Impaired Ca2 Research Articles

Plakophilin-2 Haploinsufficiency Causes Calcium Handling Deficits and Modulates the Cardiac Response Towards Stress.

Human variants in plakophilin-2 (PKP2) associate with most cases of familial arrhythmogenic cardiomyopathy (ACM). Recent studies show that PKP2 not only maintains intercellular coupling, but also regulates transcription of genes involved in Ca2+ cycling and cardiac rhythm. ACM penetrance is low and it remains uncertain, which genetic and environmental modifiers are crucial for developing the cardiomyopathy. In this study, heterozygous PKP2 knock-out mice (PKP2-Hz) were used to investigate the influence of exercise, pressure overload, and inflammation on a PKP2-related disease progression. In PKP2-Hz mice, protein levels of Ca2+-handling proteins were reduced compared to wildtype (WT). PKP2-Hz hearts exposed to voluntary exercise training showed right ventricular lateral connexin43 expression, right ventricular conduction slowing, and a higher susceptibility towards arrhythmias. Pressure overload increased levels of fibrosis in PKP2-Hz hearts, without affecting the susceptibility towards arrhythmias. Experimental autoimmune myocarditis caused more severe subepicardial fibrosis, cell death, and inflammatory infiltrates in PKP2-Hz hearts than in WT. To conclude, PKP2 haploinsufficiency in the murine heart modulates the cardiac response to environmental modifiers via different mechanisms. Exercise upon PKP2 deficiency induces a pro-arrhythmic cardiac remodeling, likely based on impaired Ca2+ cycling and electrical conduction, versus structural remodeling. Pathophysiological stimuli mainly exaggerate the fibrotic and inflammatory response.

Abstract 548: Cardiomyocyte-KLF5 Expression is Increased by FOXO1 and Accounts for Cardiomyopathy in Type-1 Diabetes

Cardiomyopathy in type 1 diabetes (T1D) is accompanied by altered cardiac energetics, impaired mitochondrial function and oxidative stress. We showed previously increased cardiac expression of Krüppel-like factor 5 (KLF5) and Peroxisome Proliferator Activated Receptor (PPAR)α at late T1D stage in mice. We confirmed that KLF5 expression is higher in cardiomyocytes of diabetic patients than in non-diabetic individuals. Mechanistic analyses in human cardiomyocyte cells (AC16) and in mice with cardiomyocyte-specific FOXO1 deletion (αMHC- Foxo1 -/- ) revealed that FOXO1 activation accounts for the increased KLF5 expression via direct binding on Klf5 promoter. Both pharmacologic and cardiomyocyte-specific inhibition of KLF5 alleviated diabetic-related cardiac dysfunction. Accordingly, mice with doxycycline-mediated cardiomyocyte-specific KLF5 constitutive expression (αMHC-rtTA- Klf5 ) recapitulated cardiomyopathy even in the absence of T1D. We also showed that diabetic PPARα -/- mice had elevated cardiac KLF5 expression and cardiac dysfunction, suggesting that KLF5-driven diabetic cardiomyopathy is PPARα-independent. Furthermore, cardiomyocyte KLF5 upregulation was associated with oxidative stress, increased NADPH oxidase (NOX)4 expression, lower NADPH levels and impaired cellular Ca 2+ handling. Conversely, KLF5 inhibition prevented NOX4 upregulation and alleviated cardiac superoxide formation. Lipidomic analysis followed by Euclidean clustering showed strong correlation of lipidome profiles between αMHC-rtTA- Klf5 mice and diabetic C57BL/6 mice, while diabetic αMHC- Klf5 -/- mice and diabetic C57BL/6 mice that were treated with pharmacologic KLF5 inhibitor grouped with the non-diabetic C57BL/6 mice. Further analysis of individual lipid species showed increased ceramide accumulation in diabetic C57BL/6 and αMHC-rtTA- Klf5 mice that was reversed upon KLF5 inhibition. Treatment of αMHC-rtTA- Klf5 mice and diabetic C57BL/6 mice with the antioxidant LGM2605 improved partially cardiac dysfunction. In conclusion, cardiomyocyte KLF5 expression is activated by FOXO1 and drives diabetic cardiomyopathy in a non-PPARα-dependent manner, suggesting KLF5 inhibition as a therapeutic intervention in T1D cardiomyopathy.

