A spectrum-of-spectrum filtering method to extract direct and multipath arrivals from simulations and measurements.

In measurements and numerical modelling of wave propagation, undesired interference between direct and multipath arrivals can be reduced using Fourier-based signal processing methods. Existing methods, such as cepstral analysis and time-signal gating, are not applicable to all cases. Here, an alternative Fourier-based signal processing method is presented, called spectrum-of-spectrum (SoS) filtering. Its main advantage over existing methods is its ability to extract single direct or multipath arrivals for relatively short propagation distances even when subsequent arrivals do not become successively weaker. The method is based on the following steps: •Apply a lowpass filter to the real and imaginary parts of an input frequency spectrum individually, using a digital finite impulse response (FIR) filter in the frequency domain.•Recombine the filtered real and imaginary parts of the frequency spectrum to get the frequency spectrum of the direct arrival.•For extraction of the first multipath arrival, subtract the filtered frequency spectrum from the input frequency spectrum and repeat the previous steps. Repeat multiple times to extract subsequent multipath arrivals.

Polyarginine Cell-Penetrating Peptides Bind and Inhibit SERCA2.

Cell-penetrating peptides (CPPs) are short peptide sequences that have the ability to cross the cell membrane and deliver cargo. Although it is critical that CPPs accomplish this task with minimal off-target effects, such actions have in many cases not been robustly screened. We presently investigated whether the commonly used CPPs TAT and the polyarginines Arg9 and Arg11 exert off-target effects on cellular Ca2+ homeostasis. In experiments employing myocytes and homogenates from the cardiac left ventricle or soleus muscle, we observed marked inhibition of Ca2+ recycling into the sarcoplasmic reticulum (SR) following incubation with polyarginine CPPs. In both tissues, the rate of SR Ca2+ leak remained unchanged, indicating that protracted Ca2+ removal from the cytosol stemmed from inhibition of the SR Ca2+ ATPase 2 (SERCA2). No such inhibition occurred following treatment with TAT, or in preparations from the SERCA1-expressing extensor digitorum longus muscle. Experiments in HEK cells overexpressing individual SERCA isoforms confirmed that polyarginine incubation specifically inhibited the activity of SERCA2a and 2b, but not SERCA1 or 3. The attenuation of SERCA2 activity was not dependent on the presence of phospholamban, and ELISA-based analyses rather revealed direct interaction between the polyarginines and the actuator domain of the protein. Surface plasmon resonance experiments confirmed strong binding within this region of SERCA2, and slow dissociation between the two species. Based on these observations, we urge caution when employing polyarginine CPPs. Indeed, as SERCA2 is expressed in diverse cell types, the wide-ranging consequences of SERCA2 binding and inhibition should be anticipated in both experimental and therapeutic settings.

Finite element-based diffraction correction for piezoelectric transducers accounting for diffraction at transmission, propagation, and reception.

Existing diffraction correction models for ultrasonic transmit-receive measurement systems rely on simplifying assumptions with respect to the boundary conditions at the transmitter or receiver. Common simplifications include approximating the sound field radiated by a piezoelectric transducer using a baffled piston model and assuming that the receiver's electrical response is proportional to the spatially averaged free-field pressure over its front surface. In many applications, such simplifications may be adequate, but their validity and accuracy need to be evaluated and quantified. Here, a diffraction correction model utilizing the full set of electrical and mechanical boundary conditions at the transmitter and receiver is presented, avoiding these simplifications. The model is based on finite element modeling of coaxially aligned piezoelectric transducers in a fluid medium. Comparison is made with existing models for an example case of cylindrical piezoelectric ceramic disk transducers operating in air at 50-300 kHz and 0.03-2 m apart, relevant for, e.g., sound velocity and absorption measurements in fluids and ultrasonic gas flow metering. In the near-field, errors introduced by the simplifications are up to 3 dB and 47° for the first radial resonance. Generally, such errors are application-specific and depend on distance, frequency, transducer construction, vibration pattern, and medium properties.

Disruption of Phosphodiesterase 3A Binding to SERCA2 Increases SERCA2 Activity and Reduces Mortality in Mice With Chronic Heart Failure.

