The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response.

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.

Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia.

COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin 6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.

vPIF-1 is an insulin-like antiferroptotic viral peptide.

Iridoviridae, such as the lymphocystis disease virus-1 (LCDV-1) and other viruses, encode viral insulin-like peptides (VILPs) which are capable of triggering insulin receptors (IRs) and insulin-like growth factor receptors. The homology of VILPs includes highly conserved disulfide bridges. However, the binding affinities to IRs were reported to be 200- to 500-fold less effective compared to the endogenous ligands. We therefore speculated that these peptides also have noninsulin functions. Here, we report that the LCDV-1 VILP can function as a potent and highly specific inhibitor of ferroptosis. Induction of cell death by the ferroptosis inducers erastin, RSL3, FIN56, and FINO2 and nonferroptotic necrosis produced by the thioredoxin-reductase inhibitor ferroptocide were potently prevented by LCDV-1, while human insulin had no effect. Fas-induced apoptosis, necroptosis, mitotane-induced cell death and growth hormone-releasing hormone antagonist-induced necrosis were unaffected, suggesting the specificity to ferroptosis inhibition by the LCDV-1 VILP. Mechanistically, we identified the viral C-peptide to be required for inhibition of lipid peroxidation and ferroptosis inhibition, while the human C-peptide exhibited no antiferroptotic properties. In addition, the deletion of the viral C-peptide abolishes radical trapping activity in cell-free systems. We conclude that iridoviridae, through the expression of insulin-like viral peptides, are capable of preventing ferroptosis. In analogy to the viral mitochondrial inhibitor of apoptosis and the viral inhibitor of RIP activation (vIRA) that prevents necroptosis, we rename the LCDV-1 VILP a viral peptide inhibitor of ferroptosis-1. Finally, our findings indicate that ferroptosis may function as a viral defense mechanism in lower organisms.

Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease.

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs.

Prompt coronary catheterization and revascularization have markedly improved the outcomes of myocardial infarction, but have also resulted in a growing number of surviving patients with permanent structural damage of the heart, which frequently leads to heart failure. There is an unmet clinical need for treatments for this condition1, particularly given the inability of cardiomyocytes to replicate and thereby regenerate the lost contractile tissue2. Here we show that expression of human microRNA-199a in infarcted pig hearts can stimulate cardiac repair. One month after myocardial infarction and delivery of this microRNA through an adeno-associated viral vector, treated animals showed marked improvements in both global and regional contractility, increased muscle mass and reduced scar size. These functional and morphological findings correlated with cardiomyocyte de-differentiation and proliferation. However, subsequent persistent and uncontrolled expression of the microRNA resulted in sudden arrhythmic death of most of the treated pigs. Such events were concurrent with myocardial infiltration of proliferating cells displaying a poorly differentiated myoblastic phenotype. These results show that achieving cardiac repair through the stimulation of endogenous cardiomyocyte proliferation is attainable in large mammals, however dosage of this therapy needs to be tightly controlled.

Cross-Linking Mass Spectrometry Uncovers Interactions Between High-Density Lipoproteins and the SARS-CoV-2 Spike Glycoprotein.

High-density lipoprotein (HDL) levels are reduced in patients with coronavirus disease 2019 (COVID-19), and the extent of this reduction is associated with poor clinical outcomes. While lipoproteins are known to play a key role during the life cycle of the hepatitis C virus, their influence on coronavirus (CoV) infections is poorly understood. In this study, we utilize cross-linking mass spectrometry (XL-MS) to determine circulating protein interactors of the severe acute respiratory syndrome (SARS)-CoV-2 spike glycoprotein. XL-MS of plasma isolated from patients with COVID-19 uncovered HDL protein interaction networks, dominated by acute-phase serum amyloid proteins, whereby serum amyloid A2 was shown to bind to apolipoprotein (Apo) D. XL-MS on isolated HDL confirmed ApoD to interact with SARS-CoV-2 spike but not SARS-CoV-1 spike. Other direct interactions of SARS-CoV-2 spike upon HDL included ApoA1 and ApoC3. The interaction between ApoD and spike was further validated in cells using immunoprecipitation-MS, which uncovered a novel interaction between both ApoD and spike with membrane-associated progesterone receptor component 1. Mechanistically, XL-MS coupled with data-driven structural modeling determined that ApoD may interact within the receptor-binding domain of the spike. However, ApoD overexpression in multiple cell-based assays had no effect upon viral replication or infectivity. Thus, SARS-CoV-2 spike can bind to apolipoproteins on HDL, but these interactions do not appear to alter infectivity.

Id genes are essential for early heart formation.

Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

Persistent SARS-CoV-2 infection in patients seemingly recovered from COVID-19.

