Loss of phospholamban phosphorylation blunts Ca²⁺ handling during the cellular adrenergic response

Adrenergic stimulation modulates excitation-contraction coupling (ECC) and cardiac function primarily through protein kinase A (PKA), which phosphorylates multiple ECC proteins. Key PKA targets include phospholamban (PLB), a critical regulator of SERCA2-mediated Ca²⁺ uptake. Previous studies using transgenic overexpression of non-phosphorylatable PLB reported that Ca²⁺ release and inotropy were partially preserved in response to adrenergic stimulation; however, this model may be confounded by transgene insertion and other compensatory mechanisms. To address these limitations, we used CRISPR/Cas9 to generate a mouse model with genomic mutations resulting in non-phosphorylatable substitutions at relevant residues in PLB (PLB-S16A/T17A, PLB-DM). Mass spectrometry confirmed the complete absence of PLB phosphorylation in PLB-DM hearts. PLB-DM mice displayed normal heart structure, mild bradycardia and reduced cardiac output. PLB-DM myocytes showed reduced Ca²⁺ transient amplitude and decay, and reduced SR Ca²⁺ load compared to WT controls under adrenergic stimulation. L-type Ca²⁺ current density and kinetics remained unaltered in PLB-DM myocytes, suggesting that previous reports of preserved inotropy likely reflected model-specific compensatory adaptations. These results suggest that PLB phosphorylation is required to achieve a complete cellular adrenergic response.