322 Chapter 12 remain unknown. The PLN-R14del cardiomyopathy has been described to cause severe fibrosis in post-mortem histology samples of both humans11 and mice63. In PLN-R14del carriers, a distinct fibrotic pattern in the subepicardial layer of the LV posterolateral wall was observed before a decrease in LV systolic function was observed.64 This suggests that fibrosis is an early feature of PLN-R14del cardiomyopathy. Moreover, recent data show that total collagen turnover correlates weakly to moderately with clinical parameters in PLN-R14del patients.5 In homozygous R14del/ R14del mice, extensive myocardial fibrosis was observed throughout the left and right ventricles, and fibrotic genes were elevated in 8-week-old mice, whereas 3-week-old mice did not show any signs of fibrosis.63 At the same time, these mice presented PLN aggregation, similar to human patients. Consistently, heterozygous WT/R14del mice did not reveal fibrosis or PLN aggregation until 18 months of age, but both pathological phenotypes co-existed at the same time, suggesting a link between the PLN aggregation and fibrotic response of the heart. Interestingly, we showed for the first time, PLN aggregation in an in vitro system (Figure 4E). Both findings in humans and mice harboring the PLN-R14del mutation could suggest that in fact, the fibrosis is linked to increased mechanical and molecular stress, consistent with the presence of PLN aggregates and an elevated UPR. However, in other mutations of the PLN gene (R9C, R9L, R9H, Leu-39stop, and R25C) no PLN aggregation was described, instead only abnormal Ca2+ cycling before the onset of overt cardiac remodeling, which leads to heart failure in human carriers.65–67 Next to the elevated UPR, the underlying molecular mechanisms of cardiomyocytes causing fibroblast activation could be caused by the mechanical remodelling of the cells. In patients, the inferolateral wall is more vulnerable due to regional molecular changes caused by the mutation or the regional susceptibility which could result from exposure to higher mechanical stress.10 The disruption in mechanical junctions like desmosomes and gap junctions have been described to increase cardiac fibrosis and arrhythmias.68 RT-qPCR revealed a significant decrease in the desmosomal gene PKP2 and gap-junction Cx43, and the geneset analysis revealed a downregulation of the cell-cell adhesion pathway in the PLN-R14del CMs. This suggests the possibility that loss of these interactions may play a contributory role in the activation of fibroblasts and the onset of the PLN-R14del disease. Given the multifactorial etiology of ‘PLN-myopathies’, it is unclear whether reduced Ca2+ uptake by SERCA, secondary to the disturbed PKA-mediated phosphorylation of PLN may represent a ‘final molecular stress pathway’ underlying the disease onset in PLN-R14del patients. Since we found the first Ca2+ abnormalities in PLN-R14del hCSs after 7 days, the massive increase in spheroid size could indicate a possible secondary response of replacement fibrosis due to Ca2+ handling dysfunction. Still, how PLN mutations affect cardiac contractility and if the Ca2+ handling is linked to fibroblast activation has not been extensively studied in human in vitro models. In pre-DCM hearts of PLN-R9 mutation mice, contractile performance was normal, yet an increased proliferation of non-myocytes, early fibroblast activation, and an altered metabolic gene expression in CMs were detected.69 The mechanism by which
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