SARS-CoV-2 infection is clinically heterogeneous, ranging from asymptomatic to deadly. A few patients with COVID-19 appear to recover from acute viral infection but nevertheless progress in their disease and eventually die, despite persistent negativity at molecular tests for SARS-CoV-2 RNA. Here, we performed post-mortem analyses in 27 consecutive patients who had apparently recovered from COVID-19 but had progressively worsened in their clinical conditions despite repeated viral negativity in nasopharyngeal swabs or bronchioalveolar lavage for 11-300 consecutive days (average: 105.5 days). Three of these patients remained PCR-negative for over 9 months. Post-mortem analysis revealed evidence of diffuse or focal interstitial pneumonia in 23/27 (81%) patients, accompanied by extensive fibrotic substitution in 13 cases (47%). Despite apparent virological remission, lung pathology was similar to that observed in acute COVID-19 individuals, including micro- and macro-vascular thrombosis (67% of cases), vasculitis (24%), squamous metaplasia of the respiratory epithelium (30%), frequent cytological abnormalities and syncytia (67%), and the presence of dysmorphic features in the bronchial cartilage (44%). Consistent with molecular test negativity, SARS-CoV-2 antigens were not detected in the respiratory epithelium. In contrast, antibodies against both spike and nucleocapsid revealed the frequent (70%) infection of bronchial cartilage chondrocytes and para-bronchial gland epithelial cells. In a few patients (19%), we also detected positivity in vascular pericytes and endothelial cells. Quantitative RT-PCR amplification in tissue lysates confirmed the presence of viral RNA. Together, these findings indicate that SARS-CoV-2 infection can persist significantly longer than suggested by standard PCR-negative tests, with specific infection of specific cell types in the lung. Whether these persistently infected cells also play a pathogenic role in long COVID remains to be addressed. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Haemorrhage of human foetal cortex associated with SARS-CoV-2 infection.

Maternal viral infection and immune response are known to increase the risk of altered development of the foetal brain. Given the ongoing global pandemic of coronavirus disease 2019 (COVID-19), investigating the impact of SARS-CoV-2 on foetal brain health is of critical importance. Here, we report the presence of SARS-CoV-2 in first and second trimester foetal brain tissue in association with cortical haemorrhages. SARS-CoV-2 spike protein was sparsely detected within progenitors and neurons of the cortex itself, but was abundant in the choroid plexus of haemorrhagic samples. SARS-CoV-2 was also sparsely detected in placenta, amnion and umbilical cord tissues. Cortical haemorrhages were linked to a reduction in blood vessel integrity and an increase in immune cell infiltration into the foetal brain. Our findings indicate that SARS-CoV-2 infection may affect the foetal brain during early gestation and highlight the need for further study of its impact on subsequent neurological development.

Regulation of endogenous cardiomyocyte proliferation: The known unknowns.

Myocardial regeneration in patients with cardiac damage is a long-sought goal of clinical medicine. In animal species in which regeneration occurs spontaneously, as well as in neonatal mammals, regeneration occurs through the proliferation of differentiated cardiomyocytes, which re-enter the cell cycle and proliferate. Hence, the reprogramming of the replicative potential of cardiomyocytes is an achievable goal, provided that the mechanisms that regulate this process are understood. Cardiomyocyte proliferation is under the control of a series of signal transduction pathways that connect extracellular cues to the activation of specific gene transcriptional programmes, eventually leading to the activation of the cell cycle. Both coding and non-coding RNAs (in particular, microRNAs) are involved in this regulation. The available information can be exploited for therapeutic purposes, provided that a series of conceptual and technical barriers are overcome. A major obstacle remains the delivery of pro-regenerative factors specifically to the heart. Improvements in the design of AAV vectors to enhance their cardiotropism and efficacy or, alternatively, the development of non-viral methods for nucleic acid delivery in cardiomyocytes are among the challenges ahead to progress cardiac regenerative therapies towards clinical application.

CARMN Loss Regulates Smooth Muscle Cells and Accelerates Atherosclerosis in Mice.

[Figure: see text].

Small non-coding RNA therapeutics for cardiovascular disease.

Novel bio-therapeutic agents that harness the properties of small, non-coding nucleic acids hold great promise for clinical applications. These include antisense oligonucleotides that inhibit messenger RNAs, microRNAs (miRNAs), or long non-coding RNAs; positive effectors of the miRNA pathway (short interfering RNAs and miRNA mimics); or small RNAs that target proteins (i.e. aptamers). These new therapies also offer exciting opportunities for cardiovascular diseases and promise to move the field towards more precise approaches based on disease mechanisms. There have been substantial advances in developing chemical modifications to improve the in vivo pharmacological properties of antisense oligonucleotides and reduce their immunogenicity. Carrier methods (e.g. RNA conjugates, polymers, and lipoplexes) that enhance cellular uptake of RNA therapeutics and stability against degradation by intracellular nucleases are also transforming the field. A number of small non-coding RNA therapies for cardiovascular indications are now approved. Moreover, there is a large pipeline of therapies in clinical development and an even larger list of putative therapies emerging from pre-clinical studies. Progress in this area is reviewed herein along with the hurdles that need to be overcome to allow a broader clinical translation.

