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1.
Kidney Int ; 105(6): 1263-1278, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38286178

ABSTRACT

Current classification of chronic kidney disease (CKD) into stages using indirect systemic measures (estimated glomerular filtration rate (eGFR) and albuminuria) is agnostic to the heterogeneity of underlying molecular processes in the kidney thereby limiting precision medicine approaches. To generate a novel CKD categorization that directly reflects within kidney disease drivers we analyzed publicly available transcriptomic data from kidney biopsy tissue. A Self-Organizing Maps unsupervised artificial neural network machine-learning algorithm was used to stratify a total of 369 patients with CKD and 46 living kidney donors as healthy controls. Unbiased stratification of the discovery cohort resulted in identification of four novel molecular categories of disease termed CKD-Blue, CKD-Gold, CKD-Olive, CKD-Plum that were replicated in independent CKD and diabetic kidney disease datasets and can be further tested on any external data at kidneyclass.org. Each molecular category spanned across CKD stages and histopathological diagnoses and represented transcriptional activation of distinct biological pathways. Disease progression rates were highly significantly different between the molecular categories. CKD-Gold displayed rapid progression, with significant eGFR-adjusted Cox regression hazard ratio of 5.6 [1.01-31.3] for kidney failure and hazard ratio of 4.7 [1.3-16.5] for composite of kidney failure or a 40% or more eGFR decline. Urine proteomics revealed distinct patterns between the molecular categories, and a 25-protein signature was identified to distinguish CKD-Gold from other molecular categories. Thus, patient stratification based on kidney tissue omics offers a gateway to non-invasive biomarker-driven categorization and the potential for future clinical implementation, as a key step towards precision medicine in CKD.


Subject(s)
Disease Progression , Glomerular Filtration Rate , Kidney , Precision Medicine , Renal Insufficiency, Chronic , Transcriptome , Humans , Precision Medicine/methods , Renal Insufficiency, Chronic/pathology , Renal Insufficiency, Chronic/urine , Renal Insufficiency, Chronic/diagnosis , Renal Insufficiency, Chronic/physiopathology , Middle Aged , Female , Male , Kidney/pathology , Kidney/physiopathology , Aged , Biopsy , Adult , Neural Networks, Computer , Case-Control Studies , Gene Expression Profiling , Unsupervised Machine Learning
2.
Int J Mol Sci ; 23(14)2022 Jul 19.
Article in English | MEDLINE | ID: mdl-35887308

ABSTRACT

(1) The cardio-reno-metabolic benefits of the SGLT2 inhibitors canagliflozin (cana), dapagliflozin (dapa), ertugliflozin (ertu), and empagliflozin (empa) have been demonstrated, but it remains unclear whether they exert different off-target effects influencing clinical profiles. (2) We aimed to investigate the effects of SGLT2 inhibitors on mitochondrial function, cellular glucose-uptake (GU), and metabolic pathways in human-umbilical-vein endothelial cells (HUVECs). (3) At 100 µM (supra-pharmacological concentration), cana decreased ECAR by 45% and inhibited GU (IC5o: 14 µM). At 100 µM and 10 µM (pharmacological concentration), cana increased the ADP/ATP ratio, whereas dapa and ertu (3, 10 µM, about 10× the pharmacological concentration) showed no effect. Cana (100 µM) decreased the oxygen consumption rate (OCR) by 60%, while dapa decreased it by 7%, and ertu and empa (all 100 µM) had no significant effect. Cana (100 µM) inhibited GLUT1, but did not significantly affect GLUTs' expression levels. Cana (100 µM) treatment reduced glycolysis, elevated the amino acids supplying the tricarboxylic-acid cycle, and significantly increased purine/pyrimidine-pathway metabolites, in contrast to dapa (3 µM) and ertu (10 µM). (4) The results confirmed cana´s inhibition of mitochondrial activity and GU at supra-pharmacological and pharmacological concentrations, whereas the dapa, ertu, and empa did not show effects even at supra-pharmacological concentrations. At supra-pharmacological concentrations, cana (but not dapa or ertu) affected multiple cellular pathways and inhibited GLUT1.


Subject(s)
Diabetes Mellitus, Type 2 , Sodium-Glucose Transporter 2 Inhibitors , Benzhydryl Compounds/pharmacology , Canagliflozin/pharmacology , Diabetes Mellitus, Type 2/drug therapy , Endothelial Cells , Glucose , Glucose Transporter Type 1 , Humans , Mitochondria , Oxidative Phosphorylation , Sodium-Glucose Transporter 2 Inhibitors/pharmacology
3.
Pulm Pharmacol Ther ; 69: 102035, 2021 08.
Article in English | MEDLINE | ID: mdl-33933611

