Tumour extracellular vesicles and particles induce liver metabolic dysfunction.

Cancer alters the function of multiple organs beyond those targeted by metastasis1,2. Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs-particularly palmitic acid-induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy.

Increased Ca2+ influx through CaV1.2 drives aortic valve calcification.

Calcific aortic valve disease (CAVD) is heritable, as revealed by recent GWAS. While polymorphisms linked to increased expression of CACNA1C - encoding the CaV1.2 L-type voltage-gated Ca2+ channel - and increased Ca2+ signaling are associated with CAVD, whether increased Ca2+ influx through the druggable CaV1.2 causes CAVD is unknown. We confirmed the association between increased CaV1.2 expression and CAVD in surgically removed aortic valves from patients. We extended our studies with a transgenic mouse model that mimics increased CaV1.2 expression within aortic valve interstitial cells (VICs). In young mice maintained on normal chow, we observed dystrophic valve lesions that mimic changes found in presymptomatic CAVD and showed activation of chondrogenic and osteogenic transcriptional regulators within these valve lesions. Chronic administration of verapamil, a CaV1.2 antagonist used clinically, slowed the progression of lesion development in vivo. Exploiting VIC cultures, we demonstrated that increased Ca2+ influx through CaV1.2 drives signaling programs that lead to myofibroblast activation of VICs and upregulation of genes associated with aortic valve calcification. Our data support a causal role for Ca2+ influx through CaV1.2 in CAVD and suggest that early treatment with Ca2+ channel blockers is an effective therapeutic strategy.

Cardiac phenotype in ATP1A3-related syndromes: A multicenter cohort study.

OBJECTIVE: To define the risks and consequences of cardiac abnormalities in ATP1A3-related syndromes. METHODS: Patients meeting clinical diagnostic criteria for rapid-onset dystonia-parkinsonism (RDP), alternating hemiplegia of childhood (AHC), and cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) with ATP1A3 genetic analysis and at least 1 cardiac assessment were included. We evaluated the cardiac phenotype in an Atp1a3 knock-in mouse (Mashl+/-) to determine the sequence of events in seizure-related cardiac death. RESULTS: Ninety-eight patients with AHC, 9 with RDP, and 3 with CAPOS (63 female, mean age 17 years) were included. Resting ECG abnormalities were found in 52 of 87 (60%) with AHC, 2 of 3 (67%) with CAPOS, and 6 of 9 (67%) with RDP. Serial ECGs showed dynamic changes in 10 of 18 patients with AHC. The first Holter ECG was abnormal in 24 of 65 (37%) cases with AHC and RDP with either repolarization or conduction abnormalities. Echocardiography was normal. Cardiac intervention was required in 3 of 98 (≈3%) patients with AHC. In the mouse model, resting ECGs showed intracardiac conduction delay; during induced seizures, heart block or complete sinus arrest led to death. CONCLUSIONS: We found increased prevalence of ECG dynamic abnormalities in all ATP1A3-related syndromes, with a risk of life-threatening cardiac rhythm abnormalities equivalent to that in established cardiac channelopathies (≈3%). Sudden cardiac death due to conduction abnormality emerged as a seizure-related outcome in murine Atp1a3-related disease. ATP1A3-related syndromes are cardiac diseases and neurologic diseases. We provide guidance to identify patients potentially at higher risk of sudden cardiac death who may benefit from insertion of a pacemaker or implantable cardioverter-defibrillator.

Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics.

Increased cardiac contractility during the fight-or-flight response is caused by ß-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and ß subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of ß-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which ß-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or ß2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during ß-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for ß subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.

The CaV1.2 L-type calcium channel regulates bone homeostasis in the middle and inner ear.