Nrf2 attenuates the innate immune response after experimental myocardial infarction.

BACKGROUND: There is compelling evidence implicating dysregulated inflammation in the mechanism of ventricular remodeling and heart failure (HF) after MI. The transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2, encoded by Nfe2l2) is a promising target in this context since it impedes transcriptional upregulation of pro-inflammatory cytokines and is anti-inflammatory in various murine models. OBJECTIVES: We aimed to investigate the contribution of Nrf2 to the inflammatory response after experimental myocardial infarction (MI). METHODS: We subjected Nrf2-/- mice and wild type (WT) controls to permanent left coronary artery (LCA) ligation. The inflammatory response was investigated with fluorescence-activated cell sorting (FACS) analysis of peripheral blood and heart cell suspensions, together with qRT-PCR of infarcted tissue for chemokines and their receptors. To investigate whether Nrf2-mediated transcription is a dedicated function of leukocytes, we interrogated publicly available RNA-sequencing (RNA-seq) data from mouse hearts after permanent LCA ligation for Nrf2-regulated gene (NRG) expression. RESULTS: FACS analysis demonstrated a profoundly inflamed phenotype in the hearts of global Nrf2-/- mice as compared to WT mice after MI. Moreover, infarcted tissue from Nrf2-/- mice displayed higher expression of mRNA coding for inflammatory cytokines, chemokines, and their receptors, including IL-6, Ccl2, and Cxcr4. RNA-seq analysis showed upregulated NRG expression in WT mice after MI compared to naive mice, which was significantly higher in bioinformatically isolated CCR2+ cells. CONCLUSIONS: Taken together, the results suggest that Nrf2 signalling in leukocytes, and possibly CCR2+ monocytes and monocyte-derived cardiac resident macrophages, may be potential targets to prevent post-MI ventricular remodeling.

Is heart failure with preserved ejection fraction a 'dementia' of the heart?

Heart failure with preserved ejection fraction (HFpEF) remains an elusive entity, due to its heterogeneous clinical profile and an arbitrarily defined nosology. Several pathophysiological mechanisms recognized as central for the development of HFpEF appear to be in common with the process of physiological aging of the heart. Both conditions are characterized by progressive impairment in cardiac function, accompanied by left ventricular hypertrophy, diastolic dysfunction, sarcomeric, and metabolic abnormalities. The neurological paradigm of dementia-intended as a progressive, multifactorial organ damage with decline of functional reserve, eventually leading to irreversible dysfunction-is well suited to represent HFpEF. In such perspective, certain phenotypes of HFpEF may be viewed as a maladaptive response to environmental modifiers, causing premature and pathological aging of the heart. We here propose that the 'HFpEF syndrome' may reflect the interplay of adverse structural remodelling and erosion of functional reserve, mirroring the processes leading to dementia in the brain. The resulting conceptual framework may help advance our understanding of HFpEF and unravel potential therapeutical targets.

Raptor is critical for increasing the mitochondrial proteome and skeletal muscle force during hypertrophy.

Loss of skeletal muscle mass and force is of critical importance in numerous pathologies, like age-related sarcopenia or cancer. It has been shown that the Akt-mTORC1 pathway is critical for stimulating adult muscle mass and function, however, it is unknown if mTORC1 is the only mediator downstream of Akt and which intracellular processes are required for functional muscle growth. Here, we show that loss of Raptor reduces muscle hypertrophy after Akt activation and completely prevents increases in muscle force. Interestingly, the residual hypertrophy after Raptor deletion can be completely prevented by administration of the mTORC1 inhibitor rapamycin. Using a quantitative proteomics approach we find that loss of Raptor affects the increases in mitochondrial proteins, while rapamycin mainly affects ribosomal proteins. Taken together, these results suggest that mTORC1 is the key mediator of Akt-dependent muscle growth and its regulation of the mitochondrial proteome is critical for increasing muscle force.

Gene Therapy for the Heart Lessons Learned and Future Perspectives.