MLP-deficient human pluripotent stem cell derived cardiomyocytes develop hypertrophic cardiomyopathy and heart failure phenotypes due to abnormal calcium handling

Muscle LIM protein (MLP, CSRP3) is a key regulator of striated muscle function, and its mutations can lead to both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, due to lack of human models, mechanisms underlining the pathogenesis of MLP defects remain unclear. In this study, we generated a knockout MLP/CSRP3 human embryonic stem cell (hESC) H9 cell line using CRISPR/Cas9 mediated gene disruption. CSRP3 disruption had no impact on the cardiac differentiation of H9 cells and led to confirmed MLP deficiency in hESC-derived cardiomyocytes (ESC-CMs). MLP-deficient hESC-CMs were found to develop phenotypic features of HCM early after differentiation, such as enlarged cell size, multinucleation, and disorganized sarcomeric ultrastructure. Cellular phenotypes of MLP-deficient hESC-CMs subsequently progressed to mimic heart failure (HF) by 30 days post differentiation, including exhibiting mitochondrial damage, increased ROS generation, and impaired Ca2+ handling. Pharmaceutical treatment with beta agonist, such as isoproterenol, was found to accelerate the manifestation of HCM and HF, consistent with transgenic animal models of MLP deficiency. Furthermore, restoration of Ca2+ homeostasis by verapamil prevented the development of HCM and HF phenotypes, suggesting that elevated intracellular Ca2+ concentration is a central mechanism for pathogenesis of MLP deficiency. In summary, MLP-deficient hESC-CMs recapitulate the pathogenesis of HCM and its progression toward HF, providing an important human model for investigation of CSRP3/MLP-associated disease pathogenesis. More importantly, correction of the autonomous dysfunction of Ca2+ handling was found to be an effective method for treating the in vitro development of cardiomyopathy disease phenotype.

Arsenite and its trivalent methylated metabolites inhibit glucose-stimulated calcium influx and insulin secretion in murine pancreatic islets.

Chronic exposure to inorganic arsenic (iAs), a common drinking water and food contaminant, has been associated with an increased risk of type 2 diabetes in population studies worldwide. Several mechanisms underlying the diabetogenic effects of iAs have been proposed through laboratory investigations. We have previously shown that exposure to arsenite (iAs(III)) or its methylated trivalent metabolites, methylarsonite (MAs(III)) and dimethylarsinite (DMAs(III)), inhibits glucose-stimulated insulin secretion (GSIS) in pancreatic islets, without significant effects on insulin expression or insulin content. The goal of the present study was to determine if iAs(III) and/or its metabolites inhibit Ca2+ influx, an essential mechanism that regulates the release of insulin from β cells in response to glucose. We found that in vitro exposures for 48h to non-cytotoxic concentrations of iAs(III), MAs(III), and DMAs(III) impaired Ca2+ influx in isolated murine pancreatic islets stimulated with glucose. MAs(III) and DMAs(III) were more potent inhibitors of Ca2+ influx than iAs(III). These arsenicals also inhibited Ca2+ influx and GSIS in islets treated with depolarizing levels of potassium chloride in the absence of glucose. Treatment with Bay K8644, a Cav1.2 channel agonist, did not restore insulin secretion in arsenical-exposed islets. Tolbutamide, a KATP channel blocker, prevented inhibition of insulin secretion in MAs(III)- and DMAs(III)-exposed islets, but only marginallyin islets exposed to iAs(III). Our findings suggest that iAs(III), MAs(III), and DMAs(III) inhibit glucose-stimulated Ca2+ influx in pancreatic islets, possibly by interfering with KATP and/or Cav1.2 channel function. Notably, the mechanisms underlying inhibition of GSIS by iAs(III) may differ from those of its trivalent methylated metabolites.

Impaired calcium homeostasis via advanced glycation end products promotes apoptosis through endoplasmic reticulum stress in human nucleus pulposus cells and exacerbates intervertebral disc degeneration in rats.

Previous studies identified advanced glycation end products (AGEs) accumulation in the intervertebral disc (IVD) as an essential risk factor associated with IVD degeneration via accelerated cell apoptosis and impeded extracellular-matrix metabolism; however, the underlying mechanisms have not been fully elucidated. Here, we investigated the effects and mechanisms of AGEs-mediated apoptosis invitro and invivo. We evaluated the effects of AGEs on endoplasmic reticulum (ER) stress, apoptosis, and subcellular calcium (Ca2+ ) redistribution. Our data indicated time- and concentration-dependent upregulation of ER-stress responses in AGEs-treated nucleus pulposus (NP) cells. Additionally, we observed marked suppression of AGEs-mediated apoptosis following the inhibition of ER stress using 4-phenylbutyric acid. Moreover, AGEs-induced sustained cytosolic Ca2+ ([Ca2+ ]c) elevation and ER luminal Ca2+ ([Ca2+ ]er) depletion in a concentration- and time-dependent manner in NP cells. Furthermore, we observed significant increases and decreases in levels of the ER-resident Ca2+ -release channels inositol 1,4,5-triphosphate receptor and ryanodine receptor and ER Ca2+ -reuptake pumps sarco/endoplasmic reticulum Ca2+ -ATPase, respectively. Pharmacologically blocking ER Ca2+ release using Ca2+ antagonists significantly ameliorated Ca2+ dyshomeostasis, ER stress, and subsequent apoptosis in NP cells and partially attenuated the progression of IVD degeneration invivo. These results demonstrated that impaired Ca2+ homeostasis plays an essential role in AGEs-mediated ER stress and subsequent apoptosis in NP cells, with blockage of ER Ca2+ release partially ameliorating subcellular Ca2+ redistribution, ER stress, and apoptosis. Our findings provide novel mechanistic insight into the role of AGEs in the pathogenesis of IVD degeneration and a potential therapeutic strategy.

