simultaneous-3d-analysis-cardiac-damage-immune-response-reperfused

Simultaneous 3D Analysis of Cardiac Damage and Immune Response in Reperfused Acute Myocardial Infarction Using Light Sheet Fluorescence Microscopy

Complex cellular interactions determine functional and structural tissue remodeling during reperfused acute myocardial infarction (repAMI). These ...

University Hospital Essen

Myocardial infarction remains the single leading cause of death worldwide. Upon reperfusion of occluded arteries, deleterious cellular mediators particularly located at the mitochondria level can be activated, thus limiting the outcome in patients. This may lead to the so-called ischemia/reperfusion (I/R) injury. Calpains are cysteine proteases and mediators of caspase-independent cell death. Recently, they have emerged as central transmitters of cellular injury in several cardiac pathologies e.g. hypertrophy and acute I/R injury.

S-nitrosation of calpains is associated with cardioprotection in myocardial I/R injury.

Cardioprotection by salvage of the infarct-affected myocardium is an unmet yet highly desired therapeutic goal. To develop new dedicated therapies, experimental myocardial ischemia/reperfusion (I/R) injury would require methods to simultaneously characterize extent and localization of the damage and the ensuing inflammatory responses in whole hearts over time. Here we present a three-dimensional (3D), simultaneous quantitative investigation of key I/R injury-components by combining bleaching-augmented solvent-based non-toxic clearing (BALANCE) using ethyl cinnamate (ECi) with light sheet fluorescence microscopy. This allows structural analyses of fluorescence-labeled I/R hearts with exceptional detail. We discover and 3D-quantify distinguishable acute and late vascular I/R damage zones. These contain highly localized and spatially structured neutrophil infiltrates that are modulated upon cardiac healing. Our model demonstrates that these characteristic I/R injury patterns can detect the extent of damage even days after the ischemic index event hence allowing the investigation of long-term recovery and remodeling processes.

Contemporaneous 3D characterization of acute and chronic myocardial I/R injury and response.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: Contemporaneous 3D characterization of acute and chronic myocardial I/R injury and response.

Childhood cancer therapy is associated with a significant risk of therapy-related cardiotoxicity. This meta-analysis aims to evaluate cardiac biomarkers for the detection of cancer therapy-related left ventricular (LV) dysfunction in childhood cancer patients.

Cardiac biomarkers for the detection of cardiotoxicity in childhood cancer-a meta-analysis.

Coronavirus disease 2019 (COVID-19) is challenging the care for cardiovascular patients, resulting in serious consequences with increasing mortality in pre-diseased heart failure patients. In the current state of the pandemic, the physiopathology of COVID-19 affecting pre-diseased hearts and the management of terminal heart failure in COVID-19 patients remain unclear. We outline the findings of a young COVID-19 patient suffering from idiopathic cardiomyopathy who was treated for acute multi-organ failure and required cardiac surgery with implantation of a temporary right ventricular and durable left ventricular assist device (LVAD). For deeper translational insights, we used in-depth tissue analysis by electron and light sheet fluorescence microscopy revealing evidence for spatial distribution of severe acute respiratory syndrome coronavirus 2 in the heart. This indicates that in-depth analysis may represent a valuable tool in understanding indistinct clinical cases. We conclude that COVID-19 directly affects pre-diseased hearts, but the consequences can be treated successfully with LVAD implantation.

Ventricular assist device for a coronavirus disease 2019-affected heart.

Cardiac immune-related adverse events (irAEs) from immune checkpoint inhibition (ICI) targeting programmed death 1 (PD1) are of growing concern. Once cardiac irAEs become clinically manifest, fatality rates are high. Cardio-oncology aims to prevent detrimental effects before manifestation of severe complications by targeting early pathological changes. We therefore aimed to investigate early consequences of PD1 inhibition for cardiac integrity to prevent the development of overt cardiac disease.

Targeting early stages of cardiotoxicity from anti-PD1 immune checkpoint inhibitor therapy.

