Glutathione-dependent depalmitoylation of phospholemman by peroxiredoxin 6.

Phospholemman (PLM) regulates the cardiac sodium pump: PLM phosphorylation activates the pump whereas PLM palmitoylation inhibits its activity. Here, we show that the anti-oxidant protein peroxiredoxin 6 (Prdx6) interacts with and depalmitoylates PLM in a glutathione-dependent manner. Glutathione loading cells acutely reduce PLM palmitoylation; glutathione depletion significantly increases PLM palmitoylation. Prdx6 silencing abolishes these effects, suggesting that PLM can be depalmitoylated by reduced Prdx6. In vitro, only recombinant Prdx6, among several peroxiredoxin isoforms tested, removes palmitic acid from recombinant palmitoylated PLM. The broad-spectrum depalmitoylase inhibitor palmostatin B prevents Prdx6-dependent PLM depalmitoylation in cells and in vitro. Our data suggest that Prdx6 is a thioesterase that can depalmitoylate proteins by nucleophilic attack via its reactive thiol, linking PLM palmitoylation and hence sodium pump activity to cellular glutathione status. We show that protein depalmitoylation can occur via a catalytic cysteine in which substrate specificity is determined by a protein-protein interaction.

Keep My Teeth: An evaluation of multi-disciplinary training in mouth care for people with intellectual developmental disorders.

AIM: To evaluate a modular didactic training intervention called Keep My Teeth designed by special care dentists, for a range of healthcare students to provide oral homecare for people with intellectual developmental disorders (PwIDD). METHODS: To evaluate the intervention a one-group pre-test post-test pre-experimental research design was utilized. The intervention was delivered by virtual platforms or face-to-face, with a sub-sample of participants also receiving practical training. Healthcare students included Speech and Language Therapy (SLT), Registered Nurse Intellectual Disability (RNID), Dental Science (DS), Dental Nursing (DN), and Dental Hygiene (DH). RESULTS: Sixty-three of the 147 trainees completed all surveys. A significant change in perspective on barriers was seen for most groups post-training, with an increase in confidence in delivering oral care to PwIDD across disciplines; 67% of DH/DN students who took part in the practical training felt that the didactic training was just as effective without the practical training, while 42% of the DS students felt that was true. CONCLUSIONS: The training interventions provided seem to have increased the awareness of study participants in relation to barriers to care, and increased their self-efficacy towards, and intention to perform, oral homecare behaviors.

Characterization of Shiga toxin-producing Escherichia coli presence, serogroups and risk factors from private groundwater sources in western Ireland.

Over recent years, Ireland has reported the highest crude incidence rates of Shiga toxin-producing Escherichia coli (STEC) enteritis in Europe. Unregulated private groundwater sources have emerged as an important potential transmission route for STEC, with up to 750,000 Irish residents reliant on these sources for domestic waters. This study aimed to investigate the prevalence and serogroup profile of STEC contamination from domestic private wells in western Ireland. Fifty-two groundwater sources were analysed during two sampling campaigns in the autumn (September/October) of 2019 (n = 21) and 2021 (n = 31). Untreated groundwater samples (30 L) were collected and analysed using the "CapE" (capture, amplify, extract) method. Extracted DNA was tested using multiplex real-time PCR for Shiga toxin stx1 and/or stx2 and eae genes. STEC positive DNA samples were tested for clinically relevant serogroups by real-time PCR. Data relating to 27 potential groundwater contamination risk factors were geospatially linked to each well and assessed for association with E. coli, stx1 and/or stx2 and eae presence/absence. Overall, 20/52 wells (38.4 %) were positive for E. coli (median concentration 8.5 MPN/100 mL as assessed by Colilert-18 method). Stx1 and/or stx2 was detected in 10/52 (19.2 %) wells overall and 8/20 E. coli positive wells, equating to a STEC to "generic" E. coli detection ratio of 40 %. Six of these wells (30 %) were also positive for eae. One or more serogroup-specific gene targets were identified in all but one stx1 and/or stx2 positive sample, with O145 (n = 6), O157 (n = 5) and O103 (n = 4) most prevalent. STEC presence was significantly associated with decreasing well depth (U = -2.243; p = 0.024) and increasing 30-day mean antecedent rainfall (U = 2.126; p = 0.034). Serogroup O104 was associated with increased sheep density (U = 2.089; p = 0.044) and detection of stx1 and/or stx2 + eae with increased septic tank density (U = 2.246 p = 0.023). Findings indicate high detection rates of clinically relevant STEC in E. coli contaminated groundwater sources in Ireland.