While clinical gene therapy celebrates its first successes, with several products already approved for clinical use and several hundreds in the final stages of the clinical approval pipeline, there is not a single gene therapy approach that has worked for the heart. Here, we review the past experience gained in the several cardiac gene therapy clinical trials that had the goal of inducing therapeutic angiogenesis in the ischemic heart and in the attempts at modulating cardiac function in heart failure. Critical assessment of the results so far achieved indicates that the efficiency of cardiac gene delivery remains a major hurdle preventing success but also that improvements need to be sought in establishing more reliable large animal models, choosing more effective therapeutic genes, better designing clinical trials, and more deeply understanding cardiac biology. We also emphasize a few areas of cardiac gene therapy development that hold great promise for the future. In particular, the transition from gene addition studies using protein-coding cDNAs to the modulation of gene expression using small RNA therapeutics and the improvement of precise gene editing now pave the way to applications such as cardiac regeneration after myocardial infarction and gene correction for inherited cardiomyopathies that were unapproachable until a decade ago.

SREBP2 gene therapy targeting striatal astrocytes ameliorates Huntington's disease phenotypes.

Brain cholesterol is produced mainly by astrocytes and is important for neuronal function. Its biosynthesis is severely reduced in mouse models of Huntington's disease. One possible mechanism is a diminished nuclear translocation of the transcription factor sterol regulatory element-binding protein 2 (SREBP2) and, consequently, reduced activation of SREBP2-controlled genes in the cholesterol biosynthesis pathway. Here we evaluated the efficacy of a gene therapy based on the unilateral intra-striatal injection of a recombinant adeno-associated virus 2/5 (AAV2/5) targeting astrocytes specifically and carrying the transcriptionally active N-terminal fragment of human SREBP2 (hSREBP2). Robust hSREBP2 expression in striatal glial cells in R6/2 Huntington's disease mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed dopamine receptor D2 (Drd2) transcript levels decline, cleared mutant huntingtin aggregates and attenuated behavioural deficits. We conclude that glial SREBP2 participates in Huntington's disease brain pathogenesis in vivo and that AAV-based delivery of SREBP2 to astrocytes counteracts key features of the disease.

Cardiovascular Damage in COVID-19: What We Know Two Years Later.

PURPOSE OF THE REVIEW: The Coronavirus disease 2019 (COVID-19) pandemic has profoundly influenced cardiological clinical and basic research in the past two years. In the present review, we summarize the current knowledge on myocardial involvement in COVID-19, providing an overview on the incidence, the pathogenetic mechanisms, and the clinical implications of cardiac injury in this setting. RECENT FINDINGS: The possibility of heart involvement in patients with COVID-19 has received great attention since the beginning of the pandemic. After more than two years, several steps have been taken in understanding the mechanisms and the incidence of cardiac injury during COVID-19 infection. Similarly, studies globally have clarified the implications of co-existing heart disease and COVID-19. Severe COVID-19 infection may be complicated by myocardial injury. To date, a direct damage from the virus has not been demonstrated. The presence of myocardial injury should be systematically assessed for a prognostication purpose and for possible therapeutic implications.

Nuclear architecture dictates HIV-1 integration site selection.

Long-standing evidence indicates that human immunodeficiency virus type 1 (HIV-1) preferentially integrates into a subset of transcriptionally active genes of the host cell genome. However, the reason why the virus selects only certain genes among all transcriptionally active regions in a target cell remains largely unknown. Here we show that HIV-1 integration occurs in the outer shell of the nucleus in close correspondence with the nuclear pore. This region contains a series of cellular genes, which are preferentially targeted by the virus, and characterized by the presence of active transcription chromatin marks before viral infection. In contrast, the virus strongly disfavours the heterochromatic regions in the nuclear lamin-associated domains and other transcriptionally active regions located centrally in the nucleus. Functional viral integrase and the presence of the cellular Nup153 and LEDGF/p75 integration cofactors are indispensable for the peripheral integration of the virus. Once integrated at the nuclear pore, the HIV-1 DNA makes contact with various nucleoporins; this association takes part in the transcriptional regulation of the viral genome. These results indicate that nuclear topography is an essential determinant of the HIV-1 life cycle.

Endothelial cell-cardiomyocyte crosstalk in heart development and disease.

The crosstalk between endothelial cells and cardiomyocytes has emerged as a requisite for normal cardiac development, but also a key pathogenic player during the onset and progression of cardiac disease. Endothelial cells and cardiomyocytes are in close proximity and communicate through the secretion of paracrine signals, as well as through direct cell-to-cell contact. Here, we provide an overview of the endothelial cell-cardiomyocyte interactions controlling heart development and the main processes affecting the heart in normal and pathological conditions, including ischaemia, remodelling and metabolic dysfunction. We also discuss the possible role of these interactions in cardiac regeneration and encourage the further improvement of in vitro models able to reproduce the complex environment of the cardiac tissue, in order to better define the mechanisms by which endothelial cells and cardiomyocytes interact with a final aim of developing novel therapeutic opportunities.