The programmed cell death protein 1 (PD1) immune checkpoint prevents inflammatory tissue damage by inhibiting immune reactions. Understanding the relevance of cardiac PD1 signaling may provide new insights into the inflammatory events under baseline conditions and disease. Here, we demonstrate distinct immunological changes upon PD1 deficiency in healthy hearts and during reperfused acute myocardial infarction (repAMI). In PD1-deficient mice, upregulated inflammatory cytokines were identified under baseline conditions including cardiac interleukins and extracellular signal-related kinase 1/2 (ERK1/2). A murine in vivo repAMI model to determine inflammatory changes in the early phase showed downregulation of the ligand PDL1, paralleled by an endothelial injury, indicated by loss of the CD31 signal. Immunofluorescence imaging showed decreased PDL1 expression specifically in the infarct zone, highlighting an involvement in PDL1 in myocardial injury response. Pharmacological depletion of PD1 prior to repAMI did not alter the area of infarction but led to increased numbers of CD8 T cells in treated mice. We conclude that PD1/PDL1 signaling plays a significant role in healthy hearts and repAMI, emphasizing the relevance of adaptive immunity during myocardial injury. The findings highlight the risk for adverse outcomes from acute myocardial infarction in the growing group of patients receiving immune checkpoint inhibitor therapy.

PD1 Deficiency Modifies Cardiac Immunity during Baseline Conditions and in Reperfused Acute Myocardial Infarction.

Cancer survival rates have increased significantly because of improvements in therapy regimes and novel immunomodulatory drugs. Recently, combination therapies of anthracyclines and immune checkpoint inhibitors (ICIs) have been proposed to maximize neoplastic cell removal. However, it has been speculated that a priori anthracycline exposure may prone the heart vulnerable to increased toxicity from subsequent ICI therapy, such as an anti-programmed cell death protein 1 (PD1) inhibitor. Here, we used a high-dose anthracycline mouse model to characterize the role of the PD1 immune checkpoint signaling pathway in cardiac tissue using flow cytometry and immunostaining. Anthracycline treatment led to decreased heart function, increased concentration of markers of cell death after six days and a change in heart cell population composition with fewer cardiomyocytes. At the same time point, the number of PD1 ligand (PDL1)-positive immune cells and endothelial cells in the heart decreased significantly. The results suggest that PD1/PDL1 signaling is affected after anthracycline treatment, which may contribute to an increased susceptibility to immune-related adverse events of subsequent anti-PD1/PDL1 cancer therapy.

Anthracycline Therapy Modifies Immune Checkpoint Signaling in the Heart.

Light-sheet fluorescence microscopy (LSFM) can produce high-resolution tomograms of tissue vasculature with high accuracy. However, data processing and analysis is laborious due to the size of the datasets. Here, we introduce VesselExpress, an automated software that reliably analyzes six characteristic vascular network parameters including vessel diameter in LSFM data on average computing hardware. VesselExpress is ∼100 times faster than other existing vessel analysis tools, requires no user interaction, and integrates batch processing and parallelization. Employing an innovative dual Frangi filter approach, we show that obesity induces a large-scale modulation of brain vasculature in mice and that seven other major organs differ strongly in their 3D vascular makeup. Hence, VesselExpress transforms LSFM from an observational to an analytical working tool.

Rapid and fully automated blood vasculature analysis in 3D light-sheet image volumes of different organs.

CD47 is a cell surface protein controlling phagocytotic activity of innate immune cells. CD47 blockade was investigated as an immune checkpoint therapy in cancer treatment, enhancing phagocytosis of tumor cells by macrophages. Anti-CD47 treatment also reduced injury size during reperfused acute myocardial infarction (repAMI) by enhancing phagocytotic acitivity of macrophages. Little is known about the impact of CD47 blockade on neutrophils, representing the main portion of early infiltrating immune cells after repAMI. Therefore, we performed 45 min of cardiac ischemia followed by 24 h of reperfusion, observing a decreased cardiac injury size measured by triphenyl tetrazolium chloride (TTC) Evan's blue staining. We were able to detect this effect with an innovative three-dimensional method based on light sheet fluorescence microscopy (LSFM). This further allowed us a simultaneous analysis of neutrophil infiltration, showing an unaltered amount of injury-associated neutrophils with reduced cardiac injury volume from repAMI. This observation suggests modulated phagocytosis of cell debris by neutrophils. Therefore, we performed flow cytometry analysis, revealing an increased phagocytotic activity of neutrophils . These findings highlight that CD47 blockade also enhances phagocytosis of cardiac cell debris by neutrophils, which might be an additional protective effect of anti-CD47 treatment after repAMI.

