Endoglin mutants retained in the endoplasmic reticulum exacerbate loss of function in hereditary hemorrhagic telangiectasia type 1 (HHT1) by exerting dominant negative effects on the wild type allele.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder affecting 1 in 5000-8000 individuals. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is the most common HHT and manifests as diverse vascular malformations ranging from mild symptoms such as epistaxis and mucosal and cutaneous telangiectases to severe arteriovenous malformations (AVMs) in the lungs, brain or liver. HHT1 is caused by heterozygous mutations in the ENG gene, which encodes endoglin, the TGFß homodimeric co-receptor. It was previously shown that some endoglin HHT1-causing variants failed to traffic to the plasma membrane due to their retention in the endoplasmic reticulum (ER) and consequent degradation by ER-associated degradation (ERAD). Endoglin is a homodimer formed in the ER, and we therefore hypothesized that mixed heterodimers might form between ER-retained variants and WT protein, thus hampering its maturation and trafficking to the plasma membrane causing dominant negative effects. Indeed, HA-tagged ER-retained mutants formed heterodimers with Myc-tagged WT endoglin. Moreover, variants L32R, V105D, P165L, I271N and C363Y adversely affected the trafficking of WT endoglin by reducing its maturation and plasma membrane localization. These results strongly suggest dominant negative effects exerted by these ER-retained variants aggravating endoglin loss of function in patients expressing them in the heterozygous state with the WT allele. Moreover, this study may help explain some of the variability observed among HHT1 patients due to the additional loss of function exerted by the dominant negative effects in addition to that due to haploinsufficiency. These findings might also have implications for some of the many conditions impacted by ERAD.

Genetic disruption of mammalian endoplasmic reticulum-associated protein degradation: Human phenotypes and animal and cellular disease models.

Endoplasmic reticulum-associated protein degradation (ERAD) is a stringent quality control mechanism through which misfolded, unassembled and some native proteins are targeted for degradation to maintain appropriate cellular and organelle homeostasis. Several in vitro and in vivo ERAD-related studies have provided mechanistic insights into ERAD pathway activation and its consequent events; however, a majority of these have investigated the effect of ERAD substrates and their consequent diseases affecting the degradation process. In this review, we present all reported human single-gene disorders caused by genetic variation in genes that encode ERAD components rather than their substrates. Additionally, after extensive literature survey, we present various genetically manipulated higher cellular and mammalian animal models that lack specific components involved in various stages of the ERAD pathway.

Oxidation-reduction imaging of myoglobin reveals two-phase oxidation in the reperfused myocardium.

Myocardial infarction (MI) is a serious acute cardiovascular syndrome that causes myocardial injury due to blood flow obstruction to a specific myocardial area. Under ischemic-reperfusion settings, a burst of reactive oxygen species is generated, leading to redox imbalance that could be attributed to several molecules, including myoglobin. Myoglobin is dynamic and exhibits various oxidation-reduction states that have been an early subject of attention in the food industry, specifically for meat consumers. However, rarely if ever have the myoglobin optical properties been used to measure the severity of MI. In the current study, we develop a novel imaging pipeline that integrates tissue clearing, confocal and light sheet fluorescence microscopy, combined with imaging analysis, and processing tools to investigate and characterize the oxidation-reduction states of myoglobin in the ischemic area of the cleared myocardium post-MI. Using spectral imaging, we have characterized the endogenous fluorescence of the myocardium and demonstrated that it is partly composed by fluorescence of myoglobin. Under ischemia-reperfusion experimental settings, we report that the infarcted myocardium spectral signature is similar to that of oxidized myoglobin signal that peaks 3 h post-reperfusion and decreases with cardioprotection. The infarct size assessed by oxidation-reduction imaging at 3 h post-reperfusion was correlated to the one estimated with late gadolinium enhancement MRI at 24 h post-reperfusion. In conclusion, this original work suggests that the redox state of myoglobin can be used as a promising imaging biomarker for characterizing and estimating the size of the MI during early phases of reperfusion.

The double whammy of ER-retention and dominant-negative effects in numerous autosomal dominant diseases: significance in disease mechanisms and therapy.

