AbstractAimsPhospholamban (PLN) and sarcolipin (SLN) are small inhibitory proteins that regulate the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) pump. Previous work from our laboratory revealed that in the soleus and gluteus minimus muscles of mice overexpressing PLN (PlnOE), SERCA function was impaired, dynamin 2 (3–5 fold) and SLN (7–9 fold) were upregulated, and features of human centronuclear myopathy (CNM) were observed. Here, we performed structural and functional experiments to evaluate whether the diaphragm muscles of the PlnOE mouse would exhibit CNM pathology and muscle weakness.MethodsDiaphragm muscles from PlnOE and WT mice were subjected to histological/histochemical/immunofluorescent staining, Ca2+‐ATPase and Ca2+ uptake assays, Western blotting, and in vitro electrical stimulation.ResultsOur results demonstrate that PLN overexpression reduced SERCA's apparent affinity for Ca2+ but did not reduce maximal SERCA activity or rates of Ca2+ uptake. SLN was upregulated 2.5‐fold, whereas no changes in dynamin 2 expression were found. With respect to CNM, we did not observe type I fiber predominance, central nuclei, or central aggregation of oxidative activity in diaphragm, although type I fiber hypotrophy was present. Furthermore, in vitro contractility assessment of PlnOE diaphragm strips revealed no reductions in force‐generating capacity, maximal rates of relaxation or force development, but did indicate that ½ relaxation time was prolonged.ConclusionsTherefore, the effects of PLN overexpression on skeletal muscle phenotype differ between diaphragm and the postural soleus and gluteus minimus muscles. Our findings here point to differences in SLN expression and type I fiber distribution as potential contributing factors.