ABSTRACT

The novel coronavirus 2019 (COVID-19) infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global pandemic that requires a multi-faceted approach to tackle this unprecedent health crisis. Therapeutics to treat COVID-19 are an integral part of any such management strategy and there is a substantial unmet need for treatments for individuals most at risk of severe disease. This perspective review provides rationale of a combined therapeutic regimen of selective endothelin-A (ET-A) receptor antagonism and sodium glucose co-transporter-2 (SGLT-2) inhibition to treat COVID-19. Endothelin is a potent vasoconstrictor with pro-inflammatory and atherosclerotic effects. It is upregulated in a number of conditions including acute respiratory distress syndrome and cardiovascular disease. Endothelin mediates vasocontractility via endothelin (ET-A and ET-B) receptors on vascular smooth muscle cells (VSMCs). ET-B receptors regulate endothelin clearance and are present on endothelial cells, where in contrast to their role on VSMCs, mediate vasodilation. Therefore, selective endothelin-A (ET-A) receptor inhibition is likely the optimal approach to attenuate the injurious effects of endothelin and may reduce ventilation-perfusion mismatch and pulmonary inflammation, whilst improving pulmonary haemodynamics and oxygenation. SGLT-2 inhibition may dampen inflammatory cytokines, reduce hyperglycaemia if present, improve endothelial function, cardiovascular haemodynamics and cellular bioenergetics. This combination therapeutic approach may therefore have beneficial effects to mitigate both the pulmonary, metabolic and cardiorenal manifestations of COVID-19. Given these drug classes include medicines licensed to treat heart failure, diabetes and pulmonary hypertension respectively, information regarding their safety profile is established. Randomised controlled clinical trials are the best way to determine efficacy and safety of these medicines in COVID-19.


Subject(s)
COVID-19 , Endothelin Receptor Antagonists , Endothelial Cells/metabolism , Endothelin-1/metabolism , Endothelins , Glucose , Humans , SARS-CoV-2 , Sodium , Sodium-Glucose Transporter 2
4.
J Mol Med (Berl) ; 102(7): 841-858, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38753041

ABSTRACT

Liver cirrhosis due to nonalcoholic steatohepatitis (NASH) is a life-threatening condition with increasing incidence world-wide. Although its symptoms are unspecific, it can lead to decompensation events such as ascites, hepatic encephalopathy, variceal hemorrhage, and hepatocellular carcinoma (HCC). In addition, an increased risk for cardiovascular events has been demonstrated in patients with NASH. Pharmacological treatments for NASH cirrhosis are not yet available, one of the reasons being the lack in surrogate endpoints available in clinical trials of NASH cirrhosis. The feasibility of non-invasive prognostic biomarkers makes them interesting candidates as possible surrogate endpoints if their change following treatment would result in better outcomes for patients in future clinical trials of NASH cirrhosis. In this systematic literature review, a summary of the available literature on the prognostic performance of non-invasive biomarkers in terms of cardiovascular events, liver-related events, and mortality is outlined. Due to the scarcity of data specific for NASH cirrhosis, this review includes studies on NAFLD whose evaluation focuses on cirrhosis. Our search strategy identified the following non-invasive biomarkers with prognostic value in studies of NASH patients: NAFLD fibrosis score (NFS), Fibrosis-4 (FIB-4), aspartate aminotransferase (AST) to platelet ratio index (APRI), enhanced liver fibrosis (ELF™), BARD (BMI, AST/ALT (alanine aminotransferase) ratio, diabetes), Hepamet Fibrosis Score (HFS), liver enzymes (AST + ALT), alpha-fetoprotein, platelet count, neutrophil to lymphocyte ratio (NLR), Lysyl oxidase-like (LOXL) 2, miR-122, liver stiffness, MEFIB (liver stiffness measured with magnetic resonance elastography (MRE) + FIB-4), and PNPLA3 GG genotype. The aim of the present systematic literature review is to provide the reader with a summary of the non-invasive biomarkers with prognostic value in NASH cirrhosis and give an evaluation of their utility as treatment monitoring biomarkers in future clinical trials.


Subject(s)
Biomarkers , Liver Cirrhosis , Non-alcoholic Fatty Liver Disease , Humans , Non-alcoholic Fatty Liver Disease/complications , Non-alcoholic Fatty Liver Disease/diagnosis , Liver Cirrhosis/diagnosis , Liver Cirrhosis/complications , Liver Cirrhosis/etiology , Prognosis
5.
J Biol Chem ; 287(27): 22584-92, 2012 Jun 29.
Article in English | MEDLINE | ID: mdl-22589548

ABSTRACT

Phosphorylation of the cardiac ß subunit (Ca(v)ß(2)) of the Ca(v)1.2 L-type Ca(2+) channel complex has been proposed as a mechanism for regulation of L-type Ca(2+) channels by various protein kinases including PKA, CaMKII, Akt/PKB, and PKG. To test this hypothesis directly in vivo, we generated a knock-in mouse line with targeted mutation of the Ca(v)ß(2) gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; ßStop mouse). This mutation prevented translation of the Ca(v)ß(2) C terminus that contains the relevant phosphorylation sites for the above protein kinases. Homozygous cardiac ßStop mice were born at Mendelian ratio, had a normal life expectancy, and normal basal L-type I(Ca). The regulation of the L-type current by stimulation of the ß-adrenergic receptor was unaffected in vivo and in cardiomyocytes (CMs). ßStop mice were cross-bred with mice expressing the Ca(v)1.2 gene containing the mutation S1928A (SAßStop) or S1512A and S1570A (SFßStop) in the C terminus of the α(1C) subunit. The ß-adrenergic regulation of the cardiac I(Ca) was unaltered in these mouse lines. In contrast, truncation of the Ca(v)1.2 at Asp(1904) abolished ß-adrenergic up-regulation of I(Ca) in murine embryonic CMs. We conclude that phosphorylation of the C-terminal sites in Ca(v)ß(2), Ser(1928), Ser(1512), and Ser(1570) of the Ca(v)1.2 protein is functionally not involved in the adrenergic regulation of the murine cardiac Ca(v)1.2 channel.