Bone remodeling of the auditory ossicles and the otic capsule is highly restricted and tightly controlled by the osteoprotegerin (OPG)/receptor activator of nuclear factor kappa-Β ligand (RANKL)/receptor activator of nuclear factor kappa-Β (RANK) system. In these bony structures, a pathological decrease in OPG expression stimulates osteoclast differentiation and excessive resorption followed by accrual of sclerotic bone, ultimately resulting in the development of otosclerosis, a leading cause of deafness in adults. Understanding the signaling pathways involved in maintaining OPG expression in the ear would shed light on the pathophysiology of otosclerosis and other ear bone-related diseases. We and others previously demonstrated that Ca2+ signaling through the L-type CaV1.2 Ca2+ channel positively regulates OPG expression and secretion in long bone osteoblasts and their precursor cells in vitro and in vivo. Whether CaV1.2 regulates OPG expression in ear bones has not been investigated. We drove expression of a gain-of-function CaV1.2 mutant channel (CaV1.2TS) using Col2a1-Cre, which we found to target osteochondral/osteoblast progenitors in the auditory ossicles and the otic capsule. Col2a1-Cre;CaV1.2TS mice displayed osteopetrosis of these bones shown by µCT 3D reconstruction, histological analysis, and lack of bone sculpting, findings similar to phenotypes seen in mice with an osteoclast defect. Consistent with those observations, we found that Col2a1-Cre;CaV1.2TS mutant mice showed reduced osteoclasts in the otic capsule, upregulated mRNA expression of Opg and Opg/Rankl ratio, and increased mRNA expression of osteoblast differentiation marker genes in the otic capsule, suggesting both an anti-catabolic and anabolic effect of CaV1.2TS mutant channel contributed to the observed morphological changes of the ear bones. Further, we found that Col2a1-Cre;CaV1.2TS mice experienced hearing loss and displayed defects of body balance in behavior tests, confirming that the CaV1.2-dependent Ca2+ influx affects bone structure in the ear and consequent hearing and vestibular functions. Together, these data support our hypothesis that Ca2+ influx through CaV1.2TS promotes OPG expression from osteoblasts, thereby affecting bone modeling/remodeling in the auditory ossicles and the otic capsule. These data provide insight into potential pathological mechanisms underlying perturbed OPG expression and otosclerosis.

Increased Ca2+ signaling through CaV1.2 promotes bone formation and prevents estrogen deficiency-induced bone loss.

While the prevalence of osteoporosis is growing rapidly with population aging, therapeutic options remain limited. Here, we identify potentially novel roles for CaV1.2 L-type voltage-gated Ca2+ channels in osteogenesis and exploit a transgenic gain-of-function mutant CaV1.2 to stem bone loss in ovariectomized female mice. We show that endogenous CaV1.2 is expressed in developing bone within proliferating chondrocytes and osteoblasts. Using primary BM stromal cell (BMSC) cultures, we found that Ca2+ influx through CaV1.2 activates osteogenic transcriptional programs and promotes mineralization. We used Prx1-, Col2a1-, or Col1a1-Cre drivers to express an inactivation-deficient CaV1.2 mutant in chondrogenic and/or osteogenic precursors in vivo and found that the resulting increased Ca2+ influx markedly thickened bone not only by promoting osteogenesis, but also by inhibiting osteoclast activity through increased osteoprotegerin secretion from osteoblasts. Activating the CaV1.2 mutant in osteoblasts at the time of ovariectomy stemmed bone loss. Together, these data highlight roles for CaV1.2 in bone and demonstrate the potential dual anabolic and anticatabolic therapeutic actions of tissue-specific CaV1.2 activation in osteoblasts.

Proteolytic cleavage and PKA phosphorylation of α1C subunit are not required for adrenergic regulation of CaV1.2 in the heart.

Calcium influx through the voltage-dependent L-type calcium channel (CaV1.2) rapidly increases in the heart during "fight or flight" through activation of the ß-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of ß-adrenergic activation of cardiac CaV1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α1C and C-terminal proteolytic cleavage of the α1C subunit. We generated transgenic mice expressing an α1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for ß-adrenergic regulation of CaV1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute ß-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α1C Prevention of C-terminal cleavage did not alter ß-adrenergic stimulation of CaV1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α1C in ß-adrenergic stimulation of CaV1.2, and show that phosphoregulatory sites on α1C are not redundant and do not each fractionally contribute to the net stimulatory effect of ß-adrenergic stimulation. Further, proteolytic cleavage of α1C is not required for ß-adrenergic stimulation of CaV1.2.

