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Cytokines present during low-grade inflammation contribute to β-cell dysfunction and diabetes. Cytokine signaling disrupts β-cell glucose-stimulated Ca influx (GSCI) and endoplasmic reticulum (ER) Ca ([Ca]) handling, leading to diminished glucose-stimulated insulin secretion (GSIS). However, cytokine-mediated changes in ion channel activity that alter β-cell Ca handling remain unknown. Here we investigated the role of K currents in cytokine-mediated β-cell dysfunction. K currents, which control the termination of intracellular Ca ([Ca]) oscillations, were reduced following cytokine exposure. As a consequence, [Ca] and electrical oscillations were accelerated. Cytokine exposure also increased basal islet [Ca] and decreased GSCI. The effect of cytokines on TALK-1 K currents were also examined as TALK-1 mediates K by facilitating [Ca] release. Cytokine exposure decreased KCNK16 transcript abundance and associated TALK-1 protein expression, increasing [Ca] storage while maintaining 2 phase GSCI and GSIS. This adaptive Ca response was absent in TALK-1 KO islets, which exhibited decreased 2 phase GSCI and diminished GSIS. These findings suggest that K and TALK-1 currents play important roles in altered β-cell Ca handling and electrical activity during low-grade inflammation. These results also reveal that a cytokine-mediated reduction in TALK-1 serves an acute protective role in β-cells by facilitating increased Ca content to maintain GSIS.
BACKGROUND - Despite increased secondary cardiovascular events in patients with ischemic cardiomyopathy (ICM), the expression of innate cardiac protective molecules in the hearts of patients with ICM is incompletely characterized. Therefore, we used a nonbiased RNAseq approach to determine whether differences in cardiac protective molecules occur with ICM.
METHODS AND RESULTS - RNAseq analysis of human control and ICM left ventricular samples demonstrated a significant decrease in expression with ICM. encodes the Kir6.2 subunit of the cardioprotective K channel. Using wild-type mice and -deficient (-null) mice, we examined the effect of expression on cardiac function during ischemia-reperfusion injury. Reactive oxygen species generation increased in -null hearts above that found in wild-type mice hearts after ischemia-reperfusion injury. Continuous left ventricular pressure measurement during ischemia and reperfusion demonstrated a more compromised diastolic function in -null compared with wild-type mice during reperfusion. Analysis of key calcium-regulating proteins revealed significant differences in -null mice. Despite impaired relaxation, -null hearts increased phospholamban Ser16 phosphorylation, a modification that results in the dissociation of phospholamban from sarcoendoplasmic reticulum Ca, thereby increasing sarcoendoplasmic reticulum Ca-mediated calcium reuptake. However, -null mice also had increased 3-nitrotyrosine modification of the sarcoendoplasmic reticulum Ca-ATPase, a modification that irreversibly impairs sarcoendoplasmic reticulum Ca function, thereby contributing to diastolic dysfunction.
CONCLUSIONS - expression is decreased in human ICM. Lack of expression increases peroxynitrite-mediated modification of the key calcium-handling protein sarcoendoplasmic reticulum Ca-ATPase after myocardial ischemia-reperfusion injury, contributing to impaired diastolic function. These data suggest a mechanism for ischemia-induced diastolic dysfunction in patients with ICM.
© 2017 American Heart Association, Inc.
The interstitial cells of Cajal (ICC) drive rhythmic pacemaking contractions in the gastrointestinal system. The ICC generate pacemaking signals by membrane depolarizations associated with the release of intracellular calcium (Ca(2+)) in the endoplasmic reticulum (ER) through inositol-trisphosphate (IP3) receptors (IP3R) and uptake by mitochondria (MT). This Ca(2+) dynamic is hypothesized to generate pacemaking signals by calibrating ER Ca(2+) store depletions and membrane depolarization with ER store-operated Ca(2+) entry mechanisms. Using a biophysically based spatio-temporal model of integrated Ca(2+) transport in the ICC, we determined the feasibility of ER depletion timescale correspondence with experimentally observed pacemaking frequencies while considering the impact of IP3R Ca(2+) release and MT uptake on bulk cytosolic Ca(2+) levels because persistent elevations of free intracellular Ca(2+) are toxic to the cell. MT densities and distributions are varied in the model geometry to observe MT influence on free cytosolic Ca(2+) and the resulting frequencies of ER Ca(2+) store depletions, as well as the sarco-endoplasmic reticulum Ca(2+) ATP-ase (SERCA) and IP3 agonist concentrations. Our simulations show that high MT densities observed in the ICC are more relevant to ER establishing Ca(2+) depletion frequencies than protection of the cytosol from elevated free Ca(2+), whereas the SERCA pump is more relevant to containing cytosolic Ca(2+) elevations. Our results further suggest that the level of IP3 agonist stimulating ER Ca(2+) release, subsequent MT uptake, and eventual activation of ER store-operated Ca(2+) entry may determine frequencies of rhythmic pacemaking exhibited by the ICC across species and tissue types.
