Despite accumulating data on underlying mechanisms, the influence of sex and prevalent cardio-metabolic co-morbidities on the manifestation and severity of immune checkpoint inhibitor (ICI)-induced cardiotoxicity has not been well defined. To elucidate whether sex and prevalent cardio-metabolic co-morbidities affect ICI-induced cardiotoxicity, we randomized 17-month-old male and female mice to receive control diet (CON) or high-fat diet (HFD) + L-NAME—a well-established mouse model of cardio-metabolic co-morbidities—for 17 weeks (n = 5–7), and evaluated markers of T-cell function in the spleen. As expected, HFD + L-NAME significantly increased body- and heart weight, and serum cholesterol levels, and caused no systolic dysfunction, however, led to diastolic dysfunction, cardiomyocyte hypertrophy, and increased fibrosis only in males compared to corresponding CON. Western blot analyses of splenic immune checkpoint protein levels showed differential expression depending on sex and prevalent cardio-metabolic co-morbidities, suggesting T-cell exhaustion in both sexes on HFD + L-NAME, but more pronounced in males. In a sub-study with a similar setup, we tested cardiotoxic manifestations of ICI by treating mice with anti-PD-1 monoclonal antibody (ICI) for the last 2 weeks of diet administration (n = 5–7). After 2 weeks of ICI treatment, cardiac systolic functions significantly decreased in CON, but not in HFD + L-NAME groups of both sexes compared to baseline (before ICI administration). In conclusion, in this exploratory study using aged mice, we describe for the first time that ICI-related systolic dysfunction is diminished in both sexes when obesity and hypercholesterolemia are present, possibly due to obesity-related T-cell exhaustion.

AbstractAimsHeart failure with reduced ejection fraction (HFrEF) is a leading cause of death worldwide; thus, therapeutic improvements are needed. In vivo preclinical models are essential to identify molecular drug targets for future therapies. Transverse aortic constriction (TAC) is a well‐established model of HFrEF; however, highly experienced personnel are needed for the surgery, and several weeks of follow‐up are necessary to develop HFrEF. To this end, we aimed (i) to develop an easy‐to‐perform mouse model of HFrEF by treating Balb/c mice with angiotensin‐II (Ang‐II) for 2 weeks by minipump and (ii) to compare its cardiac phenotype and transcriptome to the well‐established TAC model of HFrEF in C57BL/6J mice.MethodsMortality and gross pathological data, cardiac structural and functional characteristics assessed by echocardiography and immunohistochemistry and differential gene expression obtained by RNA‐sequencing and gene‐ontology analyses were used to characterize and compare the two models. To achieve statistical comparability between the two models, changes in treatment groups related to the corresponding control were compared (ΔTAC vs. ΔAng‐II).ResultsCompared with the well‐established TAC model, chronic Ang‐II treatment of Balb/c mice shares similarities in cardiac systolic functional decline (left ventricular ejection fraction: −57.25 ± 7.17% vs. −43.68 ± 5.31% in ΔTAC vs. ΔAng‐II; P = 0.1794) but shows a lesser degree of left ventricular dilation (left ventricular end‐systolic volume: 190.81 ± 44.13 vs. 57.37 ± 10.18 mL in ΔTAC vs. ΔAng‐II; P = 0.0252) and hypertrophy (cell surface area: 58.44 ± 6.1 vs. 10.24 ± 2.87 μm2 in ΔTAC vs. ΔAng‐II; P < 0.001); nevertheless, transcriptomic changes in the two HFrEF models show strong correlation (Spearman's r = 0.727; P < 0.001). In return, Ang‐II treatment in Balb/c mice needs significantly less procedural time [38 min, interquartile range (IQR): 31–46 min in TAC vs. 6 min, IQR: 6–7 min in Ang‐II; P < 0.001] and surgical expertise, is less of an object for peri‐procedural mortality (15.8% in TAC vs. 0% in Ang‐II; P = 0.105) and needs significantly shorter follow‐up for developing HFrEF.ConclusionsHere, we demonstrate for the first time that chronic Ang‐II treatment of Balb/c mice is also a relevant, reliable but significantly easier‐to‐perform preclinical model to identify novel pathomechanisms and targets in future HFrEF research.

