Role of Protein Kinase C epsilon in cardiac and skeletal muscle differentiation

La famiglia delle Protein Chinasi C è stata ampiamente studiata durante il differenziamento di diversi tipi cellulari. È noto che l'isoforma ε, appartenente al sottogruppo delle nuove PKC, esercita un essenziale ruolo cardioprotettivo e di precondizionamento in seguito a danno da riperfusione. Inoltre, nel muscolo scheletrico adulto, la PKCε fa parte della via segnaletica che regola l'internalizzazione del glucosio in seguito alla contrazione muscolare. L'obiettivo di questa ricerca è stato quello di comprendere il ruolo fisiologico che la PKCε esercita durante il differenziamento muscolare cardiaco e scheletrico e di caratterizzare le vie segnaletiche coinvolte in questi processi. Come modello di differenziamento cardiaco in vitro abbiamo scelto di utilizzare cellule staminali mesenchimali del midollo osseo. I nostri risultati dimostrano che la PKCε regola negativamente l'espressione di due fattori di trascrizione essenziali per il differenziamento cardiaco, Gata4 e Nkx2.5 attraverso l'attivazione delle chinasi ERK1/2. Abbiamo inoltre studiato il coinvolgimento della PKCε nel differenziamento muscolare scheletrico. Esperimenti effettuati in vitro dimostrano che la presenza nel nucleo della forma attiva di questa chinasi, fosforilata a livello della serina 729, inibisce la proteina di legame alla cromatina HMGA1 e promuove l'espressione dei marcatori miogenici Miogenina e MRF4. Questa cascata molecolare promuove la fusione dei mioblasti e induce il differenziamento terminale scheletrico. Infine, abbiamo dimostrato che, in seguito a danno muscolare effettuato in vivo, la PKCε è espressa nelle miofibre rigeneranti centro-nucleate. L'utilizzo di un inibitore specifico della PKCε nel muscolo danneggiato inibisce l'espressione dei fattori di trascrizione miogenici MyoD e Miogenina, modulando negativamente il processo rigenerativo. I nostri risultati dimostrano che la PKCε è un importante regolatore di geni essenziali coinvolti nel differenziamento muscolare scheletrico e cardiaco, suggerendo che l'espressione di questa chinasi deve essere finemente modulata in questi sistemi biologici.Protein kinase C has been studied in the differentiation process of several cellular types. It is well-known that a novel isoform of this family, PKCε, exerts an essential cardio-protective role and mediates the preconditioning in ischemia-reperfusion injury. Furthermore, in adult skeletal muscle, PKCε is involved in the signaling pathway that regulates glucose uptake after muscle contraction. The goal of this research was to elucidate the physiological role that PKCε plays during cardiac and skeletal muscle differentiation and to determine the molecular pathways involved in these processes. We used rat bone marrow mesenchymal stem cells (BMMSCs) as a model of in vitro cardiomyogenic differentiation. Our results show the ability of PKCε to negatively regulate the expression of two essential cardiac transcription factors, Gata4 and Nkx2.5, via activation of ERK1/2. We also studied the PKCε involvement in skeletal muscle differentiation. In vitro experiments reveal that the accumulation of phospho Ser729-PKCε in the nucleus inhibits of the chromatin binding protein HMGA1 and promotes the expression of the myogenic markers Myogenin and MRF4. This molecular cascade promotes in vitro myoblast fusion and myogenic terminal differentiation. We also found that, after in vivo muscle injury, PKCε accumulates in regenerating, centrally-nucleated myofibers. In damaged muscle, PKCε specific inhibition dramatically impairs the expression of the myogenic transcription factors, MyoD and Myogenin, affecting the regenerative process. Our findings demonstrate that PKCε is a critical regulator of essential genes involved in cardiac and skeletal muscle differentiation, suggesting that the expression of this kinase has to be finely tuned in these biological systems

Activation and nuclear translocation of PKCε promotes skeletal muscle cell differentiation via HMGA1 downregulation

The role of novel PKCs in skeletal muscle differentiation has recently emerged. PKCθ is the most expressed isoform of PKCs in muscle and it promotes the fusion of myoblasts [1]. Recently, we have demonstrated that PKCε is implicated in myocardiocyte differentiation of bone marrow mesenchymal stem cells [2] but the role of PKCε in skeletal muscle cell regeneration has only recently emerged [3]. We here demonstrate that both nuclear and cytoplasmic fractions of PKCε are up-regulated during in vitro C2C12 cell line and satellite cell differentiation. We also show that PKCε is able to modulate myogenic differentiation genes via a downmodulation of HMGA1 proteins that promotes myogenin accumulation and mature myoblast formation. To study the effects of PKCε on muscle regeneration, we have used the in vivo model of CTX-induced skeletal muscle injury. We show that the upregulation of PKCε also occurs in vivo, particularly in the centro-nucleated regenerating fibers that are derived from the fusion process of the resident satellite cells, suggesting a role for PKCε in human satellite cell-driven muscle repair and substitution, with clinically relevant implications in human muscle pathology

