Study On Temperature Response Of Voltage-gated Sodium Channels In Cardiomyocytes Of Perccottus Glenii

Animal temperature adaptation is a hot topic in ecological research,and this adaptation process may be related to many factors.In the field of fish temperature adaptation,the Oxygen-and capacity-limited thermal tolerance(OCLTT)hypothesis is a widely used mechanism framework at present,which holds that the circulatory respiratory capacity of fishes is a key factor affecting the temperature tolerance range of fishes.Among them,the cardiac capacity is an important link that determines the circulatory and respiratory capacity of fish.As a very important protein to the cardiac conduction system,the voltage-gated sodium channel(VGSC)expressed on the plasma membrane of myocardial cells may play an important role in the temperature adaptation of fishes.In this study,Perccottus glenii,an important economic fish in northeast China,was selected as the main object of temperature adaptation.According to the characteristics of tolerance to hypoxia and low temperature of this fish,the temperature response of its VGSC channel,especially Na _V1.5 channel,was preliminally studied.In this study,Symphysodon aequifasciatus was selected as the control fish adapted to the tropical environment.The temperature response of VGSC on myocardial cells of Perccottus glenii and Symphysodon aequifasciatus was studied.The primary cell acute isolation,patch-clamp electrophysiological platform,and temperature perfusion control system were used.The trend of myocardial complex sodium current in response to temperature and the relationship between the trend and the range of temperature tolerated were determined.In this study,it was found that the opening probability of VGSC of the above two fish species was high within the temperature range of their habitat,and the opening probability of VGSC decreased when the temperature range was below or beyond.These results indicate that the VGSC of Perccottus glenii and Symphysodon aequifasciatus have been specialized according to habitat temperature,and the VGSC on cardiomyocytes plays an important role in the temperature adaptation of the heart.After determining the temperature response trend of VGSC,we continued to study Na _V1.5,the subtype of VGSC mainly expressed in the plasma membrane of myocardial cells.First,three generations of transcriptome sequencing data,c DNA library,and PCR were used to obtain the gene sequence of Na _V1.5 and construct the eukaryotic expression vector.The amino acid sequence of the channel Na _V1.5 was translated and bioinformatic analysis was performed to confirm that the channel has typical VGSC characteristics.Finally,amino acid sequences of human Na _V1.5 and Perccottus glenii Na _V1.5 were compared,and 28 point mutation sites were identified by hydrophilic analysis.After successful point mutation,the function of the mutant was tested by cell transfection,patch clamp electrophysiological platform,and temperature irrigation control system.These results indicate that the human G1724L mutation is different from the wild type in its response to temperature,and the G1724L mutation corresponds to L1663 of Perccottus glenii,which may be an important site for temperature adaptation.It has been shown that VGSC in the myocardial cytoplasmic membrane is an important ion channel affecting cardiac temperature adaptation.Na _V1.5 is mainly expressed on myocardial cells.We further found that the L1663 site of Perccottus glenii Na _V1.5 may play a key role in low-temperature adaptation.