Early consequences of the phospholamban mutation PLN‐R14del^+/− in a transgenic mouse model

Abstract

AimsThe heterozygous phospholamban (PLN) mutation R14del (PLN R14del+/−) is associated with a severe arrhythmogenic cardiomyopathy (ACM) developing in the adult. “Superinhibition” of SERCA2a by PLN R14del is widely assumed to underlie the pathogenesis, but alternative mechanisms such abnormal energy metabolism have also been reported. This work aims to (1) to evaluate Ca2+ dynamics and energy metabolism in a transgenic (TG) mouse model of the mutation prior to cardiomyopathy development; (2) to test whether they are causally connected.MethodsCa2+ dynamics, energy metabolism parameters, reporters of mitochondrial integrity, energy, and redox homeostasis were measured in ventricular myocytes of 8–12 weeks‐old, phenotypically silent, TG mice. Mutation effects were compared to pharmacological PLN antagonism and analyzed during modulation of sarcoplasmic reticulum (SR) and cytosolic Ca2+ compartments. Transcripts and proteins of relevant signaling pathways were evaluated.ResultsThe mutation was characterized by hyperdynamic Ca2+ handling, compatible with a loss of SERCA2a inhibition by PLN. All components of energy metabolism were depressed; myocyte energy charge was preserved under quiescence but reduced during stimulation. Cytosolic Ca2+ buffering or SERCA2a blockade reduced O2 consumption with larger effect in the mutant. Signaling changes suggest cellular adaptation to perturbed Ca2+ dynamics and response to stress.Conclusions(1) PLN R14del+/− loses its ability to inhibit SERCA2a, which argues against SERCA2a superinhibition as a pathogenetic mechanism; (2) depressed energy metabolism, its enhanced dependency on Ca2+ and activation of signaling responses point to an early involvement of metabolic stress in the pathogenesis of this ACM model.

Unbiased complexome profiling and global proteomics analysis reveals mitochondrial impairment and potential changes at the intercalated disk in presymptomatic R14Δ/+mice hearts

“…Mitochondrial function was evaluated using the Seahorse assay. While basal oxygen consumption rates (OCRs) were not significantly decreased, maximal OCR was reduced in left- and right-ventricular cardiomyocytes isolated from 28wk-old R14 Δ/+ mice (Figure 6d-g), consistent with recent reports 14 .…”

“…Nonetheless, our core findings were observed in both datasets despite the differences in both age and cohort, indicating biological reproducibility of our results. Lastly, while our observations at the mitochondria are supported by multiple lines of evidence (complexome profiling, global proteomics, Seahorse), as well as a body of previous work in both hiPSC-CMs 50 and transgenic mice models 14 , our observations at the intercalated disk are admittedly limited to a single readout with no prior art. We believe further research into this topic, including orthogonal functional and (ultra)structural validation, to be an important direction for future work.…”

“…Cardiomyocytes have a uniquely high demand for ATP due to their constant pumping activity, and are dependent on mechanical and electrical connectivity between adjacent cells to facilitate force transduction and propagation of electrical excitation; thus, our observed alterations of mitochondrial and ICD supercomplexes could have particularly severe consequences to cardiac function. While previous studies have shown impaired mitochondrial function in hiPSC-CM models 50 , our findings are among the first to make this observation in a presymptomatic animal system 14 , and the first to demonstrate alterations at the molecular and protein levels.…”

“…The complex cellular phenotype associated with R14Δ-PLN, which includes impaired ER-mitochondrial contact 50 , mitochondrial dysregulation 14 , formation of PLN-positive perinuclear clusters 7,12 and UPR signalling 13 suggests alterations in other protein complexes/supercomplexes that may be functionally and/or spatially remote from the PLN-containing SER-Ca 2+ -handling supercomplex. Systematic analysis of CP data generally involves the use of software packages (ex.…”

“…On the molecular level, R14Δ-PLN is associated with loss of PKA binding, resul ng in PLN hypo-phosphoryla on leading to altered SERCA2a regula on and Ca 2+ -handling dysfunc on 5,9,10 . On the cellular level, R14∆-PLN is associated with a complex phenotype involving accelerated Ca 2+ -handling dynamics, forma on of PLN-posi ve perinuclear SER clusters, upregulated UPR signaling and mitochondrial dysregula on 7,[11][12][13][14] . Despite over a decade of work, the mechanisms linking R14Δ-PLN to disease pathology remain poorly understood.Many proteins assemble into mul meric and/or mul -protein complexes with well-defined molecular composi on and func on 15,16 .…”