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<p>Thrombosis is a common complication of chemotherapy for breast cancer patients. However, the specific mechanisms by which chemotherapy agents modulate these hemostatic pathways are not well understood. In this thesis, we investigated the mechanism(s) by which chemotherapy agents can upregulate procoagulant pathways (tissue factor (TF), phosphatidylserine exposure, and cell-free DNA (CFDNA) release) and impair the protein C (PC) anticoagulant pathway. We examined the effects of chemotherapy agents doxorubicin, epirubicin and the cyclophosphamide metabolite acrolein on cell surface procoagulant activity. We found that treatment of endothelial cells with the chemotherapy drugs increased phosphatidylserine exposure and TF activity on treated endothelial cells, blood monocytes and/or smooth muscle cells. This corresponded to an increase in thrombin generation on chemotherapy-treated cells exposed to recalcified, defibrinated plasma. We also found that found that doxorubicin and epirubicin can increase CFDNA release from breast cancer chemotherapy patients and healthy mice, which corresponds to an increase in thrombin-antithrombin levels. Treatment of venous whole blood and isolated neutrophils with doxorubicin and epirubicin increased CFDNA release. We found that exposure of recalcified plasma to CFDNA isolated from epirubicin-treated whole blood increased thrombin generation by activating the contact pathway. We investigated the effects of chemotherapy on the PC anticoagulant pathway. We found that acrolein decreased EPCR while increasing thrombomodulin expression on treated endothelial cells. A corresponding decrease in activated PC generation was measured on acrolein-treated endothelial cells exposed to recalcified, defibrinated plasma. Healthy mice treated with acrolein and cyclophosphamide increased PC antigen levels, but no measurable increase in plasma APC levels. Breast cancer chemotherapy drugs elevate thrombin generation by activating coagulation through the TF and contact pathways, and by promoting phosphatidylserine exposure, as well as by impairing PC activation EPCR expression. These studies provide insight into the mechanisms of breast cancer chemotherapy-induced hypercoagulation.</p> / Doctor of Philosophy (Medical Science)

Past studies of mammary tissue specific expression of transgenes using the murine whey acidic protein (WAP) promoter have shown widely variable, position-dependent and copy number-dependent expression. This study evaluates a series of three WAP transgenes containing the cDNA of human protein C (hPC) for the expression of human protein C in the milk of mice. In two of the transgenes studied, the cDNA of (hPC) was inserted at the translational start site of a 7.8 kbp mouse WAP genomic DNA Eco RI fragment containing 2.6 kbp of 5’ flanking, 3.9 kbp WAP coding (exons and introns), and 1.3 kbp 3’ untranslated region (UTR) and flanking sequences (designated WAPPC1 and WAPPC2). A third transgene consisted of only the 2.6 kbp of WAP 5’ UTR and flanking DNA, 1.4 kbp hPC cDNA, and 1.3 kbp of 3’ WAP UTR and flanking DNA with no linker sequences (designated WAPPC3). The WAPPC1 and WAPPC2 transgenes expressed up to about 10 μg/ml recombinant hPC in mouse milk while WAPPC3 expressed 30-300 (n=10, n=5, n=11, number of founder lines evaluated for each transgene, respectively). In contrast to past studies with WAP-cDNA fusion transgenes where the maximal expression was about 5% of endogenous WAP expression, the WAPPC3 transgene gave maximal expression which was about 30% of endogenous WAP expression. Thus, results from the combination in WAPPC3 of intact 5’ and 3’ WAP UTR with the cDNA of hPC suggests that introns are not necessary to enable high level expression in the mammary gland when using WAP regulatory elements. Relative specific transcript and protein levels in the transgenic animals studied suggest that the rates of translation initiation may be different for the mRNAs of each of the transgenes studied. / Ph. D.

