Deletion of Sarcolemmal Membrane-Associated Protein Isoform 3 (SLMAP3) in Cardiac Progenitors Delays Embryonic Growth of Myocardium without Affecting Hippo Pathway.

The slmap gene is alternatively spliced to generate many isoforms that are abundant in developing myocardium. The largest protein isoform SLMAP3 is ubiquitously expressed and has been linked to cardiomyopathy, Brugada syndrome and Hippo signaling. To examine any role in cardiogenesis, mice homozygous for floxed slmap allele were crossed with Nkx2.5-cre mice to nullify its expression in cardiac progenitors. Targeted deletion of the slmap gene resulted in the specific knockout (KO) of the SLMAP3 (~91 KDa) isoform without any changes in the expression of the SLMAP2 (~43 kDa) or the SLMAP1 (~35 kDa) isoforms which continued to accumulate to similar levels as seen in Wt embryonic hearts. The loss of SLMAP3 from cardiac progenitors resulted in decreased size of the developing embryonic hearts evident at E9.5 to E16.5 with four small chambers and significantly thinner left ventricles. The proliferative capacity assessed with the phosphorylation of histone 3 or with Ki67 in E12.5 hearts was not significantly altered due to SLMAP3 deficiency. The size of embryonic cardiomyocytes, marked with anti-Troponin C, revealed significantly smaller cells, but their hypertrophic response (AKT1 and MTOR1) was not significantly affected by the specific loss of SLMAP3 protein. Further, no changes in phosphorylation of MST1/2 or YAP were detected in SLMAP3-KO embryonic myocardium, ruling out any impact on Hippo signaling. Rat embryonic cardiomyocytes express the three SLMAP isoforms and their knockdown (KD) with sh-RNA, resulted in decreased proliferation and enhanced senescence but without any impact on Hippo signaling. Collectively, these data show that SLMAP is critical for normal cardiac development with potential for the various isoforms to serve compensatory roles. Our data imply novel mechanisms for SLMAP action in cardiac growth independent of Hippo signaling.

SLMAP-3 is downregulated in human dilated ventricles and its overexpression promotes cardiomyocyte response to adrenergic stimuli by increasing intracellular calcium.

Structural dilation of cardiomyocytes (CMs) imposes a decline in cardiac performance that precipitates cardiac failure and sudden death. Since membrane proteins are implicated in dilated cardiomyopathy and heart failure, we evaluated the expression of the sarcolemmal membrane-associated protein (SLMAP) in dilated cardiomyopathy and its effect on CM contraction. We found that all 3 SLMAP isoforms (SLMAP-1, -2, and -3) are expressed in CMs and are downregulated in human dilated ventricles. Knockdown of SLMAPs in cultured CMs transduced with recombinant adeno-associated viral particles releasing SLMAP-shRNA precipitated reduced spontaneous contractile rate that was not fully recovered in SLMAP-depleted CMs challenged with isoproterenol (ISO), thus phenotypically mimicking heart failure performance. Interestingly, the overexpression of the SLMAP-3 full-length isoform induced a positive chronotropic effect in CMs that was more pronounced in response to ISO insult (vs. ISO-treated naïve CMs). Confocal live imaging showed that H9c2 cardiac myoblasts overexpressing SLMAP-3 exhibit a higher intracellular calcium transient peak when treated with ISO (vs. ISO-treated cells carrying a control adeno-associated viral particle). Proteomics revealed that SLMAP-3 interacts with the regulator of CM contraction, striatin. Collectively, our data demonstrate that SLMAP-3 is a novel regulator of CM contraction rate and their response to adrenergic stimuli. Loss of SLMAPs phenotypically mimics cardiac failure and crystallizes SLMAPs as predictive of dilated cardiomyopathy and heart failure.

Case report: Primary sarcoma of the mandible with a novel SLMAP-BRAF fusion.

Primary sarcomas of the jaw are very rare tumor with unclear mechanism of tumorigenesis. Identification of genetic alterations contributes to better understanding of tumorigenesis and extension of tumor spectrum, as well as potential therapeutic targets application. Herein, we firstly report a case of primary sarcoma in the mandible with novel SLMAP-BRAF fusion. Morphologically, the tumor was composed of histiocyte-like cells, larger epithelioid cells, spindle cells and osteoclast-like giant cells with moderate atypia. Focally, it mimicked tenosynovial giant cell tumor or biphasic synovial sarcoma, and even giant cell tumor of bone. SATB2 was diffusely expressed, while p63 and p16 were locally positive with loss expression of p16 in histiocyte-like and larger epithelioid cells. SLMAP-BRAF (S11:B10) fusion was detected by both DNA and RNA NGS, and further verified by sanger sequencing, DNA electrophoresis and FISH. Then a descriptive diagnosis of BRAF rearrangement sarcoma with moderate-grade malignancy (non-specific type) was given according to the biological behavior, morphological features and gene alteration. The patient finished six cycles of chemotherapy after hemimaxillectomy. Within 7 months of follow-up, no tumor recurrence or metastasis was observed. Our case has enriched the spectrum of jaw bone tumor and BRAF rearrangement tumor.