BACKGROUND: Increasing SERCA2 (sarco[endo]-plasmic reticulum Ca2+ ATPase 2) activity is suggested to be beneficial in chronic heart failure, but no selective SERCA2-activating drugs are available. PDE3A (phosphodiesterase 3A) is proposed to be present in the SERCA2 interactome and limit SERCA2 activity. Disruption of PDE3A from SERCA2 might thus be a strategy to develop SERCA2 activators. METHODS: Confocal microscopy, 2-color direct stochastic optical reconstruction microscopy, proximity ligation assays, immunoprecipitations, peptide arrays, and surface plasmon resonance were used to investigate colocalization between SERCA2 and PDE3A in cardiomyocytes, map the SERCA2/PDE3A interaction sites, and optimize disruptor peptides that release PDE3A from SERCA2. Functional experiments assessing the effect of PDE3A-binding to SERCA2 were performed in cardiomyocytes and HEK293 vesicles. The effect of SERCA2/PDE3A disruption by the disruptor peptide OptF (optimized peptide F) on cardiac mortality and function was evaluated during 20 weeks in 2 consecutive randomized, blinded, and controlled preclinical trials in a total of 148 mice injected with recombinant adeno-associated virus 9 (rAAV9)-OptF, rAAV9-control (Ctrl), or PBS, before undergoing aortic banding (AB) or sham surgery and subsequent phenotyping with serial echocardiography, cardiac magnetic resonance imaging, histology, and functional and molecular assays. RESULTS: PDE3A colocalized with SERCA2 in human nonfailing, human failing, and rodent myocardium. Amino acids 277-402 of PDE3A bound directly to amino acids 169-216 within the actuator domain of SERCA2. Disruption of PDE3A from SERCA2 increased SERCA2 activity in normal and failing cardiomyocytes. SERCA2/PDE3A disruptor peptides increased SERCA2 activity also in the presence of protein kinase A inhibitors and in phospholamban-deficient mice, and had no effect in mice with cardiomyocyte-specific inactivation of SERCA2. Cotransfection of PDE3A reduced SERCA2 activity in HEK293 vesicles. Treatment with rAAV9-OptF reduced cardiac mortality compared with rAAV9-Ctrl (hazard ratio, 0.26 [95% CI, 0.11 to 0.63]) and PBS (hazard ratio, 0.28 [95% CI, 0.09 to 0.90]) 20 weeks after AB. Mice injected with rAAV9-OptF had improved contractility and no difference in cardiac remodeling compared with rAAV9-Ctrl after aortic banding. CONCLUSIONS: Our results suggest that PDE3A regulates SERCA2 activity through direct binding, independently of the catalytic activity of PDE3A. Targeting the SERCA2/PDE3A interaction prevented cardiac mortality after AB, most likely by improving cardiac contractility.

The female syndecan-4-/- heart has smaller cardiomyocytes, augmented insulin/pSer473-Akt/pSer9-GSK-3ß signaling, and lowered SCOP, pThr308-Akt/Akt and GLUT4 levels.

Background: In cardiac muscle, the ubiquitously expressed proteoglycan syndecan-4 is involved in the hypertrophic response to pressure overload. Protein kinase Akt signaling, which is known to regulate hypertrophy, has been found to be reduced in the cardiac muscle of exercised male syndecan-4-/- mice. In contrast, we have recently found that pSer473-Akt signaling is elevated in the skeletal muscle (tibialis anterior, TA) of female syndecan-4-/- mice. To determine if the differences seen in Akt signaling are sex specific, we have presently investigated Akt signaling in the cardiac muscle of sedentary and exercised female syndecan-4-/- mice. To get deeper insight into the female syndecan-4-/- heart, alterations in cardiomyocyte size, a wide variety of different extracellular matrix components, well-known syndecan-4 binding partners and associated signaling pathways have also been investigated. Methods: Left ventricles (LVs) from sedentary and exercise trained female syndecan-4-/- and WT mice were analyzed by immunoblotting and real-time PCR. Cardiomyocyte size and phosphorylated Ser473-Akt were analyzed in isolated adult cardiomyocytes from female syndecan-4-/- and WT mice by confocal imaging. LV and skeletal muscle (TA) from sedentary male syndecan-4-/- and WT mice were immunoblotted with Akt antibodies for comparison. Glucose levels were measured by a glucometer, and fasting blood serum insulin and C-peptide levels were measured by ELISA. Results: Compared to female WT hearts, sedentary female syndecan-4-/- LV cardiomyocytes were smaller and hearts had higher levels of pSer473-Akt and its downstream target pSer9-GSK-3ß. The pSer473-Akt inhibitory phosphatase PHLPP1/SCOP was lowered, which may be in response to the elevated serum insulin levels found in the female syndecan-4-/- mice. We also observed lowered levels of pThr308-Akt/Akt and GLUT4 in the female syndecan-4-/- heart and an increased LRP6 level after exercise. Otherwise, few alterations were found. The pThr308-Akt and pSer473-Akt levels were unaltered in the cardiac and skeletal muscles of sedentary male syndecan-4-/- mice. Conclusion: Our data indicate smaller cardiomyocytes, an elevated insulin/pSer473-Akt/pSer9-GSK-3ß signaling pathway, and lowered SCOP, pThr308-Akt/Akt and GLUT4 levels in the female syndecan-4-/- heart. In contrast, cardiomyocyte size, and Akt signaling were unaltered in both cardiac and skeletal muscles from male syndecan-4-/- mice, suggesting important sex differences.

AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. METHODS: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. RESULTS: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. CONCLUSIONS: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

The small molecule SERCA activator CDN1163 increases energy metabolism in human skeletal muscle cells.

BACKGROUND AND OBJECTIVE: A number of studies have highlighted muscle-specific mechanisms of thermogenesis involving futile cycling of Ca2+ driven by sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA) and generating heat from ATP hydrolysis to be a promising strategy to counteract obesity and metabolic dysfunction. However, to the best of our knowledge, no experimental studies concerning the metabolic effects of pharmacologically targeting SERCA in human skeletal muscle cells have been reported. Thus, in the present study, we aimed to explore the effects of SERCA-activating compound, CDN1163, on energy metabolism in differentiated human skeletal muscle cells (myotubes). METHODS: In this study, we used primary myotube cultures derived from muscle biopsies of the musculus vastus lateralis and musculi interspinales from lean, healthy male donors. Energy metabolism in myotubes was studied using radioactive substrates. Oxygen consumption rate was assessed with the Seahorse XF24 bioanalyzer, whereas metabolic genes and protein expressions were determined by qPCR and immunoblotting, respectively. RESULTS: Both acute (4 â€‹h) and chronic (5 days) treatment of myotubes with CDN1163 showed increased uptake and oxidation of glucose, as well as complete fatty acid oxidation in the presence of carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP). These effects were supported by measurement of oxygen consumption rate, in which the oxidative spare capacity and maximal respiration were enhanced after CDN1163-treatment. In addition, chronic treatment with CDN1163 improved cellular uptake of oleic acid (OA) and fatty acid ß-oxidation. The increased OA metabolism was accompanied by enhanced mRNA-expression of carnitine palmitoyl transferase (CPT) 1B, pyruvate dehydrogenase kinase (PDK) 4, as well as increased AMP-activated protein kinase (AMPK)Thr172 phosphorylation. Moreover, following chronic CDN1163 treatment, the expression levels of stearoyl-CoA desaturase (SCD) 1 was decreased together with de novo lipogenesis from acetic acid and formation of diacylglycerol (DAG) from OA. CONCLUSION: Altogether, these results suggest that SERCA activation by CDN1163 enhances energy metabolism in human myotubes, which might be favourable in relation to disorders that are related to metabolic dysfunction such as obesity and type 2 diabetes mellitus.

Probenecid Improves Cardiac Function in Subjects with a Fontan Circulation and Augments Cardiomyocyte Calcium Homeostasis.

Subjects with functionally univentricular circulation who have completed staged single ventricle palliation, with the final stage culminating in the Fontan procedure, are often living into adulthood. However, high morbidity and mortality remain prevalent in these patients, as diastolic and systolic dysfunction of the single systemic ventricle are linked to Fontan circulatory failure. We presently investigated the effects of probenecid in post-Fontan patients. Used for decades for the treatment of gout, probenecid has been shown in recent years to positively influence cardiac function via effects on the Transient Receptor Potential Vanilloid 2 (TRPV2) channel in cardiomyocytes. Indeed, we observed that probenecid improved cardiac function and exercise performance in patients with a functionally univentricular circulation. This was consistent with our findings from a retrospective cohort of patients with single ventricle physiology where TRPV2 expression was increased. Experiments in isolated cardiomyocytes associated these positive actions to augmentation of diastolic calcium homeostasis.