The current findings indicate that activated protein C (aPC) can induce FOXP3 generation and regulatory T cells (Tregs) through epigenetic modulation and metabolic reprogramming. The current findings show that preincubation of T cells with aPC increased the frequency of Treg markers, CD4+FOXP3+ T cell frequency and suppressive function of T-cells, suggesting an increase of Tregs. The emergence of FOXP3 expression and Treg-like characteristics is linked to alterations in the epigenetic profile of T cells, characterized by a decline in overall DNA methylation, a decrease in the repressive histone marks H3K27me3 , and a reduced methylation of the FOXP3 promoter region. In addition, the induction of Tregs by aPC is accompanied by changes in mitochondrial metabolism. T lymphocytes that were preincubated with aPC exhibit a decline in mitochondrial respiration, a decrease in mitochondrial membrane potential, and a transition towards metabolic quiescence. The metabolic alterations are associated with a reduction of crucial metabolites, specifically α-ketoglutarate (αKG) and glutamine, that are known to regulate T cell function and epigenetic regulation. Reversal of the aPC-induced Treg-like phenotype and associated altered mitochondrial metabolism can be achieved through the supplementation of exogenous αKG or glutamine, supporting a functional interaction of these reduced metabolites and altered T-cell function. The current results show that chronically elevated levels of aPC in transgenic APChigh mice lead to a higher incidence of CD4+FOXP3+ Tregs in the spleen without alterations in thymic Tregs (tTregs), thereby indicating that aPC facilitates the development of Tregs in peripheral lymphoid organs but does not influence primary T-cell development. T cells derived from APChigh mice display a decrease in mitochondrial metabolism, which is consistent with the findings observed in vitro. Supplementation of αKG in cultured T cells derived from APChigh mice restores mitochondrial function and decreases the frequency of CD4+FOXP3+ Tregs. These findings support a model in which aPC alters T-cell metabolism, possibly by reducing expression of glucose (GLUT1) and glutamine (ASCT2 and SNAT1) transporters and thus the availability of metabolic substrates in T-cells. The reduced availability of these substates alters epigentic gene-expression and favors Treg development. In summary, the findings indicate that activated protein C (aPC) promotes the induction of regulatory T cells (Tregs) via metabolic modulation and altered epigenetic gene-expression. The results provide new insights into the long-lasting effects of aPC and indicate the possibility of aPC as a therapeutic target for regulating immune responses and fostering immune tolerance in diverse pathologies. Additional research is necessary to investigate the potential therapeutic applications.

Despite medical advances in the last decades, diabetic kidney disease (DKD) remains a major therapeutic challenge. DKD, the major microvascular complication in diabetic patients, is the most common cause of chronic kidney disease (CKD) and end-stage kidney disease (ESKD) requiring dialysis worldwide. In recent years, new therapeutic approaches for the treatment of DKD in addition to stringent blood glucose control and inhibition of angiotensin-signaling have been established. These new therapeutic options include sodium glucose cotransporter-2 inhibitors (SGLT2i), GLP-1 agonists, and nonsteroidal mineralocorticoid receptor antagonists. Despite their promising positive outcomes regarding kidney function, their long-term effects through the course of the disease remain unknown. Additionally, recent data show that about 50% of patients with DKD do not respond to these new therapeutics even if given in combination and there is a lack of therapies that can reverse the already established DKD. The continuous progression of diabetic complications (including DKD) despite normalization of blood glucose levels is referred to as the hyperglycemic memory. This phenomenon represents an unsolved medical problem in the field