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At the mRNA level, all PKP2, DSG2, and DSC2 edited clones shared downregulation of ANK2, ATP2A2, and CASQ2 (Figure 2A). Gene-specific alterations were also found, with PKP2-KO the group with the greatest decrease in the calcium handling genes, showing significant downregulation in all the studied genes except for PLN. In contrast, DSC2-KO clones, apart from the shared alterations, only showed downregulation of TRDN and were the group with the least molecular alterations. Interestingly, although PLN did not show significant differences in any of the desmosomal KO groups, it exhibited a tendency of upregulation in all of them. RQs of the RT-PCR results are shown in Table S3.

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Taking together the massive dysregulation of calcium handling gene expression and subsequently altered calcium transient kinetics, we hypothesized putative molecular mechanisms underlying these results. PKP2-KO, DSC2-KO, and DSG2-KO clones presented shared downregulated levels of ATP2A2, CASQ2, RYR2, and ANK2. ATP2A2 encodes SERCA2, and a decrease of this protein could be responsible for the delay in the re-uptake of calcium to the sarcoplasmic reticulum. CASQ2 encodes calsequestrin-2, which is located inside the sarcoplasmic reticulum and acts as a calcium buffer, regulating the calcium release by RYR2 [40]. Thus, the downregulation of CASQ2 and RYR2 might be the mechanism underlying the dysregulation of calcium release, shown as a significantly increased rise time in DSG2-KO and DSC2-KO clones. Interestingly, although CASQ2 and RYR2 were also downregulated in PKP2-KO, the rise time was not significantly increased in this group. For this reason, the PKP2 loss might trigger other additional mechanisms associated with these functional alterations. In this sense, it was previously published that the loss of PKP2 triggered the phosphorylation of the RYR2 T2809 residue that led to an increased sarcoplasmic reticulum calcium load and higher diastolic calcium concentration due to a function gain of RYR2 [18]. This gain of function of RYR2 could compensate for the decreased levels of RYR2 protein in PKP2-KO clones, and this may explain the unaltered rise time. Therefore, our results are compatible with this previous idea that PKP2 loss may trigger specific mechanisms related to RYR2 phosphorylation that was not found in the other desmosomal gene clones.

ANK2 was commonly downregulated in PKP2-KO, DSC2-KO, and DSG2-KO clones. This protein plays an essential role in the localization and membrane stabilization of NCX1 (encoded by SLC8A1) and SERCA2 [40]. Therefore, we hypothesize that decreased ANK2 might be involved in the delayed re-uptake of calcium because NCX1 and SERCA2 may be less efficient in returning calcium to the extracellular medium and to the sarcoplasmic reticulum, respectively. In agreement with this hypothesis, DSP-KO clones, which showed detectable levels of ANK2 protein, presented a slightly shorter amplitude in the calcium peak. Therefore, ANK2 could be causing these differences in peak amplitude in the studied clones. In fact, previous studies showed that heterozygous ANK2-KO mice presented more frequent Ca2+ sparks and waves compared with WT mice [41,42]. Ca2+ waves are caused by a higher Ca2+ content in the sarcoplasmic reticulum [43], indicating that the absence of ANK2 causes dysregulation of calcium homeostasis.

Additionally, PLN is an inhibitor of the activity of ATP2A2, decreasing SERCA affinity for calcium in its unphosphorylated state [44]. An increased expression of PLN may reduce ATP2A2 activity, producing a delay in the re-uptake of calcium into the sarcoplasmic reticulum and thus causing the delayed amplitude of the peak in PKP2-KO, DSG2-KO, and DSC2-KO clones. However, in the DSP-KO clones, downregulated levels of PLN may trigger the higher activity of ATP2A2, leading to a more rapid re-uptake of calcium to the sarcoplasmic reticulum. Therefore, changes in PLN mRNA expression levels could explain the functional alterations in all clones.

Our study revealed that DSC2 downregulation was the only common alteration in desmosomal expression genes shared by the absence of PKP2, DSG2, DSC2, or DSP. Interestingly, PKP2 loss triggered major alterations in desmosomal and calcium-handling gene expression. Regarding electrical conduction alterations, PKP2, DSG2, and DSC2-KO caused the downregulation of Nav1.5, but this was not shared by DSP-KO. Moreover, upregulation of TFGB1 and PPARγ was also found to be a common molecular feature for PKP2, DSG2, and DSC2 loss, although no data was available for the loss of DSP.

Finally, our results showed massive dysregulation in calcium handling genes shared by PKP2, DSC2, or DSG2 loss associated with a slower calcium re-uptake that might be related to the ANK2, CASQ2, ATP2A2, and RYR2 decrease or PLN increase. In contrast, DSP-KO clones produced a shorter amplitude of the calcium peak, which might be associated with the downregulation of PLN, which was only shown in DSP-KO clones. More studies would be needed to corroborate these associations. Moreover, the present study demonstrated that N-DSP proteins are fully functional in terms of calcium cycling. 041b061a72