The role of calsequestrin II phosphorylation in regulating calcium leak in heart failure

Heart failure (HF) is a chronic and progressive condition characterised by the reduced ability of the heart to pump blood effectively. This condition remains a major global health burden, affecting 1-3 % of the adult population and contributing to a high mortality rate for those aged > 65 years. A hallmark of HF is impaired calcium (Ca²⁺) handling within cardiomyocytes, disrupting excitation-contraction coupling (ECC) and contributing to contractile dysfunction. At the cellular level, Ca²⁺-induced Ca²⁺ release (CICR) occurs through ryanodine receptor type II (RyR2) on the sarcoplasmic reticulum (SR). However, in HF, spontaneous Ca²⁺ release known as store-overload induced Ca²⁺ release (SOICR) becomes more frequent, driven by elevated SR Ca²⁺ content or increased sensitivity of RyR2. Calsequestrin II (CSQ2), a low-affinity, high-capacity Ca²⁺ buffering protein found at the junctional SR (jSR), can regulate SR Ca²⁺ and RyR2-mediated Ca²⁺ release. CSQ2’s function is modulated by phosphorylation at sites S385 and S393 by CK2, which is thought to promote polymerisation and enhance the ability of CSQ2 to buffer Ca²⁺ at the jSR effectively. T282 has also been suggested as a putative phosphorylation site but remains unconfirmed experimentally. Many studies have focused on RyR2 and its role in modulating SOICR, however, the role of CSQ2 and its phosphorylation in modulating SOICR in HF pathology remains unclear.

This thesis explores how CSQ2 phosphorylation influences SOICR, and whether phosphorylation of CSQ2 alters its co-localisation with RyR2 using a combination of cellular and tissue-level approaches. First, I utilised phosphomimetic (S to D) and non-phosphorylatable (S to A) CSQ2 mutants at S385 and S393 to assess the effect on SOICR using single-cell Ca²⁺ imaging in HEK293 cells. The initial hypothesis was that as I progressively increase CSQ2 phosphorylation, I would observe a cumulative effect in reducing SOICR activity. Our results showed that single-site phosphomimetic mutants did not significantly reduce SOICR activity, while non-phosphorylatable mutants increased SOICR frequency compared to CSQ2-WT. In contrast, dual site phosphomimetic CSQ2 (S385D + S393D) significantly reduced SOICR, suggesting a cooperative effect. Conversely, phosphorylation at the putative T282 site increased SOICR compared to CSQ2-WT, implying a potentially inhibitory role.

The second objective investigated the spatial relationship between CSQ2 and RyR2 in human right atrial appendage (RAA) tissue from patients with and without HF using immunofluorescence and Airyscan confocal microscopy. I hypothesised that in HF, there would be a reduction in co-localisation of RyR2 and CSQ2, which could contribute to SOICR associated with HF. The results show that CSQ2 and RyR2 co-localisation is preserved in HF, indicating that co-localisation is not a primary driver of altered Ca²⁺ handling in the context of HF. This finding shifts therapeutic interest towards CSQ2’s function, particularly its phosphorylation and buffering ability, rather than its localisation.

Lastly, I sought to characterise custom phospho-specific antibodies targeting human CSQ2-S385 and CSQ2-S393 with the end goal of assessing endogenous phosphorylation levels in cardiac tissue between non-failing and failing patient samples. While the S385 antibody lacked specificity, the S393 antibody showed greater potential for use in human samples. These tools provide a stepping stone for future studies to semi-quantify phosphorylation levels in HF and to further gain insight into the implications of these sites under typical conditions.

Overall, this project provides novel insights into how CSQ2 phosphorylation influences Ca²⁺ handling in HF, revealing that dual-site phosphomimetic mutants significantly reduce SOICR, while phosphorylation at the putative T282 site increases it. Altogether, this project employs a multi-level approach, from transfecting phosphomimetic and non-phosphorylatable CSQ2 variants in HEK293 cells, to single-cell Ca²⁺ imaging, expression analysis, and validation of site-specific phospho-antibodies to establish the functional effect of phosphorylated CSQ2 on modulating SOICR. The final translation to human cardiac tissue, using immunofluorescence combined with Airyscan confocal microscopy and co-localisation analysis, confirmed that CSQ2 and RyR2 co-localisation is preserved in HF, directing therapeutic focus toward functional modulation rather than localisation.