Secretoneurin Is an Endogenous Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor That Attenuates Ca2+-Dependent Arrhythmia.

BACKGROUND: Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia. METHODS: Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca2+ homeostasis examined by fluorescence (fluo-4) and patch-clamp recordings in isolated cardiomyocytes. RESULTS: SN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased Ca2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca2+ current. CONCLUSIONS: SN production is upregulated in conditions with cardiomyocyte Ca2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.

Finite element modeling of ultrasound measurement systems for gas. Comparison with experiments in air.

Quantitative modeling of ultrasound measurement systems is of considerable value for design, analysis, and interpretation of measurements, methods, and systems. In this work, a model is developed for description of transmit-receive measurement systems based on radial-mode transducer operation in a homogeneous fluid medium. Axisymmetric finite element (FE) modeling is used for the transmitting and receiving piezoelectric transducers and sound propagation in the medium. Transmission-line modeling is used for transmitting and receiving cabling and electronics. The model potentially accounts for the full frequency response of the transducers, including radial and thickness modes, mode coupling, and interaction with the medium. Reciprocal transducers are assumed in the model, and linearity in all parts of the system. Near field effects are accounted for using diffraction correction. Simulations are compared with measurements for the transmit-receive voltage-to-voltage transfer function of two piezoelectric ceramic disk transducers vibrating in air at 1 atm, over the frequency range of the first two radial modes of the disks, and the time domain voltage waveforms at the electric terminals of the transmitting and receiving transducers. The results demonstrate that quantitative simulations of the measurement system can be done with reasonable accuracy. Potentials of improvement are identified and discussed.

Ryanodine receptor dispersion disrupts Ca2+ release in failing cardiac myocytes.

Reduced cardiac contractility during heart failure (HF) is linked to impaired Ca2+ release from Ryanodine Receptors (RyRs). We investigated whether this deficit can be traced to nanoscale RyR reorganization. Using super-resolution imaging, we observed dispersion of RyR clusters in cardiomyocytes from post-infarction HF rats, resulting in more numerous, smaller clusters. Functional groupings of RyR clusters which produce Ca2+ sparks (Ca2+ release units, CRUs) also became less solid. An increased fraction of small CRUs in HF was linked to augmented 'silent' Ca2+ leak, not visible as sparks. Larger multi-cluster CRUs common in HF also exhibited low fidelity spark generation. When successfully triggered, sparks in failing cells displayed slow kinetics as Ca2+ spread across dispersed CRUs. During the action potential, these slow sparks protracted and desynchronized the overall Ca2+ transient. Thus, nanoscale RyR reorganization during HF augments Ca2+ leak and slows Ca2+ release kinetics, leading to weakened contraction in this disease.

Time-of-flight dependency on transducer separation distance in a reflective-path guided-wave ultrasonic flow meter at zero flow conditions.

Transit-time flow meters based on guided ultrasonic wave propagation in the pipe spool have several advantages compared to traditional inline ultrasonic flow metering. The extended interrogation field, obtained by continuous leakage from guided waves traveling in the pipe wall, increases robustness toward entrained particles or gas in the flow. In reflective-path guided-wave ultrasonic flow meters (GW-UFMs), the flow equations are derived from signals propagating solely in the pipe wall and from signals passing twice through the fluid. In addition to the time-of-flight (TOF) through the fluid, the fluid path experiences an additional time delay upon reflection at the opposite pipe wall due to specular and non-specular reflections. The present work investigates the influence of these reflections on the TOF in a reflective-path GW-UFM as a function of transducer separation distance at zero flow conditions. Two models are used to describe the signal propagation through the system: (i) a transient full-wave finite element model, and (ii) a combined plane-wave and ray-tracing model. The study shows that a range-dependent time delay is associated with the reflection of the fluid path, introducing transmitter-receiver distance dependence. Based on these results, the applicability of the flow equations derived using model (ii) is discussed.