The endoplasmic reticulum (ER) employs stringent quality control mechanisms to ensure the integrity of protein folding, allowing only properly folded, processed and assembled proteins to exit the ER and reach their functional destinations. Mutant proteins unable to attain their correct tertiary conformation or form complexes with their partners are retained in the ER and subsequently degraded through ER-associated protein degradation (ERAD) and associated mechanisms. ER retention contributes to a spectrum of monogenic diseases with diverse modes of inheritance and molecular mechanisms. In autosomal dominant diseases, when mutant proteins get retained in the ER, they can interact with their wild-type counterparts. This interaction may lead to the formation of mixed dimers or aberrant complexes, disrupting their normal trafficking and function in a dominant-negative manner. The combination of ER retention and dominant-negative effects has been frequently documented to cause a significant loss of functional proteins, thereby exacerbating disease severity. This review aims to examine existing literature and provide insights into the impact of dominant-negative effects exerted by mutant proteins retained in the ER in a range of autosomal dominant diseases including skeletal and connective tissue disorders, vascular disorders, neurological disorders, eye disorders and serpinopathies. Most crucially, we aim to emphasize the importance of this area of research, offering substantial potential for understanding the factors influencing phenotypic variability associated with genetic variants. Furthermore, we highlight current and prospective therapeutic approaches targeted at ameliorating the effects of mutations exhibiting dominant-negative effects. These approaches encompass experimental studies exploring treatments and their translation into clinical practice.

Characterization of ACE2 naturally occurring missense variants: impact on subcellular localization and trafficking.

BACKGROUND: Human angiotensin-converting enzyme 2 (ACE2), a type I transmembrane receptor physiologically acting as a carboxypeptidase enzyme within the renin-angiotensin system (RAS), is a critical mediator of infection by several severe acute respiratory syndrome (SARS) corona viruses. For instance, it has been demonstrated that ACE2 is the primary receptor for the SARS-CoV-2 entry to many human cells through binding to the viral spike S protein. Consequently, genetic variability in ACE2 gene has been suggested to contribute to the variable clinical manifestations in COVID-19. Many of those genetic variations result in missense variants within the amino acid sequence of ACE2. The potential effects of those variations on binding to the spike protein have been speculated and, in some cases, demonstrated experimentally. However, their effects on ACE2 protein folding, trafficking and subcellular targeting have not been established. RESULTS: In this study we aimed to examine the potential effects of 28 missense variants (V801G, D785N, R768W, I753T, L731F, L731I, I727V, N720D, R710H, R708W, S692P, E668K, V658I, N638S, A627V, F592L, G575V, A501T, I468V, M383I, G173S, N159S, N149S, D38E, N33D, K26R, I21T, and S19P) distributed across the ACE2 receptor domains on its subcellular trafficking and targeting through combinatorial approach involving in silico analysis and experimental subcellular localization analysis. Our data show that none of the studied missense variants (including 3 variants predicted to be deleterious R768W, G575V, and G173S) has a significant effect on ACE2 intracellular trafficking and subcellular targeting to the plasma membrane. CONCLUSION: Although the selected missense variants display no significant change in ACE2 trafficking and subcellular localization, this does not rule out their effect on viral susceptibility and severity. Further studies are required to investigate the effect of ACE2 variants on its expression, binding, and internalization which might explain the variable clinical manifestations associated with the infection.

Pharmacogenomics implementation in cardiovascular disease in a highly diverse population: initial findings and lessons learned from a pilot study in United Arab Emirates.

