Introduction:
Optical imaging has the potential to revolutionize cardiothoracic surgery by allowing for the real-time visualization of structures often inadvertently damaged due to inadequate visibility. The cardiac conduction system (CCS) consists of specialized cells embedded within the heart that are essential for cardiac function yet indistinguishable from heart muscle tissue. Intraoperative CCS injury is a major complication in cardiac surgery, representing a significant source of morbidity and mortality. To date, there exists no intraoperative method to visualize the CCS.
Hypothesis:
We hypothesized that unique, CCS-specific cell surface markers could be used for the
in vivo
labelling of the CCS.
Objectives:
Use single-cell RNA sequencing (scRNAseq) to discover cell surface markers that may serve as the basis for generating optical imaging agents for real-time CCS visualization.
Methods/Results:
Gene expression analysis of a comprehensive scRNAseq dataset of the entire murine CCS revealed significant enrichment of a host of CCS-specific cell surface genes. A subset of genes were subsequently validated in the CCS of mice and/or human tissue. In total, 7 novel cell surface markers were confirmed to have unique expression patterns throughout or within distinct components of the CCS. Next, optical imaging agents were created consisting of a near-infrared (NIR) dye conjugated to antibodies directed against two distinct CCS-specific cell surface markers. Each optical imaging agent demonstrated high sensitivity and specificity in labeling the entire CCS
in vivo
following a single intravenous injection in mice. Specificity was confirmed within intact, whole hearts using both closed-field NIR imaging and whole mount immunolabeling with volume imaging (iDISCO+). Dosage, timecourse and biodistribution analyses were performed as well as safety validation by surface ECG.
Conclusions:
In summary, we coupled scRNAseq with optical imaging to create novel tools for the real-time visualization of a complex tissue substructure. We provide a proof-of-principle for broadening the scope of optical imaging but also address a significant unmet clinical need, laying the foundation for translational opportunities in cardiac intervention and imaging.
Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay