Characterization of the PLN p.Arg14del Mutation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Beatrice Badone,Antonio Zaza,Carlotta Ronchi,Thomas Eschenhagen,Francesco Lodola,Arne Hansen,Anika E Knaust

doi:10.3390/ijms222413500

Abstract

Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca2+ ATP-ase (SERCA2a). Heterozygous PLN p.Arg14del mutation is associated with an arrhythmogenic dilated cardiomyopathy (DCM), whose pathogenesis has been attributed to SERCA2a “superinhibition”. Aim: To test in cardiomyocytes (hiPSC-CMs) derived from a PLN p.Arg14del carrier whether (1) Ca2+ dynamics and protein localization were compatible with SERCA2a superinhibition and (2) if functional abnormalities could be reverted by pharmacological SERCA2a activation (PST3093). Methods: Ca2+ transients (CaT) were recorded at 36 °C in hiPSC-CMs clusters during field stimulation. SERCA2a and PLN where immunolabeled in single hiPSC-CMs. Mutant preparations (MUT) were compared to isogenic wild-type ones (WT), obtained by mutation reversal. Results: WT and MUT differed for the following properties: (1) CaT time to peak (tpeak) and half-time of CaT decay were shorter in MUT; (2) several CaT profiles were identified in WT, “hyperdynamic” ones largely prevailed in MUT; (3) whereas tpeak rate-dependently declined in WT, it was shorter and rate-independent in MUT; (4) diastolic Ca2+ rate-dependently accumulated in WT, but not in MUT. When applied to WT, PST3093 turned all the above properties to resemble those of MUT; when applied to MUT, PST3093 had a smaller or negligible effect. Preferential perinuclear SERCA2a-PLN localization was lost in MUT hiPSC-CMs. Conclusions: Functional data converge to argue for PLN p.Arg14del incompetence in inhibiting SERCA2a in the tested case, thus weakening the rationale for therapeutic SERCA2a activation. Mechanisms alternative to SERCA2a superinhibition should be considered in the pathogenesis of DCM, possibly including dysregulation of Ca2+-dependent transcription.

Highlights

Ca2+ transients (CaT) amplitude did not differ between Mutant preparations (MUT) and wild-type ones (WT) (Figure 1a); CaT profiles differed between MUT and WT, making the comparison between CaT amplitudes less meaningful
The multiple functional differences between MUT and WT nicely converge into a pattern of accelerated systolic and diastolic Ca2+ dynamics suggestive of enhanced sarcoplasmic reticulum (SR)
This view is reinforced by the conversion of the WT pattern into the MUT one by the sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA2a) activator

Summary

Introduction

In the non-phosphorylated state, monomeric PLN partially inhibits the sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA2a), which operates Ca2+ reuptake from cytosol to the SR. Phosphorylation, by protein-kinase A (PKA), of PLN cytosolic domain (on Ser16) favors PLN pentamerization; this relieves the inhibition, increasing the rate of Ca2+ reuptake by SERCA2a under physiological conditions. One of them, consisting of deletion of arginine at position 14 (PLN p.Arg14del), confers to DCM an increased risk of sudden cardiac death and need for heart transplantation [6]. This mutation, first discovered in a Greek family affected by DCM, has been classified as the most prevalent cardiomyopathy-related mutation in the Netherlands [5,6]. More than a decade ago, Haghighi and co-workers defined PLN p.Arg14del (in the clinically occurring heterozygous form) as “superinhibitory”, based on a larger than normal shift of SERCA2a

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