BACKGROUND: Pharmacogenomic (PGx) testing has proved its utility and cost-effectiveness for some commonly prescribed cardiovascular disease (CVD) medications. In addition, PGx-guided dosing guidelines are now available for multiple CVD drugs, including clopidogrel, warfarin, and statins. The United Arab Emirates (UAE) population is diverse and multiethnic, with over 150 nationalities residing in the country. PGx-testing is not part of the standard of care in most global healthcare settings, including the UAE healthcare system. The first pharmacogenomic implementation clinical study in CVD has been approved recently, but multiple considerations needed evaluation before commencing. The current report appraises the PGx-clinical implementation procedure and the potential benefits of pursuing PGx-implementation initiatives in the UAE with global implications. METHODS: Patients prescribed one or more of the following drugs: clopidogrel, atorvastatin, rosuvastatin, and warfarin, were recruited. Genotyping selected genetic variants at genes interacting with the study drugs was performed by real-time PCR. RESULTS: For the current pilot study, 160 patients were recruited. The genotypes and inferred haplotypes, diplotypes, and predicted phenotypes revealed that 11.9% of the participants were poor CYP2C19 metabolizers, 35% intermediate metabolizers, 28.1% normal metabolizers, and 25% rapid or ultrarapid metabolizers. Notably, 46.9% of our cohort should receive a recommendation to avoid using clopidogrel or consider an alternative medication. Regarding warfarin, only 20% of the participants exhibited reference alleles at VKORC1-1639G > A, CYP2C9*2, and CYP2C9*3, leaving 80% with alternative genotypes at any of the two genes that can be integrated into the warfarin dosing algorithms and can be used whenever the patient receives a warfarin prescription. For statins, 31.5% of patients carried at least one allele at the genotyped SLCO1B1 variant (rs4149056), increasing their risk of developing myopathy. 96% of our cohort received at least one PGx-generated clinical recommendation for the studied drugs. CONCLUSION: The current pilot analysis verified the feasibility of PGx-testing and the unforeseen high frequencies of patients currently treated with suboptimal drug regimens, which may potentially benefit from PGx testing.

ACE2 Nascence, trafficking, and SARS-CoV-2 pathogenesis: the saga continues.

With the emergence of the novel coronavirus SARS-CoV-2 since December 2019, more than 65 million cases have been reported worldwide. This virus has shown high infectivity and severe symptoms in some cases, leading to over 1.5 million deaths globally. Despite the collaborative and concerted research efforts that have been made, no effective medication for COVID-19 (coronavirus disease-2019) is currently available. SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) as an initial mediator for viral attachment and host cell invasion. ACE2 is widely distributed in the human tissues including the cell surface of lung cells which represent the primary site of the infection. Inhibiting or reducing cell surface availability of ACE2 represents a promising therapy for tackling COVID-19. In this context, most ACE2-based therapeutic strategies have aimed to tackle the virus through the use of angiotensin-converting enzyme (ACE) inhibitors or neutralizing the virus by exogenous administration of ACE2, which does not directly aim to reduce its membrane availability. However, through this review, we present a different perspective focusing on the subcellular localization and trafficking of ACE2. Membrane targeting of ACE2, and shedding and cellular trafficking pathways including the internalization are not well elucidated in literature. Therefore, we hereby present an overview of the fate of newly synthesized ACE2, its post translational modifications, and what is known of its trafficking pathways. In addition, we highlight the possibility that some of the identified ACE2 missense variants might affect its trafficking efficiency and localization and hence may explain some of the observed variable severity of SARS-CoV-2 infections. Moreover, an extensive understanding of these processes is necessarily required to evaluate the potential use of ACE2 as a credible therapeutic target.

An innovative sequence of hypoxia-reoxygenation on adult mouse cardiomyocytes in suspension to perform multilabeling analysis by flow cytometry.

Cardiovascular diseases remain the leading cause of death worldwide. Although major therapeutic progress has been made during the past decades, a better understanding of the underlying mechanisms will certainly help to improve patient's prognosis. In vitro models, particularly adult mouse cardiomyocytes, have been largely used; however, their fragility and large size are major obstacles to the use of flow cytometry. Conventional techniques, such as cell imaging, require the use of large numbers of animals and are time consuming. Here, we described a new, simple, and rapid one-day protocol using living adult mouse cardiomyocytes in suspension exposed to hypoxia-reoxygenation that allows a multilabeling analysis by flow cytometry. Several parameters can be measured by fluorescent probes labeling to assess cell viability (propidium iodide, calcein-AM, and Sytox Green), mitochondrial membrane potential [DilC1(5) and TMRM], reactive oxygen species production (MitoSOX Red), and mitochondrial mass (MitoTracker Deep Red). We address the robustness and sensitivity of our model using a cardioprotective agent, cyclosporine A. Overall, our new experimental set-up offers a high-speed quantitative multilabeling analysis of adult mouse cardiomyocytes exposed to hypoxia-reoxygenation. Our model might be interesting to investigate other cellular stresses (oxidative and inflammation) or to perform pharmacological screening.

