Intracellular delivery of a phospholamban-targeting aptamer using cardiomyocyte-internalizing aptamers

The sarco (endo)plasmic reticulum Ca²⁺-ATPase 2a (SERCA2a)-phospholamban (PLN) system within the sarcoplasmic reticulum is crucial for regulating intracellular Ca²⁺ cycling in ventricular cardiomyocytes. Given that impaired Ca²⁺ cycling is associated with heart failure, modulating SERCA2a activity represents a promising therapeutic strategy. Previously, we engineered an RNA aptamer (Apt30) that binds to PLN, thereby activating SERCA2a by alleviating PLN's inhibitory effect. However, Apt30 alone cannot reach intracellular PLN, necessitating the development of a mechanism for its specific internalization into cardiomyocytes. Using the systematic evolution of ligands by exponential enrichment (SELEX) method, we isolated RNA aptamers capable of internalizing into cardiomyocytes. These aptamers demonstrated sub-micromolar EC₅₀ values for cardiomyocyte internalization and exhibited significantly reduced activity against various non-myocardial cells, highlighting their specificity for cardiomyocytes. Moreover, some of these cardiomyocyte-internalizing aptamers could be linked to Apt30 as a single RNA strand without compromising their internalization efficacy. Supplementing the culture medium with these hybrid aptamers enhanced Ca²⁺ transients and contractile function in rat cardiomyocytes. These findings provide critical insights for developing novel therapeutics directly acting on PLN in cardiomyocytes, potentially compensating for the disadvantages of conventional methods that involve viral vector-mediated intracellular transduction or alterations in endogenous protein expression.