Molecular tools for thermogenetic pacing of cardiomyocytes

We have engineered a suite of molecular tools for thermogenetic cardiac pacing. These tools are encoded within AAV (adeno-associated virus) transfer plasmids, each carrying distinct genetic constructs.The first construct, AAV cTnT hTRPV1 3xFLAG tag, comprises a constitutive hybrid promoter (CMV immediate-early enhancer fused to the cardiac troponin T (cTnT) promoter), a gene encoding the human transient receptor potential subfamily V member 1 (TRPV1), and a 3xFLAG tag for protein detection.The second construct, AAV cTnT hTRPV1 (–109 amino acids residues) P2A mRuby2, carries a truncated version of the TRPV1 gene and a red fluorescent protein mRuby2. The P2A sequence ensures bicistronic expression, allowing for simultaneous visualization of the truncated TRPV1 channel via fluorescence microscopy.The third construct, cTnT GCaMP6s, encodes a fluorescent Ca2+ sensor (GCaMP6s). This sensor fluoresces upon binding to Ca2+, thereby indicating the activation of the TRPV1 channel.These constructs provide a robust platform for the spatiotemporal control and visualization of thermogenetic cardiac pacing at the molecular level. They allow for the precise manipulation of cardiac activity, offering significant potential for advancements in cardiac research and therapeutics. We have engineered a suite of molecular tools for thermogenetic cardiac pacing. These tools are encoded within AAV (adeno-associated virus) transfer plasmids, each carrying distinct genetic constructs.The first construct, AAV cTnT hTRPV1 3xFLAG tag, comprises a constitutive hybrid promoter (CMV immediate-early enhancer fused to the cardiac troponin T (cTnT) promoter), a gene encoding the human transient receptor potential subfamily V member 1 (TRPV1), and a 3xFLAG tag for protein detection.The second construct, AAV cTnT hTRPV1 (–109 amino acids residues) P2A mRuby2, carries a truncated version of the TRPV1 gene and a red fluorescent protein mRuby2. The P2A sequence ensures bicistronic expression, allowing for simultaneous visualization of the truncated TRPV1 channel via fluorescence microscopy.The third construct, cTnT GCaMP6s, encodes a fluorescent Ca2+ sensor (GCaMP6s). This sensor fluoresces upon binding to Ca2+, thereby indicating the activation of the TRPV1 channel.These constructs provide a robust platform for the spatiotemporal control and visualization of thermogenetic cardiac pacing at the molecular level. They allow for the precise manipulation of cardiac activity, offering significant poten