Abstract Body (Do not enter title and authors here): Introduction: Intracellular calcium handling plays a central role in cardiac contractility and relaxation. Genes such as the Ca²⁺-ATPase pump (SERCA2a) and the Dihydropteridine reductase (DHPR) enzyme are especially important in the cycling of intracellular calcium. As prior research has established a link between treatment with our cardiac targeting peptide (CTP: a 12-amino acid, cardiomyocyte-specific cell penetrating peptide) and upregulation of calcium-handling genes such as SERCA2a and DHPR, we hypothesized that treatment with our novel hybrid cardiac targeting peptide (hCTP: positions #3 and 11 replaced with the D-enantiomer form of the amino acids) will improve intracellular calcium handling in human cardiomyocytes (CMCs) by increasing expression of key calcium handling genes instrumental in sequestering calcium in diastole and lowering cytosolic levels.

Methods: To test hCTP’s calcium handling abilities, CMCs were plated on day 1 and allowed to settle for 24 hours, after which they received daily treatment with various concentrations of hCTP (1-25µM) for three days. On day 5, cells underwent either intracellular calcium measurements using the FLIPR 6 Calcium assay or were prepared for western blotting. Western blotting was performed to determine the protein concentrations of SERCA2a and DHPR, as compared to a GAPDH control. To test hCTP’s intrinsic calcium handling abilities, various concentrations of hCTP were mixed with a constant amount of elemental calcium dissolved in water. After 20 min at 37^oC, the concentration of free Ca²⁺ was measured with an ABL90 Flex Plus radiometer.

Results: In CMCs, treatment with hCTP led to a decrease in cytosolic calcium in a dose dependent fashion; concentrations as low as 1µM led to a significant decrease in cytosolic calcium (p< 0.0001 for all groups). Western blots performed on the treated CMCs revealed an increase in expression of both DHPR and SERCA2a, normalized to GAPDH control. Outside of the cellular environment, hCTP did not appear to display inherent calcium sequestring properties, suggesting that improvements in calcium handling are due to upregulation of gene expression.

Conclusion: hCTP appears to lower cytosolic calcium and improve calcium handling through upregulation of SERCA2a and DHPR. Our data indicates that hCTP is worth pursuing as a stand-alone, cardio-selective therapy for cardiomyopathies involving calcium mishandling.