TRP Channels Mediated Pathological Ca2+-Handling and Spontaneous Ectopy.

Ion channel biology offers great opportunity in identifying and learning about cardiac pathophysiology mechanisms. The discovery of transient receptor potential (TRP) channels is an add-on to the opportunity. Interacting with numerous signaling pathways, being activated multimodally, and having prescribed signatures underlining acute hemodynamic control and cardiac remodeling, TRP channels regulate cardiac pathophysiology. Impaired Ca2+-handling cause contractile abnormality. Modulation of intracellular Ca2+ concentration ([Ca2+]i) is a major part of Ca2+-handling processes in cardiac pathophysiology. TRP channels including TRPM4 regulate [Ca2+]i, Ca2+-handling and cardiac contractility. The channels modulate flux of divalent cations, such as Ca2+ during Ca2+-handling and cardiac contractility. Seminal works implicate TRPM4 and TRPC families in intracellular Ca2+ homeostasis. Defective Ca2+-homeostasis through TRP channels interaction with Ca2+-dependent regulatory proteins such as sodium calcium exchanger (NCX) results in abnormal Ca2+ handling, contractile dysfunction and in spontaneous ectopy. This review provides insight into TRP channels mediated pathological Ca2+-handling and spontaneous ectopy.

Exercise attenuates maternal separation-induced mood disorder-like behaviors by enhancing mitochondrial functions and neuroplasticity in the dorsal raphe

Loss of the mother-infant relationship during early childhood affects infant development and is known to increase the infant’s vulnerability to neuropsychiatric disorders throughout life. Serotonin deficits and mitochondrial dysfunction in the dorsal raphe may underlie mood disorders such as anxiety and depression. Exercise is known to have a positive effect on brain function. In this study, we investigated the effect of exercise on mitochondrial function, apoptosis, and serotonin levels in the dorsal raphe as well as behavioral changes in cases of maternal separation. Exposure to the stress of maternal separation resulted in mitochondrial dysfunction in the dorsal raphe, including impaired Ca2+ homeostasis, an increase in reactive oxygen species such as H2O2, and a decrease in the O2 respiration rate. Exposure to maternal separation stress also decreased tryptophan hydroxylase and 5-hydroxytryptamine positive cells and increased apoptosis, anxiety, and depression. The impairments in mitochondrial function, apoptosis, and serotonin in the dorsal raphe, as well as anxiety and depression, were all improved by exercise. Exercise might alter mitochondrial function, serotonin levels, and the rate of apoptosis in the dorsal raphe. Therefore, exercise might be an important non-pharmacological intervention for the prevention and treatment of the adverse effects of maternal separation.

341-LB: Chronic ER Calcium Depletion Resulting from Sarcoendoplasmic Reticulum Ca2+ Atpase 2b (SERCA2b) Deficiency Leads to Impaired Proinsulin Processing in the Pancreatic ß Cell

Reductions in pancreatic β cell endoplasmic reticulum (ER) Ca2+ levels are associated with the development of ER stress and impaired proinsulin processing in both type 1 and type 2 diabetes. The SERCA pump transports Ca2+ into the ER lumen in order to maintain a steep Ca2+ gradient between the ER and cytosol. SERCA2 activity and expression are reduced in diabetes, however, the relationship between SERCA2 and proinsulin processing remains incompletely understood. To this end, we developed mice with β cell specific deletion of SERCA2 (βS2KO mice) and S2KO INS1 cells. βS2KO mice exhibited glucose intolerance and reduced insulin secretion in response to glucose without evidence of impaired insulin sensitivity. Islets from βS2KO mice had significantly reduced ER Ca2+ levels and impaired glucose-induced Ca2+ oscillations, while serum proinsulin/insulin (PI/I) ratios and whole pancreas PI/I content were elevated. Immunoblot analysis of βS2KO islets and S2KO INS-1 cells revealed reduced total levels of the proinsulin processing enzymes, PC1/3, PC2 and CPE, and increased levels of proPC2. Restoration of SERCA2b via adenoviral transduction in S2KO INS1 cells was sufficient to restore PC1/3 and PC2 maturation and enzyme activity. Brefeldin A treatment in INS1 cells recapitulated the impairments in PC1/3 and PC2 maturation observed in S2KO INS1 cells, suggesting a disturbance in protein trafficking between the ER and Golgi. Trafficking assays were next performed using a vesicular stomatitis virus G (VSVG) protein construct and revealed a significantly slower rate of VSVG movement from the ER to the Golgi in S2KO INS1 cells. Taken together, these data indicate that loss of SERCA2 activity and impairments in ER Ca2+ lead to reduced proinsulin processing via altered protein trafficking through the β cell secretory pathway. Disclosure H. Iida: None. T. Kono: None. X. Tong: None. I. Lindberg: None. C. Evans-Molina: None.

