Apoptosis, a useful marker in the management of hot-phase cardiomyopathy?

AIMS: 'Hot phases', characterized by chest pain and troponin release, may represent the first clinical presentation of arrhythmogenic cardiomyopathies. Differential diagnosis with acute myocarditis is an unmet challenge for the clinicians. We sought to investigate histological and genetic features in patients with cardiomyopathy presenting with hot phases. METHODS AND RESULTS: We evaluated a case series of consecutive patients hospitalized for suspected 'hot-phase cardiomyopathy' in two Italian centres from June 2017 to March 2022 (median follow-up 18 months) that underwent both endomyocardial biopsy (EMB) and genetic testing. Apoptosis was confirmed with TUNEL assay. Among the 17 enrolled patients (mean age 34 ± 15 years, 76% male), only six patients (35%) presented standard histological and immunohistochemical markers for significant cardiac inflammation at EMB. Conversely, apoptosis was found in 13 patients (77%). Genetic testing was positive for a pathogenic/likely pathogenic (P/LP) variant in genes involved in cardiomyopathies (most frequently in DSP) in eight patients (48%), rising to 62% among patients with apoptosis on EMB. Notably, all patients without apoptosis tested negative for P/LP disease-related variants. Left ventricular ejection fraction was lower in patients showing apoptosis at EMB compared to those without (p = 0.003). CONCLUSIONS: Apoptosis, rather than significant inflammation, was mostly prevalent in this case series of patients with 'hot-phase' presentation, especially in carriers of variants in cardiomyopathy-related genes. Detecting apoptosis on EMB might guide clinicians in performing genetic testing and in more tailored therapeutic choices in 'hot-phase cardiomyopathy'.

Engineered heart tissue maturation inhibits cardiomyocyte proliferative response to cryoinjury.

The cellular and molecular mechanisms that are responsible for the poor regenerative capacity of the adult heart after myocardial infarction (MI) are still unclear and their understanding is crucial to develop novel regenerative therapies. Considering the lack of reliable in vitro tissue-like models to evaluate the molecular mechanisms of cardiac regeneration, we used cryoinjury on rat Engineered Heart Tissues (rEHTs) as a new model which recapitulates in part the in vivo response after myocardial injury of neonatal and adult heart. When we subjected to cryoinjury immature and mature rEHTs, we observed a significant increase in cardiomyocyte (CM) DNA synthesis when compared to the controls. As expected, the number of mitotic CMs significantly increases in immature rEHTs when compared to mature rEHTs, suggesting that the extent of CM maturation plays a crucial role in their proliferative response after cryoinjury. Moreover, we show that cryoinjury induces a temporary activation of fibroblast response in mature EHTs, similar to the early response after MI, that is however incomplete in immature EHTs. Our results support the hypothesis that the endogenous maturation program in cardiac myocytes plays a major role in determining the proliferative response to injury. Therefore, we propose rEHTs as a robust, novel tool to in vitro investigate critical aspects of cardiac regeneration in a tissue-like asset free from confounding factors in response to injury, such as the immune system response or circulating inflammatory cytokines.

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.

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.

SARS-CoV-2, myocardial injury and inflammation: insights from a large clinical and autopsy study.

OBJECTIVE: Despite growing evidence about myocardial injury in hospitalized COronaVIrus Disease 2019 (COVID-19) patients, the mechanism behind this injury is only poorly understood and little is known about its association with SARS-CoV-2-mediated myocarditis. Furthermore, definite evidence of the presence and role of SARS-CoV-2 in cardiomyocytes in the clinical scenario is still lacking. METHODS: We histologically characterized myocardial tissue of 40 patients deceased with severe SARS-CoV-2 infection during the first wave of the pandemic. Clinical data were also recorded and analyzed. In case of findings supportive of myocardial inflammation, histological analysis was complemented by RT-PCR and immunohistochemistry for SARS-CoV-2 viral antigens and in situ RNA hybridization for the detection of viral genomes. RESULTS: Both chronic and acute myocardial damage was invariably present, correlating with the age and comorbidities of our population. Myocarditis of overt entity was found in one case (2.5%). SARS-CoV-2 genome was not found in the cardiomyocytes of the patient with myocarditis, while it was focally and negligibly present in cardiomyocytes of patients with known viral persistence in the lungs and no signs of myocardial inflammation. The presence of myocardial injury was not associated with myocardial inflammatory infiltrates. CONCLUSIONS: In this autopsy cohort of COVID-19 patients, myocarditis is rarely found and not associated with SARS-CoV-2 presence in cardiomyocytes. Chronic and acute forms of myocardial damage are constantly found and correlate with the severity of COVID-19 disease and pre-existing comorbidities.

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.

