Non-invasive biomarkers prognostic of decompensation events in NASH cirrhosis: a systematic literature review.

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.

Unbiased kidney-centric molecular categorization of chronic kidney disease as a step towards precision medicine.

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.

Differential In Vitro Effects of SGLT2 Inhibitors on Mitochondrial Oxidative Phosphorylation, Glucose Uptake and Cell Metabolism.

(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.

Endothelin antagonism and sodium glucose Co-transporter 2 inhibition. A potential combination therapeutic strategy for COVID-19.

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.

[Stroke prevention in atrial fibrillation in Germany. Situational analysis of treatment reality based on retrospective data]. / Schlaganfallprophylaxe bei Vorhofflimmern in Deutschland : Eine Statusanalyse basierend auf retrospektiven Daten der Versorgungsrealität.

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.

Genetics of NO Deficiency.

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.

Remote and reversible inhibition of neurons and circuits by small molecule induced potassium channel stabilization.

Manipulating the function of neurons and circuits that translate electrical and chemical signals into behavior represents a major challenges in neuroscience. In addition to optogenetic methods using light-activatable channels, pharmacogenetic methods with ligand induced modulation of cell signaling and excitability have been developed. However, they are largely based on ectopic expression of exogenous or chimera proteins. Now, we describe the remote and reversible expression of a Kir2.1 type potassium channel using the chemogenetic technique of small molecule induced protein stabilization. Based on shield1-mediated shedding of a destabilizing domain fused to a protein of interest and inhibition of protein degradation, this principle has been adopted for biomedicine, but not in neuroscience so far. Here, we apply this chemogenetic approach in brain research for the first time in order to control a potassium channel in a remote and reversible manner. We could show that shield1-mediated ectopic Kir2.1 stabilization induces neuronal silencing in vitro and in vivo in the mouse brain. We also validated this novel pharmacogenetic method in different neurobehavioral paradigms.The DD-Kir2.1 may complement the existing portfolio of pharmaco- and optogenetic techniques for specific neuron manipulation, but it may also provide an example for future applications of this principle in neuroscience research.

A specific role for the REV-ERBα-controlled L-Type Voltage-Gated Calcium Channel CaV1.2 in resetting the circadian clock in the late night.

Within the suprachiasmatic nucleus (SCN) of the hypothalamus, circadian timekeeping and resetting have been shown to be largely dependent on both membrane depolarization and intracellular second-messenger signaling. In both of these processes, voltage-gated calcium channels (VGCCs) mediate voltage-dependent calcium influx, which propagates neural impulses by stimulating vesicle fusion and instigates intracellular pathways resulting in clock gene expression. Through the cumulative actions of these processes, the phase of the internal clock is modified to match the light cycle of the external environment. To parse out the distinct roles of the L-type VGCCs, we analyzed mice deficient in Cav1.2 (Cacna1c) in brain tissue. We found that mice deficient in the Cav1.2 channel exhibited a significant reduction in their ability to phase-advance circadian behavior when subjected to a light pulse in the late night. Furthermore, the study revealed that the expression of Cav1.2 mRNA was rhythmic (peaking during the late night) and was regulated by the circadian clock component REV-ERBα. Finally, the induction of clock genes in both the early and late subjective night was affected by the loss of Cav1.2, with reductions in Per2 and Per1 in the early and late night, respectively. In sum, these results reveal a role of the L-type VGCC Cav1.2 in mediating both clock gene expression and phase advances in response to a light pulse in the late night.

Mutation of the calmodulin binding motif IQ of the L-type Ca(v)1.2 Ca2+ channel to EQ induces dilated cardiomyopathy and death.

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.

Deletion of the C-terminal phosphorylation sites in the cardiac ß-subunit does not affect the basic ß-adrenergic response of the heart and the Ca(v)1.2 channel.

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.

Cav 1.3 L-type Ca ( 2+) channels mediate long-term adaptation in dopamine D2L-mediated GluA1 trafficking in the dorsal striatum following cocaine exposure.

AMPA receptor (AMPAR) plasticity at glutamatergic synapses in the mesostriatal dopaminergic pathway has been implicated in persistent cocaine-induced behavioral responses; however, the precise mechanism underlying these changes remains unknown. Utilizing cocaine psychomotor sensitization in mice we find that repeated cocaine results in a basal reduction of Ser 845 GluA1 and cell surface GluA1 levels in the dorsal striatum (dStr) following a protracted withdrawal period, an adaptation that is dependent on Cav 1.3 channels but not those expressed in the VTA. We find that the basally-induced decrease in this phosphoprotein is the result of recruitment of the striatal dopamine D2 pathway, as evidenced by enhanced levels of D2 receptor (D2R) mRNA expression and D2R function as examined using the D2R antagonist, eticlopride, as well as alterations in the phosphorylation status of several downstream molecular targets of D2R's, including CREB, DARPP-32, Akt and GSK3ß. Taken together with our recently published findings examining similar phenomena in the nucleus accumbens (NAc), these results underscore the utilization of divergent molecular mechanisms in the dStr, in mediating cocaine-induced persistent behavioral changes.

Decreased cardiac L-type Ca²âº channel activity induces hypertrophy and heart failure in mice.

Antagonists of L-type Ca²âº channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C⁻/⁺ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C⁻/⁺ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C⁻/⁺ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C⁺/⁺ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocyte-specific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca²âº release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease.