Comparative phosphoproteomics reveal new candidates in the regulation of spermatogenesis of Drosophila melanogaster.

The most common phenotype induced by the endosymbiont Wolbachia in insects is cytoplasmic incompatibility, where none or fewer progenies can be produced when Wolbachia-infected males mate with uninfected females. This suggests that some modifications are induced in host sperms during spermatogenesis by Wolbachia. To identify the proteins whose phosphorylation states play essential roles in male reproduction in Drosophila melanogaster, we applied isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic strategy combined with titanium dioxide (TiO2 ) enrichment to compare the phosphoproteome of Wolbachia-infected with that of uninfected male reproductive systems in D. melanogaster. We identified 182 phosphopeptides, defining 140 phosphoproteins, that have at least a 1.2 fold change in abundance with a P-value of <0.05. Most of the differentially abundant phosphoproteins (DAPPs) were associated with microtubule cytoskeleton organization and spermatid differentiation. The DAPPs included proteins already known to be associated with spermatogenesis, as well as many not previously studied during this process. Six genes coding for DAPPs were knocked down, respectively, in Wolbachia-free fly testes. Among them, Slmap knockdown caused the most severe damage in spermatogenesis, with no mature sperm observed in seminal vesicles. Immunofluorescence staining showed that the formation of individualization complex composed of actin cones was completely disrupted. These results suggest that Wolbachia may induce wide changes in the abundance of phosphorylated proteins which are closely related to male reproduction. By identifying phospho-modulated proteins we also provide a significant candidate set for future studies on their roles in spermatogenesis.

An association between the sarcolemmal membrane-associated protein gene and microvascular endothelial diabetic retinopathy in patients with type 2 diabetes mellitus: A preliminary case control study.

BACKGROUND AND AIMS: Diabetic retinopathy (DR) is one of the most common microvascular diabetic complications. Sarcolemmal membrane-associated protein (SLMAP) has been implicated in playing a role in microvascular endothelial dysfunction. This study aimed to assess the significance of SLMAP rs17058639C > T gene polymorphism among patients with type 2 diabetes mellitus (T2DM) and its relevance to microvascular endothelial diabetic retinopathy. METHODS: We conducted this case-control study on 100 individuals divided into 60 participants with T2DM and 40 healthy controls. Patients with T2D were stratified into two groups: 40 patients with DR and 20 patients with diabetic non-retinopathy (DNR). Patients with T2DM were compared with age- and sex-matched healthy controls. Fundus examinations were conducted to detect microvascular endothelial changes. The polymorphism of SLMAP rs17058639C > T gene was identified by real-time polymerase chain reaction (RT-PCR) TaqMan allelic discrimination. RESULTS: Patients with DR have significantly increased glycated hemoglobin (HbA1c) compared to patients with DNR (P < 0.001). There was no statistically significant difference found between diabetic and control groups regarding the frequency of SLMAP rs17058639C > T genotypes. The homozygous CC genotype was the most common variant among patients with DR; however, the results did not reach statistical significance. CONCLUSIONS: Diabetic retinopathy is correlated with poor glycemic control, and SLMAP rs17058639C > T polymorphism was associated with microvascular endothelial DR in patients with T2DM, although further studies with a large sample size are needed to confirm our findings.

Sudden Death without a Clear Cause after Comprehensive Investigation: An Example of Forensic Approach to Atypical/Uncertain Findings.

Sudden death (SD) is defined as the unexpected natural death occurred within an hour after the onset of symptoms or from the last moment the subject has been seen in a healthy condition. Brugada syndrome (BrS) is one of the most remarkable cardiac causes of SD among young people. We report the case of a 20-year-old man who suddenly died after reportedly having smoked cannabis. Autopsy, toxicology, and genetic testing were performed. Autopsy found a long and thick myocardial bridging (MB) at 2 cm from the beginning of the left anterior descending coronary artery. Furthermore, at the histopathological examination, fibrosis and disarray in myocardial area above the MB, fatty tissue in the right ventricle and fibrosis of the sino-atrial node area were found. Toxicology testing was inconclusive, while genetic testing found a rare missense variant of the TTN gene, classified as likely benign, and a variant of unknown significance in the SLMAP gene (a gene that can be associated with BrS). Hence, despite several atypical features were found, no inference on the cause of the death could be made under current evidence.