Challenges and innovation: Disease modeling using human-induced pluripotent stem cell-derived cardiomyocytes.

Disease modeling using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has both challenges and promise. While patient-derived iPSC-CMs provide a unique opportunity for disease modeling with isogenic cells, the challenge is that these cells still demonstrate distinct properties which make it functionally less akin to adult cardiomyocytes. In response to this challenge, numerous innovations in differentiation and modification of hiPSC-CMs and culture techniques have been developed. Here, we provide a focused commentary on hiPSC-CMs for use in disease modeling, the progress made in generating electrically and metabolically mature hiPSC-CMs and enabling investigative platforms. The solutions are bringing us closer to the promise of modeling heart disease using human cells in vitro.

How Functional Genomics Can Keep Pace With VUS Identification.

Over the last two decades, an exponentially expanding number of genetic variants have been identified associated with inherited cardiac conditions. These tremendous gains also present challenges in deciphering the clinical relevance of unclassified variants or variants of uncertain significance (VUS). This review provides an overview of the advancements (and challenges) in functional and computational approaches to characterize variants and help keep pace with VUS identification related to inherited heart diseases.

Transformation of Clinical Nursing Practice Following a Caring-based Educational Intervention: A Qualitative Perspective.

Introduction: Hemodialysis (HD) patients experience numerous physical and psychological symptoms on a daily basis. These symptoms have a heavy impact on their quality of life, which is a key indicator of their survival in the short term. Numerous empirical studies have shown that the quality of the nurse-patient relationship (NPR) is essential in promoting positive outcomes for patients. When patients receive caring, their autonomy and independence grows, their sense of hope increases, their quality of life improves, and their sense of satisfaction with nursing care received rises. Inversely, the presence of dehumanizing practices in hemodialysis settings can contribute to delay healing for patients. In light of the importance of the quality of the relationship between nurses and HD patients and of the benefits to be had from a quality relationship, an educational intervention based on Watson's Theory of Human Caring was delivered to HD nurses. Objective: The purpose of this study was to explore qualitatively the perceptions of nurses working with HD patients in French-speaking Switzerland regarding changes to their clinical practice after receiving an educational intervention intended to reinforce caring attitudes and behaviors towards patients. Methods: The method used was that of consensual qualitative research (CQR). Sixteen semi-structured interviews were conducted with hemodialysis nurses post-intervention. Results: The results evidence a transformation of clinical nursing practice illustrated by three core ideas: (1) caring practice was reinforced; (2) new practices emerged; and (3) some limitations appeared. Conclusion: In these times of global pandemic where the issue of the humanization of nursing care is front and center, this professional development activity helped reinforce caring-based practice. This practice needs to be developed within the various care units in order to guarantee and promote quality of care and patient safety.

Effects of an educational intervention to strengthen humanistic practice on haemodialysis nurses' caring attitudes and behaviours and quality of working life: a cluster randomised controlled trial.

BACKGROUND: Nurses are trained to establish a trusting relationship with patients to create an environment promoting patients' quality of life. However, in tech-heavy care settings, such as haemodialysis units, dehumanising practices may emerge and take root for various reasons to the potential detriment of both patients and nurses. For patients, this may lead to a deterioration of quality of life and, ultimately, of health status. For nurses, it may cause a deterioration of the work environment and, in turn, of quality of working life. Based on Watson's Theory of Human Caring, we developed a brief educational intervention for haemodialysis nurses to strengthen their humanistic practice in the aim of improving the nurse-patient relationship and nurse quality of working life.. The intervention was tested by way of an experimental design. METHODS: One hundred and one haemodialysis nurses, recruited in ten hospitals in French-speaking Switzerland, were randomised into an experimental group that received the intervention and a control group. The nurse-patient relationship was measured with the Caring Nurse-Patient Interaction Scale (EIIP-70) and nurse quality of life at work was measured with the Quality of Work Life Questionnaire at four time points: pre-intervention, intervention completion, and six-month and one-year follow-ups. Random intercept regression analysis was used to evaluate change over time in the two variables under study. RESULTS: The intervention appeared to reinforce nurse attitudes and behaviours geared to a more humanistic practice. The effect seemed to fade over time but, 1 year post-intervention, six dimensions of the nurse-patient relationship (hope, sensibility, helping relationship, expression of emotions, problem solving, teaching) scored above baseline. Nurse quality of working life, too, seemed positively impacted. The cultural dimension of nurse quality of working life, that is, the degree to which everyday work activities attune with personal and cultural values, seemed positively impacted, as well, with improvement stable throughout the year following the intervention. CONCLUSIONS: Results support a positive effect of the intervention over both the short term and the medium-to-long term. A brief intervention of the sort may constitute an effective means to improve the nurse-patient relationship by preventing or reducing dehumanising practices. TRIAL REGISTRATION: NCT03283891 .

