Cardiomyocyte Targeting Peptide to Deliver Amiodarone

Abstract

Background: Amiodarone 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.

Methods: CTP 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 Tyrode's solution and blebbistatin (5μM) to minimize contractions. Voltage (RH237) and Ca ²⁺ -indicator dye (Rhod-2/AM) were injected, fluorescence from the epicardium split and focused on two cameras capturing at 570-595nm for cytosolic Ca ²⁺ 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 Ca ²⁺ transient durations at 90% recovery (CaTD ₉₀ ). mRNA was extracted from all hearts and RNA sequencing performed with results compared to control hearts.

Results: CTP-amiodarone remained stable for up to 21 days at 37°C. At ∼1/15 ^th 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 CaTD ₉₀ 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-α, IL-1β, and IL-6.

Conclusions: Our data suggests that CTP can deliver amiodarone to cardiomyocytes at ∼1/15 ^th the total molar dose of amiodarone needed to produce comparable slowing of CVs. The ability of CTP to decrease AP durations and CaTD ₉₀ may be related to its increase in expression of Ca-handling genes, and merits further study.

Figure 1:

Schematic presentation of the CTP-amiodarone chemical structure. CTP is conjugated to amiodarone via a disulfide linker (circled in red).

Figure 2:

HPLC Tracing of the CTP-amio conjugate immediately upon synthesis and purification (a), after 72 hours of incubation (b), and after 22 days of incubation (c) at 37°C.

Figure 3:

CTP-Cy5.5 transduces rat cardiomyocytes in a cardiomyocyte-specific manner. Myofibroblasts are stained green with WGA-488 and show no co-localization with the CTP-Cy5.5 (red) that is taken up exclusively by cardiomyocytes. Nuclei stained blue (DAPI).

Figure 4:

Comparison of heart rate (a), voltage conduction velocities (b), action potential duration (c), calcium conduction velocities (d) and CaT (e) between various treatment groups. All p-values are two-tailed, unpaired t-tests between each treatment group and controls.

Figure 5:

Principal component analysis showing reasonable clustering within groups and separation between different treatment groups.

Figure 6:

Differentially expressed genes in CTP treated guinea pig hearts compared with control hearts. β2-adrenergic receptor is significantly down-regulated, whereas α-adrenergic, receptor and calcium handling genes (DHPR and SERCA2a) are significantly upregulated (all circled in black).

Figure 7:

Differentially expressed genes (DEGs) in CTP treated guinea pig hearts compared with control hearts. Pro-inflammatory genes in the NF-κB pathway (IκBα), as well as TNF-α, IL-1β, and COX2 are significantly down-regulated (all circled in black).