Abstract 645 Phospholamban<SUP>R9C</SUP> mutation disturbs Ca<SUP>2+</SUP> handling with consequences for excitation/contraction coupling but also for mitochondria and the ER

Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5
Phospholamban^R9C mutation disturbs Ca²⁺ handling with consequences for excitation/contraction coupling but also for mitochondria and the ER
T. Brand¹, T. Baumgarten², S. Denzinger¹, Y. Reinders³, M. Kleindl², F. Funk⁴, N. Gedik⁵, P. Kleinbongard⁵, E. Tolstik², A. Sickmann⁶, J. Schmitt⁴, K. Lorenz¹
¹Pharmakologie, Institut für Pharmakologie und Toxikologie, Würzburg; ²Kardiovaskuläre Pharmakologie, Leibniz Institut für Analytische Wissenschaften -ISAS-e.V., Dortmund; ³Translationale Analytik, Leibniz Institut für Analytische Wissenschaften -ISAS- e.V., Dortmund; ⁴Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum Düsseldorf, Düsseldorf; ⁵Institut für Pathophysiologie, Universitätsklinikum Essen, Essen; ⁶Bioanalytik, Leibniz Institut für Analytische Wissenschaften -ISAS- e.V., Dortmund;
Background: Dysregulation of cardiac Ca²⁺ cycling leads to impaired cardiac function and is a common hallmark of heart failure. Here, we used mice that carry an Arg→Cys missense mutation in the Ca²⁺ regulatory protein phospholamban (PLN^R9C-tg) to evaluate the consequences of a “pinpoint” disruption of Ca²⁺ cycling on cellular and organelle function. Aim/Hypothesis: The aim of the study is the analysis of the impact of Ca²⁺ dysregulation on Ca²⁺-sensitive cellular functions such as excitation/contraction coupling as well as mitochondrial function and endoplasmic reticulum (ER) stress response and its respective restoration. Methods and Results: We used mice overexpressing PLN^R9C (αMHC-PLN^R9C-tg) as well as mouse models that alter β-adrenergic receptor (βAR) signalling such as the Raf kinase inhibitor protein RKIP (αMHC-RKIP-tg and αMHC-RKIP^S153A-tg), an endogenous activator of βAR signalling, and homozygous β1AR-knockout mice as well as control littermates. Further, respective double-transgenic mice (PLN^R9C/RKIP-tg, PLN^R9C/RKIP^S153A-tg, β1AR-KO/PLN^R9C/RKIP-tg) were generated. The assessment of Ca²⁺ cycling using Ca²⁺ indicator Fura-2, excitation/contraction coupling by edge detection and cardiac function by echocardiography revealed that impaired diastolic Ca²⁺ re-uptake, relaxation and cardiac contractile function of PLN^R9C were normalized to Wt levels by simultaneous RKIP-overexpression. Co-expression of RKIP also led to a doubled lifetime of PLN^R9C (T_50%: PLN^R9C-tg: 20.43 weeks vs. PLN^R9C/RKIP-tg: 41.71 weeks). The analysis of mitochondrial function and ER stress marker proteins revealed these Ca²⁺- sensitive organelles as contributors to the detrimental phenotype of PLN^R9C. ADP-stimulated respiration of isolated mitochondria derived from PLN^R9C-tg mouse hearts was significantly impaired (73.6% ±0.0852), production of reactive oxygen species (ROS) was enhanced (126%±0.195) and ATP-production was reduced (85.0%±0.035) compared to Wt. Moreover, mitochondrial membrane potential (mtMP) collapsed in isolated cardiomyocytes from PLN^R9C mice as assessed with the mtMP-sensitive dye tetramethylrhodamine (TMRM). Also, expression levels of proteins involved in ER stress response were upregulated in PLN^R9C-tg compared to wild type mice as detected in an untargeted mass spectrometry approach and subsequent Western Blots. Co-expression of RKIP led to a largely normalized mitochondrial and ER function. To validate that RKIP mediates its “rescue” function via the activation of βAR and downstream normalization of Ca²⁺, we analysed Ca²⁺ handling, contractile function, cardiac remodelling, ER stress and survival in PLN^R9C-tg crossed with RKIP^S153Amutant mice, which are deficient in βAR activation, and with β1AR-KO mice. Indeed, the rescue of the PLN^R9C phenotype by RKIP was absent in PLN^R9C/RKIP^S153A mice and in the absence of β1AR. Conclusion: The study validates the known benefit of Ca²⁺ correction on excitation/contraction coupling and highlights its central role for the functional rescue of mitochondria and ER in the pathomechanism of heart failure.
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