Molecular mechanisms in chloroquine-exposed muscle cells elucidated by combined proteomic and microscopic studies.

OBJECTIVES: Chloroquine (CQ) is an antimalarial drug with a growing number of applications as recently demonstrated in attempts to treat Covid-19. For decades, it has been well known that skeletal and cardiac muscle cells might display vulnerability against CQ exposure resulting in the clinical manifestation of a CQ-induced myopathy. In line with the known effect of CQ on inhibition of the lysosomal function and thus cellular protein clearance, the build-up of autophagic vacuoles along with protein aggregates is a histological hallmark of the disease. Given that protein targets of the perturbed proteostasis are still not fully discovered, we applied different proteomic and immunological-based studies to improve the current understanding of the biochemical nature of CQ-myopathy. METHODS: To gain a comprehensive understanding of the molecular pathogenesis of this acquired myopathy and to define proteins targets as well as pathophysiological processes beyond impaired proteolysis, utilising CQ-treated C2C12 cells and muscle biopsies derived from CQ-myopathy patients, we performed different proteomic approaches and Coherent Anti-Stokes Raman Scattering (CARS) microscopy, in addition to immunohistochemical studies. RESULTS: Our combined studies confirmed an impact of CQ-exposure on proper protein processing/folding and clearance, highlighted changes in the interactome of p62, a known aggregation marker and hereby identified the Rett syndrome protein MeCP2 as being affected. Moreover, our approach revealed-among others-a vulnerability of the extracellular matrix, cytoskeleton and lipid homeostasis. CONCLUSION: We demonstrated that CQ exposure (secondarily) impacts biological processes beyond lysosomal function and linked a variety of proteins with known roles in the manifestation of other neuromuscular diseases.

Cardiac macrophage density in Covid-19 infection: relationship to myocyte necrosis and acute lung injury.

SARS-Cov-2 infection is not limited to the respiratory tract and can involve other organs including the heart, blood vessels, kidneys, liver, gastrointestinal tract, placenta, and skin. Covid-19 patients with cardiac involvement usually have higher morbidity and mortality compared to those without cardiac involvement. The frequency and the specificity of the myocardial pathological changes in patients who die after documented infection with SARS-Cov-2 is uncertain. Macrophages can be found in the normal heart (interstitium, around the endothelial cells and in the epicardial adipose tissue), and they are considered part of the major immune cell population in the heart. In this case-control autopsy study, we compare the gross and microscopic cardiac findings, and the available clinical characteristics between a group of 10 Covid-19 decedents and a control group of 20 patients who died with non-SARS-Cov-2 severe bronchopneumonia and/or diffuse alveolar damage. The objectives of this semi-quantitative study are to study single myocyte necrosis and its relation to the strain on the heart caused by lung injury as a causative mechanism, and to study the density of myocardial and epicardial macrophages in Covid-19 hearts in comparison to the control group, and in Covid-19 hearts with single myocyte necrosis in comparison to Covid-19 hearts without single myocyte necrosis. Lymphocytic myocarditis was not identified in any of the hearts from the Covid-19 or the control group. Single myocyte necrosis is more frequent in the Covid-19 group compared to the control group, suggesting that it is unrelated to the strain on the heart caused by underlying lung injury. The density of the macrophages in the epicardium and myocardium in the hearts of the Covid-19 group is higher compared to those in the control group. The density of epicardial macrophages is higher in the Covid-19 hearts with single myocyte necrosis than in those without. These observations contribute to our increasing appreciation of the role of macrophages in the pathophysiologic response to infection by SARS-CoV-2.

Mechanism of Blood-Heart-Barrier Leakage: Implications for COVID-19 Induced Cardiovascular Injury.

Although blood-heart-barrier (BHB) leakage is the hallmark of congestive (cardio-pulmonary) heart failure (CHF), the primary cause of death in elderly, and during viral myocarditis resulting from the novel coronavirus variants such as the severe acute respiratory syndrome novel corona virus 2 (SARS-CoV-2) known as COVID-19, the mechanism is unclear. The goal of this project is to determine the mechanism of the BHB in CHF. Endocardial endothelium (EE) is the BHB against leakage of blood from endocardium to the interstitium; however, this BHB is broken during CHF. Previous studies from our laboratory, and others have shown a robust activation of matrix metalloproteinase-9 (MMP-9) during CHF. MMP-9 degrades the connexins leading to EE dysfunction. We demonstrated juxtacrine coupling of EE with myocyte and mitochondria (Mito) but how it works still remains at large. To test whether activation of MMP-9 causes EE barrier dysfunction, we hypothesized that if that were the case then treatment with hydroxychloroquine (HCQ) could, in fact, inhibit MMP-9, and thus preserve the EE barrier/juxtacrine signaling, and synchronous endothelial-myocyte coupling. To determine this, CHF was created by aorta-vena cava fistula (AVF) employing the mouse as a model system. The sham, and AVF mice were treated with HCQ. Cardiac hypertrophy, tissue remodeling-induced mitochondrial-myocyte, and endothelial-myocyte contractions were measured. Microvascular leakage was measured using FITC-albumin conjugate. The cardiac function was measured by echocardiography (Echo). Results suggest that MMP-9 activation, endocardial endothelial leakage, endothelial-myocyte (E-M) uncoupling, dyssynchronous mitochondrial fusion-fission (Mfn2/Drp1 ratio), and mito-myocyte uncoupling in the AVF heart failure were found to be rampant; however, treatment with HCQ successfully mitigated some of the deleterious cardiac alterations during CHF. The findings have direct relevance to the gamut of cardiac manifestations, and the resultant phenotypes arising from the ongoing complications of COVID-19 in human subjects.

Pathological features of COVID-19-associated myocardial injury: a multicentre cardiovascular pathology study.

AIMS: Coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been associated with cardiovascular features of myocardial involvement including elevated serum troponin levels and acute heart failure with reduced ejection fraction. The cardiac pathological changes in these patients with COVID-19 have yet to be well described. METHODS AND RESULTS: In an international multicentre study, cardiac tissue from the autopsies of 21 consecutive COVID-19 patients was assessed by cardiovascular pathologists. The presence of myocarditis, as defined by the presence of multiple foci of inflammation with associated myocyte injury, was determined, and the inflammatory cell composition analysed by immunohistochemistry. Other forms of acute myocyte injury and inflammation were also described, as well as coronary artery, endocardium, and pericardium involvement. Lymphocytic myocarditis was present in 3 (14%) of the cases. In two of these cases, the T lymphocytes were CD4 predominant and in one case the T lymphocytes were CD8 predominant. Increased interstitial macrophage infiltration was present in 18 (86%) of the cases. A mild pericarditis was present in four cases. Acute myocyte injury in the right ventricle, most probably due to strain/overload, was present in four cases. There was a non-significant trend toward higher serum troponin levels in the patients with myocarditis compared with those without myocarditis. Disrupted coronary artery plaques, coronary artery aneurysms, and large pulmonary emboli were not identified. CONCLUSIONS: In SARS-CoV-2 there are increased interstitial macrophages in a majority of the cases and multifocal lymphocytic myocarditis in a small fraction of the cases. Other forms of myocardial injury are also present in these patients. The macrophage infiltration may reflect underlying diseases rather than COVID-19.

Caspase1/11 signaling affects muscle regeneration and recovery following ischemia, and can be modulated by chloroquine.

BACKGROUND: We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ. METHODS: C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot. RESULTS: CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR. CONCLUSION: Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.