Gabris-Weber, B. A. | bioRxiv Science

bioRxiv2023

Cardiomyocyte Targeting Peptide to Deliver Amiodarone

BackgroundAmiodarone is underutilized due to significant off-target toxicities. We hypothesized that targeted delivery to the heart would lead to lowering of dose by utilizing a cardiomyocyte targeting peptide (CTP), a cell penetrating peptide identified by our prior phage display work. MethodsCTP was synthesized thiolated at the N-terminus, conjugated to amiodarone via Schiff base chemistry, HPLC purified and confirmed with MALDI/TOF. Stability of the conjugate was assessed using serial HPLCs. Guinea pigs (GP) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80mg/Kg), CTP-amiodarone (5 days;26.3mg/Kg), or CTP (5 days; 17.8mg/Kg), after which GPs were euthanized, hearts excised, perfused on a Langendorff apparatus with Tyrodes solution and blebbistatin (5M) to minimize contractions. Voltage (RH237) and Ca2+-indicator dye (Rhod-2/AM) were injected, fluorescence from the epicardium split and focused on two cameras capturing at 570-595nm for cytosolic Ca2+ and 610-750nm wavelengths for voltage. Subsequently, hearts were paced at 250ms with programmed stimulation to measure changes in conduction velocities (CV), action potential duration (APD) and Ca2+ transient durations at 90% recovery (CaTD90). mRNA was extracted from all hearts and RNA sequencing performed with results compared to control hearts. ResultsCTP-amiodarone remained stable for up to 21 days at 37{degrees}C. At [~]1/15th of the dose of amiodarone, CTP-amiodarone decreased CV in hearts significantly compared to control GPs (0.92{+/-}0.05 vs. 1.00{+/-}0.03m/s, p=0.0007), equivalent to amiodarone alone (0.87{+/-}0.08ms, p=0.0003). Amiodarone increased APD (192{+/-}5ms vs. 175{+/-}8ms for vehicle, p=0.0025), while CTP-amiodarone decreased it significantly (157{+/-}16ms, p=0.0136) similar to CTP alone (155{+/-}13ms, p=0.0039). Both amiodarone and CTP-amiodarone significantly decreased calcium transients compared to controls. CTP-amiodarone and CTP decreased CaTD90 to an extent greater than amiodarone alone (p<0.001). RNA-seq showed that CTP alone increased the expression of DHPR and SERCA2a, while decreasing expression of proinflammatory genes NF-kappa B, TNF-, IL-1{beta}, and IL-6. ConclusionsOur data suggests that CTP can deliver amiodarone to cardiomyocytes at [~]1/15th the total molar dose of amiodarone needed to produce comparable slowing of CVs. The ability of CTP to decrease AP durations and CaTD90 may be related to its increase in expression of Ca-handling genes, and merits further study.

molecular biology↗

bioRxiv2018

Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we developed a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We anatomically and functionally identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.

neuroscience↗