Molecular tools for thermogenetic pacing of cardiomyocytes

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.