Critical appraisal of STAT3 pattern in adult cardiomyocytes.

The signal transducer and activator of transcription 3, STAT3, transfers cellular signals from the plasma membrane to the nucleus, acting as a signaling molecule and a transcription factor. Reports proposed an additional non-canonical role of STAT3 that could regulate the activity of complexes I and II of the electron transport chain and the opening of the mitochondrial permeability transition pore (PTP) after ischemia-reperfusion in various cell types. The native expression of STAT3 in heart mitochondria, together with a direct versus an indirect transcriptional role in mitochondrial functions, have been recently questioned. The objective of the present study was to investigate the cellular distribution of STAT3 in mouse adult cardiomyocytes under basal and stress conditions, along with assessing its presence and activity in cardiac mitochondria using structural and functional approaches. The analysis of the spatial distribution of STAT3 signal in the cardiomyocytes interestingly showed that it is transversely distributed along the T-tubules and in the nucleus. This distribution was neither affected by hypoxia nor by hypoxia/re­oxygenation conditions. Focusing on the mitochondrial STAT3 localization, our results suggest that serine-phosphorylated STAT3 (PS727-STAT3) and total STAT3 are detected in crude but not in pure mitochondria of mouse adult cardiomyocytes, under basal and ischemia-reperfusion conditions. The inhibition of STAT3, with a pre-validated non-toxic Stattic dose, had no significant effects on mitochondrial respiration, but a weak effect on the calcium retention capacity. Overall, our results exclusively reveal a unique cellular distribution of STAT3 in mouse adult cardiomyocytes, along the T-tubules and in nucleus, under different conditions. They also challenge the expression and activity of STAT3 in mitochondria of these cells under basal conditions and following ischemia-reperfusion. In addition, our results underline technical methods, complemental to cell fractionation, to evaluate STAT3 roles during hypoxia-reoxygenation and at the interface between nucleus and endoplasmic reticulum.

TRPV1 Channels Are New Players in the Reticulum-Mitochondria Ca2+ Coupling in a Rat Cardiomyoblast Cell Line.

The Ca2+ release in microdomains formed by intercompartmental contacts, such as mitochondria-associated endoplasmic reticulum membranes (MAMs), encodes a signal that contributes to Ca2+ homeostasis and cell fate control. However, the composition and function of MAMs remain to be fully defined. Here, we focused on the transient receptor potential vanilloid 1 (TRPV1), a Ca2+-permeable ion channel and a polymodal nociceptor. We found TRPV1 channels in the reticular membrane, including some at MAMs, in a rat cardiomyoblast cell line (SV40-transformed H9c2) by Western blotting, immunostaining, cell fractionation, and proximity ligation assay. We used chemical and genetic probes to perform Ca2+ imaging in four cellular compartments: the endoplasmic reticulum (ER), cytoplasm, mitochondrial matrix, and mitochondrial surface. Our results showed that the ER Ca2+ released through TRPV1 channels is detected at the mitochondrial outer membrane and transferred to the mitochondria. Finally, we observed that prolonged TRPV1 modulation for 30 min alters the intracellular Ca2+ equilibrium and influences the MAM structure or the hypoxia/reoxygenation-induced cell death. Thus, our study provides the first evidence that TRPV1 channels contribute to MAM Ca2+ exchanges.

Unveiling the pathogenic mechanisms of NPR2 missense variants: insights into the genotype-associated severity in acromesomelic dysplasia and short stature.