Cardiac-specific overexpression of metallothionein attenuates L-NAME-induced myocardial contractile anomalies and apoptosis.

Hypertension contributes to the high cardiac morbidity and mortality. Although oxidative stress plays an essential role in hypertensive heart diseases, the mechanism remains elusive. Transgenic mice with cardiac overexpression of metallothionein, a heavy metal‐binding scavenger, were challenged with NG‐nitro‐L‐arginine methyl ester (L‐NAME) for 14 days prior to measurement of myocardial contractile and intracellular Ca2+ anomalies as well as cell signalling mechanisms using Western blot and immunofluorescence analysis. L‐NAME challenge elicited hypertension, macrophage infiltration, oxidative stress, inflammation and cardiac dysfunction manifested as increased proinflammatory macrophage marker F4/80, interleukin‐1β (IL‐1β), intracellular O2- production, LV end systolic and diastolic diameters as well as depressed fractional shortening. L‐NAME treatment reduced mitochondrial membrane potential (MMP), impaired cardiomyocyte contractile and intracellular Ca2+ properties as evidenced by suppressed peak shortening, maximal velocity of shortening/relengthening, rise in intracellular Ca2+, along with elevated baseline and peak intracellular Ca2+. These unfavourable mechanical changes and decreased MMP (except blood pressure and macrophage infiltration) were alleviated by overexpression of metallothionein. Furthermore, the apoptosis markers including BAD, Bax, Caspase 9, Caspase 12 and cleaved Caspase 3 were up‐regulated while the anti‐apoptotic marker Bcl‐2 was decreased by L‐NAME treatment. Metallothionein transgene reversed L‐NAME‐induced changes in Bax, Bcl‐2, BAD phosphorylation, Caspase 9, Caspase 12 and cleaved Caspase 3. Our results suggest that metallothionein protects against L‐NAME‐induced myocardial contractile anomalies in part through inhibition of apoptosis.

Ischemia time impacts on respiratory chain functions and Ca2+-handling of cardiac subsarcolemmal mitochondria subjected to ischemia reperfusion injury

BackgroundMitochondrial impairment can result from myocardial ischemia reperfusion injury (IR). Despite cardioplegic arrest, IR-associated cardiodepression is a major problem in heart surgery. We determined the effect of increasing ischemia time on the respiratory chain (RC) function, the inner membrane polarization and Ca2+ homeostasis of rat cardiac subsarcolemmal mitochondria (SSM).MethodsWistar rat hearts were divided into 4 groups of stop-flow induced warm global IR using a pressure-controlled Langendorff system: 0, 15, 30 and 40 min of ischemia with 30 min of reperfusion, respectively. Myocardial contractility was determined from left ventricular pressure records (dP/dt, dPmax) with an intraventricular balloon. Following reperfusion, SSM were isolated and analyzed regarding electron transport chain (ETC) coupling by polarography (Clark-Type electrode), membrane polarization (JC1 fluorescence) and Ca2+-handling in terms of Ca2+-induced swelling and Ca2+-uptake/release (Calcium Green-5 N® fluorescence).ResultsLV contractility and systolic pressure during reperfusion were impaired by increasing ischemic times. Ischemia reduced ETC oxygen consumption in IR40/30 compared to IR0/30 at complex I-V (8.1 ± 1.2 vs. 18.2 ± 2.0 nmol/min) and II-IV/V (16.4 ± 2.6/14.8 ± 2.3 vs. 2.3 ± 0.6 nmol/min) in state 3 respiration (p < 0.01). Relative membrane potential revealed a distinct hyperpolarization in IR30/30 and IR40/30 (171.5 ± 17.4% and 170.9 ± 13.5%) compared to IR0/30 (p < 0.01), wearing off swiftly after CCCP-induced uncoupling. Excess mitochondrial permeability transition pore (mPTP)-gated Ca2+-induced swelling was recorded in all groups and was most pronounced in IR40/30. Pyruvate addition for mPTP blocking strongly reduced SSM swelling in IR40/30 (relative AUC, ± pyruvate; IR0/30: 1.00 vs. 0.61, IR15/30: 1.68 vs. 1.00, IR30/30: 1.42 vs. 0.75, IR40/30: 1.97 vs. 0.85; p < 0.01). Ca2+-uptake remained unaffected by previous IR. Though Ca2+-release was delayed for ≥30 min of ischemia (p < 0.01), Ca2+ retention was highest in IR15/30 (RFU; IR0/30: 6.3 ± 3.6, IR 15/30 42.9 ± 5.0, IR30/30 15.9 ± 3.8, IR40/30 11.5 ± 6.6; p ≤ 0.01 for IR15/30 against all other groups).ConclusionsIschemia prolongation in IR injury gradually impaired SSM in terms of respiratory chain function and Ca2+-homeostasis. Membrane hyperpolarization appears to be responsible for impaired Ca2+-cycling and ETC function. Ischemia time should be considered an important factor influencing IR experimental data on subsarcolemmal mitochondria. Periods of warm global ischemia should be minimized during cardiac surgery to avoid excessive damage to SSMs.