Consolidation of remote fear memories involves Corticotropin-Releasing Hormone (CRH) receptor type 1-mediated enhancement of AMPA receptor GluR1 signaling in the dentate gyrus.

Persistent dreadful memories and hyperarousal constitute prominent psychopathological features of posttraumatic stress disorder (PTSD). Here, we used a contextual fear conditioning paradigm to demonstrate that conditional genetic deletion of corticotropin-releasing hormone (CRH) receptor 1 within the limbic forebrain in mice significantly reduced remote, but not recent, associative and non-associative fear memories. Per os treatment with the selective CRHR1 antagonist DMP696 (3 mg/kg) attenuated consolidation of remote fear memories, without affecting their expression and retention. This could be achieved, if DMP696 was administered for 1 week starting as late as 24 h after foot shock. Furthermore, by combining electrophysiological recordings and western blot analyses, we demonstrate a delayed-onset and long-lasting increase in AMPA receptor (AMPAR) GluR1-mediated signaling in the dentate gyrus (DG) of the dorsal hippocampus 1 month after foot shock. These changes were absent from CRHR1-deficient mice and after DMP696 treatment. Inactivation of hippocampal GluR1-containing AMPARs by antisense oligonucleotides or philantotoxin 433 confirmed the behavioral relevance of AMPA-type glutamatergic neurotransmission in maintaining the high levels of remote fear in shocked mice with intact CRHR1 signaling. We conclude that limbic CRHR1 receptors enhance the consolidation of remote fear memories in the first week after foot shock by increasing the expression of Ca(2+)-permeable GluR1-containing AMPARs in the DG. These findings suggest both receptors as rational targets for the prevention and therapy, respectively, of psychopathology associated with exaggerated fear memories, such as PTSD.

Cav1.2 L-type Ca²âº channels mediate cocaine-induced GluA1 trafficking in the nucleus accumbens, a long-term adaptation dependent on ventral tegmental area Ca(v)1.3 channels.

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.

Truncation of murine CaV1.2 at Asp-1904 results in heart failure after birth.

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.

Facilitation and Ca2+-dependent inactivation are modified by mutation of the Ca(v)1.2 channel IQ motif.

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.

Electrical activity suppresses axon growth through Ca(v)1.2 channels in adult primary sensory neurons.

BACKGROUND: Primary sensory neurons of the dorsal root ganglia (DRG) regenerate their spinal cord axon if the peripheral nerve axon has previously been cut. This conditioning lesion confers axon growth competence to the neurons. However, the signal that is sensed by the cell upon peripheral lesion to initiate the regenerative response remains elusive. RESULTS: We show here that loss of electrical activity following peripheral deafferentiation is an important signal to trigger axon regrowth. We first verified that firing in sensory fibers, as recorded from dorsal roots in vivo, declined after peripheral lesioning but was not altered after central lesioning. We found that electrical activity strongly inhibited axon outgrowth in cultured adult sensory neurons. The inhibitory effect depended on the L-type voltage-gated Ca(2+) channel current and involved transcriptional changes. After a peripheral lesion, the L-type current was consistently diminished and the L-type pore-forming subunit, Ca(v)1.2, was downregulated. Genetic ablation of Ca(v)1.2 in the nervous system caused an increase in axon outgrowth from dissociated DRG neurons and enhanced peripheral nerve regeneration in vivo. CONCLUSIONS: Our data indicate that cessation of electrical activity after peripheral lesion contributes to the regenerative response observed upon conditioning and might be necessary to promote regeneration after central nervous system injury.

Homeostatic switch in hebbian plasticity and fear learning after sustained loss of Cav1.2 calcium channels.

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.

Facilitation of murine cardiac L-type Ca(v)1.2 channel is modulated by calmodulin kinase II-dependent phosphorylation of S1512 and S1570.

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.

L-type calcium channel CaV 1.2 in transgenic mice overexpressing human AbetaPP751 with the London (V717I) and Swedish (K670M/N671L) mutations.

Cumulative evidence indicates that amyloid-beta peptides exert some of their neurodegenerative effects through modulation of L-type voltage gated calcium channels, which play key roles in a diverse range of CNS functions. In this study we examined the expression of CaV1.2 L-type voltage gated calcium channels in transgenic mice overexpressing human AbetaPP751 with the London (V717I) and Swedish (K670M/N671L) mutations by immunohistochemistry in light and electron microscopy. In hippocampal layers of wild type and transgenic mice, CaV1.2 channels were predominantly localized to somato-dendritic domains of neurons, and to astrocytic profiles with an age-dependent increase in labeling density. In transgenic animals, CaV1.2-like immunoreactive clusters were found in neuronal profiles in association with amyloid-beta plaques. Both the number and density of these clusters depended upon age of animals and number of plaques. The most striking difference between wild type and transgenic mice was the age-dependent expression of CaV1.2 channels in reactive astrocytes. At the age of 6 month, CaV1.2 channels were rarely detected in reactive astrocytes of transgenic mice, but an incremental number of CaV1.2 expressing reactive astrocytes was found with increasing age of animals and number of amyloid-beta plaques. This study demonstrates that CaV1.2 channels are highly expressed in reactive astrocytes of 12-months of age transgenic mice, which might be a consequence of the increasing amyloid burden. Further studies should clarify which functional implications are associated with the higher availability of CaV1.2 channels in late stage Alzheimer's disease.