SUMOylation of the cardiac sodium channel NaV1.5 modifies inward current and cardiac excitability

Background Decreased peak sodium current (INa) and increased late sodium current (INa,L), through the cardiac sodium channel NaV1.5 encoded by SCN5A, cause arrhythmias. Many NaV1.5 posttranslational modifications have been reported. A recent report concluded that acute hypoxia increases INa,L by increasing a small ubiquitin-like modifier (SUMOylation) at K442-NaV1.5. Objective The purpose of this study was to determine whether and by what mechanisms SUMOylation alters INa, INa,L, and cardiac electrophysiology. Methods SUMOylation of NaV1.5 was detected by immunoprecipitation and immunoblotting. INa was measured by patch clamp with/without SUMO1 overexpression in HEK293 cells expressing wild-type (WT) or K442R-NaV1.5 and in neonatal rat cardiac myocytes (NRCMs). SUMOylation effects were studied in vivo by electrocardiograms and ambulatory telemetry using Scn5a heterozygous knockout (SCN5A+/–) mice and the de-SUMOylating protein SENP2 (AAV9-SENP2), AAV9-SUMO1, or the SUMOylation inhibitor anacardic acid. NaV1.5 trafficking was detected by immunofluorescence. Results NaV1.5 was SUMOylated in HEK293 cells, NRCMs, and human heart tissue. HyperSUMOylation at NaV1.5-K442 increased INa in NRCMs and in HEK cells overexpressing WT but not K442R-Nav1.5. SUMOylation did not alter other channel properties including INa,L. AAV9-SENP2 or anacardic acid decreased INa, prolonged QRS duration, and produced heart block and arrhythmias in SCN5A+/– mice, whereas AAV9-SUMO1 increased INa and shortened QRS duration. SUMO1 overexpression enhanced membrane localization of NaV1.5. Conclusion SUMOylation of K442-Nav1.5 increases peak INa without changing INa,L, at least in part by altering membrane abundance. Our findings do not support SUMOylation as a mechanism for changes in INa,L. Nav1.5 SUMOylation may modify arrhythmic risk in disease states and represents a potential target for pharmacologic manipulation. Decreased peak sodium current (INa) and increased late sodium current (INa,L), through the cardiac sodium channel NaV1.5 encoded by SCN5A, cause arrhythmias. Many NaV1.5 posttranslational modifications have been reported. A recent report concluded that acute hypoxia increases INa,L by increasing a small ubiquitin-like modifier (SUMOylation) at K442-NaV1.5. The purpose of this study was to determine whether and by what mechanisms SUMOylation alters INa, INa,L, and cardiac electrophysiology. SUMOylation of NaV1.5 was detected by immunoprecipitation and immunoblotting. INa was measured by patch clamp with/without SUMO1 overexpression in HEK293 cells expressing wild-type (WT) or K442R-NaV1.5 and in neonatal rat cardiac myocytes (NRCMs). SUMOylation effects were studied in vivo by electrocardiograms and ambulatory telemetry using Scn5a heterozygous knockout (SCN5A+/–) mice and the de-SUMOylating protein SENP2 (AAV9-SENP2), AAV9-SUMO1, or the SUMOylation inhibitor anacardic acid. NaV1.5 trafficking was detected by immunofluorescence. NaV1.5 was SUMOylated in HEK293 cells, NRCMs, and human heart tissue. HyperSUMOylation at NaV1.5-K442 increased INa in NRCMs and in HEK cells overexpressing WT but not K442R-Nav1.5. SUMOylation did not alter other channel properties including INa,L. AAV9-SENP2 or anacardic acid decreased INa, prolonged QRS duration, and produced heart block and arrhythmias in SCN5A+/– mice, whereas AAV9-SUMO1 increased INa and shortened QRS duration. SUMO1 overexpression enhanced membrane localization of NaV1.5. SUMOylation of K442-Nav1.5 increases peak INa without changing INa,L, at least in part by altering membrane abundance. Our findings do not support SUMOylation as a mechanism for changes in INa,L. Nav1.5 SUMOylation may modify arrhythmic risk in disease states and represents a potential target for pharmacologic manipulation.