Both heart failure with preserved ejection fraction (HFpEF) and non-alcoholic fatty liver disease (NAFLD) develop due to metabolic dysregulation, has similar risk factors (e.g., insulin resistance, systemic inflammation) and are unresolved clinical challenges. Therefore, the potential link between the two disease is important to study. We aimed to evaluate whether NASH is an independent factor of cardiac dysfunction and to investigate the age dependent effects of NASH on cardiac function. C57Bl/6 J middle aged (10 months old) and aged mice (24 months old) were fed either control or choline deficient (CDAA) diet for 8 weeks. Before termination, echocardiography was performed. Upon termination, organ samples were isolated for histological and molecular analysis. CDAA diet led to the development of NASH in both age groups, without inducing weight gain, allowing to study the direct effect of NASH on cardiac function. Mice with NASH developed hepatomegaly, fibrosis, and inflammation. Aged animals had increased heart weight. Conventional echocardiography revealed normal systolic function in all cohorts, while increased left ventricular volumes in aged mice. Two-dimensional speckle tracking echocardiography showed subtle systolic and diastolic deterioration in aged mice with NASH. Histologic analyses of cardiac samples showed increased cross-sectional area, pronounced fibrosis and Col1a1 gene expression, and elevated intracardiac CD68+ macrophage count with increased Il1b expression. Conventional echocardiography failed to reveal subtle change in myocardial function; however, 2D speckle tracking echocardiography was able to identify diastolic deterioration. NASH had greater impact on aged animals resulting in cardiac hypertrophy, fibrosis, and inflammation.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Momentum grant of the Hungarian Academy of Sciences Introduction Immune checkpoint inhibitors (ICI), such as monoclonal antibodies targeting programmed death ligand-1 (PD-1), revolutionized cancer treatment. However, they can lead to several cardiovascular adverse effects, ranging from mild cardiac dysfunction to fulminant, lethal myocarditis. Nevertheless, the mechanisms and risk factors behind the diverse forms of ICI-induced cardiotoxicity are not entirely understood currently. Purpose In this study, we hypothesized that a prior cardiac ischemic injury, leading to acute immune cell infiltration and activation, but without subsequent heart failure, can exacerbate the cardiotoxicity and cardiac inflammation caused by anti-PD-1 monoclonal antibodies. Furthermore, we aimed to investigate in our mouse model whether abatacept, an inhibitor of T-cell co-stimulation, can ameliorate ICI-induced cardiac effects. Methods First, we treated 8 weeks-old C57BL/6J mice with isoprenaline (ISOP group, 160 mg/kg, n = 43) or with its solvent (CON group, n = 38), to induce reversible cardiac ischemia. Validation of the ischemic injury was performed in 6 randomly selected animals from each group two days after the treatment with histology and echocardiography. After this, the animals underwent 16 weeks of recovery period, followed by echocardiography to confirm cardiac functional recovery. Here, mice from both groups were randomized to three further treatment groups: isotype control, anti-PD-1 alone, or anti-PD-1 combined with abatacept and were treated for two weeks, with three weekly intraperitoneal injections (immune checkpoint inhibition phase). Echocardiography, qRT-PCR and histology was performed to evaluate cardiac function and inflammation. Results Two days after the initial ISOP treatment, mice displayed significant reduction in ejection fraction and infiltration of inflammatory cells were seen on histology. During the recovery period, 8 mice from the ISOP group and one mouse from the CON died. After the immune checkpoint inhibition phase, mice with prior ischemic injury and anti-PD-1 treatment (ISOP + anti-PD-1 alone) showed significant cardiac dysfunction on echocardiography, while animals with abatacept treatment (ISOP+anti-PD-1+abatacept) showed normal cardiac function. With qRT-PCR