Subject(s)
Calcium Channels, L-Type/chemistry , Calcium Channels, L-Type/metabolism , Heart/physiology , Myocytes, Cardiac/physiology , Receptors, Adrenergic, beta/physiology , Adrenergic beta-Agonists/pharmacology , Animals , Base Sequence , Calcium Channels, L-Type/genetics , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/metabolism , Electrocardiography , Female , Gene Deletion , Isoproterenol/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Molecular Sequence Data , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Phosphorylation/physiology , Protein Structure, Tertiary/physiology
6.
J Biol Chem ; 287(27): 22616-25, 2012 Jun 29.
Article in English | MEDLINE | ID: mdl-22589547

ABSTRACT

Cardiac excitation-contraction coupling (EC coupling) links the electrical excitation of the cell membrane to the mechanical contractile machinery of the heart. Calcium channels are major players of EC coupling and are regulated by voltage and Ca(2+)/calmodulin (CaM). CaM binds to the IQ motif located in the C terminus of the Ca(v)1.2 channel and induces Ca(2+)-dependent inactivation (CDI) and facilitation (CDF). Mutation of Ile to Glu (Ile1624Glu) in the IQ motif abolished regulation of the channel by CDI and CDF. Here, we addressed the physiological consequences of such a mutation in the heart. Murine hearts expressing the Ca(v)1.2(I1624E) mutation were generated in adult heterozygous mice through inactivation of the floxed WT Ca(v)1.2(L2) allele by tamoxifen-induced cardiac-specific activation of the MerCreMer Cre recombinase. Within 10 days after the first tamoxifen injection these mice developed dilated cardiomyopathy (DCM) accompanied by apoptosis of cardiac myocytes (CM) and fibrosis. In Ca(v)1.2(I1624E) hearts, the activity of phospho-CaM kinase II and phospho-MAPK was increased. CMs expressed reduced levels of Ca(v)1.2(I1624E) channel protein and I(Ca). The Ca(v)1.2(I1624E) channel showed "CDI" kinetics. Despite a lower sarcoplasmic reticulum Ca(2+) content, cellular contractility and global Ca(2+) transients remained unchanged because the EC coupling gain was up-regulated by an increased neuroendocrine activity. Treatment of mice with metoprolol and captopril reduced DCM in Ca(v)1.2(I1624E) hearts at day 10. We conclude that mutation of the IQ motif to IE leads to dilated cardiomyopathy and death.


Subject(s)
Calcium Channels, L-Type/genetics , Calmodulin/metabolism , Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/mortality , Amino Acid Motifs/genetics , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Anti-Arrhythmia Agents/pharmacology , Binding Sites/genetics , Calcium/metabolism , Calcium Channels, L-Type/chemistry , Calcium Channels, L-Type/metabolism , Captopril/pharmacology , Cardiomyopathy, Dilated/drug therapy , Cells, Cultured , Heart Failure/drug therapy , Heart Failure/genetics , Heart Failure/mortality , Metoprolol/pharmacology , Mice , Mice, Mutant Strains , Myocardial Contraction/physiology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/physiology , Protein Structure, Tertiary/genetics , Survival Rate
7.
Proc Natl Acad Sci U S A ; 107(22): 10285-9, 2010 Jun 01.
Article in English | MEDLINE | ID: mdl-20479240

ABSTRACT

Activity-dependent means of altering calcium (Ca(2)(+)) influx are assumed to be of great physiological consequence, although definitive tests of this assumption have only begun to emerge. Facilitation and inactivation offer two opposing, activity-dependent means of altering Ca(2+) influx via cardiac Ca(v)1.2 calcium channels. Voltage- and frequency-dependent facilitation of Ca(v)1.2 has been reported to depend on Calmodulin (CaM) and/or the activity of Calmodulin kinase II (CaMKII). Several sites within the cardiac L-type calcium channel complex have been proposed as the targets of CaMKII. Here, we generated mice with knockin mutations of alpha(1)1.2 S1512 and S1570 phosphorylation sites [sine facilitation (SF) mice]. Homocygote SF mice were viable and reproduced in a Mendelian ratio. Voltage-dependent facilitation in ventricular cardiomyocytes carrying the SF mutation was decreased from 1.58- to 1.18-fold. The CaMKII inhibitor KN-93 reduced facilitation to 1.28 in control cardiomyocytes. SF mutation negatively shifted the voltage-dependent inactivation and slowed recovery from inactivation, thereby making fewer channels available for activation. Telemetric ECG recordings at different heart rates showed that QT time decreased significantly more in SF than in control mice at higher rates. Our results strongly support the notion that CaMKII-dependent phosphorylation of Cav1.2 at S1512 and S1570 mediates Ca(2+) current facilitation in the murine heart.