Copyright © 2014 the American Physiological Society.
AIMS - Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca(2+) uptake and myofilament Ca(2+) sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in a redox-dependent manner, improving Ca(2+) handling in isolated myocytes/hearts.
RESULTS - Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln(-/-)) mice. Compared to WT, pln(-/-) myocytes displayed enhanced resting sarcomere shortening, peak Ca(2+) transient, and blunted β-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca(2+) transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln(-/-) cells/hearts. HNO enhanced SR Ca(2+) uptake in WT but not pln(-/-) SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca(2+)-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available.
INNOVATION - HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a.
CONCLUSIONS - PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca(2+) handling in failing hearts.
The treatment of heart failure (HF) is challenging and morbidity and mortality are high. The goal of this study was to determine if inhibition of the late Na(+) current with ranolazine during early hypertensive heart disease might slow or stop disease progression. Spontaneously hypertensive rats (aged 7 mo) were subjected to echocardiographic study and then fed either control chow (CON) or chow containing 0.5% ranolazine (RAN) for 3 mo. Animals were then restudied, and each heart was removed for measurements of t-tubule organization and Ca(2+) transients using confocal microscopy of the intact heart. RAN halted left ventricular hypertrophy as determined from both echocardiographic and cell dimension (length but not width) measurements. RAN reduced the number of myocytes with t-tubule disruption and the proportion of myocytes with defects in intracellular Ca(2+) cycling. RAN also prevented the slowing of the rate of restitution of Ca(2+) release and the increased vulnerability to rate-induced Ca(2+) alternans. Differences between CON- and RAN-treated animals were not a result of different expression levels of voltage-dependent Ca(2+) channel 1.2, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, ryanodine receptor type 2, Na(+)/Ca(2+) exchanger-1, or voltage-gated Na(+) channel 1.5. Furthermore, myocytes with defective Ca(2+) transients in CON rats showed improved Ca(2+) cycling immediately upon acute exposure to RAN. Increased late Na(+) current likely plays a role in the progression of cardiac hypertrophy, a key pathological step in the development of HF. Early, chronic inhibition of this current slows both hypertrophy and development of ultrastructural and physiological defects associated with the progression to HF.
Antioxidant enzymatic pathways form a critical network that detoxifies ROS in response to myocardial stress or injury. Genetic alteration of the expression levels of individual enzymes has yielded mixed results with regard to attenuating in vivo myocardial ischemia-reperfusion injury, an extreme oxidative stress. We hypothesized that overexpression of an antioxidant network (AON) composed of SOD1, SOD3, and glutathione peroxidase (GSHPx)-1 would reduce myocardial ischemia-reperfusion injury by limiting ROS-mediated lipid peroxidation and oxidative posttranslational modification (OPTM) of proteins. Both ex vivo and in vivo myocardial ischemia models were used to evaluate the effect of AON expression. After ischemia-reperfusion injury, infarct size was significantly reduced both ex vivo and in vivo, ROS formation, measured by dihydroethidium staining, was markedly decreased, ROS-mediated lipid peroxidation, measured by malondialdehyde production, was significantly limited, and OPTM of total myocardial proteins, including fatty acid-binding protein and sarco(endo)plasmic reticulum Ca(²+)-ATPase (SERCA)2a, was markedly reduced in AON mice, which overexpress SOD1, SOD3, and GSHPx-1, compared with wild-type mice. These data demonstrate that concomitant SOD1, SOD3, and GSHPX-1 expression confers marked protection against myocardial ischemia-reperfusion injury, reducing ROS, ROS-mediated lipid peroxidation, and OPTM of critical cardiac proteins, including cardiac fatty acid-binding protein and SERCA2a.
In cardiac myocytes, the activity of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is hypothesized to regulate Ca(2+) release from and Ca(2+) uptake into the sarcoplasmic reticulum via the phosphorylation of the ryanodine receptor 2 and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wild-type or PLN null backgrounds under physiologically relevant pacing conditions (frequencies from 0.2 to 10 Hz and at 37 degrees C). When compared with that of mice lacking PLN only, the combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca(2+) release rate by more than 50% at 10 Hz. Although PLN ablation increased the rate of Ca(2+) uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca(2+)](i) transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca(2+)](i) transient, indicating that the PLN-mediated feedback on [Ca(2+)](i) removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes, the combined chronic CaMKII inhibition and PLN ablation slowed Ca(2+) release at physiological frequencies: the frequency-dependent decay of the amplitude and shortening of the [Ca(2+)](i) transient occurs independent of chronic CaMKII inhibition and PLN ablation, and the PLN-mediated regulation of Ca(2+) uptake is diminished at higher stimulation frequencies within the physiological range.
