Protein phosphatase 1c associated with the cardiac sodium calcium exchanger1 regulates its activity by dephosphorylating serine 68 phosphorylated phospholemman

The sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ 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 Ca2+ 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

Lumican accumulates with fibrillar collagen in fibrosis in hypertrophic cardiomyopathy

Aims Familial hypertrophic cardiomyopathy (HCM) is the most common form of inherited cardiac disease. It is characterized by myocardial hypertrophy and diastolic dysfunction, and can lead to severe heart failure, arrhythmias, and sudden cardiac death. Cardiac fibrosis, defined by excessive accumulation of extracellular matrix (ECM) components, is central to the pathophysiology of HCM. The ECM proteoglycan lumican is increased during heart failure and cardiac fibrosis, including HCM, yet its role in HCM remains unknown. We provide an in-depth assessment of lumican in clinical and experimental HCM. Methods Left ventricular (LV) myectomy specimens were collected from patients with hypertrophic obstructive cardiomyopathy (n = 15), and controls from hearts deemed unsuitable for transplantation (n = 8). Hearts were harvested from a mouse model of HCM; Myh6 R403Q mice administered cyclosporine A and wild-type littermates (n = 8–10). LV tissues were analysed for mRNA and protein expression. Patient myectomy or mouse mid-ventricular sections were imaged using confocal microscopy, direct stochastic optical reconstruction microscopy (dSTORM), or electron microscopy. Human foetal cardiac fibroblasts (hfCFBs) were treated with recombinant human lumican (n = 3) and examined using confocal microscopy. Results Lumican mRNA was increased threefold in HCM patients (P 2 = 0.60, P 2 = 0.58, P < 0.01). Lumican protein was increased by 40% in patients with HCM (P 2 = 0.28, P = 0.05) and interstitial (R2 = 0.30, P < 0.05) fibrosis. In mice with HCM, lumican mRNA increased fourfold (P < 0.001), and lumican protein increased 20-fold (P < 0.001) in insoluble ECM lysates. Lumican and fibrillar collagen were located together throughout fibrotic areas in HCM patient tissue, with increased co-localization measured in patients and mice with HCM (patients: +19%, P < 0.01; mice: +13%, P < 0.01). dSTORM super-resolution microscopy was utilized to image interstitial ECM which had yet to undergo overt fibrotic remodelling. In these interstitial areas, collagen I deposits located closer to ( 15 nm, P < 0.05), overlapped more frequently with (+7.3%, P < 0.05) and to a larger degree with (+5.6%, P < 0.05) lumican in HCM. Collagen fibrils in such deposits were visualized using electron microscopy. The effect of lumican on collagen fibre formation was demonstrated by adding lumican to hfCFB cultures, resulting in thicker (+53.8 nm, P < 0.001), longer (+345.9 nm, P < 0.001), and fewer ( 8.9%, P < 0.001) collagen fibres. Conclusions The ECM proteoglycan lumican is increased in HCM and co-localizes with fibrillar collagen throughout areas of fibrosis in HCM. Our data suggest that lumican may promote formation of thicker collagen fibres in HCM

Regulation of cardiomyocyte T-tubular structure: opportunities for therapy

Purpose of Review Membrane invaginations called t-tubules play an integral role in triggering cardiomyocyte contraction, and their disruption during diseases such as heart failure critically impairs cardiac performance. In this review, we outline the growing understanding of the malleability of t-tubule structure and function, and highlight emerging t-tubule regulators which may be exploited for novel therapies. Recent Findings New technologies are revealing the nanometer scale organization of t-tubules, and their functional junctions with the sarcoplasmic reticulum called dyads, which generate Ca2+ sparks. Recent data have indicated that the dyadic anchoring protein junctophilin-2, and the membrane-bending protein BIN1 are key regulators of dyadic formation and maintenance. While the underlying signals which control expression and localization of these proteins remain unclear, accumulating data support an important role of myocardial workload. Summary Although t-tubule alterations are believed to be a key cause of heart failure, the plasticity of these structures also creates an opportunity for therapy. Promising recent data suggest that such therapies may specifically target junctophilin-2, BIN1, and/or mechanotransduction

Protocol for the isolation and super-resolution dSTORM imaging of RyR2 in Cardiac myocytes