Proteomic Analysis of the Functional Inward Rectifier Potassium Channel (Kir) 2.1 Reveals Several Novel Phosphorylation Sites.

Membrane proteins represent a large family of proteins that perform vital physiological roles and represent key drug targets. Despite their importance, bioanalytical methods aiming to comprehensively characterize the post-translational modification (PTM) of membrane proteins remain challenging compared to other classes of proteins in part because of their inherent low expression and hydrophobicity. The inward rectifier potassium channel (Kir) 2.1, an integral membrane protein, is critical for the maintenance of the resting membrane potential and phase-3 repolarization of the cardiac action potential in the heart. The importance of this channel to cardiac physiology is highlighted by the recognition of several sudden arrhythmic death syndromes, Andersen-Tawil and short QT syndromes, which are associated with loss or gain of function mutations in Kir2.1, often triggered by changes in the ß-adrenergic tone. Therefore, understanding the PTMs of this channel (particularly ß-adrenergic tone-driven phosphorylation) is important for arrhythmia prevention. Here, we developed a proteomic method, integrating both top-down (intact protein) and bottom-up (after enzymatic digestion) proteomic analyses, to characterize the PTMs of recombinant wild-type and mutant Kir2.1, successfully mapping five novel sites of phosphorylation and confirming a sixth site. Our study provides a framework for future work to assess the role of PTMs in regulating Kir2.1 functions.

Cardiac potassium inward rectifier Kir2: Review of structure, regulation, pharmacology, and arrhythmogenesis.

Potassium inward rectifier channel Kir2 is an important component of terminal cardiac repolarization and resting membrane stability. This functionality is part of balanced cardiac excitability and is a defining feature of excitable cardiac membranes. "Gain-of-function" or "loss-of-function" mutations in KCNJ2, the gene encoding Kir2.1, cause genetic sudden cardiac death syndromes, and loss of the Kir2 current IK1 is a major contributing factor to arrhythmogenesis in failing human hearts. Here we provide a contemporary review of the functional structure, physiology, and pharmacology of Kir2 channels. Beyond the structure and functional relationships, we will focus on the elements of clinically used drugs that block the channel and the implications for treatment of atrial fibrillation with IK1-blocking agents. We will also review the clinical disease entities associated with KCNJ2 mutations and the growing area of research into associated arrhythmia mechanisms. Lastly, the presence of Kir2 channels has become a tipping point for electrical maturity in induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) and highlights the significance of understanding why Kir2 in iPS-CMs is important to consider for Comprehensive In Vitro Proarrhythmia Assay and drug safety testing.

Genetic Loss of IK1 Causes Adrenergic-Induced Phase 3 Early Afterdepolariz ations and Polymorphic and Bidirectional Ventricular Tachycardia.