Persistence of viral RNA, pneumocyte syncytia and thrombosis are hallmarks of advanced COVID-19 pathology.

BACKGROUND: COVID-19 is a deadly pulmonary disease with peculiar characteristics, which include variable clinical course and thrombophilia. A thorough understanding of the pathological correlates of the disease is still missing. METHODS: Here we report the systematic analysis of 41 consecutive post-mortem samples from individuals who died of COVID-19. Histological analysis is complemented by immunohistochemistry for cellular and viral antigens and the detection of viral genomes by in situ RNA hybridization. FINDINGS: COVID-19 is characterized by extensive alveolar damage (41/41 of patients) and thrombosis of the lung micro- and macro-vasculature (29/41, 71%). Thrombi were in different stages of organization, consistent with their local origin. Pneumocytes and endothelial cells contained viral RNA even at the later stages of the disease. An additional feature was the common presence of a large number of dysmorphic pneumocytes, often forming syncytial elements (36/41, 87%). Despite occasional detection of virus-positive cells, no overt signs of viral infection were detected in other organs, which showed non-specific alterations. INTERPRETATION: COVID-19 is a unique disease characterized by extensive lung thrombosis, long-term persistence of viral RNA in pneumocytes and endothelial cells, along with the presence of infected cell syncytia. Several of COVID-19 features might be consequent to the persistence of virus-infected cells for the duration of the disease. FUNDING: This work was supported by a King's Together Rapid COVID-19 Call grant from King's College London. MG is supported by the European Research Council (ERC) Advanced Grant 787971 "CuRE" and by Programme Grant RG/19/11/34633 from the British Heart Foundation.

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.

Common Regulatory Pathways Mediate Activity of MicroRNAs Inducing Cardiomyocyte Proliferation.

Loss of functional cardiomyocytes is a major determinant of heart failure after myocardial infarction. Previous high throughput screening studies have identified a few microRNAs (miRNAs) that can induce cardiomyocyte proliferation and stimulate cardiac regeneration in mice. Here, we show that all of the most effective of these miRNAs activate nuclear localization of the master transcriptional cofactor Yes-associated protein (YAP) and induce expression of YAP-responsive genes. In particular, miR-199a-3p directly targets two mRNAs coding for proteins impinging on the Hippo pathway, the upstream YAP inhibitory kinase TAOK1, and the E3 ubiquitin ligase ß-TrCP, which leads to YAP degradation. Several of the pro-proliferative miRNAs (including miR-199a-3p) also inhibit filamentous actin depolymerization by targeting Cofilin2, a process that by itself activates YAP nuclear translocation. Thus, activation of YAP and modulation of the actin cytoskeleton are major components of the pro-proliferative action of miR-199a-3p and other miRNAs that induce cardiomyocyte proliferation.

High-throughput screening discovers antifibrotic properties of haloperidol by hindering myofibroblast activation.

Fibrosis is a hallmark in the pathogenesis of various diseases, with very limited therapeutic solutions. A key event in the fibrotic process is the expression of contractile proteins, including α-smooth muscle actin (αSMA) by fibroblasts, which become myofibroblasts. Here, we report the results of a high-throughput screening of a library of approved drugs that led to the discovery of haloperidol, a common antipsychotic drug, as a potent inhibitor of myofibroblast activation. We show that haloperidol exerts its antifibrotic effect on primary murine and human fibroblasts by binding to sigma receptor 1, independent from the canonical transforming growth factor-ß signaling pathway. Its mechanism of action involves the modulation of intracellular calcium, with moderate induction of endoplasmic reticulum stress response, which in turn abrogates Notch1 signaling and the consequent expression of its targets, including αSMA. Importantly, haloperidol also reduced the fibrotic burden in 3 different animal models of lung, cardiac, and tumor-associated fibrosis, thus supporting the repurposing of this drug for the treatment of fibrotic conditions.

Cellular TRIM33 restrains HIV-1 infection by targeting viral integrase for proteasomal degradation.

Productive HIV-1 replication requires viral integrase (IN), which catalyzes integration of the viral genome into the host cell DNA. IN, however, is short lived and is rapidly degraded by the host ubiquitin-proteasome system. To identify the cellular factors responsible for HIV-1 IN degradation, we performed a targeted RNAi screen using a library of siRNAs against all components of the ubiquitin-conjugation machinery using high-content microscopy. Here we report that the E3 RING ligase TRIM33 is a major determinant of HIV-1 IN stability. CD4-positive cells with TRIM33 knock down show increased HIV-1 replication and proviral DNA formation, while those overexpressing the factor display opposite effects. Knock down of TRIM33 reverts the phenotype of an HIV-1 molecular clone carrying substitution of IN serine 57 to alanine, a mutation known to impair viral DNA integration. Thus, TRIM33 acts as a cellular factor restricting HIV-1 infection by preventing provirus formation.