of diabetes management and remains without specific treatment options. Several theories have been proposed to explain the persistence of hyperglycemia-induced cellular dysfunction despite blood glucose normalization, and epigenetic regulation of gene expression has been proposed as the key pathomechanism driving the hyperglycemic memory. In this study, we used a combination of animal models, analyanalysesuman tissue and biofluid samples, in addition to in vitro mechanistic studies to identify therapeutically targetable pathways for the hyperglycemic memory in the context of DKD. To experimentally address the hyperglycemic memory, we used a model of hyperglycemia reversal in type-1 (STZ) and type-2 (db/db) diabetic mice. Hyperglycemia was reversed using SGLT2i (STZ and db/db mice) or insulin (STZ mice). We started by characterizing the functional and histological changes associated with hyperglycemic memory including persistent albuminuria, tubular hypertrophy, and fibrosis. To identify the pathways associated with the hyperglycemic memory in both models, we conducted bulk RNA sequencing from the kidneys and identified a large number of genes that were persistently up- or downregulated despite blood glucose normalization and hence possibly contributing to the hyperglycemic memory. Among these genes, we identified the cyclin-dependent kinase inhibitor p21 (Cdkn1a), which is known to regulate cellular senescence, as a primary candidate associated with the hyperglycemic memory, where its expression remained persistently upregulated despite blood glucose lowering in both diabetes models. Further investigations confirmed the “memorized” expression of p21 across DKD animal models and in cell lines in vitro, pinpointing the tubular epithelium as the specific cell type where this phenomenon occurs. Using a multimarker approach, we identified a persistent tubular senescence phenotype associated with p21 induction regardless of the intervention to reduce blood glucose levels in murine DKD models. Subsequent analyses aimed to scrutinize the relevance of this finding in the context of human DKD. We found induction of tubular p21 expression and senescence in human DKD biopsies compared to controls or to diabetic patients without kidney dysfunction. Furthermore, p21 was readily detected in the urine of DKD patients in a large cross-sectional cohort whic,h was increased with the severity of the disease and was negatively correlated with kidney function. Interestingly, urinary p21 levels remained persistently elevated despite different interventions to reduce blood glucose levels (SGLT2i or fasting-mimicking diet), corroborating its utility as a biomarker for the hyperglycemic memory in DKD. Next, we further elucidated the sequence of eventeventshyperglycemia to the induction of p21 and subsequent kidney damage, demonstrating that elevated blood glucose decreases DNA methyltransferase 1 (DNMT1) expression, resulting in hypomethylation of the p21 promoter and increased p21 expression. The induction of p21 expression triggers a senescence phenotype, that contributes to tubular damage and fibrosis. Suppression of tubular DNMT1 expression was sufficient to induce p21 in tubular cells, corroborating the importance of this epigenetic mechanism for the glucose-induced persistence of p21 expression. In order to investigate possible translational implications, we studied the potential of reversing the hyperglycemic memory in DKD. We employed the cytoprotective protease activated protein C (aPC), a disease resolving mediator associated with DKD protection. aPC in conjunction with the blood glucose lowering drug SGLT2i induced the expression of DNMT1 leading to promoter remethylation and suppression of the persistent tubular p21 expression. Notably, SGLT2i alone had no effect on p21 expression. The combined action of SGLT2i and aPC effectively counteracted albuminuria, tubular damage, senescence, and fibrosis in a murine DKD model. The protective effects of aPC were confirmed by investigating TMPro/Pro mice, in which thrombomodulin-mediated protein C activation is