BACKGROUND: Arrhythmia syndromes associated with KCNJ2 mutations have been described clinically; however, little is known of the underlying arrhythmia mechanism. We create the first patient inspired KCNJ2 transgenic mouse and study effects of this mutation on cardiac function, IK1, and Ca2+ handling, to determine the underlying cellular arrhythmic pathogenesis. METHODS: A cardiac-specific KCNJ2-R67Q mouse was generated and bred for heterozygosity (R67Q+/-). Echocardiography was performed at rest, under anesthesia. In vivo ECG recording and whole heart optical mapping of intact hearts was performed before and after adrenergic stimulation in wild-type (WT) littermate controls and R67Q+/- mice. IK1 measurements, action potential characterization, and intracellular Ca2+ imaging from isolated ventricular myocytes at baseline and after adrenergic stimulation were performed in WT and R67Q+/- mice. RESULTS: R67Q+/- mice (n=17) showed normal cardiac function, structure, and baseline electrical activity compared with WT (n=10). Following epinephrine and caffeine, only the R67Q+/- mice had bidirectional ventricular tachycardia, ventricular tachycardia, frequent ventricular ectopy, and/or bigeminy and optical mapping demonstrated high prevalence of spontaneous and sustained ventricular arrhythmia. Both R67Q+/- (n=8) and WT myocytes (n=9) demonstrated typical n-shaped IK1IV relationship; however, following isoproterenol, max outward IK1 increased by ≈20% in WT but decreased by ≈24% in R67Q+/- (P<0.01). R67Q+/- myocytes (n=5) demonstrated prolonged action potential duration at 90% repolarization and after 10 nmol/L isoproterenol compared with WT (n=7; P<0.05). Ca2+ transient amplitude, 50% decay rate, and sarcoplasmic reticulum Ca2+ content were not different between WT (n=18) and R67Q+/- (n=16) myocytes. R67Q+/- myocytes (n=10) under adrenergic stimulation showed frequent spontaneous development of early afterdepolarizations that occurred at phase 3 of action potential repolarization. CONCLUSIONS: KCNJ2 mutation R67Q+/- causes adrenergic-dependent loss of IK1 during terminal repolarization and vulnerability to phase 3 early afterdepolarizations. This model clarifies a heretofore unknown arrhythmia mechanism and extends our understanding of treatment implications for patients with KCNJ2 mutation.

A comparative descriptive analysis of perceived quality of caring attitudes and behaviours between haemodialysis patients and their nurses.

Aim: Despite its importance in nursing, perceived quality of the nurse-patient relationship has seldom been researched. This study sought to examine and compare the quality of caring attitudes and behaviours as perceived by haemodialysis patients and their nurses. Design: This comparative descriptive study involved 140 haemodialysis patients and 101 nurses caring for them in ten haemodialysis units in the French-speaking part of Switzerland. Methods: Participants completed a sociodemographic questionnaire and the Caring Nurse-Patient Interaction Scale (CNPI-70). Results: Both nurses and patients reported a high frequency of caring attitudes and behaviours. Patients gave higher ratings than nurses did on all the caring dimensions, except spirituality. Implications are discussed.

Effects on nurses' quality of working life and on patients' quality of life of an educational intervention to strengthen humanistic practice among hemodialysis nurses in Switzerland: a protocol for a mixed-methods cluster randomized controlled trial.

BACKGROUND: Humanistic nursing practice constitutes the cornerstone of the nursing profession. However, according to some authors, such practice tends to fade over time in favour of non-humanistic behaviours. To contrast this tendency, an educational intervention (EI) based on Watson's Theory of Human Caring was developed and tested in two pilot studies involving, respectively, rehabilitation nurses in Quebec (Canada) and haemodialysis (HD) nurses in Switzerland. In light of the positive results obtained in these, another study is being undertaken to examine more in depth the EI's effects on both HD nurses and patients in French Switzerland. The EI is expected to have positive effects on quality of nurse-patient relationship (NPR), team cohesion, nurse quality of working life (QoWL), and patient quality of life (QoL). METHODS/DESIGN: The study described in this protocol will use a mixed-method cluster randomised controlled trial design. For the quantitative component, nurse and patient data will be collected through questionnaires. The accessible population of 135 nurses and 430 patients will be clustered into 10 HD units. These units will be randomised into an experimental group (EG) and a waiting-list control group (WLCG). Measurements will be taken at baseline (pre-intervention) and repeatedly over time (post-intervention): immediately at EI completion and six and 12 months thereafter. For the qualitative portion of the study, 18 semi-structured interviews will be conducted with EG nurses picked at random two months after EI completion to explore perceived changes in nurse humanistic practice. Qualitative data will be analysed through the relational caring inquiry method, a phenomenological approach. Descriptive and inferential statistics will be computed from the quantitative data. DISCUSSION: The study described in this protocol will determine if and how the proposed EI promotes humanistic nursing practice and how this practice affects quality of NPR, nurse QoWL, and patient QoL. Moreover, it will lay the groundwork for offering the EI to nurses in other healthcare sectors. TRIAL REGISTRATION: This clinical study was registered with ClinicalTrials.gov [NCT03283891, 14/09/2017].

Caveolin-3 Microdomain: Arrhythmia Implications for Potassium Inward Rectifier and Cardiac Sodium Channel.