TBL1Y: a new gene involved in syndromic hearing loss.

Hereditary hearing loss (HHL) is an extremely heterogeneous disorder with autosomal dominant, recessive, and X-linked forms. Here, we described an Italian pedigree affected by HHL but also prostate hyperplasia and increased ratio of the free/total PSA levels, with the unusual and extremely rare Y-linked pattern of inheritance. Using exome sequencing we found a missense variant (r.206A>T leading to p.Asp69Val) in the TBL1Y gene. TBL1Y is homologous of TBL1X, whose partial deletion has described to be involved in X-linked hearing loss. Here, we demonstrate that it has a restricted expression in adult human cochlea and prostate and the variant identified induces a lower protein stability caused by misfolded mutated protein that impairs its cellular function. These findings indicate that TBL1Y could be considered a novel candidate for HHL.

Notch pathway activation enhances cardiosphere in vitro expansion.

Cardiospheres (CSps) are self-assembling clusters of a heterogeneous population of poorly differentiated cells outgrowing from in vitro cultured cardiac explants. Scanty information is available on the molecular pathways regulating CSp growth and their differentiation potential towards cardiac and vascular lineages. Here we report that Notch1 stimulates a massive increase in both CSp number and size, inducing a peculiar gene expression programme leading to a cardiovascular molecular signature. These effects were further enhanced using Adeno-Associated Virus (AAV)-based gene transfer of activated Notch1-intracellular domain (N1-ICD) or soluble-Jagged1 (sJ1) ligand to CSp-forming cells. A peculiar effect was exploited by selected pro-proliferating miRNAs: hsa-miR-590-3p induced a cardiovascular gene expression programme, while hsa-miR-199a-3p acted as the most potent stimulus for the activation of the Notch pathway, thus showing that, unlike in adult cardiomyocytes, these miRNAs involve Notch signalling activation in CSps. Our results identify Notch1 as a crucial regulator of CSp growth and differentiation along the vascular lineage, raising the attracting possibility that forced activation of this pathway might be exploited to promote in vitro CSp expansion as a tool for toxicology screening and cell-free therapeutic strategies.

Reversible Notch1 acetylation tunes proliferative signalling in cardiomyocytes.

Aims: The Notch signalling pathway regulates the balance between proliferation and differentiation in several tissues, including the heart. Our previous work has demonstrated that the proliferative potential of neonatal cardiomyocytes relies on Notch1 activity. A deep investigation on the biochemical regulation of the Notch signalling in cardiomyocytes is the focus of the current research. Methods and results: We show that the Notch1 intracellular domain is acetylated in proliferating neonatal rat cardiomyocytes and that acetylation tightly controls the amplitude and duration of Notch signalling. We found that acetylation extends the half-life of the protein, and enhanced its transcriptional activity, therefore counteracting apoptosis and sustaining cardiomyocyte proliferation. Sirt1 acted as a negative modulator of Notch1 signalling; its overexpression in cardiomyocytes reverted Notch acetylation and dampened its stability. A constitutively acetylated fusion protein between Notch1 and the acetyltransferase domain of p300 promoted cardiomyocyte proliferation, which was remarkably sustained over time. Viral vector-mediated expression of this protein enhanced heart regeneration after apical resection in neonatal mice. Conclusion: These results identify the reversible acetylation of Notch1 as a novel mechanism to modulate its signalling in the heart and tune the proliferative potential of cardiomyocytes.

MIB2 variants altering NOTCH signalling result in left ventricle hypertrabeculation/non-compaction and are associated with Ménétrier-like gastropathy.

We performed whole exome sequencing in individuals from a family with autosomal dominant gastropathy resembling Ménétrier disease, a premalignant gastric disorder with epithelial hyperplasia and enhanced EGFR signalling. Ménétrier disease is believed to be an acquired disorder, but its aetiology is unknown. In affected members, we found a missense p.V742G variant in MIB2, a gene regulating NOTCH signalling that has not been previously linked to human diseases. The variant segregated with the disease in the pedigree, affected a highly conserved amino acid residue, and was predicted to be deleterious although it was found with a low frequency in control individuals. The purified protein carrying the p.V742G variant showed reduced ubiquitination activity in vitro and white blood cells from affected individuals exhibited significant reductions of HES1 and NOTCH3 expression reflecting alteration of NOTCH signalling. Because mutations of MIB1, the homolog of MIB2, have been found in patients with left ventricle non-compaction (LVNC), we investigated members of our family with Ménétrier-like disease for this cardiac abnormality. Asymptomatic left ventricular hypertrabeculation, the mildest end of the LVNC spectrum, was detected in two members carrying the MIB2 variant. Finally, we identified an additional MIB2 variant (p.V984L) affecting protein stability in an unrelated isolated case with LVNC. Expression of both MIB2 variants affected NOTCH signalling, proliferation and apoptosis in primary rat cardiomyocytes.In conclusion, we report the first example of left ventricular hypertrabeculation/LVNC with germline MIB2 variants resulting in altered NOTCH signalling that might be associated with a gastropathy clinically overlapping with Ménétrier disease.

