake differences are determined by tissue-specific stoichiometry between MICUs and MCU. In fluorometric measurements of mitochondrial Ca2þ uptake, heart mitochondria displayed a decreased threshold and lesser cooperativity compared to liver mitochondria. Additionally, NAD(P)H elevation was detectable after exposure to moderate [Ca2þ] elevations only in heart mitochondria. Assessment of more direct mitochondrial Ca2þ uptake by 45Ca sequestration showed that liver mitochondria has a higher threshold and Ca2þ-dependence for uniporter activation than heart and skeletal muscle from the same donor mouse. Only the MICU1 to MCU protein expression ratio showed a complementing pattern with the mitochondrial Ca2þ uptake phenotype: lower MICU1 to MCU ratio in heart and skeletal muscle correlated with lower threshold and cooperativity of the uniporter than in liver. Increasing the MICU1 protein level in HEKs cells led to a higher MICU1 amount pulled down by MCU, showing that the abundance of MICU1 relative to MCU is reflected directly in the association between MICU1 and MCU. Overexpression of MICU1 in the heart using AAV9-MICU1 tail-vein injection increased the MICU1 to MCU ratio and caused conversion to liver-like mitochondrial Ca2þ uptake phenotype, further leading to a contractile dysfunction of the heart. In summary, our work identifies the MICU1 to MCU ratio as a physiological regulator of the mitochondrial Ca2þ uptake, contributing to the tissue-specific decoding of [Ca2þ]c oscillations towards differentially regulating oxidative metabolism in each tissue. 2652-Pos Board B259 Interactions between Transmembrane Helices in Orai1 Regulate CRAC Channel Gating Priscilla S.-W. Yeung, Megumi Yamashita, Murali Prakriya. Department of Pharmacology, Northwestern University, Chicago, IL, USA. Ca2þ release-activated Ca2þ (CRAC) channels are a family of plasma membrane-localized Ca2þ channels that are activated in response to depletion of ER Ca2þ stores, a common occurrence following stimulation of a variety of cell surface receptors. CRAC channels are assembled from two proteins: Orai1, the pore-forming subunit, and STIM1, the ER Ca2þ sensor and CRAC channel activator. Each channel consists of six Orai1 molecules with the transmembrane (TM) helices arranged in concentric rings around the pore-lining TM1 segments. The current model of channel gating postulates that the Orai1 C-terminus recruits STIM1 to the channel while STIM1 binding to the Orai1 N-terminus is exclusively responsible for channel gating. Mutations at the N-terminus abolish gating, which was attributed to a defect in STIM1 binding at the N-terminus. However, several recent studies have suggested a role for the Orai1 C-terminus in gating while calling into question the relevance of the STIM1 binding site at the N-terminus. The increasingly evident role of the C-terminus in channel activation has led us explore a conformational wave that propagates from the Orai1 C-terminus through the TMs to open the pore. In order to locate key interactions between the TMs that are involved in pore opening, we performed a cysteine scan of all four TMs using patch-clamp analysis and