In human cardiac ventricular myocytes, caveolin-3 functions as a scaffolding and regulatory protein for signaling molecules and compartmentalizes ion channels. Our lab has recently explored this sub-cellular microdomain and found that potassium inward rectifier Kir2.x is found in association with caveolin-3. The three cardiac Kir2.x isoforms (Kir2.1, Kir2.2, and Kir2.3) are the molecular correlates of IK1 in the heart, of which Kir2.1 is the dominant isoform in the ventricle. Kir2.1 channels assemble with Kir2.2 and Kir2.3 forming hetero-tetramers that modulate IK1. IK1 sets the resting membrane potential and assists with terminal phase 3 ventricular repolarization. In our studies using native human ventricular tissue, Kir2.x co-localizes with caveolin-3 and significance of the association between Kir2.x and caveolin-3 is emphasized in relation to mutations in the gene which encodes caveolin-3, CAV3, associated with Long QT Syndrome 9 (LQT9). LQT9-associated CAV3 mutations cause decreased current density in Kir2.1 and Kir2.2 as homomeric and heteromeric channels, which affects repolarization and membrane potential stability. A portion of Kir2.1 cardiac localization parallels that of the cardiac sodium channel (Nav1.5). This may have implications for Long QT9 in which CAV3 mutations cause an increase in the late current of Nav1.5 (INa-L) via nNOS mediated nitrosylation of Nav1.5. In iPS-CMs, expression of LQT9 CAV3 mutations resulted in action potential duration (APD) prolongation and early-after depolarizations (EADs), supporting the arrhythmogenicity of LQT9. To evaluate the combined effect of the CAV3 mutants on INa-L and IK1, we studied both ventricular and Purkinje myocyte mathematical modeling. Interestingly, mathematical ventricular myocytes, similar to iPS-CMs, demonstrated EADs but no sustained arrhythmia. In contrast, Purkinje modeling demonstrated delayed-after depolarizations (DADs) driven mechanism for sustained arrhythmia, dependent on the combined loss of IK1 and gain of INa-L. This finding changes the overall assumed arrhythmia phenotype for LQT9. In future studies, we are exploring caveolar micro-domain disruption in heart failure and how this effects Kir2.x and Nav1.5. Here we review the caveolae cardiac microdomain of Kir2.x and Nav1.5 and explore some of the downstream effects of caveolin-3 and caveolae disruption in specific clinical scenarios.

Pediatric Dilated Cardiomyopathy-Associated LRRC10 (Leucine-Rich Repeat-Containing 10) Variant Reveals LRRC10 as an Auxiliary Subunit of Cardiac L-Type Ca2+ Channels.

BACKGROUND: Genetic causes of dilated cardiomyopathy (DCM) are incompletely understood. LRRC10 (leucine-rich repeat-containing 10) is a cardiac-specific protein of unknown function. Heterozygous mutations in LRRC10 have been suggested to cause DCM, and deletion of Lrrc10 in mice results in DCM. METHODS AND RESULTS: Whole-exome sequencing was carried out on a patient who presented at 6 weeks of age with DCM and her unaffected parents, filtering for rare, deleterious, recessive, and de novo variants. Whole-exome sequencing followed by trio-based filtering identified a homozygous recessive variant in LRRC10, I195T. Coexpression of I195T LRRC10 with the L-type Ca2+ channel (Cav1.2, ß2CN2, and α2δ subunits) in HEK293 cells resulted in a significant ≈0.5-fold decrease in ICa,L at 0 mV, in contrast to the ≈1.4-fold increase in ICa,L by coexpression of LRRC10 (n=9-12, P<0.05). Coexpression of LRRC10 or I195T LRRC10 did not alter the surface membrane expression of Cav1.2. LRRC10 coexpression with Cav1.2 in the absence of auxiliary ß2CN2 and α2δ subunits revealed coassociation of Cav1.2 and LRRC10 and a hyperpolarizing shift in the voltage dependence of activation (n=6-9, P<0.05). Ventricular myocytes from Lrrc10-/- mice had significantly smaller ICa,L, and coimmunoprecipitation experiments confirmed association between LRRC10 and the Cav1.2 subunit in mouse hearts. CONCLUSIONS: Examination of a patient with DCM revealed homozygosity for a previously unreported LRRC10 variant: I195T. Wild-type and I195T LRRC10 function as cardiac-specific subunits of L-type Ca2+ channels and exert dramatically different effects on channel gating, providing a potential link to DCM.