hampered resulting in reduced aPC levels. Induction of persistent hyperglycemia in these mice elevated p21 expression in association with aggravated kidney damage compared to wild-type mice. Superimposed deficiency of p21 in the aforementioned aPC-deficient mice (TMPro/Pro x p21-/-) alleviated the tubular injury and senescence phenotype, providing experimental in vivo evidence for a regulation of the hyperglycemic memory in DKD by the interaction of p21 by aPC levels. To confirm that aPC regulates DNMT1 and hence p21 in an epigenetic manner, we targeted DNMT1 in aPC-treated mice using the pan DNMT inhibitor 5-aza-2'-deoxycytidine or a specific vivo morpholino against DNMT1. Both approaches abolished aPC’s protective effects in a murine DKD model and inhibited its ability to reduce p21 promoter hypomethylation and hence its expression. Exploiting the cytoprotective properties of aPC by using the mutant 3K3A-aPC, which lacks the anticoagulant ability, or the chemical compound parmodulin-2 that mimics the biased signaling of aPC via the G protein-coupled receptor PAR1, was enough to reduce the hyperglycemia-induced p21 expression and the associated tubular senescence in murine DKD. The findings of this study uncover an important role of p21 in exacerbating renal damage under diabetic conditions, suggesting that p21 not only serves as a marker of cellular senescence but actively contributes to the persistence of DKD by mediating the hyperglycemic memory. By addressing the root causes of hyperglycemic memory, such as the epigenetic modifications that perpetuate p21 expression, it may be possible to halt or even reverse the progression of DKD. The feasibility of this approach was demonstrated by exploiting cytoprotective aPC signaling, which restored DNMT1 expression and reduced p21 expression. Thus, targeting the hyperglycemic memory in DKD may be feasible in general and may be specifically achieved by targeting cytoprotective aPC signaling.:Table of contents Table of contents 2 List of figures 5 List of tables 6 List of abbreviations 7 1. Introduction 9 1.1 Diabetes mellitus 9 1.2 Diabetic complications 9 1.3 Diabetic kidney disease (DKD) 10 1.3.1 Clinical presentation and staging of DKD patients 10 1.3.2 Cellular dysfunction in DKD 12 1.4 The hyperglycemic memory: a new challenge in DM management 14 1.4.1 Hyperglycemic memory in DKD 14 1.4.2 Mechanisms of the hyperglycemic memory 18 1.5 Cellular senescence in DKD 20 1.5.1 Features of senescent cells in DKD 20 1.5.2 Tubular cell senescence in DKD 23 1.6 Therapeutic management of DKD 23 1.6.1 SGLT2 inhibitors 24 1.6.2 Other therapeutic options for DKD 25 1.7 Coagulation proteases and their receptors in DKD 26 1.7.1 Protease-activated receptors (PARs) 26 1.7.2 Activated protein C (aPC) 27 2. Aim of the study 32 3. Methods 33 3.1 Reagents 33 3.2 Mice and in vivo interventions 34 3.3 Cell culture and in vitro interventions 35 3.4 Human renal biopsies and urine samples 36 3.5 Glucose uptake assay 41 3.6 In vitro Knockdown 41 3.7 Preparation of activated protein C 42 3.8 Urine collection and processing 42 3.9 p21 ELISA for human urine samples 43 3.10 Albuminuria and in mouse urine samples 43 3.11 Methylation specific PCR (MSP) 43 3.12 Pyrosequencing 44 3.13 DNMT activity assay 44 3.14 Immunoblotting 45 3.15 Reverse transcriptase PCR (RT-PCR) 45 3.16 Quantitative real time PCR (qRT-PCR) 46 3.17 RNA expression profiling 48 3.18 Functional annotation and Pathway analysis 48 3.19 Histology, immunohistochemistry and histological analyses 49 3.20 Immunofluorescence 49 3.21 Senescence associated beta galactosidase (SA-β-gal.) staining 50 3.22 Plasma creatinine and blood urea nitrogen (BUN) 50 3.23 Cell cycle analysis 50 3.24 Statistical Analysis 51 4. Results 52 4.1 Blood glucose normalization does not reverse DKD in experimental models of DM 52 4.2 Identification of genes and pathways associated with hyperglycemic memory 54 4.3 Sustained renal tubular p21 induction in experimental DKD models despite blood glucose normalization 56 4.4 Tubular p21 induction is independent on glomerular damage 58 4.5 Sustained