CD47 blockade enhances phagocytosis of cardiac cell debris by neutrophils.

Cancer therapy-related cardiovascular toxicity (CTR-CVT) from immune checkpoint inhibitor (ICI) therapy is still incompletely characterized, and patients with pre-existing cardiovascular disease represent a particularly high-risk cohort. Valid parameters for risk stratification of these patients are missing. Neutrophil-to-lymphocyte ratio (NLR) has been shown to predict mortality and adverse events in other cardiovascular cohorts. The present study aims to examine the predictive capacity of NLR for risk stratification of patients particularly vulnerable for CTR-CVT under ICI therapy.

High neutrophil-to-lymphocyte ratio is associated with cancer therapy-related cardiovascular toxicity in high-risk cancer patients under immune checkpoint inhibitor therapy.

Post-injury dysfunction of humoral immunity accounts for infections and poor outcomes in cardiovascular diseases. Among immunoglobulins (Ig), IgA, the most abundant mucosal antibody, is produced by plasma B cells in intestinal Peyer's patches (PP) and lamina propria. Here we show that patients with stroke and myocardial ischemia (MI) had strongly reduced IgA blood levels. This was phenocopied in experimental mouse models where decreased plasma and fecal IgA were accompanied by rapid loss of IgA-producing plasma cells in PP and lamina propria. Reduced plasma IgG was detectable in patients and experimental mice 3-10 d after injury. Stroke/MI triggered the release of neutrophil extracellular traps (NETs). Depletion of neutrophils, NET degradation or blockade of NET release inhibited the loss of IgA cells and circulating IgA in experimental stroke and MI and in patients with stroke. Our results unveil how tissue-injury-triggered systemic NET release disrupts physiological Ig secretion and how this can be inhibited in patients.

Stroke and myocardial infarction induce neutrophil extracellular trap release disrupting lymphoid organ structure and immunoglobulin secretion.

Low hemoglobin values are associated with cardiotoxicity in patients with melanoma and other cancer entities receiving immune checkpoint inhibitor (ICI) therapy. However, in cancer patients under chemotherapy, enhanced incidence of cardiotoxicity events are also reported with increasing hemoglobin values. So far, the association between hemoglobin values within the normal limits and the incidence of cardiotoxicity in melanoma patients treated with ICI therapy has not been examined.

Hemoglobin is associated with cardiotoxicity in melanoma patients without anemia receiving immune checkpoint inhibitor therapy.