Cardiac electrical and contractile disorders promoted by anabolic steroid overdose are associated with late autonomic imbalance and impaired Ca2+ handling

AimInvestigate cardiac electrical and mechanical dysfunctions elicited by chronic anabolic steroid (AS) overdose. MethodsMale Wistar rats were treated with nandrolone decanoate (DECA) or vehicle (CTL) for 8 weeks. Electrocardiography and heart rate variability were assessed at weeks 2, 4, and 8. Cardiac reactivity to isoproterenol was investigated in isolated rat hearts. Action potential duration (APD) was measured from left ventricular (LV) muscle strips. L-type Ca2+ current (ICaL), and transient outward potassium current (Ito) were recorded by whole-cell patch-clamp in LV cardiomyocytes. Sarcoplasmic reticulum (SR) Ca2+ mobilization and Ca2+-induced contractile response sensitivity were evaluated in skinned cardiac fibers. Muscarinic type 2 receptor (M2R), β1-adrenergic receptor (β1AR), sarcoplasmic Ca2+ ATPase (SERCA-2a), type 2 ryanodine receptor (RyR2), L-type Ca2+ channel (CACNA1), Kv4.2 (KCND2), and Kv4.3 (KCND3) mRNA expression levels were measured by quantitative RT-PCR. ResultsCompared with CTL group, DECA group exhibited decreased high frequency band power density (HF) and increased low frequency power density (LF), Cardiac M2R mRNA level was decreased. QTc interval at 2nd, 4th, and 8th week as well as APD30 and APD90 were increased by DECA. Ito density was decreased, while ICaL density was increased by DECA. SR Ca2+ loading and release were decreased by DECA, while contractile sensitivity to Ca2+ was increased versus CTL group. ConclusionDECA overdose induced cardiac rhythmic and mechanical abnormalities that can be associated with autonomic imbalance, up-regulated ICaL and down-regulated Ito, abnormal SR Ca2+ mobilization, and increased contractile sensitivity to Ca2+.

Bronchial Epithelial Calcium Metabolism Impairment in Smokers and Chronic Obstructive Pulmonary Disease. Decreased ORAI3 Signaling.

The airway epithelium represents a fragile environmental interface potentially disturbed by cigarette smoke (CS), the major risk factor for developing chronic obstructive pulmonary disease (COPD). CS leads to bronchial epithelial damage on ciliated, goblet, and club cells, which could involve calcium (Ca2+) signaling. Ca2+ is a key messenger involved in virtually all fundamental physiological functions, including mucus and cytokine secretion, cilia beating, and epithelial repair. In this study, we analyzed Ca2+ signaling in air-liquid interface-reconstituted bronchial epithelium from control subjects and smokers (with and without COPD). We further aimed to determine how smoking impaired Ca2+ signaling. First, we showed that the endoplasmic reticulum (ER) depletion of Ca2+ stores was decreased in patients with COPD and that the Ca2+ influx was decreased in epithelial cells from smokers (regardless of COPD status). In addition, acute CS exposure led to a decrease in ER Ca2+ release, significant in smoker subjects, and to a decrease in Ca2+ influx only in control subjects. Furthermore, the differential expression of 55 genes involved in Ca2+ signaling highlighted that only ORAI3 expression was significantly altered in smokers (regardless of COPD status). Finally, we incubated epithelial cells with an ORAI antagonist (GSK-7975A). GSK-7975A altered Ca2+ influx and ciliary beating, but not mucus and cytokine secretion or epithelial repair, in control subjects. Our data suggest that Ca2+ signaling is impaired in smoker epithelia (regardless of COPD status) and involves ORAI3. Moreover, ORAI3 is additionally involved in ciliary beating.