Beam Diffraction Effects in the Backward Wave Regions of Viscoelastic Leaky Lamb Modes for Plate Transmission at Normal Incidence.

Plane-wave theory for fluid-embedded isotropic plates is often used in ultrasonic guided-wave applications, and to estimate wall thickness, corrosion, or sound velocities in plates and pipes. In such structures, measured ultrasonic transmission through the solid material is affected by acoustic beam diffraction effects, and the results may deviate from plane-wave descriptions, which are insufficient to describe the complex effects that occur. When exciting a fluid-embedded steel plate with a pulsed ultrasonic beam at normal incidence, resonance frequency downshift, axial sound pressure level increase, and beam narrowing have been observed, for measured resonance peaks in the frequency regions of certain leaky Lamb mode branches of the plate. In the ranges of other leaky Lamb mode branches, the observed effects are different. Measurements, finite element, and angular spectrum modeling are used to indicate a close connection between these beam diffraction phenomena and the backward wave characteristics of certain leaky Lamb mode pairs, in the frequency and Poisson's ratio regions around coincidence of two Lamb mode cutoff frequencies of similar symmetry. In particular, such observations made for the steel plate's fundamental thickness-extensional (TE) mode appear to be caused by acoustic beam excitation of the backward wave regions of the S-2vl and S2vl leaky Lamb modes.

Exercise training increases protein O-GlcNAcylation in rat skeletal muscle.

Protein O-GlcNAcylation has emerged as an important intracellular signaling system with both physiological and pathophysiological functions, but the role of protein O-GlcNAcylation in skeletal muscle remains elusive. In this study, we tested the hypothesis that protein O-GlcNAcylation is a dynamic signaling system in skeletal muscle in exercise and disease. Immunoblotting showed different protein O-GlcNAcylation pattern in the prototypical slow twitch soleus muscle compared to fast twitch EDL from rats, with greater O-GlcNAcylation level in soleus associated with higher expression of the modulating enzymes O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and glutamine fructose-6-phosphate amidotransferase isoforms 1 and 2 (GFAT1, GFAT2). Six weeks of exercise training by treadmill running, but not an acute exercise bout, increased protein O-GlcNAcylation in rat soleus and EDL There was a striking increase in O-GlcNAcylation of cytoplasmic proteins ~50 kDa in size that judged from mass spectrometry analysis could represent O-GlcNAcylation of one or more key metabolic enzymes. This suggests that cytoplasmic O-GlcNAc signaling is part of the training response. In contrast to exercise training, postinfarction heart failure (HF) in rats and humans did not affect skeletal muscle O-GlcNAcylation level, indicating that aberrant O-GlcNAcylation cannot explain the skeletal muscle dysfunction in HF Human skeletal muscle displayed extensive protein O-GlcNAcylation that by large mirrored the fiber-type-related O-GlcNAcylation pattern in rats, suggesting O-GlcNAcylation as an important signaling system also in human skeletal muscle.

Beam diffraction effects in sound transmission of a fluid-embedded viscoelastic plate at normal incidence.

The characteristics of a sound beam transmitted through a fluid-embedded viscoelastic plate at normal incidence can deviate significantly from those of a plane-wave. Phenomena such as frequency shift, signal amplification or reduction, and changed beam properties, are observed for resonance peaks associated with specific leaky Lamb modes. When interpreting measurements using plane-wave theory, such deviations will influence the measurement of material parameters and plate thickness. The finite-element-based models used in this study describe the signal chain from the electrical voltage excitation at the piezoelectric transducer terminals to the sound pressure propagated through the plate and fluid to the position at which it is measured by a hydrophone. The measured phenomena are described at a quantitative level.

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman.

The sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na(+)/K(+)-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca(2+) binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5-8Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.

Exhausting treadmill running causes dephosphorylation of sMLC2 and reduced level of myofilament MLCK2 in slow twitch rat soleus muscle.

