Background. Phospholamban (PLN) is a pivotal regulator of cardiac function, binding reversibly to and inhibiting the Sarcoendoplasmic Reticulum Calcium ATPase 2a (SERCA2a) pump. PLN phosphorylation at Ser16 by PKA in response to adrenergic stimulation and at Thr17 by CaMKII in response to cytosolic Ca²⁺ levels attenuates this binding. Consequently, PLN serves as a crucial link between cellular signaling pathways and SERCA2a activity. The PLN-R14del mutation is associated with arrhythmogenic and/or dilated cardiomyopathy (ACM/DCM) and severe untreatable heart failure (HF). At the molecular level, R14del disrupts the PLN-PKA interaction, leading to a loss of phosphorilation at Ser16. This may result in SERCA2a superinhibition and elevated cytosolic  Ca²⁺ level, but the precise cellular mechanisms underlying PLN-R14del cardiomyopathy (R14del-CM) are as-yet unknown. Excessive CaMKII activation contributes to CM and HF, partly due to maladaptive phosphorylation of  Ca²⁺-handling machinery and transcriptional changes. Modulating CaMKII activity has garnered recent interest as a therapeutic approach; however, its applicability to R14del-CM remains uncertain. Compensatory CaMKII activation may mitigate the loss of PKA-mediated Ser16 phosphorylation and reduce SERCA2a superinhibition, but sustained upregulated CaMKII activity is an established maladaptive response that contribute to CM. Elucidating the functional interaction between CaMKII and R14del-CM could guide therapeutic development.

 

Methods. We employed various mouse models to investigate the functional interplay between PLN-R14del and CaMKII activity. To assess whether CaMKII activity is altered in R14del mice (Aim 1), we utilised an established heterozygous PLN-R14del mouse model (R14del/+). To explore the impact of CaMKII inhibition on R14del-CM (Aim 2), we generated a novel mouse line harbouring both the PLN-R14del mutation and a tamoxifen-inducible, cardiac-specific CaMKII-delta/gamma knockout (R14del x CaMKIIdKO). In both cases, we characterised the mice at around 20 weeks of age, well before the onset of cardiomyopathy observed in R14+/del animals around 60 weeks of age. Protein expression profiles were assessed using immunoblotting, cellular morphology through immunostaining coupled with STED-nanoscopy, and cardiac function via EKG and echocardiography.

 

Results. Aim 1: biochemical analysis revealed reduced CaMKII expression in R14del/+ mice compared to WT-controls. Interestingly, CaMKII-mediated phosphorylation at PLN residue Thr17 was also diminished. These findings contradict the notion of compensatory CaMKII-mediated PLN phosphorylation following PKA binding loss and instead suggest an unexpected decrease in CaMKII activity. Future investigations will explore the phosphorylation of other CaMKII targets such as calcineurin and HDAC4. 

Aim 2: our novel R14del x CaMKIIdKO mouse model did not exhibit increased mortality or any overt changes in cardiac function. In-depth analysis of cellular morphology and cardiac functional measurements is currently underway.

 

Conclusions. Our preliminary results from Aim 1 indicate paradoxically reduced CaMKII activity in R14del/+ mice. Whether this signifies a compensatory mechanism or contributes to disease progression remains unknown. The ongoing functional characterisation of our novel R14del x CaMKIIdKO mouse model may provide additional insights into the viability of CaMKII modulation as a therapeutic strategy.