Subject(s)
Calcium Channels, L-Type/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Myocytes, Cardiac/metabolism , Amino Acid Substitution , Animals , Calcium Channels, L-Type/chemistry , Calcium Channels, L-Type/genetics , Calcium Signaling , Gene Knock-In Techniques , Ion Channel Gating , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mice, Transgenic , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Phosphorylation , Protein Subunits
8.
J Neurosci ; 31(38): 13562-75, 2011 Sep 21.
Article in English | MEDLINE | ID: mdl-21940447

ABSTRACT

AMPA receptor (AMPAR) plasticity at glutamatergic synapses in the mesoaccumbal dopaminergic pathway has been implicated in persistent cocaine-induced behavioral responses; however, the precise mechanism underlying these changes remains unknown. Utilizing cocaine psychomotor sensitization, we have examined phosphorylation of GluA1 at key residues serine 845 (S845) and S831, as well as GluA1 cell surface levels in the nucleus accumbens (NAc) of cocaine-preexposed mice and the role of brain-specific Ca(v)1.2 and Ca(v)1.3 L-type Ca²âº channels (LTCCs), therein. We found higher basal levels of S845 phospho-GluA1 (P-GluA1) and cell surface GluA1 in the NAc following protracted withdrawal from cocaine exposure, changes that occur independently of LTCCs. In contrast, we found that a cocaine challenge that elicits expression of the cocaine-sensitized response increases S831 P-GluA1 that further increases surface GluA1 beyond the higher basal levels. Intra-NAc pharmacological manipulations indicate that the Ca(v)1.2-activated CaM kinase II (CaMKII) mediates cocaine-induced increase in S831 P-GluA1 and that both Ca(v)1.2-activated CaMKII and extracellular signal-regulated kinase 2 (ERK2) mediate the increase in GluA1 cell surface levels specific to the sensitized response. Experiments using adenoassociated viral vectors expressing Ca(v)1.3 and ERK2 siRNA further indicate that recruitment of the Ca(v)1.2 pathway in the NAc is dependent on ventral tegmental area Ca(v)1.3 LTCCs and ERK2. Together, these results identify candidate pathways that mediate cocaine-induced AMPAR plasticity in the NAc and provide a mechanism linking LTCCs and GluA1 plasticity to cocaine-induced persistent behavioral changes.


Subject(s)
Adaptation, Physiological/physiology , Calcium Channels, L-Type/physiology , Cocaine/pharmacology , Nucleus Accumbens/physiology , Receptors, AMPA/metabolism , Ventral Tegmental Area/physiology , Adaptation, Physiological/drug effects , Animals , Calcium Channels, L-Type/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/physiology , Dependovirus/genetics , Genetic Vectors , Male , Membrane Proteins/metabolism , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/physiology , Motor Activity/drug effects , Motor Activity/physiology , Nucleus Accumbens/drug effects , Nucleus Accumbens/metabolism , Phosphorylation , RNA, Small Interfering/genetics , Receptors, AMPA/physiology , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiology , Ventral Tegmental Area/drug effects , Ventral Tegmental Area/metabolism
9.
J Biol Chem ; 286(30): 26702-7, 2011 Jul 29.
Article in English | MEDLINE | ID: mdl-21665954

ABSTRACT

The heart muscle responds to physiological needs with a short-term modulation of cardiac contractility. This process is determined mainly by properties of the cardiac L-type Ca(2+) channel (Ca(v)1.2), including facilitation and Ca(2+)-dependent inactivation (CDI). Both facilitation and CDI involve the interaction of calmodulin with the IQ motif of the Ca(v)1.2 channel, especially with Ile-1624. To verify this hypothesis, we created a mouse line in which Ile-1624 was mutated to Glu (Ca(v)1.2(I1624E) mice). Homozygous Ca(v)1.2(I1624E) mice were not viable. Therefore, we inactivated the floxed Ca(v)1.2 gene of heterozygous Ca(v)1.2(I1624E) mice by the α-myosin heavy chain-MerCreMer system. The resulting I/E mice were studied at day 10 after treatment with tamoxifen. Electrophysiological recordings in ventricular cardiomyocytes revealed a reduced Ca(v)1.2 current (I(Ca)) density in I/E mice. Steady-state inactivation and recovery from inactivation were modified in I/E versus control mice. In addition, voltage-dependent facilitation was almost abolished in I/E mice. The time course of I(Ca) inactivation in I/E mice was not influenced by the use of Ba(2+) as a charge carrier. Using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid as a chelating agent for intracellular Ca(2+), inactivation of I(Ca) was slowed down in control but not I/E mice. The results show that the I/E mutation abolishes Ca(2+)/calmodulin-dependent regulation of Ca(v)1.2. The Ca(v)1.2(I1624E) mutation transforms the channel to a phenotype mimicking CDI.