Cardiac Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca(2+) current (I(Ca)) facilitation, enhanced sarcoplasmic reticulum (SR) Ca(2+) release and frequency-dependent acceleration of relaxation (FDAR) via enhanced SR Ca(2+) uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaMKII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide AIP selectively in the SR membrane. Wild type mice (WT) and mice expressing AIP exclusively in the nucleus (NLS-AIP) served as controls. Increasing stimulation frequency produced typical FDAR in WT and NLS-AIP, but FDAR was markedly inhibited in SR-AIP. Quantitative analysis of cytosolic Ca(2+) removal during [Ca(2+)](i) decline revealed that FDAR is due to an increased apparent V(max) of SERCA. CaMKII-dependent RyR phosphorylation at Ser2815 and SR Ca(2+) leak was both decreased in SR-AIP vs. WT. This decrease in SR Ca(2+) leak may partly balance the reduced SERCA activity leading to relatively unaltered SR-Ca(2+) load in SR-AIP vs. WT myocytes. Surprisingly, CaMKII regulation of the L-type Ca(2+) channel (I(Ca) facilitation and recovery from inactivation) was abolished by the SR-targeted CaMKII inhibition in SR-AIP mice. Inhibition of CaMKII effects on I(Ca) and RyR function by the SR-localized AIP places physical constraints on the localization of these proteins at the junctional microdomain. Thus SR-targeted CaMKII inhibition can directly inhibit the activation of SR Ca(2+) uptake, SR Ca(2+) release and I(Ca) by CaMKII, effects which have all been implicated in triggered arrhythmias.
Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a and SERCA2a, two isoforms of the sarco(endo)plasmic Ca-ATPase, by lowering their apparent Ca(2+) affinity and thereby enabling muscle relaxation. SLN is expressed in both fast-twitch and slow-twitch muscle fibers with significant expression levels also found in the cardiac muscle. SLN shares approximately 30% identity with the transmembrane domain of phospholamban (PLN), and recent solution NMR studies carried out in detergent micelles indicate that the two polypeptides bind to SERCA in a similar manner. Previous 1D solid-state NMR experiments on selectively (15)N-labeled sites showed that SLN crosses the lipid bilayer with an orientation nearly parallel to the bilayer normal. With a view toward the characterization of SLN structure and its interactions with both lipids and SERCA, herein we report our initial structural and topological assignments of SLN in mechanically oriented DOPC/DOPE lipid bilayers as mapped by 2D (15)N PISEMA experiments. The PISEMA spectra obtained on uniformly (15)N-labeled protein as well as (15)N-Leu, (15)N-Ile and (15)N-Val map the secondary structure of SLN and, simultaneously, reveal that SLN exists in two distinct topologies. Both the major and the minor populations assume an orientation with the helix axis tilted by approximately 23 degrees with respect to the lipid bilayer normal, but vary in the rotation angle about the helix axis by approximately 5 degrees . The existence of the multiple populations in model membranes may be a significant requirement for SLN interaction with SERCA.
Low oxygen pressures exist in many solid tissues, including primary and secondary lymphoid organs. One key element in cellular adaptation to hypoxia is induced expression of hypoxia inducible factor (Hif) 1alpha. Here, we have examined the effect of Hif-1alpha, isolated from the myriad other effects of hypoxia, on T cell receptor (TCR) signaling in thymocytes. Because pVHL (von Hippel-Lindau protein) directs the proteolysis of Hif-1alpha under "normoxic" conditions, we achieved constitutive stabilization of Hif-1alpha through thymic deletion of Vhlh and reversed Hif-1alpha stabilization with double deletion of Vhlh and Hif-1alpha. We found that constitutive activity of Hif-1alpha resulted in diminished Ca(2+) response upon TCR crosslinking despite equivalent activation of phospholipase C(gamma1), normal intracellular Ca(2+) stores, and normal entry of Ca(2+) across the plasma membrane. Altered Ca(2+) response was instead due to accelerated removal of Ca(2+) from the cytoplasm into intracellular compartments, which occurred in association with Hif-1alpha-dependent overexpression of the calcium pump SERCA2 (sarcoplasmic/endoplasmic reticulum calcium ATPase 2). These data suggest a unique mechanism for control of TCR signaling through Hif-1alpha, which may be operative at the physiologic oxygen tensions seen in solid lymphoid organs.