The SLMAP/Striatin complex: An emerging regulator of normal and abnormal cardiac excitation-contraction coupling.

The excitation-contraction (E-C) module involves a harmonized correspondence between the sarcolemma and the sarcoplasmic reticulum. This is provided by membrane proteins, which primarily shape the caveolae, the T-tubule/Sarcoplasmic reticulum (TT/SR) junction, and the intercalated discs (ICDs). Distortion of either one of these structures impairs myocardial contraction, and subsequently translates into cardiac failure. Thus, detailed studies on the molecular cues of the E-C module are becoming increasingly necessary to pharmacologically eradicate cardiac failure Herein we reviewed the organization of caveolae, TT/SR junctions, and the ICDs in the heart, with special attention to the Sarcolemma Membrane Associated Protein (SLMAP) and striatin (STRN) in cardiac membranes biology and cardiomyocyte contraction. We emphasized on their in vivo and in vitro signaling in cardiac function/dysfunction. SLMAP is a cardiac membrane protein that plays an important role in E-C coupling and the adrenergic response of the heart. Similarly, STRN is a dynamic protein that is also involved in cardiac E-C coupling and ICD-related cardiomyopathies. Both SLMAP and STRN are linked to cardiac conditions, including heart failure, and their role in cardiomyocyte function was elucidated in our laboratory. They interact together in a protein complex that holds therapeutic potentials for cardiac dysfunction. This review is the first of its kind to conceptualize the role of the SLMAP/STRN complex in cardiac function and failure. It provides in depth information on the signaling of these two proteins and projects their interaction as a novel therapeutic target for cardiac failure.

Homeostatic Control of Hpo/MST Kinase Activity through Autophosphorylation-Dependent Recruitment of the STRIPAK PP2A Phosphatase Complex.

The Hippo pathway controls organ size and tissue homeostasis through a kinase cascade leading from the Ste20-like kinase Hpo (MST1/2 in mammals) to the transcriptional coactivator Yki (YAP/TAZ in mammals). Whereas previous studies have uncovered positive and negative regulators of Hpo/MST, how they are integrated to maintain signaling homeostasis remains poorly understood. Here, we identify a self-restricting mechanism whereby autophosphorylation of an unstructured linker in Hpo/MST creates docking sites for the STRIPAK PP2A phosphatase complex to inactivate Hpo/MST. Mutation of the phospho-dependent docking sites in Hpo/MST or deletion of Slmap, the STRIPAK subunit recognizing these docking sites, results in constitutive activation of Hpo/MST in both Drosophila and mammalian cells. In contrast, autophosphorylation of the Hpo/MST linker at distinct sites is known to recruit Mats/MOB1 to facilitate Hippo signaling. Thus, multisite autophosphorylation of Hpo/MST linker provides an evolutionarily conserved built-in molecular platform to maintain signaling homeostasis by coupling antagonistic signaling activities.

STRIPAK complexes: structure, biological function, and involvement in human diseases.

The mammalian striatin family consists of three proteins, striatin, S/G2 nuclear autoantigen, and zinedin. Striatin family members have no intrinsic catalytic activity, but rather function as scaffolding proteins. Remarkably, they organize multiple diverse, large signaling complexes that participate in a variety of cellular processes. Moreover, they appear to be regulatory/targeting subunits for the major eukaryotic serine/threonine protein phosphatase 2A. In addition, striatin family members associate with germinal center kinase III kinases as well as other novel components, earning these assemblies the name striatin-interacting phosphatase and kinase (STRIPAK) complexes. Recently, there has been a great increase in functional and mechanistic studies aimed at identifying and understanding the roles of STRIPAK and STRIPAK-like complexes in cellular processes of multiple organisms. These studies have identified novel STRIPAK and STRIPAK-like complexes and have explored their roles in specific signaling pathways. Together, the results of these studies have sparked increased interest in striatin family complexes because they have revealed roles in signaling, cell cycle control, apoptosis, vesicular trafficking, Golgi assembly, cell polarity, cell migration, neural and vascular development, and cardiac function. Moreover, STRIPAK complexes have been connected to clinical conditions, including cardiac disease, diabetes, autism, and cerebral cavernous malformation. In this review, we discuss the expression, localization, and protein domain structure of striatin family members. Then we consider the diverse complexes these proteins and their homologs form in various organisms, emphasizing what is known regarding function and regulation. Finally, we explore possible roles of striatin family complexes in disease, especially cerebral cavernous malformation.