Subject(s)
Calcium Channels, L-Type/metabolism , Calcium/metabolism , Heart Ventricles/metabolism , Mutation, Missense , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Amino Acid Motifs , Amino Acid Substitution , Animals , Antineoplastic Agents, Hormonal/pharmacology , Calcium Channels, L-Type/genetics , Cells, Cultured , Heart Ventricles/pathology , Mice , Mice, Mutant Strains , Myocardium/pathology , Myocytes, Cardiac/pathology , Tamoxifen/pharmacology
10.
J Biol Chem ; 286(39): 33863-71, 2011 Sep 30.
Article in English | MEDLINE | ID: mdl-21832054

ABSTRACT

The carboxyl-terminal intracellular tail of the L-type Ca(2+) channel CaV1.2 modulates various aspects of channel activity.For example, deletion of the carboxyl-terminal sequence at Ser-1905 increased CaV1.2 currents in an expression model. To verify this finding in an animal model, we inserted three stop codons at the corresponding Asp-1904 in the murine CaV1.2 gene. Mice homozygous for the Stop mutation (Stop/Stop mice)were born at a Mendelian ratio but died after birth. Stop/Stop hearts showed reduced beating frequencies and contractions.Surprisingly, Stop/Stop cardiomyocytes displayed reduced IBa and a minor expression of the CaV1.2Stop protein. In contrast,expression of the CaV1.2Stop protein was normal in pooled smooth muscle samples from Stop/Stop embryos. As the CaV1.2 channel exists in a cardiac and smooth muscle splice variant, HK1 and LK1, respectively, we analyzed the consequences of the deletion of the carboxyl terminus in the respective splice variant using the rabbit CaV1.2 clone expressed in HEK293 cells.HEK293 cells transfected with the HK1Stop channel showed a reduced IBa and CaV1.2 expression. Treatment with proteasome inhibitors increased the expression of HK1Stop protein and IBa in HEK293 cells and in Stop/Stop cardiomyocytes indicating that truncation of CaV1.2 containing the cardiac exon 1a amino terminus results in proteasomal degradation of the translated protein. In contrast, HEK293 cells transfected with the LK1Stop channel had normal IBa and CaV1.2 expression. These findings indicate that absence of the carboxyl-terminal tail differentially determines the fate of the cardiac and smooth muscle splice variant of the CaV1.2 channel in the mouse.


Subject(s)
Calcium Channels, L-Type/metabolism , Codon, Terminator , Heart Failure/metabolism , Infant, Newborn, Diseases/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Smooth Muscle/metabolism , Alternative Splicing/genetics , Animals , Calcium Channels, L-Type/genetics , Disease Models, Animal , Embryo, Mammalian/metabolism , HEK293 Cells , Heart Failure/genetics , Humans , Infant, Newborn , Infant, Newborn, Diseases/genetics , Mice , Myocardial Contraction/genetics , Organ Specificity/genetics , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Structure, Tertiary , Rabbits
11.
Circ Res ; 106(7): 1285-9, 2010 Apr 16.
Article in English | MEDLINE | ID: mdl-20299662

ABSTRACT

RATIONALE: In arterial myocytes, membrane depolarization-induced Ca(2+) release (DICR) from the sarcoplasmic reticulum (SR) occurs through a metabotropic pathway that leads to inositol trisphosphate synthesis independently of extracellular Ca(2+) influx. Despite the fundamental functional relevance of DICR, its molecular bases are not well known. OBJECTIVE: Biophysical and pharmacological data have suggested that L-type Ca(2+) channels could be the sensors coupling membrane depolarization to SR Ca(2+) release. This hypothesis was tested using smooth muscle-selective conditional Ca(v)1.2 knockout mice. METHODS AND RESULTS: In aortic myocytes, the decrease of Ca(2+) channel density was paralleled by the disappearance of SR Ca(2+) release induced by either depolarization or Ca(2+) channel agonists. Ca(v)1.2 channel deficiency resulted in almost abolition of arterial ring contraction evoked by DICR. Ca(2+) channel-null cells showed unaltered caffeine-induced Ca(2+) release and contraction. CONCLUSION: These data suggest that Ca(v)1.2 channels are indeed voltage sensors coupled to the metabolic cascade, leading to SR Ca(2+) release. These findings support a novel, ion-independent, functional role of L-type Ca(2+) channels linked to intracellular signaling pathways in vascular myocytes.


Subject(s)
Calcium Channels, L-Type/deficiency , Calcium Signaling , Muscle, Smooth, Vascular/metabolism , Sarcoplasmic Reticulum/metabolism , Vasoconstriction , Animals , Aorta/metabolism , Caffeine/pharmacology , Calcium Channels, L-Type/genetics , Calcium Signaling/drug effects , Membrane Potentials , Mice , Mice, Knockout , Muscle, Smooth, Vascular/drug effects , Potassium/metabolism , Sarcoplasmic Reticulum/drug effects , Sodium/metabolism , Time Factors , Vasoconstriction/drug effects
12.
J Neurosci ; 30(25): 8367-75, 2010 Jun 23.
Article in English | MEDLINE | ID: mdl-20573883

ABSTRACT

Ca(2+) influx through postsynaptic Ca(v)1.x L-type voltage-gated channels (LTCCs) is particularly effective in activating neuronal biochemical signaling pathways that might be involved in Hebbian synaptic plasticity (i.e., long-term potentiation and depression) and learning and memory. Here, we demonstrate that Ca(v)1.2 is the functionally relevant LTCC isoform in the thalamus-amygdala pathway of mice. We further show that acute pharmacological block of LTCCs abolishes Hebbian plasticity in the thalamus-amygdala pathway and impairs the acquisition of conditioned fear. On the other hand, chronic genetic loss of Ca(v)1.2 triggers a homeostatic change of the synapse, leading to a fundamental alteration of the mechanism of Hebbian plasticity by synaptic incorporation of Ca(2+)-permeable, GluA2-lacking AMPA receptors. Our results demonstrate for the first time the importance of the Ca(v)1.2 LTCC subtype in synaptic plasticity and fear memory acquisition.