Heart failure is associated with low cardiac output (CO) and low brain perfusion that imposesa significant risk for accelerated brain ageing and Alzheimer’s disease (AD) development. Al-though clinical heart failure can emerge several years following acute myocardial infarction(AMI), the impact of AMI on cerebral blood flow (CBF) at early stages and up to 30 daysfollowing MI is unknown. Sixteen months old male mice underwent left anterior descending(LAD) coronary artery ligation. Hemodynamics analyses were performed at baseline and atdays 1, 7, and 30 post-MI. Left ventricular (LV) ejection fraction (EF), LV volumes, CO, andright common carotid artery (RCCA) diameter were recorded by echocardiography. RCCAflow (RCCA FL) was measured by Doppler echocardiography. LV volumes consistently in-creased (P<0.0012) and LV systolic function progressively deteriorated (P<0.0001) post-MI.CO and RCCA FL showed a moderate but significant decrease over the course of MI withsimilar fluctuation pattern such that both variables were decreased at day 1, increased atday 7, and decreased at 30 days post-MI. Correlation and regression analyses between COand RCCA FL showed a strong correlation with significance at baseline and day 30 post-MI(R=0.71,P=0.03, andR=0.72,P=0.03, respectively). Days 1 and 7 analyses betweenCO and RCCA FL showed moderate correlation with non-significance post-MI (R=0.51,P=0.2, andR=0.56,P=0.12, respectively). In summary, CBF significantly decreased fol-lowing AMI and remained significantly decreased for up to 30 days, suggesting a potentialrisk for brain damage that could contribute to cognitive dysfunction later in life

The physiology and pathophysiology of T-tubules in the heart

In cardiomyocytes, invaginations of the sarcolemmal membrane called t-tubules are critically important for triggering contraction by excitation-contraction (EC) coupling. These structures form functional junctions with the sarcoplasmic reticulum (SR), and thereby enable close contact between L-type Ca 2+ channels (LTCCs) and Ryanodine Receptors (RyRs). This arrangement in turn ensures efficient triggering of Ca 2+ release, and contraction. While new data indicate that t-tubules are capable of exhibiting compensatory remodeling, they are also widely reported to be structurally and functionally compromised during disease, resulting in disrupted Ca 2+ homeostasis, impaired systolic and/or diastolic function, and arrhythmogenesis. This review summarizes these findings, while highlighting an emerging appreciation of the distinct roles of t-tubules in the pathophysiology of heart failure with reduced and preserved ejection fraction (HFrEF and HFpEF). In this context, we review current understanding of the processes underlying t-tubule growth, maintenance, and degradation, underscoring the involvement of a variety of regulatory proteins, including junctophilin-2 (JPH2), amphiphysin-2 (BIN1), caveolin-3 (Cav3), and newer candidate proteins. Upstream regulation of t-tubule structure/function by cardiac workload and specifically ventricular wall stress is also discussed, alongside perspectives for novel strategies which may therapeutically target these mechanisms

Electrolyte imbalances in an unselected population in an emergency department: A retrospective cohort study

Background: Although electrolyte imbalances (EIs) are common in the emergency department (ED), few studies have examined the occurrence of such conditions in an unselected population. Objectives: To investigate the frequency of EI among adult patients who present to the ED, with regards to type and severity, and the association with age and sex of the patient, hospital length of stay (LOS), readmission, and mortality. Methods: A retrospective cohort study. All patients ≥18 years referred for any reason to the ED between January 1, 2010, and December 31, 2015, who had measured blood electrolytes were included. In total, 62 991 visits involving 31 966 patients were registered. Results: EIs were mostly mild, and the most common EI was hyponatremia (glucose-corrected) (24.6%). Patients with increasing severity of EI had longer LOS compared with patients with normal electrolyte measurements. Among all admitted patients, there were 12928 (20.5%) readmissions within 30 days from discharge during the study period. Hyponatremia (glucose-corrected) was associated with readmission, with an adjusted odds ratio (OR) of 1.25 (95% CI, 1.18–1.32). Hypomagnesemia and hypocalcemia (albumin-corrected) were also associated with readmission, with ORs of 1.25 (95% CI, 1.07–1.45) and 1.22 (95% CI, 1.02–1.46), respectively. Dysnatremia, dyskalemia, hypercalcemia, hypermagnesemia, and hyperphosphatemia were associated with increased in-hospital mortality, whereas all EIs except hypophosphatemia were associated with increased 30-day and 1-year mortality. Conclusions: EIs were common and increasing severity of EIs was associated with longer LOS and increased in-hospital, 30-days and 1-year mortality. EI monitoring is crucial for newly admitted patients, and up-to-date training in EI diagnosis and treatment is essential for ED physicians