and histology, increased infiltration of T-cells and macrophages was seen in the myocardium of the ISOP+anti-PD-1 treated group compared to CON animals, with increased expression of pro-inflammatory cytokines, including Il17a, Il23 and Ifng. However, no cardiac infiltration was seen in mice without prior ischemic injury and the pro-inflammatory cytokine response was less pronounced as well. Conclusions Prior cardiac ischemic injury without overt cardiac dysfunction exacerbates cardiac inflammation and cardiotoxicity induced by anti-PD-1 immune checkpoint inhibition therapy. Patients with pre-existing ischemic heart disease may be at greater risk for developing ICI-induced severe cardiac adverse events.Hypothesis

Background and PurposeImmune checkpoint inhibitors (ICI), such as anti-PD-1 monoclonal antibodies, have revolutionized cancer therapy by enhancing the cytotoxic effects of T-cells against tumours. However, enhanced T-cell activity also may cause myocarditis and cardiotoxicity. Our understanding of the mechanisms of ICI-induced cardiotoxicity is limited. Here, we aimed to investigate the effect of PD-1 inhibition on cardiac function and explore the molecular mechanisms of ICI-induced cardiotoxicity. Experimental ApproachC57BL6/J and BALB/c mice were treated with isotype control or anti-PD-1 antibody.Echocardiography was used to assess cardiac function. Cardiac transcriptomic changes were investigated by bulk RNA sequencing. Inflammatory changes were assessed by qRT-PCR and immunohistochemistry in heart, thymus, and spleen of the animals. In follow-up experiments, anti-CD4 and anti-IL-17A antibodies were used along with PD-1 blockade in C57BL/6J mice. Key ResultsAnti-PD-1 treatment led to cardiac dysfunction and left ventricular dilation in C57BL/6J mice, with increased nitrosative stress. Only mild inflammation was observed in the heart. However, PD-1 inhibition resulted in enhanced thymic inflammatory signalling, where Il17a increased most prominently. In BALB/c mice, cardiac dysfunction was not evident, and thymic inflammatory activation was more balanced. Inhibition of IL-17A prevented anti-PD-1-induced cardiac dysfunction in C57BL6/J mice. Comparing myocardial transcriptomic changes in C57BL/6J and BALB/c mice, differentially regulated genes (Dmd, Ass1, Chrm2, Nfkbia, Stat3, Gsk3b, Cxcl9, Fxyd2, and Ldb3) were revealed, related to cardiac structure, signalling, and inflammation. ConclusionsPD-1 blockade induces cardiac dysfunction in mice with increased IL-17 signalling in the thymus. Pharmacological inhibition of IL-17A treatment prevents ICI-induced cardiac dysfunction.

Interleukin-1β (IL-1β) is a key mediator of non-alcoholic steatohepatitis (NASH), a chronic liver disease, and of systemic inflammation-driven aging. IL-1β contributes to cardio-metabolic decline, and may promote hepatic oncogenic transformation. Therefore, IL-1β is a potential therapeutic target in these pathologies. We aimed to investigate the hepatic and cardiac effects of an IL-1β targeting monoclonal antibody in an aged mouse model of NASH. 24 months old male C57Bl/6J mice were fed with control or choline deficient (CDAA) diet and were treated with isotype control or anti-IL-1β Mab for 8 weeks. Cardiac functions were assessed by conventional—and 2D speckle tracking echocardiography. Liver samples were analyzed by immunohistochemistry and qRT-PCR. Echocardiography revealed improved cardiac diastolic function in anti-IL-1β treated mice with NASH. Marked hepatic fibrosis developed in CDAA-fed group, but IL-1β inhibition affected fibrosis only at transcriptomic level. Hepatic inflammation was not affected by the IL-1β inhibitor. PCNA staining revealed intensive hepatocyte proliferation in CDAA-fed animals, which was not influenced by neutralization of IL-1β. IL-1β inhibition increased hepatic expression of Pd-1 and Ctla4, while Pd-l1 expression increased in NASH. In conclusion, IL-1β inhibition improved cardiac diastolic function, but did not ameliorate features of NASH; moreover, even promoted hepatic immune checkpoint expression, with concomitant NASH-related hepatocellular proliferation.