α1-Syntrophin Variant Identified in Drug-Induced Long QT Syndrome Increases Late Sodium Current.

Drug-induced long-QT syndrome (diLQTS) is often due to drug block of IKr, especially in genetically susceptible patients with subclinical mutations in the IKr-encoding KCHN2. Few variants in the cardiac NaV1.5 Na+ channel complex have been associated with diLQTS. We tested whether a novel SNTA1 (α1-syntrophin) variant (p.E409Q) found in a patient with diLQTS increases late sodium current (INa-L), thereby providing a disease mechanism. Electrophysiological studies were performed in HEK293T cells co-expressing human NaV1.5/nNOS/PMCA4b with either wild type (WT) or SNTA1 variants (A390V-previously reported in congenital LQTS; and E409Q); and in adult rat ventricular cardiomyocytes infected with SNTA1 expressing adenoviruses (WT or one of the two SNTA1 variants). In HEK293T cells and in cardiomyocytes, there was no significant difference in the peak INa densities among the SNTA1 WT and variants. However, both variants increased INa-L (% of peak current) in HEK293T cells (0.58 ± 0.10 in WT vs. 0.90 ± 0.11 in A390V, p = 0.048; vs. 0.88 ± 0.07 in E409Q, p = 0.023). In cardiomyocytes, INa-L was significantly increased by E409Q, but not by A390V compared to WT (0.49 ± 0.14 in WT vs.0.94 ± 0.23 in A390V, p = 0.099; vs. 1.12 ± 0.24 in E409Q, p = 0.019). We demonstrated that a novel SNTA1 variant is likely causative for diLQTS by augmenting INa-L. These data suggest that variants within the NaV1.5-interacting α1-syntrophin are a potential mechanism for diLQTS, thereby expanding the concept that variants within congenital LQTS loci can cause diLQTS.

Long-term in vivo imaging of multiple organs at the single cell level.

Two-photon microscopy has enabled the study of individual cell behavior in live animals. Many organs and tissues cannot be studied, especially longitudinally, because they are located too deep, behind bony structures or too close to the lung and heart. Here we report a novel mouse model that allows long-term single cell imaging of many organs. A wide variety of live tissues were successfully engrafted in the pinna of the mouse ear. Many of these engrafted tissues maintained the normal tissue histology. Using the heart and thymus as models, we further demonstrated that the engrafted tissues functioned as would be expected. Combining two-photon microscopy with fluorescent tracers, we successfully visualized the engrafted tissues at the single cell level in live mice over several months. Four dimensional (three-dimensional (3D) plus time) information of individual cells was obtained from this imaging. This model makes long-term high resolution 4D imaging of multiple organs possible.

A VGF-derived peptide attenuates development of type 2 diabetes via enhancement of islet ß-cell survival and function.

Deterioration of functional islet ß-cell mass is the final step in progression to Type 2 diabetes. We previously reported that overexpression of Nkx6.1 in rat islets has the dual effects of enhancing glucose-stimulated insulin secretion (GSIS) and increasing ß-cell replication. Here we show that Nkx6.1 strongly upregulates the prohormone VGF in rat islets and that VGF is both necessary and sufficient for Nkx6.1-mediated enhancement of GSIS. Moreover, the VGF-derived peptide TLQP-21 potentiates GSIS in rat and human islets and improves glucose tolerance in vivo. Chronic injection of TLQP-21 in prediabetic ZDF rats preserves islet mass and slows diabetes onset. TLQP-21 prevents islet cell apoptosis by a pathway similar to that used by GLP-1, but independent of the GLP-1, GIP, or VIP receptors. Unlike GLP-1, TLQP-21 does not inhibit gastric emptying or increase heart rate. We conclude that TLQP-21 is a targeted agent for enhancing islet ß-cell survival and function.