Liu Sun

Cardiomyopathies constitute a heterogeneous group of myocardial disorders characterized by structural and/or functional abnormalities of the heart. These abnormalities can lead to progressive contractile dysfunction, arrhythmias, heart failure, and sudden cardiac death. A significant portion of these conditions has a genetic cause. Within this group, phospholamban (PLN)-related cardiomyopathy holds a prominent place. In particular, the pathogenic PLN-R14del variant is of great clinical importance, as it occurs relatively frequently in the Netherlands and is associated with an increased risk of both arrhythmogenic and dilated cardiomyopathy. However, clinical expression is highly variable: some carriers develop severe disease at a young age, while others develop symptoms late or only in a mild form. This heterogeneity indicates that the pathogenesis of PLN-R14del cardiomyopathy is complex and cannot be fully explained by a single mechanism.

PLN is a small regulatory membrane protein that modulates the activity of the calcium pump SERCA2a in the sarco(endo)plasmic reticulum (SR/ER) in cardiac muscle cells, thereby influencing calcium uptake from the cytoplasm into the SR/ER. For a long time, it was assumed that the disease was primarily the result of disturbed calcium homeostasis due to abnormal inhibition of SERCA2a by PLN. However, recent results, including those in this thesis, show that this classic explanation is insufficient. Instead, experimental and conceptual findings point to a broader clinical picture in which disruption of SR/ER structure, abnormal protein organization, dysregulation of proteostasis, and mutation-specific cellular stress responses are central. This shifts the perspective from a purely calcium-oriented model to a model in which structural and proteotoxic processes play a decisive role in the development of the disease.

The central question in this thesis was therefore which molecular and pathophysiological mechanisms underlie PLN-R14del cardiomyopathy, and to what extent these mechanisms offer points of contact for targeted therapy. To this end, five coherent lines of research have been developed: 1) an overview of clinically described PLN variants and their suspected mechanisms of action; 2) an analysis of cardiac stress as a possible disease modifier; 3) the development of an inducible mouse model to study early disease processes; 4) an evaluation of dose-dependent effects of PLN-targeted antisense oligonucleotides (ASOs); 5) an in silico analysis of the consequences of PLN silencing for different PLN complexes.

The overview of known pathogenic PLN variants in Chapter 2 shows that PLN-related cardiomyopathy cannot be considered a single uniform condition. Different variants, including truncations, missense mutations, and deletions such as R14del, R9C, R9H, R9L, and L39stop, lead to diverse phenotypes and probably also to different underlying molecular defects. The differences concern, among other things, oligomerization, phosphorylation, membrane insertion, stability, and protein-protein interactions. These findings underscore that not all PLN variants are pathogenic via the same mechanism, and that a uniform therapeutic approach will probably not be optimal for all carriers. This has important implications for future precision medicine within the field of inherited cardiomyopathies.

Subsequently, Chapter 3 investigated whether increased cardiac load can accelerate the onset of PLN-R14del cardiomyopathy. Since carriers show a strong variation in the age of disease onset and severity, it was hypothesized that additional stressors could potentially lower the threshold for disease activation. In a heterozygous PLN-R14del mouse model, pressure overload was induced for this purpose with transverse aortic constriction (TAC). Although TAC, as expected, led to hypertrophy, fibrosis, and a decrease in cardiac function, it did not cause accelerated formation of the characteristic PLN-positive SR/ER clusters, nor any additional mutation-specific worsening on top of the general response to pressure overload. These results show that acute biomechanical stress in itself is insufficient to initiate or accelerate the specific disease process of PLN-R14del. Thus, general cardiac stress does not appear to be a simple explanation for the clinical heterogeneity in carriers.

To better study the earliest events in the disease process, a new inducible PLN-R14del mouse model was developed in this thesis, as described in Chapter 4. In this model, the expression of wild-type PLN in adult cardiomyocytes could be synchronously converted to the expression of PLN-R14del by means of a tamoxifen-dependent MerCreMer/LoxP strategy. This made it possible to monitor disease initiation in a time-controlled manner, without interference from developmental processes or asynchronous expression. Using this model, it was demonstrated that the first detectable abnormality consists of the appearance of small PLN-positive clusters in the SR/ER, which over time grow into the characteristic perinuclear structures that are hallmark of this cardiomyopathy. Importantly, these structural changes were already visible before clear functional decline or pronounced tissue abnormalities occurred. This suggests that SR/ER malformation is an early and probably primary event in the pathogenesis of PLN-R14del cardiomyopathy.

