Jakob Ogrodnik

SAP97 and Dystrophin Macromolecular Complexes Determine Two Pools of Cardiac Sodium Channels Nav1.5 in Cardiomyocytes

Rationale: The cardiac sodium channel Nav1.5 plays a key role in excitability and conduction. The 3 last residues of Nav1.5 (Ser-Ile-Val) constitute a PDZ-domain binding motif that interacts with the syntrophin–dystrophin complex. As dystrophin is absent at the intercalated discs, Nav1.5 could potentially interact with other, yet unknown, proteins at this site. Objective: The aim of this study was to determine whether Nav1.5 is part of distinct regulatory complexes at lateral membranes and intercalated discs. Methods and Results: Immunostaining experiments demonstrated that Nav1.5 localizes at lateral membranes of cardiomyocytes with dystrophin and syntrophin. Optical measurements on isolated dystrophin-deficient mdx hearts revealed significantly reduced conduction velocity, accompanied by strong reduction of Nav1.5 at lateral membranes of mdx cardiomyocytes. Pull-down experiments revealed that the MAGUK protein SAP97 also interacts with the SIV motif of Nav1.5, an interaction specific for SAP97 as no pull-down could be detected with other cardiac MAGUK proteins (PSD95 or ZO-1). Furthermore, immunostainings showed that Nav1.5 and SAP97 are both localized at intercalated discs. Silencing of SAP97 expression in HEK293 and rat cardiomyocytes resulted in reduced sodium current (INa) measured by patch-clamp. The INa generated by Nav1.5 channels lacking the SIV motif was also reduced. Finally, surface expression of Nav1.5 was decreased in silenced cells, as well as in cells transfected with SIV-truncated channels. Conclusions: These data support a model with at least 2 coexisting pools of Nav1.5 channels in cardiomyocytes: one targeted at lateral membranes by the syntrophin-dystrophin complex, and one at intercalated discs by SAP97.

PDZ Domain–Binding Motif Regulates Cardiomyocyte Compartment-Specific NaV1.5 Channel Expression and Function

Background— Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain–binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. Methods and Results— To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (&Dgr;SIV). &Dgr;SIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of &Dgr;SIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and &Dgr;SIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued &Dgr;SIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. Conclusions— Our results demonstrate the in vivo significance of the PDZ domain–binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Pathways of abnormal stress-induced Ca2+ influx into dystrophic mdx cardiomyocytes.

In Duchenne muscular dystrophy, deficiency of the cytoskeletal protein dystrophin leads to well-described defects in skeletal muscle, but also to dilated cardiomyopathy, accounting for about 20% of the mortality. Mechanisms leading to cardiomyocyte cell death and cardiomyopathy are not well understood. One hypothesis suggests that the lack of dystrophin leads to membrane instability during mechanical stress and to activation of Ca2+ entry pathways. Using cardiomyocytes isolated from dystrophic mdx mice we dissected the contribution of various putative Ca2+ influx pathways with pharmacological tools. Cytosolic Ca2+ and Na+ signals as well as uptake of membrane impermeant compounds were monitored with fluorescent indicators using confocal microscopy and photometry. Membrane stress was applied as moderate osmotic challenges while membrane current was quantified using the whole-cell patch-clamp technique. Our findings suggest a major contribution of two primary Ca2+ influx pathways, stretch-activated membrane channels and short-lived microruptures. Furthermore, we found evidence for a secondary Ca2+ influx pathway, the Na+-Ca2+ exchange (NCX), which in cardiac muscle has a large transport capacity. After stress it contributes to Ca2+ entry in exchange for Na+ which had previously entered via primary stress-induced pathways, representing a previously not recognized mechanism contributing to subsequent cellular damage. This complexity needs to be considered when targeting abnormal Ca2+ influx as a treatment option for dystrophy.

NO-dependent CaMKII activation during β-adrenergic stimulation of cardiac muscle.

AIMS During β-adrenergic receptor (β-AR) stimulation, phosphorylation of cardiomyocyte ryanodine receptors by protein kinases may contribute to an increased diastolic Ca(2+) spark frequency. Regardless of prompt activation of protein kinase A during β-AR stimulation, this appears to rely more on activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), by a not yet identified signalling pathway. The goal of the present study was to identify and characterize the mechanisms which lead to CaMKII activation and elevated Ca(2+) spark frequencies during β-AR stimulation in single cardiomyocytes in diastolic conditions. METHODS AND RESULTS Confocal imaging revealed that β-AR stimulation increases endogenous NO production in cardiomyocytes, resulting in NO-dependent activation of CaMKII and a subsequent increase in diastolic Ca(2+) spark frequency. These changes of spark frequency could be mimicked by exposure to the NO donor GSNO and were sensitive to the CaMKII inhibitors KN-93 and AIP. In vitro, CaMKII became nitrosated and its activity remained increased independent of Ca(2+) in the presence of GSNO, as assessed with biochemical assays. CONCLUSIONS β-AR stimulation of cardiomyocytes may activate CaMKII by a novel direct pathway involving NO, without requiring Ca(2+) transients. This crosstalk between two established signalling pathways may contribute to arrhythmogenic diastolic Ca(2+) release and Ca(2+) waves during adrenergic stress, particularly in combination with cardiac diseases. In addition, NO-dependent activation of CaMKII is likely to have repercussions in many cellular signalling systems and cell types.