Mosaicism in ATP1A3-related disorders: not just a theoretical risk.

Mutations in ATP1A3 are involved in a large spectrum of neurological disorders, including rapid onset dystonia parkinsonism (RDP), alternating hemiplegia of childhood (AHC), and cerebellar ataxia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS), with recent descriptions of overlapping phenotypes. In AHC, a few familial cases of autosomal dominant inheritance have been reported, along with cases of de novo sporadic mutations. In contrast, autosomal dominant inheritance has frequently been associated with RDP and CAPOS. Here, we report on two unrelated sets of full siblings with ATP1A3 mutations, (c.2116G>A) p. Gly706Arg in the first family, and (c.2266C>T) p. Arg756Cys in the second family, presenting with familial recurrence of the disease. Both families displayed parental germline mosaicism. In the first family, the brother and sister presented with severe intellectual deficiency, early onset pharmacoresistant epilepsy, ataxia, and autistic features. In the second family, both sisters demonstrated severe encephalopathy with ataxia and dystonia following a regression episode during a febrile episode during infancy. To our knowledge, mosaicism has not previously been reported in ATP1A3-related disorders. This report, therefore, provides evidence that germline mosaicism for ATP1A3 mutations is a likely explanation for familial recurrence and should be considered during recurrence risk counseling for families of children with ATP1A3-related disorders.

Palmitoylation of the Na/Ca exchanger cytoplasmic loop controls its inactivation and internalization during stress signaling.

The electrogenic Na/Ca exchanger (NCX) mediates bidirectional Ca movements that are highly sensitive to changes of Na gradients in many cells. NCX1 is implicated in the pathogenesis of heart failure and a number of cardiac arrhythmias. We measured NCX1 palmitoylation using resin-assisted capture, the subcellular location of yellow fluorescent protein-NCX1 fusion proteins, and NCX1 currents using whole-cell voltage clamping. Rat NCX1 is substantially palmitoylated in all tissues examined. Cysteine 739 in the NCX1 large intracellular loop is necessary and sufficient for NCX1 palmitoylation. Palmitoylation of NCX1 occurs in the Golgi and anchors the NCX1 large regulatory intracellular loop to membranes. Surprisingly, palmitoylation does not influence trafficking or localization of NCX1 to surface membranes, nor does it strongly affect the normal forward or reverse transport modes of NCX1. However, exchangers that cannot be palmitoylated do not inactivate normally (leading to substantial activity in conditions when wild-type exchangers are inactive) and do not promote cargo-dependent endocytosis that internalizes 50% of the cell surface following strong G-protein activation or large Ca transients. The palmitoylated cysteine in NCX1 is found in all vertebrate and some invertebrate NCX homologs. Thus, NCX palmitoylation ubiquitously modulates Ca homeostasis and membrane domain function in cells that express NCX proteins.

Substrate recognition by the cell surface palmitoyl transferase DHHC5.

The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a ∼ 120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.

A separate pool of cardiac phospholemman that does not regulate or associate with the sodium pump: multimers of phospholemman in ventricular muscle.

BACKGROUND: Phospholemman regulates the plasmalemmal sodium pump in excitable tissues. RESULTS: In cardiac muscle, a subpopulation of phospholemman with a unique phosphorylation signature associates with other phospholemman molecules but not with the pump. CONCLUSION: Phospholemman oligomers exist in cardiac muscle. SIGNIFICANCE: Much like phospholamban regulation of SERCA, phospholemman exists as both a sodium pump inhibiting monomer and an unassociated oligomer. Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the related ATPase SERCA, PLM is reported to oligomerize. We investigated subpopulations of PLM in adult rat ventricular myocytes based on phosphorylation status. Co-immunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at Ser(63) and one associated with the pump, both phosphorylated at Ser(68) and unphosphorylated. Phosphorylation of PLM at Ser(63) following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localized to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for Ser(63)-phosphorylated PLM, and cross-linking reagents also failed to identify any partner proteins for this pool. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, Ser(63)-phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, confirming the existence of PLM multimers. Dephosphorylation of the PLM multimer does not change sodium pump activity. Hence like phospholamban, PLM exists as a pump-inhibiting monomer and an unassociated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases rather than a direct effect of phosphorylation on PLM association with the pump.