Additional transcriptome and proteome analyses of isolated cardiomyocytes from this inducible model showed that early molecular changes manifest themselves mainly at the protein level. While relatively few changes were observed at the mRNA level, the proteome showed clear disruptions in SR/ER-related processes. Changes were found that match activation or overload of quality control mechanisms in the endoplasmic reticulum, including accumulation of proteins related to ER-associated degradation (ERAD). A discrepancy was also visible between transcription and protein expression, suggesting an important role for post-translational and/or degradation-related regulation. Together, these findings support the concept that PLN-R14del cardiomyopathy is characterized early on by disturbed proteostasis and a dysregulated intracellular quality control system, followed by later transcriptome changes.

A next central question was how reduction of PLN expression intervenes therapeutically in this disease process. In a homozygous PLN-R14del mouse model, the effect of different doses of PLN-targeted antisense oligonucleotides was therefore investigated, as described in Chapter 5. These experiments showed a clear dose-dependent protection: ASO treatment led to improvement in cardiac function, reduction in remodeling, extension of survival, and reduction of the pathological PLN-positive SR clusters. Strikingly, however, the influence on calcium dynamics in mutant heart muscle cells remained limited. In wild-type cells, PLN depletion did lead to the expected acceleration of calcium and contractile dynamics, but in PLN-R14del heart muscle cells these parameters were already accelerated and could hardly be improved further. This supports the conclusion that the therapeutic effect of PLN-ASO in this mutation is not primarily based on restoration of calcium regulation, but mainly on restoration of SR structure.

To better understand why PLN silencing primarily seems to affect the structural pathology, an additional in silico model was developed in Chapter 6 in which different molecular forms of PLN were simulated: free monomers, pentamers, and SERCA-bound complexes. These simulations showed that pentameric PLN complexes are more sensitive to a decrease in total PLN concentration than free or SERCA-bound PLN forms. This is relevant because the pentamer or clustered PLN pool presumably contributes strongly to the abnormal intracellular accumulation seen in PLN-R14del. The model results thus provide a mechanistic explanation for the experimental observation that PLN silencing is accompanied by a decrease in abnormal clustering and restoration of SR architecture, while the functional SERCA-related pool remains relatively longer preserved. These findings support the idea that not all PLN pools are equally sensitive to a decrease in total PLN concentration and that the pentamer form, which responds most sensitively, is perhaps the most pathological component of the system.

Taken together, the results in this thesis call for a recalibration of the disease concept of PLN-R14del cardiomyopathy. Instead of a condition driven primarily by abnormal calcium inhibition of SERCA2a, a picture emerges of a structural proteinopathy of the sarco(endo)plasmic reticulum. A central early event is the formation of abnormal PLN-positive SR/ER clusters, followed by dysregulation of proteostasis, activation of intracellular stress pathways, and ultimately progressive cardiac remodeling and loss of function. This interpretation connects the histological, molecular, functional, and therapeutic findings within this thesis into one coherent whole.

The results also have clear translational implications. First, they support the further development of PLN-targeted silencing strategies as a promising therapeutic route, where optimizing the correct dosage in humans will be an important point of attention. Second, our results emphasize the importance of early detection, as intervention before irreversible structural damage occurs will probably be most effective. Third, they suggest that future biomarker development should not focus exclusively on global cardiac function measurements, but also on early indicators of SR/ER stress. Finally, we have described that there may be PLN-variant-specific differences in underlying pathological mechanisms. This must be taken into account in the development of new therapies.

In summary, this thesis demonstrates that the pathogenic PLN-R14del variant leads to an early onset dysregulation of intracellular SR/ER organization, with subsequent disruption of proteostasis and progressive decline in cardiomyocyte function. The findings shift the pathogenetic paradigm from a predominantly calcium-oriented explanation to a model in which structural dysregulation and abnormal protein organization are central. Thus, this thesis provides not only new insights into the biology of PLN-R14del cardiomyopathy, but also a basis for the further development of mechanism-driven and mutation-specific treatment strategies.