renal tubular p21 expression is associated with induction of senescence 59 4.6. Induction of renal tubular p21 expression in human DKD patients is associated with kidney dysfunction 61 4.7. Sustained p21 expression despite glucose normalization in human DKD patients 63 4.8 aPC reverses glucose induced p21 promoter methylation and sustained p21 expression 64 4.9 Impaired protein C activation increases tubular p21 expression and senescence in vivo 66 4.10 p21 mediates enhanced tubular senescence in aPC-deficient mice 68 4.11 High glucose differentially regulates renal DNMTs expression and activity 70 4.12 Hyperglycemia-induced DNMT1 suppression is part of the hyperglycemic memory 72 4.13 aPC reverses glucose-induced and sustained renal p21 expression via DNMT1 in vivo 74 4.14 aPC reverses glucose-induced and sustained renal tubular senescence via DNMT1 in vivo 76 4.15 aPC requires PAR1 and EPCR to regulate p21 expression 78 4.16 aPC regulates the epigenetically sustained p21 expression independent of its anticoagulant function in vivo 79 4.17 aPC enhances the regenerative capacity of DM kidneys after acute injury by reversing the hyperglycemic memory 81 5. Discussion 85 6. Future perspectives 92 7. Summary of the work 93 8. References 96 Declaration on the independent preparation of the dissertation 104 Curriculum Vitae 106 List of publications 107 Acknowledgement 109

Thesis (PhD ) -- Stellenbosch University, 2011. / Bibliography / ENGLISH ABSTRACT: The regulation of cardiac contractility is dependent on cooperative interaction between the thick and thin filaments, as well as their accessory proteins, within the cardiac sarcomere. Alteration in cardiac contractility due to a defective sarcomere typically results in cardiomyopathies, such as hypertrophic cardiomyopathy (HCM). One of the sarcomeric genes frequently mutated and which accounts for the second most common form of HCM encodes cardiac myosin binding protein C (cMyBPC), a thick filament accessory protein whose physiological function is poorly understood. However, studies have implicated cMyBPC in thick filament structure and function as well as in the regulation of contractility. The N-terminal region of cMyBPC houses the cMyBPC-motif, which contains three phosphorylation sites, between domains C1 and C2. The hierarchical phosphorylation of this motif, by first calcium/calmodulin kinase II (CamKII) and then by cyclic AMP-activated protein kinase (PKA), is cardinal in the role of cMyBPC in the regulation of cardiac contractility in response to ß-adrenergic stimulation. Moreover, phosphorylation of this motif is inversely correlated to cMyBPC proteolysis and has been shown to be cardioprotective. Thus, proteins that have an effect on cMyBPC function or turnover may also influence filament structure and hence affect contractility, which, in turn, affects the structure of the cardiac muscle. One such protein is the Copper metabolism MURR1-domain containing protein 4 (COMMD4), which was previously identified as a novel interactor of cMyBPC during a yeast two-hybrid (Y2H) library screen in our laboratory. COMMD4 binds specifically to the cMyBPC motif in a phosphorylation-dependent manner. The exact function of COMMD4 is unknown; however, it is a member of the COMM family of proteins that has been linked to copper metabolism as well as to the ubiquitin-proteasome pathway (UPS). Intriguingly, recent studies have shown that the UPS plays a role in cMyBPC-derived HCM, while dietary copper depletion is also known to cause cardiac hypertrophy. Based on these findings, COMMD4 was considered an interesting candidate regulator of sarcomeric function and contractility, and by extension, a candidate modifier of cardiac hypertrophy. Thus, the aim of the present study was two-fold. Firstly, COMMD4 was used as bait in a Y2H library screen to determine its distal ligands, with a view to further elucidate its function, particularly in the context of MyBPC functioning, and identified interactors were subjected to further in vitro and in vivo verification studies. Also, the phosphorylation-dependent nature