Duchenne muscular dystrophy (DMD) is characterized by wasting of muscles that leads to difficulty moving and premature death, mainly from heart failure. Glucocorticoids are applied in the management of the disease, supporting the hypothesis that inflammation may be driver as well as target. However, the inflammatory mechanisms during progression of cardiac and skeletal muscle dysfunction are still not well characterized. Our objective was to characterize the inflammasomes in myocardial and skeletal muscle in rodent models of DMD. Gastrocnemius and heart samples were collected from mdx mice and DMDmdx rats (3 and 9–10 months). Inflammasome sensors and effectors were assessed by immunoblotting. Histology was used to assess leukocyte infiltration and fibrosis. In gastrocnemius, a tendency towards elevation of gasdermin D irrespective of the age of the animal was observed. The adaptor protein was elevated in the mdx mouse skeletal muscle and heart. Increased cleavage of the cytokines was observed in the skeletal muscle of the DMDmdx rats. Sensor or cytokine expression was not changed in the tissue samples of the mdx mice. In conclusion, inflammatory responses are distinct between the skeletal muscle and heart in relevant models of DMD. Inflammation tends to decrease over time, supporting the clinical observations that the efficacy of anti-inflammatory therapies might be more prominent in the early stage.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union’s Horizon 2020 Research and Innovation Programme under grant agreement no. 739593 “Semmelweis 250+ Kiválósági PhD Ösztöndíj” (EFOP-3.6.3-VEKOP-16-2017-00009) Gedeon Richter Talentum Foundation’s scholarship Background Immune checkpoint inhibitors have revolutionized the treatment of several form of malignancies (including metastatic melanoma) by enhancing the cytotoxic effects of T cells against cancer cells. Cancer cells evade immune surveillence by increasing the expression of T cell inhibitory molecules, also known as immune checkpoints, such as programmed cell death-1 (PD-1). Pharmacological inhibition of these molecules by immune checkpoint inhibitors (ICI) will enhance the antitumor activity of T cells. However, enhanced T cell activity may cause immune related adverse effects, including cardiotoxicity. Aims We aimed to investigate the effect of PD-1 inhibition on cardiac function and the underlying mechanisms in mice. Methods 8-10 weeks old C57BL6/J mice were treated with isotype control or anti-PD-1 antibody for 2 or 4 weeks. Cardiac function and morphology was assessed by echocardiography and histology, while the transcriptomic changes were analyzed via RNA sequencing. Nitrosative stress in the heart was assessed by immunohistochemistry and qRT-PCR. Inflammatory gene expression alterations were determined by qRT-PCR in the heart and thymus. Results Small animal echocardiography revealed cardiac dysfunction even after 2 weeks of anti-PD-1 treatment, with distinct transcriptomic changes. Nitrosative stress was found to be elevated in the myocardium due to anti-PD-1 treatment, however, histological and qRT-PCR analysis did not reveal T cell infiltration into the myocardium and only mild inflammation was seen in the heart. In contrast, inflammatory gene expression was significantly enhanced in the thymus of anti-PD-1-treated animals, where interleukin-17 showed the most prominent increase. Conclusions These findings characterize cardiac dysfunction as a form of ICI-induced cardiotoxicity, which may be mediated by increased thymic inflammatory activation and cytokine production.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union's Horizon 2020 Research and Innovation Programme, grant agreement no. 739593 New National Excellence Program of the Ministry of Human Capacities (ÚNKP-21-3-II) Introduction Heart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction, left ventricular hypertrophy, left atrial enlargement and increased serum levels of NT-pro-BNP. HFpEF accounts for 50% of heart failure cases, and typically develops in patients with metabolic comorbidities. Non-alcoholic fatty liver disease and subsequent steatohepatitis (NAFLD, NASH) is the most common chronic liver disease developing due to obesity. Although clinical/epidemiological data exists in humans showing that NASH may lead to cardiac dysfunction per se, experimental data in this regard is lacking. Purpose We aimed to evaluate whether NASH is an independent factor of cardiac dysfunction and to investigate the age-dependent effects of NASH on cardiac function. Methods Middle aged (10 months old) and aged (24 months old) C57Bl/6J mice were fed either control diet or Choline Deficient (CDAA) diet over a period of eight weeks. Young (2 months old) mice were used as a control. Before termination, echocardiography was performed. Upon termination, organs were isolated for further analysis. Results CDAA diet lead to the development of NASH in both age groups, without inducing weight gain, allowing us to investigate the direct effects of NASH on cardiac function. Left ventricular end-diastolic volume (EDV) was increased in aged animals, compared to young and middle aged animals, suggesting increased ventricular pressure. Aged animals were characterized by increased posterior wall thickness (PWT) during diastole and by increased LV mass, indicating left ventricular hypertrophy. Assessment of ejection fraction showed an age-dependent decline. Pulse wave and tissue Doppler measurements showed no difference in E/e’ ratio between the groups. However, strain analysis showed that diastolic dysfunction developed only in aged mice due to NASH. Conclusion We conclude that there were no observed changes in cardiac diastolic function due to NASH when using standard echocardiographic evaluation; however, the more sensitive method of strain analysis with 2D speckle tracking was able to show evidence of diastolic dysfunction due to NASH in aging animals.