Progress in understanding mitochondrial calcium uniporter complex-mediated calcium signalling: A potential target for cancer treatment.

Due to its Ca2+ buffering capacity, the mitochondrion is one of the most important intracellular organelles in regulating Ca2+ dynamic oscillation. Mitochondrial calcium uniporter (MCU) is the primary mediator of Ca2+ influx into mitochondria, manipulating cell energy metabolism, ROS production, and programmed cell death, all of which are critical for carcinogenesis. The understanding of the uniporter complex was significantly boosted by recent groundbreaking discoveries that identified the uniporter pore-forming subunit MCU and its regulatory molecules, including MCU-dominant negative β subunit (MCUb), essential MCU regulator (EMRE), MCU regulator 1 (MCUR1), mitochondrial calcium uptake (MICU) 1, MICU2, and MICU3. These provide the means and molecular platform to investigate MCU complex (uniplex)-mediated impaired Ca2+ signalling in physiology and pathology. This review aims to summarize the progress of the understanding regulatory mechanisms of uniplex, roles of uniplex-mediated Ca2+ signalling in cancer, and potential pharmacological inhibitors of MCU.

Mucin 1 is Necessary for TrpV5 Localization in the Kidney's Distal Convoluted Tubule and Normal Bone Architecture

Mucin 1 (Muc1 in mice and MUC1 in humans) is a heavily glycosylated transmembrane protein expressed in numerous epithelia. Muc1 interacts with other glycoproteins via interaction with galectins, which bind β‐galactoside sugars. Genome‐wide association data suggest a link between MUC1 polymorphism and bone density. MUC1 exogenously expressed in cultured cells physically interacts with calcium‐selective ion channel TRPV5, increasing cell surface expression and activity in a galectin 3‐dependent fashion. TrpV5 knock‐out (KO) mice exhibit impaired reabsorption of filtered Ca2+ in the kidney's distal convoluted tubule (DCT), resulting in hypercalciuria and diminished trabecular and cortical bone thickness. We hypothesize that interaction between Muc1 and TrpV5 is necessary for normal Ca2+ homeostasis and bone architecture. We examined density and architecture in Muc1 KO mice using μCT of lumbar vertebrae. Male Muc1 KO mice exhibit reduced lumbar vertebral cortical and trabecular thickness, with increased surface area/volume. Muc1 KO mice exhibit similar whole‐blood ionized Ca2+ levels, but increased kidney 1‐α‐hydroxylase levels, suggestive of bodily Ca2+ depletion. We observe that TrpV5 is mis‐localized in the DCT of Muc1 KO mice, suggesting that impaired renal tubular Ca2+ reabsorption may contribute to this Ca2+ depletion. Additionally, TrpV5 is mis‐localized in the DCT in galectin‐3 KO mice, consistent with a role for galectin 3 in mediating Muc1‐TrpV5 interaction. Taken together, these findings suggest that Muc1 plays a role in bodily Ca2+ homeostasis and bone physiology by enhancing cell surface expression of TrpV5.Support or Funding InformationNIDDK K08 DK110332, K01 DK109038, P30 DK079307This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardiomyocyte‐Specific EGFR Deletion Differentially Alters Cardiac Contractility and Remodeling

Epidermal Growth Factor Receptor (EGFR), a member of the Erbb receptor tyrosine kinase family, is essential to the development of multiple tissues and organs. Due to the embryonic lethality of global EGFR deletion, the impact of EGFR signaling in the adult heart, normally or in response to pathological stimuli, has not been well‐explored. Using mice with floxed EGFR alleles crossed with αMHC‐Cre mice, we have generated a cardiomyocyte‐specific constitutive EGFR knockout mouse model (CM‐EGFR‐KO) to address its role in the heart. Compared to their wild‐type (WT) littermate controls, CM‐EGFR‐KO mice displayed age‐related development of cardiac contractile dysfunction, marked by impaired diastolic relaxation and Ca2+ handling at the individual myocyte level, as monitored via echocardiographic, hemodynamic, myocyte contractility and Ca2+ transient analyses. RNASeq analysis of CM‐EGFR‐KO versus WT littermate left ventricular samples revealed temporally‐dependent alterations in the expression of a number of myofilament, Ca2+ handling and fetal genes. The contractile defect initially occurs between 7 and 9 weeks of age and is maintained over time without overt cardiac hypertrophic or fibrotic remodeling until 10 months of age, by which time the mice ultimately succumb to severe heart failure and reduced lifespan. Chronic administration of the β‐adrenergic receptor (βAR) agonist isoproterenol effectively compensated for the contractile dysfunction in CM‐EGFR‐KO mice, an effect that was reversible upon cessation of infusion. Notably, βAR‐mediated cardiomyocyte hypertrophy was attenuated in CM‐EGFR‐KO mice compared to WT littermate controls. Treatment of 16 week‐old CM‐EGFR‐KO mice with AAV9‐cTnT‐EGFR permanently rescued cardiomyocyte‐specific EGFR expression and contractile function, suggesting that the contractile dysfunction phenotype observed in these mice is not secondary to aberrant cardiac development, but is specific to EGFR‐dependent regulation of contractile signaling.Support or Funding InformationNIH grant HL105414 to DGTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Relationships between three and twelve month outcomes in children enrolled in the therapeutic hypothermia after pediatric cardiac arrest trials