Subject(s)
Amygdala/physiology , Calcium Channels, L-Type/physiology , Conditioning, Classical/physiology , Fear/physiology , Long-Term Potentiation/physiology , Thalamus/physiology , Analysis of Variance , Animals , Blotting, Western , Electrophysiology , Homeostasis/physiology , In Situ Hybridization , Mice , Mice, Transgenic , Neural Pathways/physiology , Neurons/physiology , Receptors, AMPA/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Silver Staining , Synapses/physiology , Synaptic Transmission/physiology , Video Recording
13.
FASEB J ; 24(3): 700-11, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19850773

ABSTRACT

Ectopic expression of defined sets of genetic factors can reprogram somatic cells to create induced pluripotent stem (iPS) cells. The capacity to direct human iPS cells to specific differentiated lineages and to their progenitor populations can be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. During mouse cardiogenesis, the major lineages of the mature heart, cardiomyocytes, smooth muscle cells, and endothelial cells arise from a common, multipotent cardiovascular progenitor expressing the transcription factors Isl1 and Nkx2.5. Here we show, using genetic fate-mapping, that Isl1(+) multipotent cardiovascular progenitors can be generated from mouse iPS cells and spontaneously differentiate in all 3 cardiovascular lineages in vivo without teratoma. Moreover, we report the identification of human iPS-derived ISL1(+) progenitors with similar developmental potential. These results support the possibility to use patient-specific iPS-generated cardiovascular progenitors as a model to elucidate the pathogenesis of congenital and acquired forms of heart diseases.-Moretti, A., Bellin, M., Jung, C. B., Thies, T.-M., Takashima, Y., Bernshausen, A., Schiemann, M., Fischer, S., Moosmang, S., Smith, A. G., Lam, J. T., Laugwitz, K.-L. Mouse and human induced pluripotent stem cells as a source for multipotent Isl1(+) cardiovascular progenitors.


Subject(s)
Homeodomain Proteins/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Multipotent Stem Cells/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Line , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Flow Cytometry , Heterozygote , Humans , Immunohistochemistry , LIM-Homeodomain Proteins , Mice , Mice, Inbred C57BL , Multipotent Stem Cells/cytology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Polymerase Chain Reaction , Transcription Factors
14.
J Comp Neurol ; 506(4): 569-83, 2008 Feb 01.
Article in English | MEDLINE | ID: mdl-18067152

ABSTRACT

In the hippocampal formation, Ca(v)1.2 (L-type) voltage-gated Ca(2+) channels mediate Ca(2+) signals that can trigger long-term alterations in synaptic efficacy underlying learning and memory. Immunocytochemical studies indicate that Ca(v)1.2 channels are localized mainly in the soma and proximal dendrites of hippocampal pyramidal neurons, but electrophysiological data suggest a broader distribution of these channels. To define the subcellular substrates underlying Ca(v)1.2 Ca(2+) signals, we analyzed the localization of Ca(v)1.2 in the hippocampal formation by using antibodies against the pore-forming alpha(1)-subunit of Ca(v)1.2 (alpha(1)1.2). By light microscopy, alpha(1)1.2-like immunoreactivity (alpha(1)1.2-IR) was detected in pyramidal cell soma and dendritic fields of areas CA1-CA3 and in granule cell soma and fibers in the dentate gyrus. At the electron microscopic level, alpha(1)1.2-IR was localized in dendrites, but also in axons, axon terminals, and glial processes in all hippocampal subfields. Plasmalemmal immunogold particles representing alpha(1)1.2-IR were more significant for small- than large-caliber dendrites and were largely associated with extrasynaptic regions in dendritic spines and axon terminals. These findings provide the first detailed ultrastructural analysis of Ca(v)1.2 localization in the brain and support functionally diverse roles of these channels in the hippocampal formation.


Subject(s)
Calcium Channels, L-Type/metabolism , Calcium Signaling/physiology , Hippocampus/metabolism , Neurons/metabolism , Synapses/metabolism , Synaptic Transmission/physiology , Animals , Calcium Channels, L-Type/genetics , Cell Line , Dendritic Spines/metabolism , Dendritic Spines/ultrastructure , Dentate Gyrus/metabolism , Dentate Gyrus/ultrastructure , Hippocampus/ultrastructure , Humans , Male , Mice , Microscopy, Immunoelectron , Neurons/ultrastructure , Presynaptic Terminals/metabolism , Presynaptic Terminals/ultrastructure , Protein Subunits/metabolism , Pyramidal Cells/metabolism , Pyramidal Cells/ultrastructure , Rats , Rats, Sprague-Dawley , Synapses/ultrastructure , Synaptic Membranes/metabolism , Synaptic Membranes/ultrastructure
15.
Circ Res ; 98(1): 105-10, 2006 Jan 06.
Article in English | MEDLINE | ID: mdl-16306443