Gene transfer to promote cardiac regeneration.

There is an impelling need to develop new therapeutic strategies for patients with myocardial infarction and heart failure. Leading from the large quantity of new information gathered over the last few years on the mechanisms controlling cardiomyocyte proliferation during embryonic and fetal life, it is now possible to devise innovative therapies based on cardiac gene transfer. Different protein-coding genes controlling cell cycle progression or cardiomyocyte specification and differentiation, along with microRNA mimics and inhibitors regulating pre-natal and early post-natal cell proliferation, are amenable to transformation in potential therapeutics for cardiac regeneration. These gene therapy approaches are conceptually revolutionary, since they are aimed at stimulating the intrinsic potential of differentiated cardiac cells to proliferate, rather than relying on the implantation of exogenously expanded cells to achieve tissue regeneration. For efficient and prolonged cardiac gene transfer, vectors based on the Adeno-Associated Virus stand as safe, efficient and reliable tools for cardiac gene therapy applications.

Mesenchymal stromal cells reset the scatter factor system and cytokine network in experimental kidney transplantation.

BACKGROUND: In former studies we showed in a rat model of renal transplantation that Mesenchymal Stromal Cells (MSC) prevent acute rejection in an independent way of their endowing in the graft. In this study we investigated whether MSC operate by resetting cytokine network and Scatter Factor systems, i.e. Hepatocyte Growth Factor (HGF), Macrophage Stimulating Protein (MSP) and their receptors Met and RON, respectively. METHODS: MSC were injected into the renal artery soon after reperfusion. Controls were grafted untreated and normal rats. Rats were sacrificed 7 days after grafting. Serum and renal tissue levels of IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-10, MSP/RON, HGF/Met systems, Treg lymphocytes were investigated. RESULTS: In grafted untreated rats IFN-γ increased in serum and renal tissue and IL-6 rose in serum. MSC prevented both the phenomena, increased IL-10 serum levels and Treg number in the graft. Furthermore MSC increased serum and tissue HGF levels, Met tubular expression and prevented the suppression of tubular MSP/RON expression. CONCLUSIONS: Our results demonstrate that MSC modify cytokine network to a tolerogenic setting, they suppress Th1 cells, inactivate monocytes/macrophage, recruit Tregs. In addition, MSC sustain the expression of the Scatter Factor systems expression, i.e. systems that are committed to defend survival and stimulate regeneration of tubular cells.

Epigenetic modification at Notch responsive promoters blunts efficacy of inducing notch pathway reactivation after myocardial infarction.

RATIONALE: The Notch pathway plays a key role in stimulating mammalian cardiomyocyte proliferation during development and in the early postnatal life; in adult zebrafish, reactivation of this pathway is also essential to drive cardiac regeneration after injury. OBJECTIVE: We wanted to assess efficacy of Notch pathway stimulation in neonatal and adult hearts as a means to induce cardiac regeneration after myocardial infarction in mice. METHODS AND RESULTS: In early postnatal life, cardiomyocyte exit from the cell cycle was paralleled by decreased Notch signaling and the establishment of a repressive chromatin environment at Notch-responsive genes, characterized by recruitment of the polycomb group enhancer of zeste homolog 2 methyltransferase and the acquisition of the histone 3 Lysine 27 trimethylation histone mark, as detected by chromatin immunoprecipitation. Forced Notch pathway activation by adenoassociated virus gene transfer of activated Notch1 or its ligand Jagged1 expanded the proliferative capacity of neonatal cardiomyocytes; this correlated with increased transcription of Notch target genes and maintenance of an open chromatin conformation at their promoters. The same adenoassociated virus vectors, however, were largely ineffective in stimulating cardiac repair after myocardial infarction in adult mice, despite optimal and long-lasting transgene expression. Analysis of Notch-responsive promoters in adult cardiomyocytes showed marks of repressed chromatin and irreversible CpG DNA methylation. Induction of adult cardiomyocyte re-entry into the cell cycle with microRNAs was independent from Notch pathway reactivation. CONCLUSIONS: Notch pathway activation is crucial in regulating cardiomyocyte proliferation during the early postnatal life, but it is largely ineffective in driving cardiac regeneration in adults, because of permanent epigenetic modification at Notch-responsive promoters.