AimTo inform design aspects of future trials by comparing 3 and 12-month neurobehavioural outcomes in children enrolled in Therapeutic Hypothermia After Pediatric Cardiac Arrest Out-Of-Hospital and In-Hospital (THAPCA-OH, THAPCA-IH) trials. MethodsThe THAPCA trials evaluated two targeted temperature management interventions (hypothermia, 32.0–34.0 °C; normothermia, 36.0–37.5 °C). Children, aged 2 days to <18 years, were enrolled from 2009–2015. Three and 12-month post-cardiac arrest (CA) outcomes included the Vineland Adaptive Behavior Scales, Second Edition (VABS-II) (population mean = 100, SD = 15) and the pediatric cerebral performance category (PCPC) scale. Children without significant pre-existing neurodevelopmental deficits were included in primary outcome analyses. Among survivors, favorable 12-month outcome was defined as VABS-II ≥ 70. ResultsVABS-II and PCPC were available at 3 and 12 months in 204 of 222 eligible survivors (THAPCA-OH, n = 82; THAPCA-IH, n = 122). Relative to THAPCA-IH, THAPCA-OH had significantly less pre-CA disability and significantly greater 12-month CA impairment, based on both VABS-II and PCPC. Correlations between 3 and 12-month VABS-II scores were strong for THAPCA-OH (r = 0.95) and THAPCA-IH (r = 0.72), and lower (p ≤ 0.001) in THAPCA-IH. Between time-points correlations were lower, but still significant in children <1 year at CA (p < 0.001). In both cohorts, 3-month VABS-II and PCPC categorical outcomes had high sensitivity (≥70%) for predicting favorable 12-month VABS-II outcomes, but specificity was lower for THAPCA-IH (68–89%) relative to THAPCA-OH (≥95%). Overall, 12-month diagnostic accuracy was ≥80% for both VABS-II and PCPC in both cohorts. ConclusionsIn future paediatric cardiac arrest clinical trials that enroll similar cohorts, integration of 3-month neurobehavioral outcome measures should be considered.

Mitochondrial transport serves as a mitochondrial quality control strategy in axons: Implications for central nervous system disorders.

SummaryAxonal mitochondrial quality is essential for neuronal health and functions. Compromised mitochondrial quality, reflected by loss of membrane potential, collapse of ATP production, abnormal morphology, burst of reactive oxygen species generation, and impaired Ca2+ buffering capacity, can alter mitochondrial transport. Mitochondrial transport in turn maintains axonal mitochondrial homeostasis in several ways. Newly generated mitochondria are anterogradely transported along with axon from soma to replenish axonal mitochondrial pool, while damaged mitochondria undergo retrograde transport for repair or degradation. Besides, mitochondria are also arrested in axon to quarantine damages locally. Accumulating evidence suggests abnormal mitochondrial transport leads to mitochondrial dysfunction and axon degeneration in a variety of neurological and psychiatric disorders. Further investigations into the details of this process would help to extend our understanding of various neurological diseases and shed light on the corresponding therapies.