ABSTRACT

The role of T-type Ca2+ channels for cardiovascular physiology, in particular blood pressure regulation, is controversial. Selective blockade of T-type Ca2+ channels in resistance arteries has been proposed to explain the effect of the antihypertensive drug mibefradil. In the present study, we used a third generation, time- and tissue-specific conditional knockout model of the L-type Ca2+ channel Cav1.2 (Cav1.2SMAKO mice) to genetically dissect the effects of mibefradil on T- and L-type Ca2+ channels. Myogenic tone and phenylephrine-induced contraction in hindlimb perfusion experiments were sensitive to mibefradil in control mice, whereas the drug showed no effect in Cav1.2-deficient animals. Mean arterial blood pressure in awake, freely moving control mice was reduced by 38+/-2.5 mm Hg at a dose of 1.25 mg/kg bodyweight mibefradil, but not changed in Cav1.2SMAKO mice. These results demonstrate that the effect of the putative T-type Ca2+ channel-selective blocker mibefradil on blood pressure and small vessel myogenic tone is mediated by the Cav1.2 L-type Ca2+ channel.


Subject(s)
Antihypertensive Agents/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/physiology , Calcium Channels, T-Type/physiology , Mibefradil/pharmacology , Animals , Blood Pressure/drug effects , Male , Membrane Potentials , Mice , Mice, Inbred C57BL , Muscle, Smooth, Vascular/physiology
16.
Life Sci ; 82(1-2): 41-9, 2008 Jan 02.
Article in English | MEDLINE | ID: mdl-18045623

ABSTRACT

Ca(v)1.2 L-type calcium channels support hippocampal synaptic plasticity, likely by facilitating dendritic Ca2+ influx evoked by action potentials (AP) back-propagated from the soma. Ca2+ influx into hippocampal neurons during somatic APs is sufficient to activate signalling pathways associated with late phase LTP. Thus, mechanisms controlling AP firing of hippocampal neurons are of major functional relevance. We examined the excitability of CA1 pyramidal cells using somatic current-clamp recordings in brain slices from control type mice and mice with the Ca(v)1.2 gene inactivated in principal hippocampal neurons. Lack of the Ca(v)1.2 protein did not affect either affect basic characteristics, such as resting membrane potential and input resistance, or parameters of single action potentials (AP) induced by 5 ms depolarising current pulses. However, CA1 hippocampal neurons from control and mutant mice differed in their patterns of AP firing during 500 ms depolarising current pulses: threshold voltage for repetitive firing was shifted significantly by about 5 mV to more depolarised potentials in the mutant mice (p<0.01), and the latency until firing of the first AP was prolonged (73.2+/-6.6 ms versus 48.1+/- 7.8 ms in control; p<0.05). CA1 pyramidal cells from the mutant mice also showed a lowered initial spiking frequency within an AP train. In control cells, isradipine had matching effects, while BayK 8644 facilitated spiking. Our data demonstrate that Ca(v)1.2 channels are involved in regulating the intrinsic excitability of CA1 pyramidal neurons. This cellular mechanism may contribute to the known function of Ca(v)1.2 channels in supporting synaptic plasticity and memory.


Subject(s)
Action Potentials/physiology , Calcium Channels, L-Type/physiology , Calcium/metabolism , Pyramidal Cells/physiology , Animals , Calcium Channels, L-Type/genetics , Electrophysiology , In Vitro Techniques , Mice , Mice, Knockout , Neuronal Plasticity/physiology , Patch-Clamp Techniques , Pyramidal Cells/metabolism
17.
Am J Cardiol ; 120(8S): S80-S88, 2017 Oct 15.
Article in English | MEDLINE | ID: mdl-29025574

ABSTRACT

The nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway plays a key role in regulating cardiovascular homeostasis, and genetic variants allocated to NO-cGMP pathway genes, leading to NO-cGMP deficiency, may influence the prevalence or course of cardiovascular disease. NO-cGMP deficiency can be caused by nitric oxide synthase substrate deficiency, substrate competition, defects, or uncoupling; endogenous inhibitors of nitric oxide synthase; decreased cGMP production; or increased cGMP degradation. This review presents evidence supporting the role of NO-cGMP deficiency in cardiovascular disease, including findings from genetic association studies for particular polymorphisms, haplotypes, and racial disparities. NO-cGMP pathway components including arginases, guanosine-5'-triphosphate cyclohydrolase 1, nitric oxide synthase, dimethylarginine dimethylaminohydrolases, soluble guanylyl cyclase, protein kinase G, phosphodiesterase 5, and natriuretic peptides will be discussed.