PKCε expression is required during proplatelet formation in murine model

Megakaryocytes (MK) remodel their cytoplasm into long proplatelet extensions to generate platelets [1]. We have previously demonstrated that PKCepsilon expression is strictly regulated during megakaryocytopoiesis (MKpoiesis), and its forced expression in the late phases of MK differentiation impairs platelet production [2,3]. However, our preliminary data suggest that PKCepsilon positive platelets may be released around the acute event of myocardial infarction, affecting their aggregation potential. Primary fetal liver (FL) cells isolated from CD1 pregnant mice are the preferential model to study the platelet formation mechanism [4]. Therefore, here we focused on the mouse PKCepsilon positive model to elucidate the role of PKCepsilon in MK maturation. Our data show that not only PKCepsilon expression increases during mouse MK differentiation, but also its forced down-regulation strongly reduces pro-platelet formation. Therefore, PKCepsilon is strongly required for murine proplatelet production. On the basis of these results and other known model systems, we show that PKCepsilon has a relevant role in the completion of platelet release

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage

An explainable model of host genetic interactions linked to COVID-19 severity

We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k-fold screening, to rank variants more associated with severity, with the training of multiple supervised classifiers, to predict severity based on screened features. Feature importance analysis from tree-based models allowed us to identify 16 variants with the highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with high accuracy (ACC = 81.88%; AUCROC = 96%; MCC = 61.55%). Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome-Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response. It also identified additional processes cross-talking with immune pathways, such as GPCR signaling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as "Respiratory or thoracic disease", supporting their link with COVID-19 severity outcome.A multifaceted computational strategy identifies 16 genetic variants contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing dataset of a cohort of Italian patients

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19

A genome-wide association study for survival from a multi-centre European study identified variants associated with COVID-19 risk of death

: The clinical manifestations of SARS-CoV-2 infection vary widely among patients, from asymptomatic to life-threatening. Host genetics is one of the factors that contributes to this variability as previously reported by the COVID-19 Host Genetics Initiative (HGI), which identified sixteen loci associated with COVID-19 severity. Herein, we investigated the genetic determinants of COVID-19 mortality, by performing a case-only genome-wide survival analysis, 60 days after infection, of 3904 COVID-19 patients from the GEN-COVID and other European series (EGAS00001005304 study of the COVID-19 HGI). Using imputed genotype data, we carried out a survival analysis using the Cox model adjusted for age, age2, sex, series, time of infection, and the first ten principal components. We observed a genome-wide significant (P-value < 5.0 × 10-8) association of the rs117011822 variant, on chromosome 11, of rs7208524 on chromosome 17, approaching the genome-wide threshold (P-value = 5.19 × 10-8). A total of 113 variants were associated with survival at P-value < 1.0 × 10-5 and most of them regulated the expression of genes involved in immune response (e.g., CD300 and KLR genes), or in lung repair and function (e.g., FGF19 and CDH13). Overall, our results suggest that germline variants may modulate COVID-19 risk of death, possibly through the regulation of gene expression in immune response and lung function pathways

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways. Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor

How future surgery will benefit from SARS-COV-2-related measures: a SPIGC survey conveying the perspective of Italian surgeons

COVID-19 negatively affected surgical activity, but the potential benefits resulting from adopted measures remain unclear. The aim of this study was to evaluate the change in surgical activity and potential benefit from COVID-19 measures in perspective of Italian surgeons on behalf of SPIGC. A nationwide online survey on surgical practice before, during, and after COVID-19 pandemic was conducted in March-April 2022 (NCT:05323851). Effects of COVID-19 hospital-related measures on surgical patients' management and personal professional development across surgical specialties were explored. Data on demographics, pre-operative/peri-operative/post-operative management, and professional development were collected. Outcomes were matched with the corresponding volume. Four hundred and seventy-three respondents were included in final analysis across 14 surgical specialties. Since SARS-CoV-2 pandemic, application of telematic consultations (4.1% vs. 21.6%; p &lt; 0.0001) and diagnostic evaluations (16.4% vs. 42.2%; p &lt; 0.0001) increased. Elective surgical activities significantly reduced and surgeons opted more frequently for conservative management with a possible indication for elective (26.3% vs. 35.7%; p &lt; 0.0001) or urgent (20.4% vs. 38.5%; p &lt; 0.0001) surgery. All new COVID-related measures are perceived to be maintained in the future. Surgeons' personal education online increased from 12.6% (pre-COVID) to 86.6% (post-COVID; p &lt; 0.0001). Online educational activities are considered a beneficial effect from COVID pandemic (56.4%). COVID-19 had a great impact on surgical specialties, with significant reduction of operation volume. However, some forced changes turned out to be benefits. Isolation measures pushed the use of telemedicine and telemetric devices for outpatient practice and favored communication for educational purposes and surgeon-patient/family communication. From the Italian surgeons' perspective, COVID-related measures will continue to influence future surgical clinical practice

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types