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 (Nav1.5) in mdx mice. Methods and results Co-immunoprecipitation studies in HEK293 cells showed that utrophin interacts with Nav1.5 via syntrophin proteins, an interaction abolished by deletion of the PDZ (PSD-95, Dlg, and Zona occludens) domain-binding motif of Nav1.5. We also provide evidence for such interaction in mouse heart using Nav1.5 C-terminus fusion proteins. In hearts of DKO mice, Nav1.5 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 Nav1.5 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 Nav1.5 in mdx mice. These findings provide support for therapeutic strategies aimed at overexpressing utrophin in the hopes of reducing cardiac pathology in DMD patient

Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Nav1.5 expressed in HEK293 cells

Background: The pore-forming subunit of the cardiac sodium channel, Nav1.5, has been previously found to be mutated in genetically determined arrhythmias. Nav1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Nav1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Nav1.5 can be generated. Methods: In this short study, we (1) compared the biophysical properties of the sodium current (INa) generated by the mouse Nav1.5 (mNav1.5) and human Nav1.5 (hNav1.5) constructs that were expressed in HEK293 cells, and (2) investigated the possible alterations of the biophysical properties of the human Nav1.5 construct that was modified with specific epitopes. Results: The biophysical properties of mNav1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNav1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of INa 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 hNav1.5. 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 mNav1.5

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 Ca2+ spark frequency. Regardless of prompt activation of protein kinase A during β-AR stimulation, this appears to rely more on activation of Ca2+/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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ transients. This crosstalk between two established signalling pathways may contribute to arrhythmogenic diastolic Ca2+ release and Ca2+ 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 type

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

Increased Ca2+ leak and spatiotemporal coherence of Ca2+ release in cardiomyocytes during β-adrenergic stimulation

β-Adrenergic receptor (β-AR) stimulation of cardiac muscle has been proposed to enhance Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs). However, the anticipated increase in RyR Ca2+ sensitivity has proven difficult to study in intact cardiomyocytes, due to accompanying alterations in SR Ca2+ content, inward Ca2+ current (ICa) and diastolic cytosolic Ca2+ concentration ([Ca2+]i). Here, we studied whole-cell Ca2+ release and spontaneous Ca2+ leak (Ca2+ sparks) in guinea-pig ventricular myocytes with confocal Ca2+ imaging before and during β-AR stimulation by isoproterenol (Iso), but under otherwise nearly identical experimental conditions. The extent of SR Ca2+ loading was controlled under whole-cell voltage-clamp conditions. UV flash-induced uncaging of Ca2+ from DM-nitrophen was employed as an invariant trigger for whole-cell Ca2+ release. At matched SR Ca2+ content, we found that Iso enhanced the spatiotemporal coherence of whole-cell Ca2+ release, evident from spatially intercorrelated release and accelerated release kinetics that resulted in moderately (∼20%) increased release amplitude. This may arise from higher RyR Ca2+ sensitivity, and was also reflected in spontaneous SR Ca2+ leak. At comparable SR Ca2+ content and cytosolic [Ca2+]i, we observed a ∼4-fold increase in Ca2+ spark frequency in Iso that also appeared in quiescent cells within 2 min without increased SR Ca2+ content. This was likely to have been mediated by Ca2+/calmodulin-dependent protein kinase (CaMKII), rather than cAMP dependent protein kinase (PKA). We conclude that Iso increases the propensity of RyRs to open, both in response to rapid elevations of [Ca2+]i and at diastolic [Ca2+]i. While this could be beneficial in enhancing and synchronizing systolic whole-cell SR Ca2+ release, the same behaviour could also be proarrhythmogenic during diastole