of the interaction between COMMD4 and cMyBPC was further investigated using a domain/phosphorylation assay. Secondly, COMMD4 and its Y2H-identified putative interactors were assessed as possible modifiers of hypertrophy in a family-based association study, using three cohorts of South African HCM-families in which one of three founder mutations segregate. Six putative interactors, viz. cardiac actin (ACTC1), Down syndrome critical region 3 (DSCR3), enolase 1 (ENO1), F-box and leucine rich repeat protein 10 (FBXL10), legumain (LGMN) and sorting nexin3 (SNX3) were identified and confirmed as COMMD4 interactors using Y2H analyses, followed by in vitro and in vivo co-immunoprecipitation and 3D co-localisation assays. Moreover, as some COMMD protein family members and the newly-identified interactors of COMMD4 have previously been linked to the UPS, the functional effect of siRNA-mediated knockdown of COMMD4 on cMyBPC turnover was also investigated. Data revealed accumulation of cMyBPC in the endosomes upon COMMD4 knockdown, suggesting a functional role for COMMD4 in the turnover of cMyBPC. In addition, association analysis revealed strong evidence of association between various single nucleotide polymorphisms (SNPs) in SNX3 and a number of hypertrophy traits, thus suggesting a role for SNX3 as a candidate modifier of hypertrophy in HCM. No evidence of association was observed for any of the genes encoding the other COMMD4 interactors implicated in protein turnover. The present study demonstrates that COMMD4, a little understood member of the COMM family of proteins, binds to the cMyBPC motif of cMyBPC in a phosphorylation-dependent manner. Furthermore, based on the functions of its protein interactions, we hypothesise that COMMD4 plays a role in protein trafficking and turnover. More specifically, COMMD4 seems to help to facilitate formation of protein complexes with the Skp1-Cul1-Fbxl (SCF) E3 ubiquitin ligase and probably helps to stabilise the target substrate for subsequent ubiquitin-conjugation. As COMMD4 seems to affect the protein turnover of cMyBPC and possibly other sarcomeric proteins, such as actin, these results establish a novel association between the sarcomere, HCM and the UPS. In addition, identification of SNX3 as a hypertrophy modifier will allow for the improved understanding of HCM patho-aetiology. SNX3 thus adds to the growing body of sarcomeric modifier genes, which, eventually, may improve risk profiling in HCM. Furthermore, as genetic modifiers appear sufficient to completely prevent disease expression in some HCM carriers, the identification of SNX3 may point to the protein turnover pathway as a potential new target for intervention. / AFRIKAANSE OPSOMMING: Die regulering van kardiale kontraktiliteit is afhanklik van die koöperatiewe interaksie tussen die dik en dun filamente, asook hul geassosieerde proteïene, in die kardiale sarkomeer. Veranderinge in kardiale kontraktiliteit as gevolg van 'n defektiewe sarkomeer lei tot kardiomiopatieë soos hipertrofiese kardiomiopatie (HKM). Een van die sarkomeriese gene wat dikwels gemuteer is en wat verantwoordelik is vir die tweede algmeenste vorm van HKM,is dié van kardiale miosien-bindingsproteïen C (cMyBPC),'n proteïen geassosieer met die dik filament waarvan die fisiologiese funksie nog nie goed bekend is nie. Studies betrek cMyBPC in dik filament struktuur en funksie asook in die regulering van kontraktiliteit. Die N-terminale gebied van cMyBPC huisves die cMyBPC-motief, wat drie fosforilerings-setels tussen domeine C1 en C2 bevat. Die hiërargiese fosforilering van hierdie motief, eerstens deur kalsium/kalmodulien-gereguleerde kinase II (CamKII), gevolg deur siklies AMP-geaktiveerde proteïen kinase (PKA), is kardinaal in die rol van cMyBPC in die regulering van kardiale kontraktiliteit in reaksie op ß-adrenergiese stimulasie. Verder, fosforilering van hierdie motief is omgekeerd gekorreleer aan cMyBPC proteolise en is ook bewys om kardiobeskermend te wees. Dus, proteïene wat 'n uitwerking het op die funksie van cMyBPC mag ook filament struktuur en kontraktiliteit beïnvloed, wat