BackgroundThe prevalence of non-alcoholic steatohepatitis (NASH) rapidly increases with associated metabolic disorders such as dyslipidemia; therefore, NASH is now considered an independent risk factor of cardiovascular diseases. NASH displays sex-linked epidemiological, phenotypical, and molecular differences; however, little is known about the background of these sex-specific differences on the molecular level.ObjectivesWe aimed to assess sex-specific differences in the expression of inflammatory and fibrotic genes, as well as in cholesterol metabolism, focusing on the expression of Pcsk9 in several tissues in a mouse model of NASH that shows the typical features of the human condition.Methods and ResultsWe fed 10-months-old male and female C57Bl/6J mice with a NASH-inducing CDAA or corresponding control diet for 8 weeks. We found that, compared to the control male mice baseline, hepatic Pcsk9 expression as well as serum PCSK9 level was significantly higher in females, and both circulating PCSK9 level and the hepatic Pcsk9 gene were markedly decreased in female mice during NASH development. Histological analysis revealed that male and female mice develop a similar degree of steatosis; however, fibrosis was more pronounced in males upon CDAA diet feeding. Strikingly, female mice have higher hepatic expression of the pro-inflammatory cytokines (Il1b, Ifng), and increased IL-1β cleavage by the NLRP3 inflammasome, and a decrease in Clec4f+ resident Kupffer cell population in comparison to males in the CDAA-fed groups.ConclusionThis is the first demonstration that there are critical sex-specific differences during NASH development in middle-aged mice regarding inflammation, fibrosis, and cholesterol metabolism and that changes in PCSK9 and IL-1β are likely important contributors to sex-specific changes during the transition to NASH.

AIMS Interleukin-1β (IL-1β) is an important pathogenic factor in cardiovascular diseases including chronic heart failure (HF). The CANTOS trial highlighted that inflammasomes as primary sources of IL-1 β are promising new therapeutic targets in cardiovascular diseases. Therefore, we aimed to assess inflammasome activation in failing hearts to identify activation patterns of inflammasome subtypes as sources of IL-1β. METHODS AND RESULTS Out of the 4 major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human heart failure regardless of the etiology (ischemic or dilated cardiomyopathy) while the NLRP1/NALP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change in HF samples. AIM2 expression was primarily detected in monocytes/macrophages of failing hearts. Translational animal models of HF (pressure or volume overload, and permanent coronary artery ligation in rat, as well as ischemia/reperfusion-induced HF in pigs) demonstrated activation pattern of AIM2 similar to that of observed in end-stages of human HF. In vitro AIM2 inflammasome activation in human THP-1 monocytic cells and human AC16 cells was significantly reduced by pharmacological blockade of pannexin-1 channels by the clinically used uricosuric drug probenecid. Probenecid was also able to reduce pressure overload-induced mortality and restore indices of disease severity in a rat chronic HF model in vivo. CONCLUSIONS This is the first report showing that AIM2 and NLRC4 inflammasome activation contribute to chronic inflammation in heart failure and that probenecid alleviates chronic HF by reducing inflammasome activation. The present translational study suggests the possibility of repositioning of probenecid for HF indications.

The administration of immune checkpoint inhibitors (ICIs) often leads to immune-related adverse events. However, their effect on auditory function is largely unexplored. Thorough preclinical studies have not been published yet, only sporadic cases and pharmacovigilance reports suggest their significance. Here we investigated the effect of anti-PD-1 antibody treatment (4 weeks, intraperitoneally, 200 μg/mouse, 3 times/week) on hearing function and cochlear morphology in C57BL/6J mice. ICI treatment did not influence the hearing thresholds in click or tone burst stimuli at 4–32 kHz frequencies measured by auditory brainstem response. The number and morphology of spiral ganglion neurons were unaltered in all cochlear turns. The apical-middle turns (<32 kHz) showed preservation of the inner and outer hair cells (OHCs), whilst ICI treatment mitigated the age-related loss of OHCs in the basal turn (>32 kHz). The number of Iba1-positive macrophages has also increased moderately in this high frequency region. We conclude that a 4-week long ICI treatment does not affect functional and morphological integrity of the inner ear in the most relevant hearing range (4–32 kHz; apical-middle turns), but a noticeable preservation of OHCs and an increase in macrophage activity appeared in the >32 kHz basal part of the cochlea.