Electrolyte imbalances in an unselected population in an emergency department: A retrospective cohort study

Background Although electrolyte imbalances (EIs) are common in the emergency department (ED), few studies have examined the occurrence of such conditions in an unselected population. Objectives To investigate the frequency of EI among adult patients who present to the ED, with regards to type and severity, and the association with age and sex of the patient, hospital length of stay (LOS), readmission, and mortality. Methods A retrospective cohort study. All patients ≥18 years referred for any reason to the ED between January 1, 2010, and December 31, 2015, who had measured blood electrolytes were included. In total, 62 991 visits involving 31 966 patients were registered. Results EIs were mostly mild, and the most common EI was hyponatremia (glucose-corrected) (24.6%). Patients with increasing severity of EI had longer LOS compared with patients with normal electrolyte measurements. Among all admitted patients, there were 12928 (20.5%) readmissions within 30 days from discharge during the study period. Hyponatremia (glucose-corrected) was associated with readmission, with an adjusted odds ratio (OR) of 1.25 (95% CI, 1.18–1.32). Hypomagnesemia and hypocalcemia (albumin-corrected) were also associated with readmission, with ORs of 1.25 (95% CI, 1.07–1.45) and 1.22 (95% CI, 1.02–1.46), respectively. Dysnatremia, dyskalemia, hypercalcemia, hypermagnesemia, and hyperphosphatemia were associated with increased in-hospital mortality, whereas all EIs except hypophosphatemia were associated with increased 30-day and 1-year mortality. Conclusions EIs were common and increasing severity of EIs was associated with longer LOS and increased in-hospital, 30-days and 1-year mortality. EI monitoring is crucial for newly admitted patients, and up-to-date training in EI diagnosis and treatment is essential for ED physicians

Energy-efficiency of Cardiomyocyte Stimulation with Rectangular Pulses

In cardiac pacemaker design, energy expenditure is an important issue. This work aims to explore whether varying stimulation pulse configuration is a viable optimization strategy for reducing energy consumption by the pacemaker. A single cardiomyocyte was used as an experimental model. Each cardiomyocyte was stimulated with different stimulation protocols using rectangular waveforms applied in varying number, in short succession. The amplitude, the width of each pulse, and the interval between consecutive pulses were modified. The application of multiple pulses in a short sequence led to a reduction of the threshold voltage required for stimulation when compared to a single pulse. However, none of the employed multi-pulse sequences reduced the overall energy expenditure of cell stimulation when compared to a single pulse stimulation. Among multiple pulse protocols, a combination of two short pulses (1 ms) separated with a short interval (0.5 ms) had the same energy requirements as a single short pulse (1 ms), but required the application of significantly less voltage. While increasing the number of consecutive pulses does not reduce the energy requirements of the pacemaker, the reduction in threshold voltage can be considered in practice if lower stimulation voltages are desired

Energy-efficiency of Cardiomyocyte Stimulation with Rectangular Pulses

In cardiac pacemaker design, energy expenditure is an important issue. This work aims to explore whether varying stimulation pulse configuration is a viable optimization strategy for reducing energy consumption by the pacemaker. A single cardiomyocyte was used as an experimental model. Each cardiomyocyte was stimulated with different stimulation protocols using rectangular waveforms applied in varying number, in short succession. The amplitude, the width of each pulse, and the interval between consecutive pulses were modified. The application of multiple pulses in a short sequence led to a reduction of the threshold voltage required for stimulation when compared to a single pulse. However, none of the employed multi-pulse sequences reduced the overall energy expenditure of cell stimulation when compared to a single pulse stimulation. Among multiple pulse protocols, a combination of two short pulses (1 ms) separated with a short interval (0.5 ms) had the same energy requirements as a single short pulse (1 ms), but required the application of significantly less voltage. While increasing the number of consecutive pulses does not reduce the energy requirements of the pacemaker, the reduction in threshold voltage can be considered in practice if lower stimulation voltages are desired