op hul beurt die struktuur van die kardiale spier affekteer. Die koper metabolisme MURR1-domein bevattende protein 4 (COMMD4), was voorheen geïdentifiseer as 'n nuwe bindingsgenoot van cMyBPC tydens gis twee-hibried (G2H) analise in ons laboratorium. COMMD4 bind spesifiek aan die cMyBPC motief in 'n fosforilasie afhanklike wyse. Die presiese funksie van COMMD4 is onbekend; maar dit is 'n lid van die COMM domein familie van proteine wat geassosieerd is met koper metabolisme sowel as die “ubiquitin” proteosoom pad (UPP). Interesant genoeg, onlangse studies het bewys dat die UPP 'n rol speel in cMyBPC-afgeleide HKM, terwyl koper uitputting in die dieet ook bekend is om kardiale hipertrofie te veroorsaak. Gebaseer op hierdie bevindinge was COMMD4 oorweeg as 'n interessante kandidaat reguleerder van sarkomeries funksie en kontraktiliteit, asook 'n kandidaat modifiseerder van kardiale hipertrofie. Dus, die doel van die huidige studie was tweeledig. Eerstens, was COMMD4 as aas gebruik in 'n G2H biblioteek sifting om sy distale ligande te bepaal, met die oog om verdere lig te werp op sy funksie, veral in die konteks van MyBPC funksionering, en geïdentifiseerde bindingsgenote was onderwerp aan verdere 'in vitro’ en 'in vivo’ verifikasie studies. Daarbenewens was die fosforilering-afhanklike aard van die interaksie tussen COMMD4 en cMyBPC verder ondersoek met behulp van 'n domein/fosforilasie toets. Tweedens, COMMD4 en sy G2H-geïdentifiseerde vermeende bindingsgenote was geassesseer as moontlik modifiseerders van hipertrofie in 'n familie-gebaseerde assosiasie studie, met behulp van drie kohorte van Suid-Afrikaanse HKM-families waarin een van die drie stigter mutasies segregeer. Ses vermeende interaktors, nl. kardiale aktien (ACTC1), Down-sindroom kritiese streek 3 (DSCR3), enolase 1 (ENO1), F-boks en leusien ryke herhalings proteïen 10 (FBXL10), legumain (LGMN) en sorteer nexin3 (SNX3) is geïdentifiseer en bevestig as COMMD4 bindingsgenote deur G2H analises, gevolg deur in vitro en in vivo ko-immunopresipitasie en 3D ko-lokalisasie toetse. Die funksionele effek van siRNA-bemiddelde uitklop van COMMD4 op cMyBPC omset was ook ondersoek omdat 'n paar COMMD proteïen familielede, asook die nuut-geïdentifiseerde bindingsgenote van COMMD4, geassosieerd is met die UPP. Data toon ophoping van cMyBPC in die endosome by COMMD4 uitklop, wat dus aandui op 'n funksionele rol vir COMMD4 in die omset van cMyBPC. Daarbenewens, toon assosiasie analise sterk bewyse van assosiasie tussen die verskillende enkele nukleotied polimorfismes (SNPs) in SNX3 en 'n aantal hipertrofiese kenmerke,wat aandui op 'n rol vir SNX3 as 'n kandidaat modifiseerder van hipertrofie in HKM. Geen bewyse van assosiasie was waargeneem vir enige van die gene wat kodeer vir die ander COMMD4 bindingsgenote wat geïmpliseer word in die proteïen omset. Die huidige studie toon dat COMMD4, 'n min verstaande lid van die COMM familie van proteïene, aan die cMyBPC motief van cMyBPC in'n fosforilasie-afhanklike wyse bind. Verder, gebasseer op die funksies van die proteïen interaksies, hipotiseer ons dat COMMD4 'n rol speel in proteïen vervoer en omset. Meer spesifiek, COMMD4 blyk om die vorming van proteïene komplekse met die Skp1-Cul1-Fbxl (SCF) E3 "ubiquiti". ligase te fasiliteer en help waarskynlik om die teiken-substraat vir die daaropvolgende ubiquitin-konjugasie te stabiliseer. Omdat dit lyk asof COMMD4 die proteïen-omset van cMyBPC en moontlik ander sarkomeriese proteïene, soos aktien, ook beïnvloed, vestig die resultate dus 'n nuwe assosiasie tussen die sarkomeer, HKM en die UPP. Daarbenewens sal die identifisering van SNX3 as 'n hipertrofie modifiseerder voorsiening maak vir die verbeterde begrip van HKM pato-etiologie. SNX3 voeg dus by tot die groeiende ?getal van sarkomeriese modifiseerende gene, wat uiteindelik, die risiko-ontleding in HKM mag verbeter. Verder, omdat dit blyk dat genetiese modifiseerders voldoende is om die siekte-uitdrukking heeltemal te verhoed in sekere HKM draers, kan die identifikasie van SNX3 na die proteïen-omset roete dui as 'n potensiële nuwe teiken vir intervensie.