Molecular And Functional Changes In Voltage-gated Na~+ Channels In Cardiomyocytes During Mouse Embryogenesis

Background:Cardiac arrhythmia is common in the clinical practice, and its mechanism is complex. Fundamentally, the abnormalities of expression and function of the membrane ion channels can contribute to the development of cardiac arrhythmias.Na+ channel plays a key role in the generation and conduction of action potential in the heart. It has been showed that altered Na+ channel gating might underlie multiple cardiac diseases, such as long QT syndrome (LQTS), the Brugada syndrome and some inheritable cardiac conduction disorders. Na+ channel gating is altered in acquired diseases such as cardiac ischemia and heart failure. In addition, some signaling pathways that regulate Na+ channel function are altered in the diseased heart, such as cAMP-dependent protein kinase A (PKA).An experimental strategy to improve cardiac function in failing hearts is cell transplant. Some investigator has attempted to engraft fetal cardiomyocytes, and bone marrow stem cell. However, progress in this approach is hampered by lack of knowledge about normal differentiation of cardiomyocytes especially at embryonic stage. Thus, it would be of great interest to determine the developmental changes in gating properties of Na+ channel from a pathophysiological and therapeutic standpoint.Objective:To study the developmental changes in gating properties of Na+ channel during embryogenesis and to determine the changes in Na+ channel subunits expression that might be associated with functional changes.Methods:Single ventricular myocytes from embryos of early developmental stage (10.5 days postcoitum) and late developmental stage (17.5 days postcoitum) were obtained by enzymatic dissociation method and kept in the incubator for 24-48 hours until use. Whole-cell voltage-clamp technique was used to record Na+ currents in ventricular myocytes of early (EDS) and late (LDS) developmental stages in embryonic mice。Additionally, RT-PCR was performed to determine the transcripts of six Na+ channel a subunits (Nav1.1-Nav1.6) and threeβsubunits (Navβ1-Navβ3)。Results:Na+ current could be recorded in the isolated ventricular myocytes of both developmental stages, which indicated these myocytes had satisfactory electrophysiological properties.1. Developmental changes in peak INa current density:Peak Na+ current density was significantly larger in LDS cells (-60 to +20 mV) than in EDS cells (P<0.01). Na+ current density at -30 mV increased significantly from -88.1±8.0 pA/pF (n=8) in EDS to -287.9±9.7 pA/pF (n=11,P<0.01) in LDS.2. Developmental changes in activation properties of Na+ channels:The voltage dependence of activation in both cell types were similar. There was no significant difference in the voltage of half activation (Va) and slope factor (k) between EDS and LDS myocytes (Va:-46.1±3.4 mV EDS vs.-47.0±3.4 mV LDS, P>0.05; k:5.5±0.4 mV EDS vs.5.8±0.7 mV LDS, P>0.05). In addition, the time-to-peaks in both cell types were similar at potentials over -60 to 0 mV.3. Developmental changes in inactivation properties of Na+ channels:The voltage of half inactivation (V;) was shifted to more negative potentials in LDS than EDS cells (Vi:-82.7±4.4 mV LDS vs.-71.6±2.4 mV EDS, P<0.01). K values for EDS and LDS cells were not significantly different (k:7.2±0.7 mV EDS vs.6.5±0.9 mV LDS, P>0.05).In addition, the time course of inactivation at a test potential of-30 mV was well described by a bi-exponential fit in both cell types, containing a large fast component and a small slow component. Both the time constants of Na+ channel inactivation in both cell types were similar. But LDS myocytes had significantly larger amplitude of fast (Af) inactivation component and smaller amplitude of slow (As) inactivation component than EDS myocytes.4. Developmental changes in recovery properties of Na+ channels:Similarly, the time course of recovery from inactivation in both cell types also included a large fast component and a small slow component. The fast (if) and slow (τs) time constants for Na+ channel recovery were significantly smaller in LDS (τf:5.5±0.4 ms LDS vs. 7.5±0.5 ms EDS, P<0.01;τs:122.5±4.5 ms LDS vs.198.6±12.3 ms EDS, P<0.01) than in EDS cells.5. Expression of Na+ channel a subunits andβsubunits in embryonic cardiomyocytes:Transcripts of Navl.1, Nav1.2 and Nav1.3 were absent or present at very low levels in embryonic hearts. The amount of Nav1.4, Nav1.5 and Nav1.6 mRNA were increased with age during embryogenesis. Additionally, three Na+ channel 3 subunits (Navβ1-Navβ3) were upregulated during embryogenesis.6. Sensitivity of total Na+ currents to TTX in EDS and LDS cardiocytes:To determine the sensitivity of total Na+ current in both embryonic cardiomyocytes types, we analyzed the relationship between the dose of TTX and the blocking effect of TTX on total INa·Total Na+ currents were decreased in a dose-dependent manner by TTX in both myocyte types. Fitting the dose-response relationship with Hill equation yielded an IC50 of 5.2μM (EDS) and 6.6μM (LDS).Conclusions:These results suggest significantly functional changes in Na+ channels occur in cardiomyocytes of mouse embryo and that different Na+ channel subunits genes are strongly regulated during embryogenesis, and further support a physiological role for voltage-gated Na+ channels during heart development.