Myosin light chain 2 (MLC2) is a small protein in the myosin complex, regulating muscle contractile function by modulating Ca(2+) sensitivity of myofilaments. MLC2 can be modified by phosphorylation and O-GlcNAcylation, two reversible and dynamic posttranslational modifications. The slow isoform of MLC2 (sMLC2) is dephosphorylated in soleus muscle during in situ loaded shortening contractions, which correlates with reduction in shortening capacity. Here, we hypothesize that exhausting in vivo treadmill running induces dephosphorylation of MLC2 in slow twitch soleus, but not in fast twitch EDL muscle, and that there are reciprocal changes in MLC2 O-GlcNAcylation. At rest, both phosphorylation and O-GlcNAcylation of MLC2 were lower in slow than fast twitch muscles. One bout of exhausting treadmill running induced dephosphorylation of sMLC2 in soleus, paralleled by reduced levels of the kinase MLCK2 associated to myofilaments, suggesting that the acute reduction in phosphorylation is mediated by dissociation of MLCK2 from myofilaments. O-GlcNAcylation of MLC2 did not change significantly, and seems of limited importance in the regulation of MLC2 phosphorylation during in vivo running. After 6 weeks of treadmill running, the dephosphorylation of sMLC2 persisted in soleus along with reduction in MLCK2 both in myofilament- and total protein fraction. In EDL on the contrary, phosphorylation of MLC2 was not altered after one exercise bout or after 6 weeks of treadmill running. Thus, in contrast to fast twitch muscle, MLC2 dephosphorylation occurs in slow twitch muscle during in vivo exercise and may be linked to reduced myofilament-associated MLCK2 and reduced shortening capacity.

Lack of collagen VIII reduces fibrosis and promotes early mortality and cardiac dilatation in pressure overload in mice.

AIMS: In pressure overload, left ventricular (LV) dilatation is a key step in transition to heart failure (HF). We recently found that collagen VIII (colVIII), a non-fibrillar collagen and extracellular matrix constituent, was reduced in hearts of mice with HF and correlated to degree of dilatation. A reduction in colVIII might be involved in LV dilatation, and we here examined the role of reduced colVIII in pressure overload-induced remodelling using colVIII knock-out (col8KO) mice. METHODS AND RESULTS: Col8KO mice exhibited increased mortality 3-9 days after aortic banding (AB) and increased LV dilatation from day one after AB, compared with wild type (WT). LV dilatation remained increased over 56 days. Forty-eight hours after AB, LV expression of main structural collagens (I and III) was three-fold increased in WT mice, but these collagens were unaltered in the LV of col8KO mice together with reduced expression of the pro-fibrotic cytokine TGF-ß, SMAD2 signalling, and the myofibroblast markers Pxn, α-SMA, and SM22. Six weeks after AB, LV collagen mRNA expression and protein were increased in col8KO mice, although less pronounced than in WT. In vitro, neonatal cardiac fibroblasts from col8KO mice showed lower expression of TGF-ß, Pxn, α-SMA, and SM22 and reduced migratory ability possibly due to increased RhoA activity and reduced MMP2 expression. Stimulation with recombinant colVIIIα1 increased TGF-ß expression and fibroblast migration. CONCLUSION: Lack of colVIII reduces myofibroblast differentiation and fibrosis and promotes early mortality and LV dilatation in response to pressure overload in mice.

Vascular Ehlers-Danlos Syndrome in siblings with biallelic COL3A1 sequence variants and marked clinical variability in the extended family.

Vascular Ehlers-Danlos Syndrome (vEDS), also known as EDS type IV, is considered to be an autosomal dominant disorder caused by sequence variants in COL3A1, which encodes the chains of type III procollagen. We identified a family in which there was marked clinical variation with the earliest death due to extensive aortic dissection at age 15 years and other family members in their eighties with no complications. The proband was born with right-sided clubfoot but was otherwise healthy until he died unexpectedly at 15 years. His sister, in addition to signs consistent with vascular EDS, had bilateral frontal and parietal polymicrogyria. The proband and his sister each had two COL3A1 sequence variants, c.1786C>T, p.(Arg596*) in exon 26 and c.3851G>A, p.(Gly1284Glu) in exon 50 on different alleles. Cells from the compound heterozygote produced a reduced amount of type III procollagen, all the chains of which had abnormal electrophoretic mobility. Biallelic sequence variants have a significantly worse outcome than heterozygous variants for either null mutations or missense mutations, and frontoparietal polymicrogyria may be an added phenotype feature. This genetic constellation provides a very rare explanation for marked intrafamilial clinical variation due to sequence variants in COL3A1.