Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway.
K.G. Jebsen Center for Cardiac Research, University of Oslo, 0313 Oslo, Norway.
Cells. 2024 May 30;13(11):947. doi: 10.3390/cells13110947.
The transmembrane proteoglycan syndecan-4 is known to be involved in the hypertrophic response to pressure overload. Although multiple downstream signaling pathways have been found to be involved in this response in a syndecan-4-dependent manner, there are likely more signaling components involved. As part of a larger syndecan-4 interactome screening, we have previously identified MLP as a binding partner to the cytoplasmic tail of syndecan-4. Interestingly, many human MLP mutations have been found in patients with hypertrophic (HCM) and dilated cardiomyopathy (DCM). To gain deeper insight into the role of the syndecan-4-MLP interaction and its potential involvement in MLP-associated cardiomyopathy, we have here investigated the syndecan-4-MLP interaction in primary adult rat cardiomyocytes and the H9c2 cell line. The binding of syndecan-4 and MLP was analyzed in total lysates and subcellular fractions of primary adult rat cardiomyocytes, and baseline and differentiated H9c2 cells by immunoprecipitation. MLP and syndecan-4 localization were determined by confocal microscopy, and MLP oligomerization was determined by immunoblotting under native conditions. Syndecan-4-MLP binding, as well as MLP self-association, were also analyzed by ELISA and peptide arrays. Our results showed that MLP-WT and syndecan-4 co-localized in many subcellular compartments; however, their binding was only detected in nuclear-enriched fractions of isolated adult cardiomyocytes. In vitro, syndecan-4 bound to MLP at three sites, and this binding was reduced in some HCM-associated MLP mutations. While MLP and syndecan-4 also co-localized in many subcellular fractions of H9c2 cells, these proteins did not bind at baseline or after differentiation into cardiomyocyte-resembling cells. Independently of syndecan-4, mutated MLP proteins had an altered subcellular localization in H9c2 cells, compared to MLP-WT. The DCM- and HCM-associated MLP mutations, W4R, L44P, C58G, R64C, Y66C, K69R, G72R, and Q91L, affected the oligomerization of MLP with an increase in monomeric at the expense of trimeric and tetrameric recombinant MLP protein. Lastly, two crucial sites for MLP self-association were identified, which were reduced in most MLP mutations. Our data indicate that the syndecan-4-MLP interaction was present in nuclear-enriched fractions of isolated adult cardiomyocytes and that this interaction was disrupted by some HCM-associated MLP mutations. MLP mutations were also linked to changes in MLP oligomerization and self-association, which may be essential for its interaction with syndecan-4 and a critical molecular mechanism of MLP-associated cardiomyopathy.
细胞表面蛋白聚糖连接蛋白-4 已被证实参与了压力超负荷引起的肥厚反应。虽然已经发现了多个下游信号通路以依赖于连接蛋白-4 的方式参与这种反应，但可能还有更多的信号成分参与其中。作为更大的连接蛋白-4 相互作用组筛选的一部分，我们之前已经鉴定出 MLP 是连接蛋白-4 胞质尾的结合伙伴。有趣的是，许多人类 MLP 突变已在肥厚性（HCM）和扩张型心肌病（DCM）患者中被发现。为了更深入地了解连接蛋白-4-MLP 相互作用及其在 MLP 相关心肌病中的潜在作用，我们在此研究了原代成年大鼠心肌细胞和 H9c2 细胞系中的连接蛋白-4-MLP 相互作用。通过免疫沉淀分析原代成年大鼠心肌细胞总裂解物和亚细胞部分以及基础和分化的 H9c2 细胞中的连接蛋白-4 和 MLP 的结合情况。通过共聚焦显微镜确定 MLP 和连接蛋白-4 的定位，并在天然条件下通过免疫印迹确定 MLP 寡聚化。通过 ELISA 和肽阵列分析连接蛋白-4-MLP 结合以及 MLP 自缔合。我们的结果表明，MLP-WT 和连接蛋白-4 在许多亚细胞区室中共同定位；然而，它们的结合仅在分离的成年心肌细胞的核富集部分中被检测到。在体外，连接蛋白-4 结合到 MLP 的三个位点，并且这种结合在一些 HCM 相关的 MLP 突变中减少。虽然 MLP 和连接蛋白-4 也在 H9c2 细胞的许多亚细胞部分中共定位，但这些蛋白质在基础状态或分化为心肌细胞样细胞后不结合。独立于连接蛋白-4，与 DCM 和 HCM 相关的 MLP 突变，W4R、L44P、C58G、R64C、Y66C、K69R、G72R 和 Q91L，改变了 MLP 的亚细胞定位，与 MLP-WT 相比，单体增加，而三聚体和四聚体重组 MLP 蛋白减少。最后，确定了两个对 MLP 自缔合至关重要的位点，这些位点在大多数 MLP 突变中减少。我们的数据表明，连接蛋白-4-MLP 相互作用存在于分离的成年心肌细胞的核富集部分中，并且这种相互作用被一些与 HCM 相关的 MLP 突变破坏。MLP 突变还与 MLP 寡聚化和自缔合的变化有关，这可能对其与连接蛋白-4 的相互作用以及 MLP 相关心肌病的关键分子机制至关重要。