Introduction: Natriuretic peptide receptor 2 (NPR2 or NPR-B) plays a central role in growth development and bone morphogenesis and therefore loss-of-function variations in NPR2 gene have been reported to cause Acromesomelic Dysplasia, Maroteaux type 1 and short stature. While several hypotheses have been proposed to underlie the pathogenic mechanisms responsible for these conditions, the exact mechanisms, and functional characteristics of many of those variants and their correlations with the clinical manifestations have not been fully established. Methods: In this study, we examined eight NPR2 genetic missense variants (p.Leu51Pro, p.Gly123Val, p.Leu314Arg, p.Arg318Gly, p.Arg388Gln, p.Arg495Cys, p.Arg557His, and p.Arg932Cys) Acromesomelic Dysplasia, Maroteaux type 1 and short stature located on diverse domains and broadly classified as variants of uncertain significance. The evaluated variants are either reported in patients with acromesomelic dysplasia in the homozygous state or short stature in the heterozygous state. Our investigation included the evaluation of their expression, subcellular trafficking and localization, N-glycosylation profiles, and cyclic guanosine monophosphate (cGMP) production activity. Results and Discussion: Our results indicate that variants p.Leu51Pro, p.Gly123Val, p.Leu314Arg, p.Arg388Gln have defective cellular trafficking, being sequestered within the endoplasmic reticulum (ER), and consequently impaired cGMP production ability. Conversely, variants p.Arg318Gly, p.Arg495Cys, and p.Arg557His seem to display a non-statistically significant behavior that is slightly comparable to WT-NPR2. On the other hand, p.Arg932Cys which is located within the guanylyl cyclase active site displayed normal cellular trafficking profile albeit with defective cGMP. Collectively, our data highlights the genotype-phenotype relationship that might be responsible for the milder symptoms observed in short stature compared to acromesomelic dysplasia. This study enhances our understanding of the functional consequences of several NPR2 variants, shedding light on their mechanisms and roles in related genetic disorders which might also help in their pathogenicity re-classification.

ANT2-Mediated ATP Import into Mitochondria Protects against Hypoxia Lethal Injury.

Following a prolonged exposure to hypoxia-reoxygenation, a partial disruption of the ER-mitochondria tethering by mitofusin 2 (MFN2) knock-down decreases the Ca2+ transfer between the two organelles limits mitochondrial Ca2+ overload and prevents the Ca2+-dependent opening of the mitochondrial permeability transition pore, i.e., limits cardiomyocyte cell death. The impact of the metabolic changes resulting from the alteration of this Ca2+crosstalk on the tolerance to hypoxia-reoxygenation injury remains partial and fragmented between different field of expertise. >In this study, we report that MFN2 loss of function results in a metabolic switch driven by major modifications in energy production by mitochondria. During hypoxia, mitochondria maintain their ATP concentration and, concomitantly, the inner membrane potential by importing cytosolic ATP into mitochondria through an overexpressed ANT2 protein and by decreasing the expression and activity of the ATP hydrolase via IF1. This adaptation further blunts the detrimental hyperpolarisation of the inner mitochondrial membrane (IMM) upon re-oxygenation. These metabolic changes play an important role to attenuate cell death during a prolonged hypoxia-reoxygenation challenge.

A Dynamic Transcriptional Analysis Reveals IL-6 Axis as a Prominent Mediator of Surgical Acute Response in Non-ischemic Mouse Heart.

BACKGROUND: Ischemic heart diseases are a major cause of death worldwide. Different animal models, including cardiac surgery, have been developed over time. Unfortunately, the surgery models have been reported to trigger an important inflammatory response that might be an effect modifier, where involved molecular processes have not been fully elucidated yet. OBJECTIVE: We sought to perform a thorough characterization of the sham effect in the myocardium and identify the interfering inflammatory reaction in order to avoid misinterpretation of the data via systems biology approaches. METHODS AND RESULTS: We combined a comprehensive analytical pipeline of mRNAseq dataset and systems biology analysis to characterize the acute phase response of mouse myocardium at 0 min, 45 min, and 24 h after surgery to better characterize the molecular processes inadvertently induced in sham animals. Our analysis showed that the surgical intervention induced 1209 differentially expressed transcripts (DETs). The clustering of positively co-regulated transcript modules at 45 min fingerprinted the activation of signalization pathways, while positively co-regulated genes at 24 h identified the recruitment of neutrophils and the differentiation of macrophages. In addition, we combined the prediction of transcription factors (TF) regulating DETs with protein-protein interaction networks built from these TFs to predict the molecular network which have induced the DETs. By mean of this retro-analysis of processes upstream gene transcription, we revealed a major role of the Il-6 pathway and further confirmed a significant increase in circulating IL-6 at 45 min after surgery. CONCLUSION: This study suggests that a strong induction of the IL-6 axis occurs in sham animals over the first 24 h and leads to the induction of inflammation and tissues' homeostasis processes.