Altered reticulum-mitochondria interactions contribute to mitochondrial Ca2 + signaling dysfunction in the diabetic mice heart

Diabetic cardiomyopathy has been linked to Ca2 + signaling alterations, notably a decreased mitochondrial Ca2 + uptake. Uncovering the changes occurring at Ca2 + microdomains between reticulum and mitochondria in the heart has launched a new area of investigation for cardiometabolic diseases. We here aimed to study if the impairment of mitochondrial Ca2 + handling could be due to a dysregulation of the reticulum-mitochondria interactions or of the mitochondrial Ca2 + uniporter in the diabetic mice heart. Mice were either fed with a standard diet (SD: 16.9% proteins, 4.3% lipids) or a high-fat high sucrose diet (HFHSD: 20% proteins, 36% lipids) for 16 weeks. Cardiac mitochondria associated membranes (MAM) composition was analyzed by proteomics and immunoblotting. Proximity ligation assay, calcium imaging and hypoxia/reoxygenation were performed on isolated cardiomyocytes. Our HFHSD mice displayed a cardiac insulin resistance and hypertrophy. Decreased MAM/pure mitochondria content in the HFHSD vs SD heart was observed, with an elevated level of proteins involved in lipid metabolism and a decrease in tethering proteins. Decreased IP3R-VDAC proximity upon HFHSD was concomitant to a reduced IP3R-stimulated Ca2 + transfer to mitochondria, with no changes in mitochondrial calcium uniporter protein expression and function. Additionally, decreased amplitude of cytosolic Ca2 + transients was seen in the HFHSD cardiomyocytes, with no significant changes in the reticular Ca2 + release level. Besides, increased cell death susceptibility was measured after both in vitro hypoxia/reoxygenation and in vivo ischemia/reperfusion under HFHSD. Our data, therefore, indicate that decreased reticulum-mitochondria interactions trigger an impaired mitochondrial Ca2 + handling in the diabetic mice heart, with no alteration of the mitochondrial Ca2 + uniporter, while the enhanced susceptibility to cell death could be referred to lipid toxicity.

LRRK2 deficiency induced mitochondrial Ca2+ efflux inhibition can be rescued by Na+/Ca2+/Li+ exchanger upregulation

Variants of leucine-rich repeat kinase 2 (lrrk2) are associated with an increased risk in developing Parkinson’s disease (PD). Mitochondrial dysfunction and specifically mitochondrial Ca2+ handling has been linked to the pathogenesis of PD. Here we describe for the second time a mitochondrial Ca2+ efflux deficiency in a model displaying alterations in a PD-associated risk protein. LRRK2 deletion, inhibition and mutations led to an impaired mitochondrial Ca2+ extrusion via Na+/Ca2+/Li+ exchanger (NCLX) which in turn lowered mitochondrial permeability transition pore (PTP) opening threshold and increased cell death. The mitochondrial membrane potential was found not to be the underlying cause for the Ca2+ extrusion deficiency. NCLX activity was rescued by a direct (phosphomimetic NCLX mutant) and indirect (protein kinase A) activation which in turn elevated the PTP opening threshold. Therefore, at least two PD-associated risk protein pathways appear to converge on NCLX controlling mitochondrial Ca2+ extrusion and therefore mitochondrial health. Since mitochondrial Ca2+ overload has been described in many neurological disorders this study warrants further studies into NCLX as a potential therapeutic target.

Cardiac-specific overexpression of caveolin-3 preserves t-tubular ICa during heart failure in mice.

New Findings What is the central question of this study? What is the cellular basis of the protection conferred on the heart by overexpression of caveolin‐3 (Cav‐3 OE) against many of the features of heart failure normally observed in vivo? What is the main finding and its importance? Cav‐3 overexpression has little effect in normal ventricular myocytes but reduces cellular hypertrophy and preserves t‐tubular I Ca, but not local t‐tubular Ca2+ release, in heart failure induced by pressure overload in mice. Thus Cav‐3 overexpression provides specific but limited protection following induction of heart failure, although other factors disrupt Ca2+ release. Caveolin‐3 (Cav‐3) is an 18 kDa protein that has been implicated in t‐tubule formation and function in cardiac ventricular myocytes. During cardiac hypertrophy and failure, Cav‐3 expression decreases, t‐tubule structure is disrupted and excitation–contraction coupling (ECC) is impaired. Previous work has suggested that Cav‐3 overexpression (OE) is cardio‐protective, but the effect of Cav‐3 OE on these cellular changes is unknown. We therefore investigated whether Cav‐3 OE in mice is protective against the cellular effects of pressure overload induced by 8 weeks’ transverse aortic constriction (TAC). Cav‐3 OE mice developed cardiac dilatation, decreased stroke volume and ejection fraction, and hypertrophy and pulmonary congestion in response to TAC. These changes were accompanied by cellular hypertrophy, a decrease in t‐tubule regularity and density, and impaired local Ca2+ release at the t‐tubules. However, the extent of cardiac and cellular hypertrophy was reduced in Cav‐3 OE compared to WT mice, and t‐tubular Ca2+ current (I Ca) density was maintained. These data suggest that Cav‐3 OE helps prevent hypertrophy and loss of t‐tubular I Ca following TAC, but that other factors disrupt local Ca2+ release.