Subject(s)
Cardiovascular Diseases/etiology , Nitric Oxide/deficiency , Nitric Oxide/genetics , Cyclic GMP/physiology , Humans , Signal Transduction
18.
MMW Fortschr Med ; 159(Suppl 7): 26-32, 2017 Dec.
Article in German | MEDLINE | ID: mdl-29204949

ABSTRACT

BACKGROUND: Guideline-based, risk-adjusted therapy with anticoagulants reduce thromboembolic stroke risk in patients with atrial fibrillation (AF). METHOD: This study analyzed use of oral anticoagulation in German AF-patients. Access to anonymized patient records was made via IMS Health Disease Analyzer database (sample size: 113,619 patients with ICD-10 Code I48.-; observation period: 11/2010-10/2013). Results were subsequently extrapolated to all general practitioners' (GPs) and cardiological practices in Germany. RESULTS: In 2011 12-month AF-prevalence was extrapolated to 2.1 million patients (first diagnosed: n = 537.548). In 2012 AF-prevalence gone up to 2.2 million cases (first diagnosed: n = 537.548) and in 2013 to 2.8 million (first diagnosed: n = 636.571). Commonly prescribed oral anticoagulants (OAC) were vitamin K antagonists (VKA). Unstable INR setting, private health insurance, hospital admission, heart failure or hypertension increased probability of change from VKA to non-vitamin K antagonist oral anticoagulants (NOAC). 17.3-36.5% of patients with CHA2DS2-VASc-score ≥ 2 did not receive any thromboembolism prophylaxis; 38.5% with CHA2DS2-VASc-score = 0 received unnecessarily OACs. For 2013 a potential of 29.749 ischemic strokes in GP practices was calculated, which possibly can be avoided by thromboembolism prophylaxis according to guidelines. CONCLUSIONS: Risk-based anticoagulation showed requirements for optimization. Use of OACs, according to guideline recommendations, would minimize bleeding risks, reduce ischemic strokes and could release resources.


Subject(s)
Anticoagulants/therapeutic use , Atrial Fibrillation/complications , Brain Ischemia/prevention & control , Stroke/prevention & control , Brain Ischemia/complications , Humans , Hypertension , Retrospective Studies , Thromboembolism
19.
J Neurosci ; 25(43): 9883-92, 2005 Oct 26.
Article in English | MEDLINE | ID: mdl-16251435

ABSTRACT

Current knowledge about the molecular mechanisms of NMDA receptor (NMDAR)-independent long-term potentiation (LTP) in the hippocampus and its function for memory formation in the behaving animal is limited. NMDAR-independent LTP in the CA1 region is thought to require activity of postsynaptic L-type voltage-dependent Ca2+ channels (Cav1.x), but the underlying channel isoform remains unknown. We evaluated the function of the Cav1.2 L-type Ca2+ channel for spatial learning, synaptic plasticity, and triggering of learning-associated biochemical processes using a mouse line with an inactivation of the CACNA1C (Cav1.2) gene in the hippocampus and neocortex (Cav1.2(HCKO)). This model shows (1) a selective loss of protein synthesis-dependent NMDAR-independent Schaffer collateral/CA1 late-phase LTP (L-LTP), (2) a severe impairment of hippocampus-dependent spatial memory, and (3) decreased activation of the mitogen-activated protein kinase (MAPK) pathway and reduced cAMP response element (CRE)-dependent transcription in CA1 pyramidal neurons. Our results provide strong evidence for a role of L-type Ca2+ channel-dependent, NMDAR-independent hippocampal L-LTP in the formation of spatial memory in the behaving animal and for a function of the MAPK/CREB (CRE-binding protein) signaling cascade in linking Cav1.2 channel-mediated Ca2+ influx to either process.


Subject(s)
Calcium Channels, L-Type/physiology , Hippocampus/physiology , Memory/physiology , Neuronal Plasticity/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Spatial Behavior/physiology , 2-Amino-5-phosphonovalerate/pharmacology , Animals , Anisomycin/pharmacology , Behavior, Animal , Butadienes/pharmacology , Calcium Channels, L-Type/deficiency , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Drug Interactions , Electric Stimulation/methods , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Fluorescent Antibody Technique/methods , Gene Expression Regulation/drug effects , Hippocampus/cytology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Membrane Potentials/radiation effects , Mice , Mice, Knockout , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/physiology , Neuronal Plasticity/drug effects , Neuronal Plasticity/radiation effects , Nitriles/pharmacology , Patch-Clamp Techniques/methods , Potassium Channel Blockers/pharmacology , Protein Synthesis Inhibitors/pharmacology , Pyramidal Cells/drug effects , Pyramidal Cells/physiology , Pyramidal Cells/radiation effects , Tetraethylammonium/pharmacology , Time Factors
20.
Pharmacol Ther ; 106(3): 347-55, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15922017

ABSTRACT

Calcium influx through voltage gated L-type Ca2+ channels has evolved as one of the most widely used transmembrane signalling mechanisms in eukaryotic organisms. Although pharmacological inhibitors of L-type Ca2+ channels have an important place in medical therapy, the full therapeutic potential of the 4 L-type Ca2+ channel subtypes has not been explored yet. To dissect the physiological relevance of the L-type Ca2+ channel subtype diversity, gene-targeted mouse models carrying deletions of these channels ("knockout mice") have been generated. This review focuses on recent data from studies in mice lacking the Ca(v)1.2 and Ca(v)1.3 pore subunits, which have elucidated some of the roles of L-type Ca2+ channels as mediators of signalling between cell membrane and intracellular processes like blood pressure regulation, smooth muscle contractility, insulin secretion, cardiac development, and learning and memory.


Subject(s)
Calcium Channels, L-Type/physiology , Models, Animal , Animals , Blood Pressure/physiology , Calcium Channels, L-Type/genetics , Mice , Mice, Knockout , Mice, Transgenic , Muscle, Smooth, Vascular/physiology , Uterine Contraction/physiology , Vasoconstriction/physiology
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