Synthesis and Two-photon Photolysis of 6-(ortho-Nitroveratryl)-Caged IP3 in Living Cells

The synthesis of a photolabile derivative of inositol‐1,4,5‐trisphosphate (IP3) is described. This new caged second messenger (6‐ortho‐nitroveratryl)‐IP3 (6‐NV‐IP3) has an extinction coefficient of 5000 M−1 cm−1 at 350 nm, and a quantum yield of photolysis of 0.12. Therefore, 6‐NV‐IP3 is photolyzed with UV light about three times more efficiently than the widely used P4(5)‐1‐(2‐nitrophenyl)ethyl‐caged IP3 (NPE‐IP3). 6‐NV‐IP3 has a two‐photon cross‐section of about 0.035 GM at 730 nm. This absorbance is sufficiently large for effective two‐photon excitation in living cells at modest power levels. Using near‐IR light (5 mW, 710 nm, 80 MHz, pulse‐width 70 fs), we produced focal bursts of IP3 in HeLa cells, as revealed by laser‐scanning confocal imaging of intracellular Ca2+ concentrations. Therefore, 6‐NV‐IP3 can be used for efficient, subcellular photorelease of IP3, not only in cultured cells but also, potentially, in vivo. It is in the latter situation that two‐photon photolysis should reveal its true forte.

Regulation of the Cardiac Sodium Channel Nav1.5 by Utrophin in Dystrophin Deficient Mice

AIMS Duchenne muscular dystrophy (DMD) is a severe striated muscle disease due to the absence of dystrophin. Dystrophin deficiency results in dysfunctional sodium channels and conduction abnormalities in hearts of mdx mice. Disease progression in the mdx mouse only modestly reflects that of DMD patients, possibly due to utrophin up-regulation. Here, we investigated mice deficient in both dystrophin and utrophin [double knockout (DKO)] to assess the role of utrophin in the regulation of the cardiac sodium channel (Na(v)1.5) in mdx mice. METHODS AND RESULTS Co-immunoprecipitation studies in HEK293 cells showed that utrophin interacts with Na(v)1.5 via syntrophin proteins, an interaction abolished by deletion of the PDZ (PSD-95, Dlg, and Zona occludens) domain-binding motif of Na(v)1.5. We also provide evidence for such interaction in mouse heart using Na(v)1.5 C-terminus fusion proteins. In hearts of DKO mice, Na(v)1.5 protein levels were decreased by 25 ± 8%, together with a 42 ± 12% reduction of syntrophins compared with mdx, where utrophin was up-regulated by 52 ± 9% compared with C57BL/10 control mice. Sodium current was found to be reduced by 41 ± 5% in DKO cardiomyocytes compared with mdx, representing a loss of 63 ± 3% when compared with C57BL/10 wild-type control mice. Decreased Na(v)1.5 protein and current in DKO were reflected in a significant slowing of 27 ± 6% of maximal upstroke velocity of the cardiac action potential compared with mdx. CONCLUSION Utrophin plays a central role in the regulation of Na(v)1.5 in mdx mice. These findings provide support for therapeutic strategies aimed at overexpressing utrophin in the hopes of reducing cardiac pathology in DMD patients.

Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Na v1.5 expressed in HEK293 cells.

Background: The pore-forming subunit of the cardiac sodium channel, Na v1.5, has been previously found to be mutated in genetically determined arrhythmias. Na v1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Na v1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Na v1.5 can be generated. Methods: In this short study, we (1) compared the biophysical properties of the sodium current (I Na) generated by the mouse Na v1.5 (mNa v1.5) and human Na v1.5 (hNa v1.5) constructs that were expressed in HEK293 cells, and (2) investigated the possible alterations of the biophysical properties of the human Na v1.5 construct that was modified with specific epitopes. Results: The biophysical properties of mNa v1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNa v1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of I Na and slightly altered its biophysical properties. Adding green fluorescent protein (GFP) to the N-terminus did not modify any of the measured biophysical properties of hNa v1.5. Conclusions: These findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the gene encoding mNa v1.5.

Activation of Calcium Sparks in Resting Cardiomyocytes by β-Adrenergic Stimulation May Involve CaMKII and nNOS

It has been reported that during β-adrenergic stimulation of cardiac myocytes, phosphorylation of Ca2+ release channels (ryanodine receptors, RyRs) by PKA and/or CaMKII may result in arrhythmogenic diastolic Ca2+ leak (as elementary Ca2+ release events, Ca2+ sparks) from intracellular Ca2+ stores (the sarcoplasmic reticulum, SR). Using confocal Ca2+ imaging, we have recently shown that β-adrenergic stimulation by 1 µM isoproterenol (ISO) increases the Ca2+ spark frequency several-fold in quiescent, whole-cell voltage-clamped guinea-pig myocytes, without altering SR Ca2+ content. As this occurs without variations of the diastolic intracellular Ca2+ concentration, this observation suggests a sensitization of the RyRs. Experiments with protein kinase inhibitors (KN-93 and H89) indicated an involvement of CaMKII in the change of spark frequency. Surprisingly, but in line with the kinase inhibitor experiments, increasing cAMP production and PKA activity by direct stimulation of adenylate cyclase with forskolin (1 µM) did not significantly elevate Ca2+ spark frequencies under the same experimental conditions. Further experiments revealed that the change in sensitivity of the RyRs upon β-adrenergic stimulation may be linked to nitric oxide (NO), as pre-incubation of the cells with the NOS inhibitor L-NAME (500 µM) prevented the increase of the Ca2+ spark frequency without dramatic changes of SR Ca2+ content. Using the nNOS specific inhibitor AAAN (100 µM) resulted in analogous observations, suggesting that the nNOS isoform, located in close proximity of the RyRs, may be involved in this signaling pathway. Taken together, the results suggest the presence of a non-classical pathway linking β-adrenergic stimulation of cardiac myocytes to enhanced activity of the RyRs. Preliminary pharmacological evidence indicates that the pathway includes both, CaMKII and nNOS as important components. Supported by SNF.