Reactive fibrosis is a complex response to chronic myocardial insults, contributing to heart failure progression. Fibroblast activation protein inhibitor (FAPI) PET shows promise in distinguishing active from established fibrosis. Although antifibrotic therapies may improve left ventricular (LV) function in preclinical studies, their clinical application is limited by the lack of noninvasive imaging methods to assess fibrosis regression. This study investigates the potential of FAPI PET to track the therapeutic transition of activated fibroblast activation protein (FAP)-positive fibroblasts toward a FAP-negative phenotype.  Mice were implanted with minipumps, infused with angiotensin-II/phenylephrine (Ang-II/PE) for 6 wk and scanned with Ga-FAPI-46 PET/CT longitudinally. Control mice received saline. Ga-FAPI-46 biodistribution studies were conducted at preselected time points, and FAPI uptake in the major organs was measured ex vivo. To assess the potential reversibility of the FAPI PET signal in the myocardium and liver, Ang-II/PE infusion was discontinued in a group of animals at 1 and 2 wk, respectively. LV structural and functional changes were assessed via echocardiography, tissue fibrosis via histology, and FAP expression via immunohistochemistry.  Significant Ga-FAPI-46 uptake in the myocardium of treated mice peaked at 1 wk. An increase of Ga-FAPI-46 uptake was also observed in the liver, peaking at 2 wk, and decreased significantly at 4 wk. The PET signal declined to an indiscernible level in the heart and liver early after Ang-II/PE withdrawal. Three weeks after the removal of the minipumps, the hearts of mice previously exposed to Ang-II/PE for 1 wk exhibited a significant reduction in fibrosis compared with mice that were sacrificed immediately after 1 wk of Ang-II/PE infusion, without the 3-wk recovery period. Coinjection with excess unlabeled FAPI-46 reduced uptake in the heart, liver, and kidneys. Despite an increase in LV wall thickness at 1 wk, the ejection fraction remained stable initially but dropped significantly by 4 wk.  The rapid decline in PET signal after Ang-II/PE withdrawal shows that FAPI PET effectively visualizes dynamic changes in FAP expression, making it a valuable tool for quickly assessing treatment responses targeting activated fibroblasts. The cardiac FAPI signal precedes functional myocardial changes, indicating that FAPI PET could detect early fibrosis in cardiac remodeling leading to heart failure. FAPI PET may also visualize cardiac cirrhosis, a serious complication of cardiac disorders.

Article	Year
S-nitrosation of calpains is associated with cardioprotection in myocardial I/R injury. Nitric oxide : biology and chemistry\| PubMed ID: 28392447	2017
Contemporaneous 3D characterization of acute and chronic myocardial I/R injury and response. Nature communications\| PubMed ID: 31127113	2019
Publisher Correction: Contemporaneous 3D characterization of acute and chronic myocardial I/R injury and response. Nature communications\| PubMed ID: 31217436	2019
Cardiac biomarkers for the detection of cardiotoxicity in childhood cancer-a meta-analysis. ESC heart failure\| PubMed ID: 32069386	2020
Ventricular assist device for a coronavirus disease 2019-affected heart. ESC heart failure\| PubMed ID: 33219613	2021
Targeting early stages of cardiotoxicity from anti-PD1 immune checkpoint inhibitor therapy. European heart journal\| PubMed ID: 34389849	2022
PD1 Deficiency Modifies Cardiac Immunity during Baseline Conditions and in Reperfused Acute Myocardial Infarction. International journal of molecular sciences\| PubMed ID: 35886878	2022
Anthracycline Therapy Modifies Immune Checkpoint Signaling in the Heart. International journal of molecular sciences\| PubMed ID: 37047026	2023
Rapid and fully automated blood vasculature analysis in 3D light-sheet image volumes of different organs. Cell reports methods\| PubMed ID: 37056368	2023
CD47 blockade enhances phagocytosis of cardiac cell debris by neutrophils. International journal of cardiology. Heart & vasculature\| PubMed ID: 37731517	2023
High neutrophil-to-lymphocyte ratio is associated with cancer therapy-related cardiovascular toxicity in high-risk cancer patients under immune checkpoint inhibitor therapy. Clinical research in cardiology : official journal of the German Cardiac Society\| PubMed ID: 37955712	2024
Stroke and myocardial infarction induce neutrophil extracellular trap release disrupting lymphoid organ structure and immunoglobulin secretion. Nature cardiovascular research\| PubMed ID: 39195931	2024
Hemoglobin is associated with cardiotoxicity in melanoma patients without anemia receiving immune checkpoint inhibitor therapy. International journal of cardiology. Heart & vasculature\| PubMed ID: 40851558	2025
How Rapidly Does the FAPI PET Signal Reverse Following Therapy? Assessing the FAPI PET Signal in Hypertensive Cardiac Injury and Fibrosis in Mice. Journal of nuclear medicine : official publication, Society of Nuclear Medicine\| PubMed ID: 40935615	2025

Sebastian Korste

Biography

JoVE Journal Publications

Other Publications